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GHANA’S MANGROVE WETLAND 
ENDOPHYTIC FUNGI: CYTOTOXICITY, ANTI-
PLASMODIAL AND APOPTOTIC ACTIVITY OF 
QUINOLACTACIN A1/A2, CITRINADIN A AND 
BUTRECITRINADIN 
 
 
 
BY 
 
KOFI BAFFOUR-AWUAH OWUSU 
 
 
 
THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, 
LEGON, IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR 
THE AWARD OF A MASTER OF PHILOSOPHY DEGREE IN 
CHEMISTRY 
 
 
 
JULY, 2015
 
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DECLARATION 
I, Kofi Baffour-Awuah Owusu, declare that the work contained in this thesis was 
undertaken solely by me under supervision and has neither wholly nor partially been 
presented elsewhere for another degree. 
 
 
………………………………………. 
Kofi Baffour-Awuah Owusu (BSc.) 
10442006 
 
 
………………………………………. 
Kwaku Kyeremeh (PhD) 
(PRINCIPAL SUPERVISOR) 
 
 
………………………………………. 
Mark Ofosuhene (PhD) 
(Co-Supervisor) 
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ABSTRACT 
Marine-derived endophytic fungi isolated from mangrove plants are an important and 
novel resource of natural bioactive compounds with their potential applications in 
medicine, agriculture and food industry. There are many instances that seem to 
suggest that, some of the novel metabolites previously isolated from certain species of 
marine plants and invertebrates are actually metabolites of marine-derived endophytic 
fungi. In this project, chemical screening of mangrove plantsConocarpus erectus, 
Laguncularia racemosa and Rhizophora racemosacollected along the banks of the 
River Butre showed that the compound 14S-bromo-1S-hydroxy-1,2,13,14-
tetrahydrosphaerococenol A is not a biosynthetic product of the red alga 
Sphaerococcus coronopifolius but a product of possible endophytic fungi living inside 
the alga tissues. The compound 3-(phenethylamino)demethyl(oxy)aaptamine and its 
derivatives like aaptamine are not necessarily the biosynthetic products of the sea 
sponge Aaptos aaptos but originate from endophytic fungi within the tissues of the 
sponge. The mycospurine amino acid N-methylpalythine-serine originally deemed to 
be the metabolites of the stony coral Pocillopora eydouxicould actually be the 
biosynthetic products of marine-derived endophytic fungi. 
Mangrove plants sampled along the banks of River Butre were investigated for new or 
novel secondary metabolites. The HRESI/HPLC-DAD-MSn dereplication technique 
was employed in identifying and isolating new secondary metabolites from the 
mangrove plants. Out of twenty-four (24) marine-derived endophytic fungi isolated 
from various parts of three major mangrove plants growing in and along the banks of 
the River Butre in the Western Region of Ghana, six (6) different species were 
selected for chemical profiling, prioritization, identification and subsequent isolation 
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of compounds. Measurement and analysis of HRESI/HPLC-DAD-MSn data of the 
small scale culture of these six (6) species led to the prioritization of Penicillum sp. 
BRS2A-AR2. Penicillum sp. BRS2A-AR2 was prioritized due to its ability to make a 
wide range of interesting secondary metabolites with biosynthesis prowess and 
ingenuity. 
The HRESI/HPLC-DAD-MSn analysis of Penicillum sp. BRS2A-AR2 was prioritised 
for further investigation because it produced metabolites which did not give any 
relevant hit when their masses were entered in the marine natural product based 
commercial database MarinLit, AntiMarin and Antibase database for novelty. The 
structures of these metabolites were elucidated by 1D and 2D NMR data 
interpretation. 
Quinolactacin A1/A2 was isolated with m/z of 270.1362. Citrinadin A and a new 
compound Butrecitrinadin were also isolated with m/z of 625.3962 and 681.4226 
respectively. Citrinadin A and Butrecitrinadin were virtually identical with similar 
mass fragmentation pattern, eluting at the same time, having same retention time and 
collected together. 
Biological activity studies on the compounds and other compounds from the research 
group showed the anti-proliferative ability of the two steroidal compounds to inhibit 
human prostate cancer cell line with IC50 of 5.58 and 5.28 µM. Butrepyrazinone, 
Citrinadin Aand Butrecitrinadin showed cytotoxicity against cell lines tested. 
Quinolactacins isolated also showed anti-plasmodial activity with IC50 of 24.80 µM 
and also, showed apoptotic activity via the loss of mitochondrion membrane potential 
of the plasmodium parasite. 
 
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DEDICATION 
This thesis is dedicated to my parents Mr. & Mrs. Owusu-Berko for teaching me the 
value of hard work and perseverance. Your prayers, motivation, love and support 
inspired me in the course of the study. 
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ACKNOWLEDGEMENTS 
I am grateful to the almighty God for giving me life, strength and knowledge to 
undertake this study. 
I am most grateful to my supervisors, Dr. Kwaku Kyeremeh and Dr. Mark Ofosuhene 
for their support, mentorship, guidance, encouragement, sacrifice and immense 
contribution during this study. I thank them for their constructive comments and 
criticism which helped to improve the quality of this thesis. I am grateful to them also 
for their wholeheartedness with which they always received me whenever I needed 
assistance. I appreciate the informative lessons each of them gave me on the art of 
writing scientific manuscripts. 
I appreciate the support I had from Messrs Kojo Sakyi Acquah, Cephas Ziwu, Roland 
Gyampoh and all the members of the MAPRELOG research group as well as my 
colleagues, and the entire staff, both teaching and non- teaching, of the Chemistry 
Department, University of Ghana for their assistance. 
I am greatly indebted to Dr. Regina Appiah-Opong, head of the Department of 
Clinical Pathology, Noguchi Memorial Institute for Medical Research (NMIMR) for 
permitting to study whilst working on the JICA/JST Medicinal Plants Project. My 
thanks also goes to Dr. T. Uto of the Nagasaki International University, Japan for 
providing the panel of human cancerous cell lines used for the study. I appreciate Dr. 
Mitsuko Suzuki, Dr. Patrick Tsouh Fokou, Mr. Jeffrey Agyapong and Ms. Abena 
Adomah Kissi Twum all of NMIMR for their support and scientific guidance in the 
course of the study and also to the staff of the Department of Clinical Pathology, 
NMIMR. 
I am very grateful to Professor Marcel Jaspars and researchers at the Marine Bio-
discovery Center, Chemistry Department, University of Aberdeen, Scotland, for their 
invaluable assistance in acquiring spectrometric and spectroscopic data and also to the 
Royal Society-Leverhulme Trust Africa Award AA090088 for financial assistance. 
Finally and most importantly, I thank my beloved family for their continuous support, 
prayers and encouragement in the course of my work and studies. 
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TABLE OF CONTENTS 
DECLARATION ............................................................................................................ i 
ABSTRACT ................................................................................................................... ii 
DEDICATION .............................................................................................................. iv 
ACKNOWLEDGEMENTS ........................................................................................... v 
TABLE OF CONTENTS .............................................................................................. vi 
LIST OF FIGURES ....................................................................................................... x 
LIST OF TABLES ..................................................................................................... xiii 
LIST OF SCHEMES................................................................................................... xiv 
LIST OF PLATES ....................................................................................................... xv 
LIST OF ABBREVIATIONS ..................................................................................... xvi 
CHAPTER ONE ............................................................................................................ 1 
1.0 INTRODUCTION ............................................................................................... 2 
1.1 Problem Statement ............................................................................................... 8 
1.2 Overall Goal ......................................................................................................... 9 
1.3 Hypothesis............................................................................................................ 9 
1.4 Objectives .......................................................................................................... 10 
CHAPTER TWO ......................................................................................................... 12 
2.0 LITERATURE REVIEW .................................................................................. 13 
2.1 Mangrove wetlands ............................................................................................ 13 
2.2 Brief introduction to mangrove plant endophytic fungi .................................... 15 
2.3 Bioactive compounds from marine-derived endophytic fungi .......................... 18 
2.3.1 Alkaloids isolated from marine-derived fungi ............................................ 19 
2.3.2 Peptides isolated from marine-derived fungi .............................................. 25 
2.3.3 Polyketides isolated from marine-derived fungi ......................................... 28 
2.3.4 Steroids isolated from marine-derived fungi .............................................. 31 
2.3.5 Terpenes isolated from marine-derived fungi ............................................. 33 
2.4 Terminologies in bioactivity measurements ...................................................... 35 
2.4.1 Half maximal inhibitory concentration (IC50) ............................................. 35 
2.4.2 Half maximal effective concentration (EC50) ............................................. 36 
2.4.3 Half maximal Effective dose (ED50) ........................................................... 36 
2.4.4 Lethal dose for 50% (LD50) ........................................................................ 37 
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2.4.5 Lethal concentration for 50% (LC50) .......................................................... 38 
2.4.6 Minimum inhibitory concentration (MIC) .................................................. 38 
2.4.7 Minimum bactericidal concentration (MBC) .............................................. 38 
2.4.8 Growth inhibition concentration for 50% (GI50) ........................................ 40 
2.5 Malaria ............................................................................................................... 40 
2.5.1 Introduction, statistics and interventions .................................................... 40 
2.5.2 Life cycle of the plasmodium parasites ...................................................... 42 
2.5.3 Antimalarials in current use ........................................................................ 45 
2.5.4 Development of drug-resistant plasmodium parasites ................................ 47 
2.5.5 Preliminary screening of antimalarial activity ............................................ 48 
CHAPTER THREE ..................................................................................................... 49 
3.0 MATERIALS AND METHODS ................................................................... 50 
3.1 Mangrove plant sample collections ................................................................... 50 
3.2 Preparation of culture media .............................................................................. 55 
3.2.1 Malt extract solid media .............................................................................. 55 
3.2.2 Malt extract liquid media ............................................................................ 55 
3.3.3 Treatment of mangrove plant and culture of endophytic fungi .................. 55 
3.3.4 Isolation of pure endophytic fungi .............................................................. 56 
3.3.5 Small scale culture of pure endophytic fungi ............................................. 56 
3.3.6 Chemical profiling and prioritization of pure endophytic fungi ................. 57 
3.3.7 Large scale culture of high priority endophytic fungi ................................ 59 
3.3.8 Solvent partitioning by a modification of Kupchan’s method .................... 59 
3.3.9 Purification of BRS2A-AR2-FM and isolation of compounds................... 61 
3.3.10 1D and 2D NMR analysis of Quinolactacin A1 and A2 and Citrinadin A
.............................................................................................................................. 62 
3.3 Biological activity test ....................................................................................... 62 
3.3.1 Chemicals and reagents............................................................................... 62 
3.3.2 Compounds tested for possible biological activity ..................................... 63 
3.3.3 Cell lines tested in the cytotoxicity assay ................................................... 65 
3.3.4 Preparation of compounds for bioactivity testing ....................................... 65 
3.3.5 Cytotoxicity studies .................................................................................... 66 
3.3.6 Anti-malaria activity study ......................................................................... 69 
3.3.7 Apoptosis study ........................................................................................... 72 
3.3.8 Anti-buruli ulcer activity assay ................................................................... 73 
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CHAPTER FOUR ........................................................................................................ 74 
4.0 RESULTS AND DISCUSSION ........................................................................ 75 
4.1 Dereplication of marine-derived endophytic fungi crude extracts .................... 75 
4.1.1 Dereplication of crude extracts of BRS3A-T2 ............................................ 76 
4.1.2 Dereplication of crude extracts of BRS2A-AR .......................................... 80 
4.1.3 Dereplication of crude extracts of BRS1A-B ............................................. 82 
4.1.4 Dereplication of crude extracts of BUSUA2B-T ........................................ 86 
4.1.5 Dereplication of crude extracts of BUSUA2B-F ........................................ 88 
4.1.6 Dereplication of crude extracts of BRS2A-AR2 ........................................ 90 
4.2 Structure determination of Citrinadin A and Butrecitrinadin ............................ 93 
4.3 Structure determination of Quinolactacins A1 and A2 .................................... 104 
4.3.1 Acid-base rapid epimerization of quinolactacins ...................................... 109 
4.4 Anti-proliferative activity of compounds......................................................... 110 
4.5 Anti-Plasmodial Activity of Compounds......................................................... 115 
4.5.1 Anti-Plasmodial activity ofcompounds to chloroquine sensitive ............. 115 
4.6 Plasmodial Apoptotic Activity of Quinolactacins A1/A2 ............................... 117 
4.7 Anti-Buruli ulcer Activity of Compounds ....................................................... 120 
CHAPTER FIVE ....................................................................................................... 122 
5.0 CONCLUSION AND RECOMMENDATIONS ............................................ 123 
5.1 Conclusion ....................................................................................................... 123 
5.2 Recommendations ............................................................................................ 124 
REFERENCES .......................................................................................................... 125 
APPENDICES ........................................................................................................... 148 
Appendix 1a: 13CNMR spectrum of quinolactacins .............................................. 148 
Appendix 1b: HSQC spectrumof quinolactacins ................................................... 149 
Appendix 1c:  1H NMR spectrum of quinolactacins ............................................. 150 
Appendix 1d: 1H-1H COSY spectrum of quinolactacins ....................................... 151 
Appendix 1e: 1H-1H COSY spectrum of quinolactacins ....................................... 152 
Appendix 1f: 1H-1H COSY spectrum of quinolactacins ........................................ 153 
Appendix 1g: HMBC spectrum of quinolactacins ................................................. 154 
Appendix 1h: HMBC spectrum of quinolactacins ................................................. 155 
Appendix 1i: HMBC spectrum of quinolactacins .................................................. 156 
Appendix 1j: HMBC spectrum of quinolactacins .................................................. 157 
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Appendix 1k: HMBC spectrum of quinolactacins ................................................. 158 
Appendix 1l: 1H-1H TOCSY spectrum quinolactacin ........................................... 159 
Appendix 1m: 1H-1H TOCSY spectrum quinolactacin ......................................... 160 
Appendix 2a:  HSQCspectrum of citrinadins ........................................................ 161 
Appendix 2b:  HSQCspectrum of citrinadins ........................................................ 162 
Appendix 2c: 1H NMR spectrum of citrinadins ..................................................... 163 
Appendix 2d:  1H NMR spectrum of citrinadins ................................................... 164 
Appendix 2e: 1H-1H COSY spectrum of citrinadins.............................................. 165 
Appendix 2f: 1H-1H COSY spectrum of citrinadins .............................................. 166 
Appendix 2g: 1H-1H COSY spectrum of citrinadin ............................................... 167 
Appendix 2h: HMBC spectrum of quinolactacin .................................................. 168 
Appendix 2i: HMBC spectrum of citrinadins ........................................................ 169 
Appendix 2j: HMBC spectrum of citrinadins ........................................................ 170 
Appendix 2k: HMBC spectrum of citrinadins ....................................................... 171 
Appendix 2l: HMBC spectrum of citrinadins ........................................................ 172 
Appendix 2m: HMBC spectrum of citrinadins ...................................................... 173 
Appendix 2n: HMBC spectrum of citrinadins ....................................................... 174 
Appendix 2o: TOCSY spectrum of citrinadin ....................................................... 175 
Appendix 2p: HMBC spectrum of citrinadins ....................................................... 176 
Appendix 2r: TOCSY spectrum of citrinadins ...................................................... 178 
Appendix 2s:  1H-1H TOCSY spectrum of citrinadin ............................................ 179 
Appendix 2t:  1H NMR spectrum of citrinadin ...................................................... 180 
 
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LIST OF FIGURES 
Figure 1.1: All new approved drugs over a period of thirty years 1981-2010. .............. 2 
Figure 2.1: Illustration of Half Inhibitory Concentration (IC50). ................................. 35 
Figure 2.2: Illustration of Half Maximal Effect Concentration (EC50). ....................... 36 
Figure 2.3: Illustration of Effective Dose (ED50)......................................................... 37 
Figure 2.4: Illustration of Lethal Dose (LD50). ............................................................ 37 
Figure 2.5: Illustration of Minimum Inhibitory Concentration (MIC) and Minimum 
Bactericidal Concentration (MBC). ............................................................................. 39 
Figure 2.6: Life cycle of Plasmodium parasite showing the stages at which current 
drugs act. ...................................................................................................................... 44 
Figure 3.1: Map of Ghana showing Butre River running through communities. ........ 51 
Figure 3.2: Illustration of the HRESI/HPLC-DAD-MSn spectrum.............................. 58 
Figure 3.3: Flow chart for the isolation of BRS2A-AR2-FD and FM. ........................ 60 
Figure 4.1: HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of BRS3A-T2 
liquid culture in malt extract. ....................................................................................... 78 
Figure 4.2: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia of 
BRS3A-T2 cultured in malt extract. ............................................................................ 78 
Figure 4.3: HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of BRS2A-
AR liquid culture in malt extract. ................................................................................ 81 
Figure 4.4: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia of 
BRS2A-AR cultured in malt extract. ........................................................................... 81 
Figure 4.5: HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of BRS1A-B 
liquid culture in malt extract. ....................................................................................... 84 
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Figure 4.6: MSn fragmentation pattern of m/z 331.1321 seen in both the ethyl acetate 
and methanol extracts of BRS1A-B. ............................................................................ 84 
Figure 4.7: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia of 
BRS1A-B cultured in malt extract. .............................................................................. 85 
Figure 4.8: HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of 
BUSUA2B-T liquid culture in malt extract. ................................................................ 87 
Figure 4.9: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia of 
BUSUA2B-T cultured in malt extract. ........................................................................ 88 
Figure 4.10: HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of 
BUSUA2B-F liquid culture in malt extract. ................................................................ 89 
Figure 4.11: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia of 
BUSUA2B-F cultured in malt extract.......................................................................... 90 
Figure 4.12: HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of BRS2A-
AR2 liquid culture in malt extract. .............................................................................. 92 
Figure 4.13: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia of 
BRS2A-AR2 cultured in malt extract. ......................................................................... 92 
Figure 4.14: Substructures obtained solely from the analysis of 1H-1H-gCOSY data of 
citrinadin A. ................................................................................................................. 94 
Figure 4.15: Full 1H-1H-gCOSY data of citrinadin A.................................................. 95 
Figure 4.16: Full HMBC correlation data of citrinadin A provides extension for the 
substructures obtained from 1H-1H-gCOSY data analysis........................................... 95 
Figure 4.17: Full 2D TOCSY correlations of citrinadin A. ......................................... 96 
Figure 4.18: HRESI/HPLC-DAD-MSn profile of citrinadin A and butrecitrinadin. ... 98 
Figure 4.19: Mass fragmentation pattern of citrinadin A. ........................................... 99 
Figure 4.20: Mass fragmentation pattern of butrecitrinadin ...................................... 101 
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Figure 4.21: HMBC spectrum of butrecitrinadin ....................................................... 103 
Figure 4.22: 1H NMR spectrum of butrecitrinadin .................................................... 104 
Figure 4.23: Substructures obtained solely from the analysis of 1H-1H-gCOSY data of 
quinolactacin A1 and A2. .......................................................................................... 106 
Figure 4.24: Full 1H-1H-gCOSY data of quinolactacin A1/A2. ................................ 106 
Figure 4.25: Full HMBC correlation data of quinolactacin A1/A2 provides extension 
for the substructures obtained from 1H-1H-gCOSY data analysis. ............................ 107 
Figure 4.26: Full 2D TOCSY correlations of quinolactacin A1/A2. ......................... 107 
Figure 4.27: Acid-base epimerization interconversions of quinolactacin A2 to A1.. 110 
Figure 4.28 A-F: Anti-proliferative activities of pure compounds and standard on 
human cancer cell lines. ............................................................................................. 113 
Figure 4.29: Anti-plasmodial activity of compounds on 3D7 plasmodial strain.. ..... 116 
Figure 4.30 A-F: Effect of quinolactacin on the loss of mitochondrion membrane 
potential...................................................................................................................... 119 
Figure 4.31: Apoptotic effect of quinolactacins on the plasmodial strain. ................ 120 
 
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LIST OF TABLES 
Table 3.1: Name and structure of compounds studied for biological activity ............. 64 
Table 4.1: 1H and 13C NMR data of citrinadins in CD3OD. ........................................ 97 
Table 4.2:1H and 13C NMR data of quinolactacins in CD3OD. ................................ 108 
Table 4.3: Cell growth-inhibitory potencies of pure compounds expressed as IC50 
values. ........................................................................................................................ 114 
Table 4.4: Effective concentration of the six compounds on the 3D7 plasmodial strain.
.................................................................................................................................... 115 
Table 4.5 Minimum Inhibition Concentration of the compounds on Mycobacterium 
ulcerans MN209. ........................................................................................................ 121 
 
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LIST OF SCHEMES 
Scheme 3.1: Conversion of yellow MTT dye to dark blue formazan by cellular 
mitochondrion reductase. ............................................................................................. 67 
Scheme 4.1: Proposed structures for the mass spectrometry fragments of 14S-bromo-
1S-hydroxy-1,2,13,14-tetrahydrosphaerococenol A. ................................................... 77 
Scheme 4.2: Biosynthesis of 14S-bromo-1S-hydroxy-1,2,13,14-
tetrahydrosphaerococenol. ........................................................................................... 79 
Scheme 4.3: Biosynthesis of 3-(phenethylamino)demethyl(oxy)aaptamine. .............. 86 
Scheme 4.4: Proposed structures for the mass fragments of citrinadin A ................. 100 
Scheme 4.5: Proposed structures for the mass fragments of butrecitrinadin ............. 102 
 
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LIST OF PLATES 
Plate 3.1: Pictures of three mangrove plants particularly common in the Western 
Region of Ghana. ......................................................................................................... 52 
Plate 3.2: Boat riding on the Butre River to the site where samples were collected. .. 53 
Plate 3.3: Collection and Identification of mangrove plants along the Butre River. ... 53 
Plate 3.4: Collection and bagging of mangrove plants along Butre River................... 54 
Plate 3.5: Butre River running through the mangrove where plants were collected. .. 54 
Plate 3.6: Small scale fermentation broth of BUSUA 2A-L. ....................................... 57 
Plate 3.7: Sephadex LH-20 size exclusion chromatography of BRS2A-AR2-FM with 
fractions starting to separate into different molecular weights. ................................... 61 
Plate 3.8: Cancer cells in incubation at the temperature of 37 °C. .............................. 66 
Plate 3.9: 96 well plate showing conversion of yellow MTT to Formazan ................. 69 
Plate 4.1: Pure endophytic fungi BRS3A-T2 isolated from the trunk of an indigenous 
mangrove plant sampled at Butre River....................................................................... 76 
Plate 4.2: Pure endophytic fungi BRS2A-AR isolated from the aerial root of an 
indigenous mangrove plant sampled at Butre River. ................................................... 80 
Plate 4.3: Pure endophytic fungi BRS1A-B isolated from the bud of an indigenous 
mangrove plant sampled at Butre River....................................................................... 83 
Plate 4.4: Pure endophytic fungi BUSUA 2B-T isolated from the trunk of an 
indigenous mangrove plant sampled at Busia beach. .................................................. 87 
Plate 4.5: Pure endophytic fungi BUSUA 2B-F isolated from the fruit of an 
indigenous mangrove plant sampled at Busia beach. .................................................. 89 
Plate 4.6: Pure endophytic fungi BRS2A-AR2 isolated from the aerial root of an 
indigenous mangrove plant sampled at Butre River. ................................................... 91 
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LIST OF ABBREVIATIONS 
AIDS    Acquired Immune Deficiency Syndrome 
COSY    Correlation Spectroscopy 
CD    Circular dicroism 
DCM    Dichloromethane 
DMSO    Dimethyl sulfoxide 
DNA    Deoxyribonucleic acid 
EC50    Median Effective Concentration 
ED50     Median Effective Dose 
EtOAc    Ethyl acetate 
ESMS    Electrospray Mass Spectrometry 
FD    Dichloromethane Fraction 
FH    Hexane Fraction 
FM    50% aqueous methanol fraction  
G8    Group of Eight Nations 
GDP    Gross Domestic Product 
GI50    Median Growth Inhibition  
GPS    Global Positioning System 
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HIV    Human Immunodeficiency Virus 
HMBC   Heteronuclear Multiple Bond Correlation 
HPLC     High Performance Liquid Chromatography  
HSQC    Heteronuclear Single Quantum Coherence 
HTS     High throughput screening 
IC50    Median Inhibition Concentration 
ICPMS   Inductively Coupled Mass Spectrometry 
LC    Liquid Chromatography 
LC50    Median Lethal Concentration 
LD50    Median Lethal Dose 
MeOH    Methanol 
MBC    Minimum Bactericidal Concentration 
MIC    Minimum Inhibition Concentration 
MRSA    Methicillin-Resistant Staphylococcus aureus 
MS    Mass Spectrometry 
NMR     Nuclear Magnetic Resonance Spectroscopy 
PDA    Photodiode Array Detector 
PTM    Polycyclic Tetramic Acid Macrolactams  
RNA    Ribonucleic acid 
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SASW    Sterile Artificial Sea Water 
TCE    Total Crude Extract 
TOCSY   Total Correlation Spectroscopy 
UV    Ultraviolet 
WB    Water/Sec-butanol fraction 
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CHAPTER ONE 
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1.0 INTRODUCTION 
A review by Newman and Cragg in 2012 provided a solid proof with appropriate 
statistics of the fact that natural products play an indubitable role in drug discovery at 
all disease frontiers. Within the period 1981-2010, close to 61% of our approved 
drugs had some kind of a linkage to natural product sources.1 
 
 
Figure 1.1: All new approved drugs over a period of thirty years 1981-2010. 
 
However, the percentages change when individual disease areas are considered 
because, while natural product sources provide several leads every year for some 
diseases, in some disease areas leads from natural sources are particularly rare.1For 
example, natural products have been very successful in the discovery of anticancer 
drugs with more than 128 new chemical entities approved within 1981-2010 
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period.1In sharp contrast, no natural product drugs were approved as antihistamines, 
diuretics or hypnotics within the same study period.1 
Amongst all the parasitic diseases that are endemic to most developing countries 
malaria is the one that is most researched worldwide.2-4 Other parasitic diseases like 
African sleeping sickness, Chagas disease, leishmaniasis, cryptosporidiosis, 
amoebiasis etc have very low research inputs worldwide.5 Malaria continues to be a 
major threat in the world with 500 million infections and 1 million deaths every year. 
Currently, approximately 40% of the world’s population reside in areas of active 
malaria transmission with the disease and its symptoms most severe in children and 
pregnant women.6-13 A licensed vaccine for malaria has not materialised and although 
chloroquine, the first synthetic drug developed as an antimalarial with inspiration 
from the naturally occurring quinine provided an almost magical cure for over 30 
years, the emergence of the chloroquine-resistant parasites has made it virtually 
ineffective in most parts of the world.14-18 
 
 
 
Currently, the plant-derived antimalarial artemisinin is the only available drug that is 
globally effective against malarial parasites.19Although several new drugs have been 
introduced in the past 30 years, widespread or isolated cases of resistance indicate that 
their window of effectiveness is most definitely limited.20, 21 
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Hence, there is the need to discover new antimalarials and the previous successes of 
chloroquine a synthetic derivative of quinine and artemisinin and its synthetic 
derivatives suggests that nature could prove to be the best source of our future 
antimalarials.22-26 Our future antimalarials must be: 
 effective and promiscuous by acting through more than one mechanisms of 
action 
 low cost and affordable to the poorest inhabitants on this planet 
 novel chemotypes that are entirely different from the currently available 
structures. 
Microbes mainly bacteria and fungi have provided a phenomenal contribution to the 
health and well-being of people throughout the world. In addition to producing many 
post-biosynthetically modified primary metabolites such as amino acids, vitamins and 
nucleotides, they have a tremendous ability to produce secondary metabolites which 
have become an important constituent of pharmaceuticals on the market and provide 
industry and agriculture with many essential products.27-30 However, a comparison of 
the success rate of bacteria and fungi shows a big skew where discovery and 
subsequent approval of antifungal drugs have been relatively rare.31-35Only three 
classes of molecules are currently used to treat invasive, life-threatening fungal 
infections in clinical practice. Out of these three, two of them azolesand polyenes 
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were already in the clinic before 1980. From 1980-to date, only one new class of 
antifungal have emerged in the clinic and these are known as the echinocandins.35 
 
 
 
 
 
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Three main factors account for this very slow pace in the development and approval 
of new antifungals and these are as outlined below. 
 The first fundamental and philosophical challenge is that, fungal pathogens 
unlike bacteria are very closely related to their host which in this case 
represents humans. Many inherent physiological, biochemical and cell 
biological processes are conserved from fungi to humans. The direct 
implication is that, many small molecules that are toxic to yeast are likewise 
toxic to humans through similar mechanisms of action. Prototype antifungals 
must possess a high level of selectivity and target processes that are only 
unique to fungi of which there are only a few. Fortunately, such high 
selectivity also finds application in other drug discovery pipe lines including 
anticancer and antiparasitic drug development. 
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 Secondly, the evaluation of new antifungals has challenges directly related 
to the effective design of the clinical trials stages as a result of the higher risks 
involved.  
 Thirdly, the two challenges mentioned here add to the routine fundamental 
challenges including well-documented scientific, economic and regulatory 
factors that are inherent to the discovery of anti-infectives in general.35-37 
Notwithstanding the problems outlined above, invasive life-threatening fungal 
infections are an important cause of mobidity and mortality, particularly for patients 
with heavily compromised immune function like those leaving with HIV-AIDS and 
primary immune deficiency or those that are on some kind of cancer chemotherapy, 
immune-modulatory medications or patients of hematologic and solid organ 
transplantation.35 Hence, the need to identify new or novel antifungal chemical 
structures that can also find application as anticancer or antiparasitic drugs is eminent 
and cannot be overemphasized. 
However, as a result of the difficulties already outlined herein, very potent strategies 
are needed in order to ensure that in the next 5-10 years, new or novel chemical 
entities will emerge in the drug discovery pipeline for the treatment of yeast 
infections. 
A look at the currently available drugs shows two natural products derived molecular 
entities polyenes (bacteria derived) and echinocandins (fungi derived) versus one 
entirely synthetic chemical class, the azoles. Hence, detailed scout of new natural 
sources (niches or habitats) could yield new or novel antifungal drugs possibly with 
some anticancer or antiparasitic activity.38-41The exact natural source to be 
investigated must also be chosen in such a way as to maximise the frequency of 
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obtaining hits which could evolve into leads and eventually become drugs. The two 
main sources where maximised efforts are most likely to yield good results are 
bacteria and the fungi themselves. 
The philosophy behind interests in these two is no different from the current 
philosophy that drives the discovery of anti-infectives in general. Competition for 
substrates amongst these microbes forces them to make secondary metabolites that 
possess great selectivity and target very specific physiological, biochemical and 
cellular processes of some microbes. These molecules when recruited directly or 
altered through semi-synthesis can provide novel leads for the development of new 
antifungals. 
This thesis represents an attempt using Ghanaian mangrove plant-derived endophytic 
fungi to fulfil the need to feed the current drug discovery pipeline with antifungals, 
antiparasitics and anticancer drug prototypes and ensure that, these molecules will in 
the next 5-10 years provide new treatments for clinical practice. Several different 
kinds of endophytic fungi were isolated from a collection of mangrove plants/plant 
parts that were collected along the banks of the Butre River in the Western Region of 
Ghana. Small scale culture and subsequent screening of their metabolites using high 
resolution electrospray ionization liquid chromatography tandem mass spectrometry 
(HRESI-LCMSn) led to the prioritization and detailed chemical investigation of one 
species,Penicillium sp. BRS2A-AR2. 
1.1 Problem Statement 
Current medical practices have been very successful to curtail a majority of the 
diseases that previously made life on earth very difficult. However, currently there are 
many problems that seem to suggest a necessary speed-up in the discovery of novel 
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drug entities with entirely different mechanisms of action. There are still diseases that 
have no cures and this comes amidst the current widespread of new diseases, the 
spreading geographically of known diseases with increased virulence alongside the 
development of strong resistance to treatment by known diseases. The current world-
wide philosophy is to intensify the search of new un- or underexplored extreme 
environments or habitats that harbour novel organisms capable of producing novel 
chemistry. Ghana represents one of the world’s biodiversity hotspots with several un- 
or underexplored extreme habitats where explorations on for example, mangrove 
plant derived endophytic fungi for possible future drug prototypes is an entirely new 
subject but has a lot of potential to provide future drug leads. 
1.2 Overall Goal 
The overall goal of this project is to examine the ‘talent profile’ of Ghanaian marine 
endophytic fungi to produce new or novel secondary metabolites that possess the 
ability to act as future drugs for the treatment of yeast and parasite infections or 
cancer. 
1.3 Hypothesis 
The Western region of Ghana is characterized by several mangrove patches that are 
un- or underexplored with respect to their novel microbial species. Ghanaian 
mangrove plants harbour a wide variety of new or novel endophytic fungi strains that 
like their counterparts elsewhere biosynthesise a wide range of structurally intriguing 
and medicinally important secondary metabolites. 
 
 
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1.4 Objectives 
1) To collect indigenous mangrove plant samples from several well chosen 
sites on the Butre River, starting from the source to about 1000 m up-river 
where there is little or no human activity.  
2) To mark the positions of all the plants collected in a GPS and store data on 
the Google Earth GPS satellite. 
3) To identify all the plants sampled and store voucher specimens at the 
University of Ghana in Legon. 
4) To choose different mangrove plant parts and isolate the endophytic fungi 
that can be found within the tissues of these parts using a standard protocol 
and malt extract media. 
5) To purify isolated endophytic fungi by repeated sub-cultures until very 
pure strains are obtained on malt extract agar plates. 
6) To obtain the chemistry profile of the isolated species of mangrove 
endophytic fungi through small scale fermentation, subsequent extraction of 
metabolites and acquisition of high resolution HPLC-Mass spectrometry data. 
7) To single out high priority endophytic fungi that are capable of making 
new or novel secondary metabolites by entering UV and accurate mass data 
into two marine natural product databases; Marin-Lit and Antimarin. 
8) To do a large scale fermentation of high priority species of endophytic 
fungi and obtain enough extracts that allows the subsequent isolation of 
compounds of interest in reasonable quantities.  
9) To extract secondary metabolites from the large scale fermentation media 
using HP-20 resin.  
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10) To extract secondary metabolites from the HP-20 resin and fungal mycelia 
using an alternating sequence of methanol and dichloromethane extractions 
(Total Crude Extract or TCE). 
11) To subject TCE to a modified Kupchan solvent partitioning process that 
eventually results into four fractions FH, FD, WB and FM. 
12) To obtain high resolution HPLC-Mass spectrometry data for Kupchan 
solvent partitioning fractions and determine which fractions contain 
compounds of interests. 
13) To isolate compounds of interest from high priority Kupchan fractions 
using size-exclusion, gravity column and HPLC chromatography. 
14) To obtain spectroscopic and spectrometric data of the isolated compounds 
for the elucidation of their structures. 
15) To test for anticancer, antimalarial and anti buruli ulcer activity of the pure 
compounds and determine the mechanisms of action in the case of any 
positive tests.  
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CHAPTER TWO
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2.0 LITERATURE REVIEW 
2.1 Mangrove wetlands 
Collectively, mangroves are a group of trees and shrubs that live in the coastal inter-
tidal zone. In order to survive in this habitat, these plants are normally very tolerant to 
salt, high humidity, low oxygen content of sediments, and constantly varying 
conditions of temperature, pressure and nutrients.42-44 Typically, there are about 
eighty (80) different kinds of trees that inhabit mangroves worldwide but, each 
mangrove habitat has its own collection of trees and shrubs that can be found to recur 
throughout the area occupied by the mangrove. Some of the floras in mangroves are 
true mangrove plants while others are basically normal plant species that are 
associated with the mangroves.45,46 
Mangrove forests only grow at tropical and sub-tropical latitudes near the equator 
because; they are not able to withstand freezing temperatures. Many mangroves can 
easily be recognized by their dense tangle of prop and buttress breathing roots that 
make the trees appear to be standing on stilts above the water. This tangle of roots 
allows the trees to take up oxygen and handle the daily rise and fall of tides, which 
means that most mangroves get flooded at least twice per day. The roots also slow the 
movement of tidal waters, causing sediments to settle out of the water and build up a 
very muddy bottom. Mangrove forest generally stabilizes the coastline, reducing 
erosion from storm surges, currents, waves and tides.42-44 
Mangroves have become an important extreme habitat in the continued research to 
discover new drug entities from marine derived organisms as a result of the following 
reasons: 
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These wetlands are a great assembly point of novel marine microbes including 
bacteria and fungi. As a result of the rather harsh environmental conditions in these 
wetlands, microbes that have been isolated and studied in these habitats have proven 
to exhibit a lot of biosynthetic prowess. 
Compared to the deep oceans, mangrove habitats are much easier to access and 
sample. Small boats, canoes, rafts and other small vessels can be used to access the 
most remote of mangrove wetlands and in addition sample grabs, shovels or much 
simpler equipment’s can be used for sampling. The deep oceans require ships, larger 
boats, paid crew and divers with expensive remotely operated submarines to 
effectively access invertebrates and sediments. 
Large sections of mangroves are under constant threat of extinction worldwide and 
their novel species if not studied might be lost forever.40,47-51 
In Ghana, mangrove wetlands cover close to 140 km2but are largely limited to a very 
narrow, non-continuous coastal area that surrounds lagoons on the western and 
eastern sections of the country and the lower fringes of the Volta River. Mangrove 
wetlands are most extensive to the west in the stretch between Cape Three Points and 
Côte d’Ivoire, especially in areas around Half Assini, Sekondi Tarkoradi, Amanzure 
lagoon, Axim, Princes Town and Shama. To the east of the country, they are best 
developed at Apam, Muni lagoon, Winneba, Sakumono lagoon, Bortiano, Korle 
lagoon, Teshie, Ada, Sroegbe and Keta lagoon.52-54 Six species of mangroves that are 
typical of West-Central African mangroves are found in Ghana and these include: 
Acrostichum aureum, Avicennia germinans, Conocarpus erectus, Laguncularia 
racemosa, Rhizophora harrisonii, and Rhizophora racemosa. The open lagoons tend 
to be dominated by Rhizophora racemosa, whilst closed lagoons with an elevated 
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salinity contain Avicennia germinans, Conocarpus erectus, Laguncularia racemosa 
and Acrostichum aureum.52-55 
Ghanaian mangrove wetlands have not been previously explored for the possible 
isolation of new endophytic fungi and subsequent chemical screening thereof. This 
project represents the first ever attempt to screen these wetlands for possible isolation 
of new or novel species that have the ability to make biologically active compounds. 
2.2 Brief introduction to mangrove plant endophytic fungi 
Endophytic fungi are indeed eukaryotic fungal microorganisms which spend all or 
part of their lifecycle inter- and/or intra-cellular colonizing healthy tissues of their 
hosts plants.56-63They constitute a polyphyletic group of highly diverse, primarily 
ascomycetous fungi defined functionally by their occurrence within asymptomatic 
tissues of plants. Typically, they cause no disease symptoms and their relationships 
with the respective plant hosts have been mostly described as symbiotic although this 
idea has in some cases proved contentious.56-63Endophytic fungi have been found in 
each plant species examined, and it is estimated that, there are over 1.5 million 
endophytic fungi existing in nature out of which only about 7% are known.64 They are 
found in both the roots and aerial plant parts or for those types of marine flora which 
are entirely submerged by sea water in the cells and within the intercellular spaces of 
the plant. They are found in all plant types from the arctic to the tropics and from 
agricultural fields to the most biotic and diverse tropical forests.56-63 
Their ecological significance is particularly complex and has most of the times been 
simplified to the scenario where the host plant provides good shelter, water, wide 
array of ready available nutrients and biosynthetic raw materials while in return the 
fungi protects its host from predators both micro and macro. As endophytes, they 
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have an inherent ability to adapt to several sometimes rapidly changing environmental 
factors. For example, leaves and roots represent the most dynamic interfaces between 
plants and their environments. Endophytic fungi that inhabit these biologically active 
and fast growing tissues may share characteristics that allow them to grow and persist 
in an ever-changing biochemical milieu or in the context of rapidly changing gene 
expression as host tissues grow and age.65,66 In contrast to this, endophytic fungi that 
colonize the submerged roots of mangrove plants, constantly find themselves in a 
matrix that in many cases contains lots of moisture, organic and inorganic materials, 
high salt content, low nutrient, temperatures or pressures and low to no light 
conditions. As a result of these harsh environmental stresses and changes, endophytic 
fungi have developed the most biosynthetically versatile and bio-diverse genomes 
allowing them to produce a very wide range of secondary metabolites that exhibit 
very important bioactivities.65,66 
Since the first endophytic fungus was identified, a lot of attention has been given to 
the potential of exploitation of these fungi for the production of novel antibacterial, 
antifungal, anti-parasitic and anticancer agents. In some cases, in addition to 
producing a great number of diverse bioactive compounds which have been 
implicated in the protection of their host against pathogens and herbivores, some 
endophytic fungi have evolved and possess the ability to produce the same or similar 
compounds characteristic of their host plants.56,67,68 
An important example is the anticancer agent taxol, which is currently used to treat 
different types of cancer including ovarian, breast, lung and pancreatic cancer 
amongst others. Taxol acts primarily by interfering with the normal breakdown of 
microtubules during cell division. This compound was originally discovered in 1962 
as a result of a U.S. National Cancer Institute-funded screening program. It was 
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isolated from the bark of the Pacific yew tree, Taxus brevifolia. Thirty-one years later, 
the taxol producing endophytic fungus Taxomyces andreanae was identified.62 
 
 
The ability of some endophytes to produce the same rare and important bioactive 
compounds originally deemed characteristic of their host plant is a very important 
revelation. This phenomenon is of great importance and offers a prudent solution to 
the drug resupply issues. It effectively reduces the need to harvest plant or various 
plant parts which are rather slow-growing and possibly rare in occurrence and 
therefore, helps to preserve the world’s ever diminishing biodiversity. Moreover, the 
production of a high value phytochemical by exploiting a microbial source is easier 
and more economical and it leads to increased availability and the reduced market 
price of the drug products.69 
In conclusion, mangrove plant endophytic fungi represent a niche that should be 
meticulously investigated and used as a base for sustainable research and 
development of new antibacterial, antifungal, anti-parasitic and anticancer agents that 
can respond to current pathogenic, parasitic or cancer resistance and anticipate 
evolving resistance. 
 
 
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2.3 Bioactive compounds from marine-derived endophytic fungi 
Since the discovery of penicillin in the last century, the diverse and bioactive fungal 
metabolites have played an important role in drug development. By the year 2005, 
nearly 35 drugs that originated from fungi were identified as antibacterials, immune 
suppressives and other therapeutics. Some were also found in clinical trials like the 
diketopiperazine compound phenylahistine which was projected as a future anticancer 
drug.70,71Furthermore, it is worth mentioning the antibacterial drug cephalosporin C 
which was originally discovered in 1945 from the marine fungus Cephalosporium 
acremonium.72 
 
 
Despite the discovery of such important drugs from fungi, the number of bioactive 
natural products originated from marine fungi increased extremely slowly for a very 
long time. It is only during the last decade that researchers have returned focus on 
marine-derived fungi to search for novel bioactive secondary metabolites that could 
potentially be used as drugs.73,74 Between the years 2006-2011, intensified research on 
marine derived fungi yielded many compounds of different functionalities and 
bioactivity. 
 
 
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2.3.1 Alkaloids isolated from marine-derived fungi 
Marine-derived endophytic fungi have produced quite a number of alkaloids with 
fascinating structures and excellent biological activities. A detailed description of 
some of the structures is given below to provide a good comparison with the 
compounds presented in the results of this project. 
The compound acremolin is a novel alkaloid obtained from the culture broths of 
Acremonium strictum. Structurally, the molecule constitutes a methyl guanine base 
containing an isoprene unit and a 1H-azirine moiety, which is entirely unique in 
natural product chemistry. Acremolin exhibited weak cytotoxicity against A549 cell 
line with IC50 value of 45.90 µg/ml. It is however inactive against several strains of 
Gram-positive and Gram-negative bacteria.75,76 
Generally, diketopiperazines are widespread in the chemistry of microbes especially 
fungi. Apart from the already mentioned phenylahistine, some important examples 
include the compounds gliocladride A and B and deoxymycelianamide which were 
isolated from the mycelia of marine fungus Gliocladium sp. Gliocladride A and B 
were found to possess moderate activity against HL-60, U937 and T47D with IC50 
values from 11.6 µg/ml to 52.83 µg/ml. Deoxymycelianamide on the other hand, 
showed strong cytotoxic activity against U937 cell line with IC50 value 0.785 µg/ml.77 
 
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The same fungus Gliocladium sp. produced the compounds PJ157 and glioclaride 
which appears to be an artefact. However, glioclaride exhibited strong cytotoxicity 
with an IC50 value of 3.86 µg/ml against human A375-S2 melanoma cell line. 
Looking at their structures therefore, it is easy to realise that the glioclaride moiety 
present in the structures of these compounds are responsible for the cytotoxicity 
activity generally seen in these compounds.78,79 Also, it appears that the N-OH moiety 
in gliocladride A and B is likely to be responsible for the decreased cytotoxicity seen 
in these two compounds.77 
 
 
 
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The compound p-hydroxyphenopyrrozin isolated from the culture broths of the 
marine-derived fungi Chromocleista sp. exhibited potent antifungal properties by 
killing the yeast cells Candida albicans with MIC of 25 µg/ml. Interestingly, its 
dehydroxy derivative, phenopyrrozin did not produce any relevant activity against 
yeast cells.80 
 
 
 
The culture extracts of a marine-derived fungus Stachylidium sp. isolated from the 
sponge Callyspongia flammea, produced four phthalimidine derivatives that were 
named marilines A1 and A2, marilines B and C. Marilines A1 and A2 are enantiomers 
which were identified by a combination of circular dichroism (CD) analysis and 
quantum chemical CD calculations. Marilines afford an unusual skeleton, which is 
most definitely derived from some uncommon biochemical reactions characteristic 
only of fungal secondary metabolism. The two enantiomers marilines A1 and A2, 
inhibited human leukocyte elastase (HLE) with an IC50 value of 0.86 µM. 
Furthermore, mariline A1 also moderately inhibited the growth of pathogens of the 
tropical infectious disease, African sleeping sickness and leishmaniasis.81 
 
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Cytochalasins are a group of fungal metabolites that possess the ability to bind to 
actin and thereby alter its ability to polymerize. This class of fungal metabolites have 
been widely used to study the role of actin in biological processes and as models for 
actin-binding proteins. By virtue of their mechanisms of action, the cytochalasins 
display a wide array of biological effects which include the inhibition of cytoplasm 
division, reversible inhibition of cell movement, nuclear extrusion, platelet 
aggregation and clot retraction, glucose transport and thyroid secretion, antimicrobial 
and anticancer properties, HIV-1 integrase inhibition and various other activities. Due 
to their commercial availability during the last four decades, several cytochalasin 
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derivatives have been synthesized and sold for various cytological research 
programs.82-98 
The compound cytochalasin D represents a typical example of the cytochalasins. It 
was originally obtained from the mold Xylaria sp. SCSIO156 collected from South 
China Sea Marine sediments. It is a cell permeable potent inhibitor of actin 
polymerization and disrupts actin filaments through activation of the p53-dependent 
pathways causing arrest of the cell cycle at the G1-S transition. It is also believed to 
bind F-actin polymer and prevent polymerization of actin monomers. 99,100 
 
 
 
The pyrrolyl-4-quinolinone alkaloid possesses an unprecedented ring system and was 
named penicinoline. This compound was obtained from a mangrove endophytic 
fungus and its complete structure was determined by spectroscopic methods and in 
comparison to its penicinotam, an unexpected lactam that was obtained from 
penicinoline by intramolecular dehydration. Eventually, the structure ofpenicinoline 
was confirmed by X-ray analysis and it was shown to possess potent in vitro 
cytotoxicity towards 95-D and HepG2 cell lines with IC50 values of 0.57 and 6.5 
µg/ml respectively.101 
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Penicinoline and its derivative penicinotam where further evaluated to check their 
potential activity against the insects Aphis gossypii, Plutella xylostella, Heliothis 
virescens, Septoria tritici and Uromyces fabae. Penicinoline was found to possess an 
impressively strong activity against Aphis gossypii with 100% mortality at 1000 ppm 
whereas penicinotam not only killed completely all pests tested but, it also showed 
total control of the larvae of the pests Plutella xylostella at 500 ppm concentration. 
Penicinotam also moderately controlled the pests Heliothis virescens.101 
Marine-derived fungus Penicillium janthinellum is the source of the three alkaloids 
shearinines D-F. Shearinine E inhibited EGF-induced malignant transformation of 
JB6 P + Cl41 cells in a soft agar with INCC50 (inhibition of number of colonies) of 
13 µM. Shearinines D and F induced apoptosis in human leukemia HL-60 cells at 100 
µM concentration by 10%, 39% and 34% of the apoptotic cells respectively.102 
 
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2.3.2 Peptides isolated from marine-derived fungi 
Marine-derived fungi produce a lot of peptides that includes cyclic peptides, cyclic 
depsipeptides, and straight chain peptides. Some of these have interesting biological 
activity and a few examples are mentioned here. 
Microsporin A and B were isolated from the marine fungus Microsporum gypseum 
and found to possess cytotoxicity activity. The unique structural feature of these two 
peptides is the presence of-amino acids in their structure resulting in a 12-membered 
cyclic peptide ring. Three of the amino acids have L-configurations, while the 
pipecolic acid has a D-configuration. Both microsporin A and B have been reported as 
potent inhibitors of histone deacetylase and demonstrate activity against human colon 
adenocarcinoma (HCT-116) with IC50 0.6 µg/ml. They also proved potent in the NCI 
60 cancer cell panel with average IC50 value of 8.5 µg/ml. In all these assays, 
microsporin A was more active than microsporin B and this was attributed to the 
importance of the ketone carbonyl group in the bioactivity observed.103 
 
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Zygosporamide is a cyclic pentadepsipeptide that was discovered in the fermentation 
broth of the marine fungus Zygosporium masonii. Despite a rather simple structure 
compared to other cyclic peptides, this compound showed significant cytotoxicity 
GI50 of 9.1 µM against NCI 60 cell line panel. Zygosporamide showed highly 
enhanced selectivity with GI50 of 6.5 nM against the CNS cancer cell line SF-268 and 
the renal cancer cell line RXF 393, GI50 6.0 nM.104 
 
The cyclodepsipeptide IB-01.212 was obtained from the mycelium of Clonostachys 
sp. and found to exhibit leishmanicidal activity on promastigote and amastigote forms 
of the parasite with LC50 of 10.5 and 7.5 µM respectively. Other derivatives of this 
compound isolated from the same organism were also tested but, cyclodepsipeptide 
IB-01.212 showed double potential compared to the other analogues.105 
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Apart from the cyclic peptides mentioned above, marine-derived fungi also produce a 
very wide variety of linear peptides that possess very interesting biological activity. 
Examples of such peptides are efrapeptin E, efrapeptin H, N-methylated octapeptide 
RHM3 and RHM4 which were separated from a marine-derived fungus Acremonium 
sp. Efrapeptin E displayed IC50 of 1.3 nM against H125 cells. The other analogues of 
this peptide separated from the same species have been reported to possess the same 
activity with different potential.106 
 
 
 
  
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2.3.3 Polyketides isolated from marine-derived fungi 
Polyketides are a highly diverse group of natural products with structurally intriguing 
carbon skeletons which are assembled from a very simple building block which is 
acetate. Many polyketides have become important therapeutics in clinical use for 
example; the fungal polyketide lovastatin is a very well-known cholesterol-lowering 
agent on the pharmaceutical market and represents one of the success stories of 
natural products in drug discovery.107 
 
 
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In addition to this, several marine-derived fungal species also make a large number of 
polyketides and their derivatives. The medicinal importance of this capability is very 
important and only further buttress the importance of marine fungi as a source of our 
future drug prototypes. A few examples are mentioned below to emphasise the 
importance of this resource. 
The azaphilone polyketides, chaetomugilins P-R and 11-epi-chaetomugilin I were 
isolated from the broth culture of Chaetomium globosum which was retrieved from 
the marine fish Mugil cephalus.108 
 
Chaetomugilins P and 11-epi-chaetomugilin I significantly inhibited the P388, HL-60, 
L1210 and KB cancer cell lines with highest activity in the range of IC50 0.7-1.8 pM. 
The reported results suggested the importance of the enone functionality within the 
acyclic side chain for strong cytotoxicity.108 
Marine derived fungus Phoma sp. produced the two compounds epoxyphomalin A 
and B. The cytotoxicity of the two compounds was evaluated with 36 different cancer 
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cell lines. Epoxyphomalin A showed significant in vitro tumour cell selectivity. IC50 
values in the tested above-average sensitive cell lines ranged from 0.010 µg/ml for 
breast cancer MAXF 401NL to 0.038 µg/ml in adeno lung cancer LXFA 629L. 
Epoxyphomalin B exhibited 22% of selectivity with IC50 values in the eight above-
average sensitive cell lines ranging from 0.251 µg/ml for pleuramesothelioma PXF 
1752 L to 0.402 µg/ml for bladder cancer BXF T24. However, summary of the results 
indicated that epoxyphomalin A showed superior cytotoxicity at nanomolar 
concentrations for pleuramesothelioma PXF 1752 L and bladder cancer BXF T24.109 
 
The marine fungus Aspergillus glaucus produced the cytotoxic anthraquinone-derived 
polyketide aspergiolide A. Aspergiolide A exhibited selective cytotoxicities against 
A-549, HL-60, BEL-7402 and P388 cell lines with IC50 values of 0.13, 0.28, 7.5, and 
35.0 µM respectively.110 
 
 
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Xanthone-derivatives chaetoxanthones A-C were isolated from the culture extracts of 
the fungus Chaetomium sp. Chaetoxanthones A and B bear a dioxane/tetrahydropyran 
moiety, which is not so common in natural product chemistry. Chaetoxanthones C in 
turn is a chlorinated xanthone substituted with a tetrahydropyran ring. 
Chaetoxanthones B was selectively active against Plasmodium falciparum with an 
IC50 value of 0.5 µg/ml and it did not show any cytotoxicity toward cultured 
eukaryotic cells. On the other hand, chaetoxanthones C was found to be moderately 
active against Trypanosoma cruzi with an IC50 value of 1.5 µg/ml.111 
 
 
2.3.4 Steroids isolated from marine-derived fungi 
Steroids constitute an important class of natural products that have produced many 
drugs currently on the pharmaceutical market. Interestingly, marine derived fungi also 
produce several new compounds, derivatives or analogues of this class of natural 
products. For example, ergosterol and its peroxy derivative5,8-epidioxy-24(S)-
methylcholesta-6,22-diene-3-ol are common fungal metabolites routinely found and 
isolated from the liquid broths of Guignardia sp. recovered from the marine alga 
Undaria pinnatifida. Ergosterol and 5,8-epidioxy-24(S)-methylcholesta-6,22-
diene-3-ol inhibited the growth of the pathogenic yeast cells Microsporum canis 
with MICs of 10 and 20 µg/ml and of Trichophyton rubrum with MICs 15 and 20 
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µg/ml respectively.Ergosterol also showed some activity against Epidermophyton 
floccosum with MIC of 20 µg/ml.112 
 
 
A Pennicillium sp. isolated from a strain of moss in the South Pole, produced the 
steroids ergosta-8(14),22-diene-3,5,6,7-tetraol-(3,5,6,7,22E), ergosta-8(9),22-
diene-3,5,6,7-tetraol-(3,5,6,7,22E), 5,8-epidioxy-24(S)-methylcholesta-6,22-
diene-3-ol, 5,8-epidioxy-24(S)-methylcholesta-6,9(11),22-triene-3-ol and 
3,5,9-trihydroxyergosta-7,22-diene-6-one.113,114 
 
 
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These compounds were tested against human liver cancer cell (Hep G2), and all of 
them exhibited potent activity with ergosta-8(14),22-diene-3,5,6,7-tetraol-
(3b,5a,6b,7a,22E) showing the lowest IC50 value of 10.4 µg/ml.113,114 
 
2.3.5 Terpenes isolated from marine-derived fungi 
Terpenes are the most numerous and structurally diverse group of natural products. 
Even though a majority of these compounds have come from plants, marine derived 
fungi also produce a number of very important terpenes. In some cases, terpenes 
isolated from plants have been attributed to the endophytic fungi which are directly 
associated with these plants.  
A marine fungus, Penicillium sp. BL27-2 produces eremophilane sesquiterpenes, 
which were characterized as 3-acetyl-9,7(11)-dien-7-hydroxy-8-oxoeremophilane, 
3-acetyl-13-deoxyphomenone, sporogen-AO1, 7-hydroxypetasol, 8-hydroxy-13-
deo-xyphomenone and 6-dehydropetasol.115 
 
 
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Reports indicate that, 3-acetyl-9,7(11)-dien-7-hydroxy-8-oxoeremophilane, 3-acetyl-
13-deoxyphomenone and 8-hydroxy-13-deo-xyphomenone with epoxide 
functionalities exhibit cytotoxicity activity for P388, A549, HL60, BEL7402 and 
K562 cell lines with IC50 values ranging between 0.073 to 11.8 µM/L, while the 
compounds sporogen-AO1, 7-hydroxypetasol and 6-dehydropetasol without epoxide 
functionalities show weak cytotoxicity activity. These results suggested that, the 
epoxide rings are absolutely essential for their activity. Also, it appears as if 
acetylation may enhance activity.115 
Hirsutanol A, E and F were obtained from the culture extracts of Chondrostereum sp. 
which is a fungus recovered from the soft coral Sarcophyton tortuosum. Biological 
activity tests showed interesting results for only hirsutanol A which showed activity 
against human colon, human hepatic, human lungs, human breast and human cervical 
cancer cell lines with IC50 values ranging from 0.58 to 8.27 µg/ml.116 
 
 
 
*The literature reviewed here for natural product compounds produced by marine 
derived fungi is by no means a comprehensive list of all the molecules obtained from 
these microbes to-date. However, the literature serves to confirm the following: 
 Different species of marine derived flora and fauna indeed harbour within 
their tissues a wide variety of fungi. 
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 These different types of fungi produce a very diverse range of secondary 
metabolites including alkaloids, peptides, polyketides, steroids and terpenes. 
 These diverse ranges of metabolites have bioactivity with a large number of 
them possessing cytotoxicity activity, a few with antimicrobial, anti-parasitic 
and antifungal activities.  
 Chemical investigation of marine-derived fungi is very likely to yield 
interesting drug leads in many disease areas. 
2.4 Terminologies in bioactivity measurements 
2.4.1 Half maximal inhibitory concentration (IC50) 
This is a quantitative measure in vitro that indicates how much of a particular 
substance or drug is needed to inhibit by half a specific biological or biochemical 
processor a component of that process occurring within a cell.117-123 
 
 
Figure 2.1: Illustration of Half Inhibitory Concentration (IC50). 
 
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2.4.2 Half maximal effective concentration (EC50) 
The term EC50 is commonly used to depict or measure the potency of a drug. It 
represents the concentration of a compound, drug, antibody or toxicant at which 50% 
of its maximal effect is observed after a specified exposure time.123-128 
 
 
Figure 2.2: Illustration of Half Maximal Effective Concentration (EC50). 
 
2.4.3 Half maximal Effective dose (ED50) 
This is the dose or amount of drug that produces a specific effect or therapeutic 
response in some fraction (50%) of the subjects or test sample that have taken it. It is 
a measure of average effectiveness of the drug or the tolerance level of toxin in a 
sampled set of individuals.129-134 
 
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Figure 2.3: Illustration of Effective Dose (ED50). 
2.4.4 Lethal dose for 50% (LD50) 
This is the quantity of a substance (toxin, drug, pesticide etc) which is enough to 
cause the death of 50% of a group of test animals in a specified period. It is 
commonly used in bioassay assessment to measure the acute toxicity of a chemically 
active agent.135-138 
 
 
Figure 2.4: Illustration of Lethal Dose (LD50). 
 
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2.4.5 Lethal concentration for 50% (LC50) 
This is the quantity of a substance (toxin, drug, pesticide etc) present in the air or 
water which is enough to cause the death of 50% of a group of test animals in a 
specified period.135,137,139 
2.4.6 Minimum inhibitory concentration (MIC) 
This is the minimum concentration of an antimicrobial that will inhibit the visible 
growth of a microorganism after overnight incubation. MICs are important in 
diagnostic assessments of the potency a new antimicrobial agents or to detect 
resistance of microbes to already existing antimicrobial agents.140-143 
 
2.4.7 Minimum bactericidal concentration (MBC) 
This is the minimum concentration of an antimicrobial that will kill and stop the 
growth of a microorganism after overnight incubation.144-148 
 
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Figure 2.5: Illustration of Minimum Inhibitory Concentration (MIC) and 
Minimum Bactericidal Concentration (MBC). 
 
 
 
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2.4.8 Growth inhibition concentration for 50% (GI50) 
This is the concentration of a substance or drug that inhibits the growth cells by 50%. 
For an anticancer agent, it is the concentration of the drug that inhibits the growth of 
cancer cells by 50% or produces a 50% reduction in cell proliferation.149,150 
2.5 Malaria 
2.5.1 Introduction, statistics and interventions 
Parasites are organisms that live within or on other organisms at whose expense they 
obtain some advantage such as nourishment and shelter.151Parasites range from the 
pathogenic single celled microscopic organisms (protozoans) to larger multi-cellular 
worms that reach three feet long and are responsible for some of the world’s deadliest 
diseases including leishmaniasis, trypanosomiasis, schistosomiasis, filariasis and 
enteric protozoans that cause infections such as diarrhoea and dysentery. Among all 
the parasitic agents causing diseases in the world, Plasmodium parasites continue to 
be the most destructive by virtue of the widespread occurrence and difficult to avoid 
nature of its vector, the female Anopheles mosquito.152It affects the developing world 
to a large extent but, almost half of the world’s population stand a chance of being 
infected. Malaria is capable of serious economic damage since it is able to completely 
weaken its victims and make them completely non-productive.153 Hence, it is mostly 
linked to the economic deterioration of most developing countries especially in Sub-
Saharan Africa. 
The unicellular protozoan parasite Plasmodium belongs to the phylum Apicomplexa 
which constitute a large group of eukaryotic microorganisms that possess a unique 
organelle known as the apicoplast and the apical complex that are involved in cell 
division within the host.154 The different species of plasmodium that commonly infect 
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humans are P. falciparum, P. vivax, P. ovale, P. malariae and the recently discovered 
South-East Asian P. knowlesi.155 P. falciparum however, is the most virulent and 
mostly associated with severe cases of malaria and malaria-associated complications 
or subsequent death. 
Currently, malaria is the most prevalent disease in the world, killing 1-2 million 
people each year.153Approximately 3.3 billion people which is about half of the 
world’s population are at risk of being infected with malaria. In 2013, 198 million 
cases of malaria occurred globally and the disease led to 584,000 deaths. Most of the 
deaths are amongst children under five who, together with pregnant women, are 
particularly vulnerable to the disease.153In 2013, malaria was one of the causes that 
led to death of 6.3 million children under the age of five.156South and Central 
America, South and East Asia, the Caribbean, Oceania, Central Asia and the Middle 
East are all affected by malaria, but the burden is heaviest in Africa where an 
estimated 90% of malaria deaths occur with most cases in sub-Saharan Africa.153 The 
disease does not only significantly affect morbidity and mortality but, the economic 
burden caused by malaria is estimated to cost Africa US$ 12 billion in lost Gross 
Domestic Product (GDP). It is estimated to have slowed economic growth in Africa 
by 1.3% per year as a result of lost life and lower productivity. 
The global impact of the disease has caused International agencies and bodies to help 
eradicate and improve the socio economic challenges associated with it. In 1998, the 
Group of Eight (G8) nations who met in Birmingham, UK, endorsed an international 
initiative to control malaria. The leaders agreed to improve mutual cooperation on 
infectious and parasitic diseases and offered support for the new “Roll Back Malaria” 
movement to reduce levels of malaria-related mortality by 2010. The G8 leaders 
sought to achieve the following: 
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 Collaborate with governments, private sector companies and non-
governmental organizations in public-private partnerships to expand malaria 
interventions and programs. 
 Work with African-countries to scale up malaria control interventions, reduce 
the burden of the disease and eventually defeat malaria on the continent to 
meet the Abuja target of halving the burden of malaria by 2010. 
 Support the development of new, safe and effective drugs or the creation of a 
vaccine and promote the widest possible availability of prevention and 
treatment to people in need. 
 Support activities of public and private entities to save children from the 
disease. 
 Contribute to the additional US$ 1.5 billion a year needed annually to help 
ensure access to anti-malarial insecticide treated mosquito nets, adequate and 
sustainable supplies of combination therapies, preventive treatment for 
pregnant women and babies and household residual spraying.157 
2.5.2 Life cycle of the plasmodium parasites 
The life cycle of the plasmodium parasite involves both humans and the female 
Anopheles mosquito hosts. The human part of the life cycle normally begins with the 
bite of an infected Anopheles mosquito leading to the introduction of the parasites 
which at this stage are known as sporozoites into the human blood stream. The 
haploid sporozoites immediately move to the liver and invade a liver cell or 
hepatocyte which subsequently reproduces by mitotic cell division. The sporozoites 
within the hepatocytes transform into schizonts. Each schizoint contains thousands of 
haploid cells called merozoites. The schizonts swell and rupture in about seven (7) 
days, killing the hepatocytes and hence releasing millions of merozoites into the blood 
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stream. This stage of replication is referred to as asexual exo-erythrocytic schizogony. 
At the parasite development stages within the liver, hypnozoites which are dormant 
forms of the parasite that refuse to continue the life cycle but lie dormant for a long 
time may develop. The development of malaria relapse has been attributed to these 
latent hepatic forms of the parasite which are mostly characteristic of P. vivax and P. 
ovale infections. 
The merozoites that are released from the raptured hepatocyte into the bloodstream 
quicklyinvade erythrocytes initiating the erythyroctic stage of malaria parasitic 
infections. P. Falciparum normally invades the erythrocytes of all ages whereas P. 
vivax and P. ovale prefer the erythrocytes of younger people in contrast to P. 
malaraiae which prefers the erythrocytes of older and more matured individuals. 
Subsequently, the merozoites undergo a period of mitotic amplification during 
erythrocytic schizogony. The early ring forms of the parasites mature into 
trophozoites that feed on the haemoglobin found in erythrocytes. Trophozoites then 
mature into schizonts that contain newly formed merozoites. The schizonts along with 
the erythrocytes rupture after 2-3 days releasing the merozoites which quickly invade 
uninfected erythrocytes. The symptoms associated with this continue destruction of 
red blood cells and the release of toxins into the blood include anaemia, fever and 
chills. 
A portion of the erythrocytic merozoites can then develop into gamete producing cells 
termed gametocytes. This takes place in the female Anopheles host of the parasite and 
commence when the Anopheles receives a blood meal containing gametocytes. The 
gametocytes transform into male and female haploids gametes minutes after they 
enter the mosquito mid-gut. The male gamete fertilizes the female gamete to produce 
a diploid zygote which undergoes meiotic division to become an ookinete and travels 
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toward the mosquito gut wall and invade the semi-permeable membrane called the 
peritrophic matrix. The ookinete upon traversing the gut wall transforms into an 
oocyst occupying the space between the gut wall and the basal lamina. The oocyst 
bursts and releases sporozoites into the hemocoel cavity. The sporozoites proceed to 
invade and establish residence in the salivary glands of the mosquito and are released 
from the salivary glands of the female Anopheles host to the human host during blood 
meals and hence completing the cycle.155 
 
 
Figure 2.6: Life cycle of Plasmodium parasite showing the stages at which 
current drugs act.   
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2.5.3 Antimalarials in current use 
Chemotherapeutic strategies to combat malaria aim to target different stages of the 
life cycles in humans or total elimination of the vector which is the female Anopheles 
mosquito. The matured trophozoites and schizonts at the human blood stage have 
previously served as good targets for many different antimalarials.155 Different 
compound structures have been discovered and used to treat malaria in the past and 
indeed many different drugs are currently available but, the ability of the parasites to 
develop resistance and subsequently render useless a particular structure or skeleton at 
any time demands a continued investigation to discover new drugs. 
Quinine the first quinoline antimalarial drug acts as a blood schizonticide and weak 
gametocide against P. vivax and P. malariae. The drug is also capable of 
accumulating in the food vacuoles of plasmodium species such as P.falciparum and 
act by inhibiting the heme polymerase enzyme, thus facilitating anaggregation of 
cytotoxic heme which is not useful to the parasite. Quinine is still used in the 
treatment of acute cases of severe P. falciparum infection and sometimes prescribed 
as a prophylactic in infants that is taken every Sunday. Until recently, chloroquine 
was very effective and the drug of choice for over three decades. 
Chloroquine is believed to reach high concentration in the vacuoles of the parasites 
which due to its alkaline nature, raises the internal pH. It inhibits the conversion of 
toxic heme a by-product of the digestion of haemoglobin to haemozoin by preventing 
the biocrystallization of haemozoin. The parasites subsequently poison themselves by 
feeding on human haemoglobin and are thus destroyed. However, the resistance of P. 
falciparum to chloroquine has spread from Asia to Africa making the drug 
ineffective.158 
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Currently, artemisinin based drugs are the first line of treatment for the parasitic 
disease and has been proven to be effective against all forms of multi-drug resistant P. 
falciparum. The drug has demonstrated the fastest clearance of all antimalarials 
currently used and acts primarily on the trophozoite phase of the plasmodium life 
cycle, thus preventing further progression of the disease. Other derivatives of 
artemisinin have also proven to be efficacious. For example, artemether is a methyl 
derivative of dihydroartemesinin and it has a similar mode of action to that of 
artemesinin but, it has demonstrated a reduced ability as a hypnozoiticidal 
(hypnozoite-activating) compound, instead acting more significantly to decrease 
gametocyte carriage.  
 
 
 
Artesunate is a water-soluble semi-synthetic hemi-succinate derivative of artemesinin. 
It is the most effective because of its instantaneous bioavailability. A combination of 
mefloquine and artesunate is highly effective in multi-drug resistant malaria.159 
 
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2.5.4 Development of drug-resistant plasmodium parasites 
The most intimidating problem in the fight against malaria is the widespread 
occurrence of drug resistance especially in the species of P. falciparum which as 
mentioned earlier, infects all ages in humans. The three other species have no 
documented resistance apart from the recorded chloroquine resistance in Papua New 
Guinea and Indonesian regions.159 
The reasons for this widespread development and spread of resistance are: 
 Drug-use pattern such as wrong diagnosis and incorrect dosage. 
 Physical and chemical characteristics of the drug itself, for example poor drug 
quality and poor absorption. 
 Human host factors including diet and habits that speed up the quick 
breakdown of the drug in the blood stream. 
 Parasite characteristics, which leads to gene mutation that results in the over-
expression of some genes eventually leading to the development of resistance 
by the parasite. 
 Parasite-vector combinations of factors that are not entirely documented 
enhance the transmission of resistant parasites. 
 Environmental factors.159 
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The development of drug resistance is one of the problems that necessitate the 
continued discovery of antimalarials with novel backbones or structures. 
2.5.5 Preliminary screening of antimalarial activity 
Most of the preliminary assessments of the antimalarial efficacy of molecules are 
made in vitro using a number of bioassays. Plasmodium parasites require a source of 
hypoxanthine for nucleic acid synthesis and energy metabolism and they also use 
ethanolamine, normally derived from serine in the synthesis of glycolipids. Hence, by 
quantifying parasite uptake of radioactive substrates [3H] hypoxanthine and [3H] 
ethanolamine as a measure of growth and viability in the presence of the test drug, the 
effects of many drugs on the parasites were previously studied. These radioactive 
bioassays were accurate and reliable but, their reliance on very expensive radio-
isotopes and multi-step procedures that is increasingly problematic and impractical 
with large numbers of drugs to be tested, made them gradually unpopular. Also, since 
the late 1970s, regulations regarding handling of radioactive materials have become 
considerably more restrictive causing isotopic assays to gradually give way to other in 
vitro tests. 
For example, fluorescence based in vitro method; the SYBR Green I assay has been 
designed for the high-throughput screening of the activities of malaria parasites. The 
advantages of the assay include simplicity, lower costs, robust performance, 
applicability to automated analysis and speed.160,161 
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CHAPTER THREE 
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3.0 MATERIALS AND METHODS 
3.1 Mangrove plant sample collections 
The Western Region of Ghana is noted for its bio-diversely rich indigenous mangrove 
plants. Out of the six indigenous plants that are characteristic of the mangroves found 
in Ghana, three of them are particularly common in the Western Region and these are 
Conocarpus erectus, Laguncularia racemosa and Rhizophora racemosa. Collection of 
mangrove plants was done along the banks of the River Butre with five (5) sampling 
sites chosen at 100 m apart from the shore to where the river meets the sea. The three 
main plant samples collected were Conocarpus erectus, Laguncularia racemosa and 
Rhizophora racemosa but, different plant parts were sampled at different sections of 
the river. The plant parts sampled were leaves, buds, submerged roots, aerial roots, 
aerial stems, fruit shoots and flowers. The exact positions of plants from which 
different parts were sampled were stored in a GPS and the data uploaded into Google 
Earth satellite database. The samples were videoed, photographed, bagged, labelled 
and stored at 4 °C in an ice-chest and transported to the Department of Chemistry, 
University of Ghana, Legon. Identification of the plants was done at the Department 
and specimens of all plant parts collected were dried in newspapers for onward 
submission to the herbarium. 
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Figure 3.1:Map of Butre River running through communities.
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Rhizophora racemosa    Cornacarpus erectus 
 
 
 
Langucularia racemosa 
Plate 3.1: Pictures of three mangrove plants particularly common in the Western 
Region of Ghana. 
 
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Plate 3.2: Boat riding on the Butre River to the site where samples were 
collected. 
 
 
Plate 3.3: Collection and Identification of mangrove plants along the Butre 
River. 
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Plate 3.4: Collection and bagging of mangrove plants along Butre River. 
 
 
Plate 3.5: Butre River running through the mangrove where plants were 
collected. 
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3.2 Preparation of culture media 
3.2.1 Malt extract solid media 
Approximately 15g of malt extract and 15g of bacteriological agar were weighed into 
a 1L autoclave bottle containing 900 ml of water and stirred to mix with a magnetic 
stirrer. An autoclave tape was pasted on the bottle and the mixture autoclaved. The 
autoclaved mixture was cooled to about 55°C and then poured into petri dishes (90 
mm) to fill about 2/3 of the volume of each Petri dish under sterile conditions (25 
plates for each 900 ml preparation). The plates were left half-open under a clean 
bench until almost all the water had evaporated from the plates. Dry plates were 
parafilmed and kept in a fridge at 4°C until needed. 
 
 
3.2.2 Malt extract liquid media 
Approximately 15g of malt extract was weighed into a 1L autoclave bottle containing 
900 ml of water and stirred to mix with a magnetic stirrer. An autoclave tape was 
pasted on the bottle and the mixture autoclaved. The autoclaved media was allowed to 
cool and then stored in a fridge at 4°C until needed. 
3.3.3 Treatment of mangrove plant and culture of endophytic fungi 
In the laboratory, pieces of each plant part including leaves, buds, submerged roots, 
aerial roots, aerial stems, fruit shoots and flowers was surface sterilized under sterile 
conditions by first rinsing it with sterile artificial sea water (SASW) and then 
immersing it in 70% ethanol for 1 minute. 
The plant part was then cut transversely at all sides into a smaller piece with a pair of 
flame sterilized scissors in a bio-safety cabinet. This piece was again sterilized under 
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sterile conditions by first rinsing it with SASW and then immersing it in 70% ethanol 
for 1 minute. The piece was again cut at all sides into a much smaller piece with a 
flame sterilized scalpel. This piece was again sterilized by rinsing with SASW, then 
immersing in 2% sodium hypochlorite for 1 minute and then rinsed again with SASW 
3 times. The treated plant piece was afterwards placed on a malt extract agar plate 
(parent or master plate) and the plate was labelled, parafilmed and incubated at 28 °C 
for three weeks with daily observations to detect the spring-up of new fungal colonies. 
3.3.4 Isolation of pure endophytic fungi 
Observation of the parent or master plates obtained for each plant part cultured, 
starting from day one (1) to day twenty-one (21) saw the appearance on the parent 
plates of several different colonies of marine endophytic fungi which were 
subsequently picked one at a time, re-cultured on fresh malt extract plates and kept at 
an incubation temperature of 28°C. All the colonies initially sub-cultured from the 
parent or master plates were subsequently sub-cultured until very pure strains were 
obtained for each species.  
3.3.5 Small scale culture of pure endophytic fungi 
The malt extract liquid broth prepared earlier was thawed in a water bath and 100ml 
was transferred into an already autoclaved 250 ml Erlenmeyer flasks with non-
absorbent cotton wool stuck in the mouth under sterile conditions. A single colony 
from one of the pure strains of endophytic fungi obtained was picked and inoculated 
into the broth under sterile conditions. The inoculated flasks were labelled and 
incubated in an incubator at 28°C for two weeks. After two weeks, the culture broth 
was filtered by suction filtration to separate fungal mycelia from the broth itself. The 
broth was extracted once with ethyl acetate and the mycelia repeatedly and 
alternatively extracted with methanol and dichloromethane. The ethyl acetate, 
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methanol and dichloromethane extracts were dried under vacuum and subsequently 
combined to give a total crude extract or TCE. The same steps were repeated for all 
the pure fungal strains that looked interesting and worthy of chemical investigations. 
 
 
Plate 3.6: Small scale fermentation broth of BUSUA 2A-L. 
 
3.3.6 Chemical profiling and prioritization of pure endophytic fungi 
About 1-1.5 mg of TCEs obtained for pure species of mangrove endophytic fungi 
isolated from different plant parts were dissolved in 1-1.5 ml of HPLC grade 
methanol and sent to the Marine Biodiscovery Centre, Department of Chemistry, 
University of Aberdeen, Scotland for HRESI/HPLC-DAD-MSn analysis. 
High resolution mass spectrometric data were obtained using a Thermo Instruments 
MS system (LTQ XL/LTQ Orbitrap Discovery) coupled to a Thermo Instruments 
HPLC system (Accela PDA detector, Accela PDA autosampler, and Accela pump). 
The following conditions were used: capillary voltage 45V, capillary temperature 320 
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C, auxiliary gas flow rate 10–20 arbitrary units, sheath gas flow rate 40–50 arbitrary 
units, spray voltage 4.5 kV, mass range 100 2000 amu (maximum resolution 30,000). 
HPLC separations were carried out using a Phenomenex reversed-phase (C18, 250 6 
10 mm, L x i.d.) column connected to an Agilent 1200 series binary pump and 
monitored using an Agilent photodiode array detector. Detection was carried out at 
227 nm. 
This data provided the photodiode array profile, mass ion counts, specific UV and 
fragmentation pattern of all the metabolites present in each TCE. Input of this 
information into the MarinLit, AntiMarin and Antibase databases provided detailed 
information on already known compounds and prior information about the structures 
of all new compounds.  
 
 
Figure 3.2: Illustration of the HRESI/HPLC-DAD-MSn spectrum. 
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3.3.7 Large scale culture of high priority endophytic fungi 
After the HRESI/HPLC-DAD-MSn analysis, it was detected that marine-derived 
fungi, Penicillium sp. BRS2A-AR2 produced interesting compounds. Therefore, large 
scale fermentation in about 1 L broth was carried out to isolate these compounds. This 
was done by inoculating two already autoclaved 1 L Erlenmeyer flasks, sealed with 
non-adsorbent cotton wool, containing 500 ml of malt extract liquid broth with 
Penicillium sp. BRS2A-AR2under sterile conditions. These flasks were labelled and 
incubated at 28 °C for a period of 12 months. At the end of the incubation period, the 
cultures were filtered under suction and the mycelia separated from the broth. The 
broths were extracted once with ethylacetate and the mycelia were combined and 
repeatedly and alternatively extracted with methanol and dichloromethane. The 
extracts obtained after drying under vacuum from the ethylacetate, methanol and 
dichloromethane fractions were combined to give a TCE (1.30 mg).  
3.3.8 Solvent partitioning by a modification of Kupchan’s method 
The next step which followed solvent extraction of the liquid broth and mycelia of 
Penicillium sp. BRS2A-AR2 was solvent partitioning. The procedures adopted were a 
modification of Kupchan’s method (1973),162 employing a combination of solvent 
systems with polarities ranging from the relatively non-polar hexane to the extremely 
polar water solvent. First, the TCE was suspended in water and extracted with an 
equal volume of dichloromethane three times. The result was two fractions, an 
aqueous fraction and a dichloromethane fraction. The aqueous fraction was extracted 
with an equal volume of sec-butanol once. Two separate fractions were obtained, the 
aqueous fraction from this end was discarded and the sec-butanol layer was rotary 
evaporated to dryness and labelled BRS2A-AR2-WB (390.04 mg). The 
dichloromethane fraction obtained initially from the first partitioning process was 
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rotary evaporated to dryness under reduced pressure. This fraction was then 
suspended in 90% methanol: water and extracted with an equal volume of hexane 
(1:1) three times. Resulting hexane layer was rotary evaporated to dryness and 
labelled BRS2A-AR2-FH (248.78 mg). The 90% methanol: water layer was then 
phase adjusted to 50% methanol: water and extracted with an equal volume of 
dichloromethane (1:1) three times. Both resultant fractions were rotary evaporated to 
dryness under reduced pressure and labelled BRS2A-AR2-FD dichloromethane 
fraction (396.95 mg) and BRS2A-AR2-FM 50% methanol: water fraction (256.48 
mg) accordingly. Fractions that separated out in the solvent partitioning process 
possessed defined polarities.  
 
TCE
Suspend in water
extract with DCM 
three times
DCM
Aqueous Layer
layer rotary evaporated 
To dryness
Partition  with Dissolve in 90% MeOH
sec-butanol once and extract with n-hexane 
three times
Butanol layer 90% MeOH Hexane layer
Aqueous layer rotary evaporated Layer phase rotary evaporated 
discarded To dryness Adjusted to 50% To dryness
WB WATER FH
Extract with DCM 
three times
DCM layer 50% MeOH 
rotary evaporated Layer
To dryness rotary evaporated 
FD To dryness
FM
 
 
Figure 3.3: Flow chart for the isolation of BRS2A-AR2-FD and FM.
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Between 1-1.5 mg of each of the resulting four fractions FH, FD, FM, and WB were 
then sent to our collaborators in the Aberdeen for HRESI/HPLC-DAD-MSn analysis 
to determine which fractions had retained the compounds of interest. 
 
3.3.9 Purification of BRS2A-AR2-FM and isolation of compounds 
HRESI/HPLC-DAD-MSn analysis of the Kupchan solvent partitioning extracts 
showed that, most of the compounds of interest were in BRS2A-AR2-FM. Hence, this 
fraction was subjected to a size exclusion column chromatography using Sephadex-
LH20 as stationary phase and methanol as mobile phase as shown in the Figure 
below.  
 
 
Plate 3.7: Sephadex LH-20 size exclusion chromatography of BRS2A-AR2-FM 
with fractions starting to separate into different molecular weights. 
 
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Five Sephadex LH-20 fractions were collected in all and these fractions were 
concentrated using a rotary evaporator, transferred into vials and labelled BRS2A-
AR2-FM-SF1-5. About 1.0 mg of each fraction was dissolved in 1.5 ml HPLC grade 
methanol and submitted for HRESI/HPLC-DAD-MSn analysis. From the analysis, the 
SF2, SF3, SF4 and SF5 fractions all contained interesting compounds but only the 
isolation of compounds in SF2 (58.00 mg) is subsequently described in this thesis.  
Fraction BRS2A-AR2-FM-SF2 was subjected to alternating HPLC separation and 
purification using a Phenomenex Luna C18 column (C18 250 × 10 mm, L × i.d.) and 
column chromatography by gravity using silica as stationary phase and hexane-ethyl 
acetate mixtures as eluents. Gradients of H2O:CH3CN (1:1 in 30 min and hold for 20 
min) were used as eluents with column flow rates set at 1.5 mL/min to afford 3.90 mg 
of Quinolactacin A1 and A2 as an intimate mixture and 3.03 mg of Citrinadin A from 
58.00 mg of sample BRS2A-AR2-FM-SF2. About 1.0 mg of each of the fairly pure 
compounds was subjected to HRESI/HPLC-DAD-MSn analysis to ascertain the 
purity, molecular formula and the fragmentation pattern. 
3.3.10 1D and 2D NMR analysis of Quinolactacin A1 and A2 and Citrinadin A 
NMR data were acquired on a Varian VNMR 600 MHz and Bruker 500 MHz 
spectrometers with either CDCl3 or CD3OD as solvents. Data acquired include 
1H, 
13C, DEPT-135°, 1H-1H-COSY, HSQC, HMBC and gHSQC-TOCSY.  
3.3 Biological activity test 
3.3.1 Chemicals and reagents 
RPMI-1640, HEPES, Dulbecco Modified Eagle’s culture Media (DMEM), Foetal 
Bovine Serum (FBS),Trypsin, Gentamycin, Penicillin-Streptomycin-L-Glutamine 
(PSG), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), Curcumin, 
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Artesunate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 
dye, Dimethyl sulphoxide (DMSO), Sodium citrate, Adenine,  Sodium bicarbonate 
(NaHCO3), AlbuMax II, Sodium chloride (NaCl), Potassium chloride (KCl), Sodium 
Phosphate Dibasic (Na2HPO4), Sodium Phosphate Monobasic (KH2PO4), Sodium 
bicarbonate (NaHCO3), Sodium hydroxide (NaOH), Hydrochloric acid 
(HCl),5,5’,6,6’-tetrachloro-1, 1’,3,3’-tetramethyl-benzimidalylcarbocyanine iodide 
and 7-Aminoactinomycin D were purchased from Sigma-Aldrich, USA.Malt extract, 
and other chemicals and reagents used were of analytical grade or of the highest 
purity commercially available. 
3.3.2 Compounds tested for possible biological activity 
Apart from the compounds isolated in the course of this project, some of the 
compounds previously isolated by other students within the research group were also 
tested alongside to make the study more interesting. All the compounds tested are 
therefore summarized in Table3.1 as shown below. 
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Table 3.1: Name and structure of compounds studied for biological activity 
Compound name/Source Structure 
Cholest-5-ene-3,25,26-triol 
(3 ,25 ) 
 
Axinella sp. 
*new compound 
SF2C5HG  
20-Ethyl-5-pregnen-3-ol 
 
Axinella sp. 
 
SF2C5HH 
 
-Sitosterol 
 
Axinella sp. 
 
SF2C5HI 
 
Butrepyrazinone 
 
Verrucosispora sp. K51G 
*new compound 
  
 
Quinolactacin A1 
 
Penicillium sp. BRS2A-AR2 
 
 
Quinolactacin A2 
 
Penicillium sp. BRS2A-AR2 
 
 
Citrinadin A 
 
Penicillium sp. BRS2A-AR2 
 
 
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Butrecitrinadin 
 
Penicillium sp. BRS2A-AR2 
 
 
3.3.3 Cell lines tested in the cytotoxicity assay 
The under listed human cancerous cell lines used in the cytotoxicity assays conducted 
during the course of this project were kind gifts from Dr. Takuhiro Uto of Nagasaki 
International University, Japan. 
Jurkat  HumanT-lymphoblastic leukemia cells (Suspension cells) 
HepG2  Human hepatocellular carcinoma (Adhesive cells) 
HL-60  Human promyelocytic leukemia cells (Suspension cells) 
LNCap Human prostate cancer (Adhesive cells) 
MCF-7 Human breast cancer (Adhesive cells) 
PC-3  Human prostate cancer (Adhesive cells) 
 
3.3.4 Preparation of compounds for bioactivity testing 
Stock solutions of all the compounds were prepared at a concentration of 10mM. This 
was achieved by drying the compounds with nitrogen gas, weighing to ascertain the 
mass of the compound and dissolving in appropriate amount of DMSO in order to 
attain desired concentration. The solutions were vortexed and filter sterilized into 
vials through 0.45µm millipore filters under sterile conditions and stored at -20°C 
until use. 
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3.3.5 Cytotoxicity studies 
3.3.5.1 Cell culture and cell treatments 
Jurkat, HL-60, LNCap and PC-3cells were cultured in RPMI-1640 medium, while the 
MCF-7 and HepG2 cells were maintained in DMEM medium. All cell cultures were 
supplemented with 10% FBS, 1% Penicillin-Streptomycin-L-Glutamine and 
incubated at 37˚C in a 5% CO2under humidified atmosphericconditions. 
All the compounds in addition to Curcumin (used as positive control) were dissolved 
in DMSO and stored at -20˚C until used. The DMSO concentration in the test wells 
did not exceed 0.1% (v/v), and the control cells were treated with the same amount of 
DMSO. 
 
 
Plate 3.8: Cancer cells in incubation. 
 
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3.3.5.2 Cell viability assay 
The growth inhibition of the cell lines due to the test compounds or standard was 
examined using the tetrazolium-based colorimetric assay (MTT Assay) to determine 
the viability of the cells at any particular time. 
PRINCIPLE: The assay is based on the capacity of the cellular mitochondrial 
reductase enzyme in living cells to reduce the yellow water-soluble substrate 3-(4, 5-
dimethylthiazol- 2yl)-2, 5-diphenyl tetrazolium bromide (MTT) into apurple 
formazan crystals which is soluble in acidified isopropanol. Since reduction of MTT 
can only occur in metabolically active cells, the level of activity is a measure of the 
viability of the cells. The colour development from yellow to purple ismonitored at 
570nm using a spectrophotometer. 
 
 
Scheme 3.1: Conversion of yellow MTT dye to dark blue formazan by cellular 
mitochondrion reductase. 
 
PROCEDURE: The procedures described by Ayisi et. al. (1991) were followed with 
modifications. Dilution of the 10mM stock solution of each compound was made in 
1% DMSO to obtain five different concentrations ranging from0to 100µM.163 
Cultured suspension cells (HL-60 and Jurkat cells) were transferred into centrifuge 
tubes. The mono-layer adhesive cells (LNCap, PC3, HepG2, and MCF7) in the 
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culture flasks were washed with PBS, detached with trypsin solution and transferred 
into centrifuge tubes. Tubes were centrifuged at 1000rpm for 5 minutes and the pellets 
were re-suspended in growth media. Cells were counted using a haemocytometer 
(MARIENFELD, Germany) and a cell suspension of 1x105 cells/ml was prepared by 
diluting with a growthmedia.100µL (1 × 105 cells/mL) of cell suspensions were 
seeded into 96-well plates and incubated overnight before treatment. 
10 µL of the compounds at different concentrations were each added to the wells with 
the cell suspensions and incubated under the condition stated above for 72 hours. 
Curcumin was used as a positive control in all assays and a colour control plate was 
also setup for each compound. 20µL of 2.5mg/mL MTT solution was added to the 
wells and incubated further for4 hours in the CO2 incubator. 150µL of acidified 
isopropanol containing Triton-X was then added to stop the reaction. The reaction 
plates were incubated in the dark at room temperature overnight and absorbance read 
at 570nm using a micro-plate spectrophotometer (Tecan Infinite M200 Pro plate 
reader, Austria).   
Percent Cell viability was determined as: 
 
Aα=   Mean absorbance of treated cells 
Aβ=   Mean absorbance of blank 
Aγ =   Mean absorbance of untreated cells 
The average percentage cell viability determined at each concentration was plotted as 
a dose response curve using Graph Pad Prism Version 5.02. The inhibition 
concentration at fifty percent (IC50) values, that is, concentration of compounds or 
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standard drug inducing 50% inhibition of cells, determined from the dose response 
curve by nonlinear regression analysis. 
 
 
 
Plate 3.9: 96 well plate showing conversion of yellow MTT to Formazan 
 
3.3.6 Anti-malaria activity study 
3.3.6.1 Blood collection and erythrocytes preparation 
Erythrocytes were obtained from blood of consented volunteers (Blood group O+). 
Venous blood was drawn and collected into containers containing citrate phosphate 
dextrose (CPD) solution and kept at 4 oC overnight. It was centrifuged at 2,000 rpm 
for 10 minutes to separate the serum and buffy coat. Packed erythrocytes were washed 
three times with parasite washing medium (RPMI 1640, buffered with,50 µg/mL 
gentamicin and 2 mM L-glutamine) Each washing step involved addition of wash 
medium, pipetting up and down thrice, centrifuging at 2,000 rpm for 10 minutes and 
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then discarding suspended medium. After washing, medium was added to the packed 
erythrocytesand stored at 4 oC until ready for use. Washed RBCs were stored and 
used for up to 2 weeks maximum after which new blood was collected. 
3.3.6.2 Giemsa stained thin blood smear and parasitaemia determination 
A drop of infected erythrocytes cultured medium was placed on a microscope slide 
and spread with the aid of another slide. Slides were air dried, dipped into methanol 
for some seconds to fix and air dry again. A 10% Giemsa stain was added to cover 
completely the surface of the fixed slides for at least 10 minutes and then gently 
rinsed off with running water. Stained slides were air dried and then viewed under a 
light microscope (OLYMPUS CK30) (with immersion oil at 100x objective) to 
determine parasitaemia. Percent parasitaemia was determined by counting the number 
of infected cells in a total of 500 erythrocytes in the Giemsa-stained thin blood smear. 
Parasitaemia was expressed as a percentage of the number of infected erythrocytes to 
the total number of erythrocytes counted. 
3.3.6.3 In vitro cultivation of Malaria Parasite 
Erythrocytic stages of malaria parasite (Plasmodium falciparum-chloroquine sensitive 
strain 3D7) were cultured in 25 cm2flasks using the method of Trager and Jensen 
(1976) with modifications. Erythrocytes were maintained at 2% haematocrit (v/v) cell 
suspension in complete malaria parasite medium (RPMI 1640, buffered with 25 mM 
HEPES, supplemented with 7.5% NaHCO3, 25 ug/ml gentamycin, 5% heat-
inactivated human O+ serum (from consented subject) and 5 mg/mlAlbuMax II) and 
incubated at 37oC under gas condition of 2% O2, 5% CO2 and 93% N2. 
Parasite growth and development were monitored with Giemsa stained thin blood 
smear. Parasite culture was purified by using 5% sorbitol to obtain matured 
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erythrocyte parasitic stages (late trophozoites and schizonts) from uninfected cells. 
The matured erythrocyte parasitic stages (purity > 90%) obtained were used to screen 
the compounds for anti-malaria activity. 
3.3.6.4 Screening for anti-malaria activity using the SYBR Green I assay 
A total of six(6) compounds were screened for anti-malaria activity by using the 
SYBR Green I fluorescence assay as described by Smilktein et. al. (2004) with some 
modifications.164 Serial dilution of the standard (artesunate) which served as an 
experimental control and stock solution of compounds to yield final concentrations 
ranging from 100 ng/ml to 400 ng/ml and 3.13 µg/ml to 25 µg/ml respectively were 
prepared. The matured erythrocyte parasitic stages were treated with compounds and 
washed erythrocytes in 96 well plates (Nunc) and incubated with complete malaria 
parasite medium for 24 hrs.  
Slides were then prepared and percent parasitaemia was determined by counting the 
number of infected cells in a total of 500 erythrocytes in the Giemsa-stained thin 
blood smear. Briefly an aliquot of 5 µL per each concentration of the standard drug 
and compounds was dispensed into test wells. 95µL of complete malaria parasite 
medium with washed erythrocytes at 2% haematocrit and the purified matured 
erythrocyte parasitic stages (1% parasitaemia) were added, and incubated at 37oC 
under gaseous conditions as stated above and untreated erythrocytes were used as 
control. 
Wells containing erythrocytes at 2% haematocrit, infected erythrocytes at 2% 
haematocrit and complete parasite medium alone served as negative controls, positive 
controls and blank controls respectively. Furthermore, wells containing infected 
parasites and 0.1% DMSO served as reference controls. Final volume per well was 
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100 µL. Plates were then incubated for 24 hr as described above in the cultivation of 
malaria parasites. 100 µL aliquot of 2.5x buffered SYBR Green I (0.25 µL of SYBR 
Green I/mL of phosphate buffer saline) was added to each well after the incubation 
period and incubated in the dark for 30 min at 37 oC. Fluorescence was detected by 
Guava EasyCyte HT FACS machine (Millipore, USA). 
Principle: The contrast between host erythrocytes which lack DNA and RNA and the 
malaria parasites which have DNA and RNA form the basis of the SYBR Green 
experiment. The plasmodium parasites can easily be stained with the SYBR Green 
dye and quantified by fluorescence spectroscopy.  
3.3.7 Apoptosis study 
3.3.7.1 Mitochondrial Potential 
Determination of apoptosis-inducing capabilities of compounds as indication of high 
anti- malaria activities was done by the use of the Guava MitoPotential(R) kit (Guava 
Technologies, USA) as described by the manufacturer’s instructions. A cationic dye, 
5,5’,6,6’-tetrachloro-1, 1’,3,3’-tetrathylbenzimidalylcarbocyanine iodide (JC-1), was 
used to evaluate mitochondrial membrane potential changes and 7-Aminoactinomycin 
D (7-AAD), a cell-impermeant DNA intercalator, was also used to monitor cell 
membrane permeability changes. Synchronized culture parasites and preparation of 
96-well micro-titer plates were taken through the same procedure described above for 
the screening of the compounds to. 2x staining solution (4 µL of 50x staining 
solution/100 µL of complete parasite medium) of the Guava MitoPotential(R) kit was 
then added to each well in a ratio of 1:1 (v/v) and incubated for 30 min at 37 oC in the 
dark. Stained cells were analyzed on a Guava EasyCyte HT system. 
 
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3.3.8 Anti-buruli ulcer activity assay 
3.3.8.1 Resazurin Microtiter Assays (REMA) 
Preparation of Inoculum: The Resazurin based assay as described by Yemoa et. al. 
(2011) was followed.165The assay used in detecting growth inhibition of 
microorganisms including mycobacteria was used to access the ability of the 
compounds to exhibit inhibition against Mycobacterium ulcerans.The inoculum was 
prepared by making a direct broth suspension of a full loop of isolated colonies 
selected from a 6 weeks Middlebrook 7H9 solid medium fresh Mycobacterium 
ulcerans MN209 characterized isolate culture adjusted to achieve a turbidity of 1.0 
McFarland turbidity standard which is equivalent to 3 x 108 colony forming units 
(CFU)/ml. The adjusted inoculum suspension was further diluted 1:1000 in 7H9broth 
up to approximately 1 x 105 CFU/ml. 
Inoculation: Within 15 minutes after the inoculum has been standardized as 
described above, 100 µL of the adjusted inoculum was added to each tube containing 
4 µL of the compounds in the dilution series with a positive control well containing 
only broth and the plates were incubated.  
Procedure: After 15 days of incubation, resazurin was added and a colour change 
observed after 48 hours incubation. 
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CHAPTER FOUR 
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4.0 RESULTS AND DISCUSSION 
4.1 Dereplication of marine-derived endophytic fungi crude extracts 
Dereplication is a process in natural product drug discovery that is designed to 
maximise all laboratory efforts and prioritise the isolation of new or novel chemistry 
from crude extracts of nature’s flora or fauna.166,167 When structural novelty is not a 
priority but, biological activity constitute the main driving force for a specific natural 
product drug discovery project, biological activity guided screening of crude extracts 
is preferred. In some cases, both techniques have been used hand-in-hand to yield 
compounds that have both structural novelty and biological activity. 
In all, twenty-four (24) different species of marine-derived endophytic fungi were 
isolated from various plant parts of three major mangrove plants growing in and along 
the banks of the Butre River in the Western Region of Ghana. The three (3) main 
plants sampled were Conocarpus erectus, Laguncularia racemosa and Rhizophora 
racemosa. Six (6) different species out of the twenty-four (24) isolated fungi were 
selected for chemical profiling, prioritization, identification and subsequent isolation 
of compounds from high priority extracts. 
After small scale culture of these six (6) endophytic fungal species with measurement 
and analysis of HRESI/HPLC-DAD-MSn data, two fungi i.e. Strain BRS2A-AR 
andBRS2A-AR2 were prioritized for identification and further isolation and 
characterization of secondary metabolites. 
However, due to time constraints, only Penicillium sp. BRS2A-AR2 was further 
fractionated to isolate some of the component secondary metabolites. 
It is important to note that, the remaining four (4) species were also interesting but, set 
aside for two main reasons: 
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 These species did not make a wide range of secondary metabolites. They seem 
to possess low biosynthetic prowess and ingenuity. 
 The secondary metabolites detected in their small scale culture extracts were 
interesting but already known compounds. 
4.1.1 Dereplication of crude extracts of BRS3A-T2 
The marine-derived fungi BRS3A-T2 (Plate4.1) was isolated as a fast-growing fluffy 
white microbe with granules of dark stones interspersed within its structural matrix. 
Careful analysis of the crude extracts obtained from small scale culture of this species 
showed a rather low biosynthetic prowess. This observation was true for this species, 
at least for the malt extract medium in which it was cultured.  
 
  
 
Plate 4.1: Pure endophytic fungi BRS3A-T2 isolated from the trunk of an 
indigenous mangrove plant sampled at Butre River. 
 
After small scale culture of BRS3A-T2, the culture was suction filtered and the 
mycelium was separated from the broth as outlined in detail in Chapter 3. The broth 
was extracted once in a separating funnel with ethyl acetate while the mycelium was 
extracted with methanol. The two extracts were then dried under vacuum and 1.0 
mg/ml of each was submitted for the HRESI/HPLC-DAD-MSn analysis. The 
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MarinLit database search for the two measurements shown in Figures 4.1 and 4.2 
revealed that, BRS3A-T2 predominantly makes the already isolated and characterized 
brominated diterpene 14S-bromo-1S-hydroxy-1,2,13,14-tetrahydrosphaerococenol A 
which was previously isolated from the red alga Sphaerococcus coronopifolius 
(Scheme 4.1).168 In this article, Smyrniotopoulos et. al. (2008) obtained five other 
derivatives of this compound all of which were based essentially on the brominated 
diterpene backbone. These compounds were tested and found to possess antibacterial 
activity against a panel of multidrug-resistant and methicillin-resistant Staphylococcus 
aureus with MICs in the range of 0.5-128 µg/ml.168 
 
 
Scheme 4.1: Proposed structures for the mass spectrometry fragments of 14S-
bromo-1S-hydroxy-1,2,13,14-tetrahydrosphaerococenol A. 
 
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Figure 4.1: HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of 
BRS3A-T2 liquid culture in malt extract. 
 
Figure 4.2: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia 
of BRS3A-T2 cultured in malt extract. 
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In order to confirm the identity of this known compound, the fragmentation pattern 
produced in theHRESI/HPLC-DAD-MSn was investigated and sub-structures were 
proposed as shown in Scheme 4.2. 
This species BRS3A-T2 was therefore abandoned for fear of re-isolating a bunch of 
already characterized marine-derived fungal metabolites. However, the very important 
discovery here is that, it appears as if the red alga Sphaerococcus coronopifolius is not 
really responsible for the biosynthesis of 14S-bromo-1S-hydroxy-1,2,13,14-
tetrahydrosphaerococenol A or any of its derivatives. Rather, marine-derived 
endophytic fungi colonizing the inner tissues of the red alga might be responsible for 
making these compounds as an attempt to protect their host which in this case is 
Sphaerococcus coronopifolius.  
 
 
Scheme 4.2: Biosynthesis of 14S-bromo-1S-hydroxy-1,2,13,14-
tetrahydrosphaerococenol. 
 
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4.1.2 Dereplication of crude extracts of BRS2A-AR 
The species BRS2A-AR was purified and found to grow as a crispy dark green carpet 
which formed mostly uniform lawn on the agar plates (Plate4.4). Small scale culture 
of the marine-derived endophytic fungal species BRS2A-AR followed by a work-up 
process as described above, gave two extracts; an ethyl acetate extract of the broth 
and methanol extract of the mycelia. 1.5 mg/ml of these two extracts were submitted 
for HRESI/HPLC-DAD-MSn analysis. The data obtained showed that, BRS2A-
ARpredominantly makes two main compounds at m/z 456.2260 and 421.1258 (Figure 
4.3 and 4.4). The accurate masses and fragmentation patterns of these two molecules 
did not give any hits in the MarinLit, Antimarin and Antibase databases. This species 
was therefore important and characterized as high priority for further large scale 
culture and subsequent isolation and characterization of compounds. 
 
  
 
Plate 4.2: Pure endophytic fungi BRS2A-AR isolated from the aerial root of an 
indigenous mangrove plant sampled at Butre River. 
 
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Figure 4.3:HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of 
BRS2A-AR liquid culture in malt extract. 
 
Figure 4.4: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia 
of BRS2A-AR cultured in malt extract. 
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4.1.3 Dereplication of crude extracts of BRS1A-B 
The marine-derived fungi BRS1A-B was very similar to BRS3A-T2 in morphology 
but, the two produced entirely different secondary metabolites (Plate4.7). Ethyl 
acetate and methanol extracts of BRS1A-B were dominated by the known 
metabolite3-(phenethylamino)demethyl(oxy)aaptamine and its derivatives (Figures 
4.5-4.7). This compound along with the first member of the group to be characterized 
which is aaptaminewas previously isolated by Shaari et. al. (2009) from the sea 
sponge Aaptos aaptos belonging to the Phylum Porifera.169 It is most surprising 
therefore to find out that, these metabolites constitute the main secondary metabolites 
produced by the marine-derived endophytic fungus BRS1A-B. It is therefore possible 
that, the marine sponge Aaptos aaptos is not responsible for the biosynthesis of 
aaptamine and its derivatives but, harbours endophytic fungi which biosynthesise 
these secondary metabolites to protect their host which in this case is the 
sponge.Aaptamine, 3-(phenethylamino)demethyl(oxy)aaptamine and their derivatives 
were found to be cytotoxic to CEM-SS cells which are T-lymphoblastic leukemia 
cells with IC50 ranging from 5-15 µg/ml.169 
Further confirmation of the known structure of 3-
(phenethylamino)demethyl(oxy)aaptamine was achieved through the interpretation of 
the fragmentation pattern of this compound in Scheme4.3. 
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Plate 4.3:Pure endophytic fungi BRS1A-B isolated from the bud of an indigenous 
mangrove plant sampled at Butre River. 
 
 
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Figure 4.5: HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of 
BRS1A-B liquid culture in malt extract. 
 
Figure 4.6: MSn fragmentation pattern of m/z 331.1321 seen in both the ethyl 
acetate and methanol extracts of BRS1A-B. 
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Figure 4.7: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia 
of BRS1A-B cultured in malt extract. 
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Scheme 4.3: Biosynthesis of 3-(phenethylamino)demethyl(oxy)aaptamine. 
 
4.1.4 Dereplication of crude extracts of BUSUA2B-T 
Marine-derived endophytic fungus BUSUA2B-T was isolated as pure species which 
grew as a thick grey carpet on solid media (Plate4.4). Crude ethyl acetate and 
methanol extracts of this fungus did not show the presence of any interesting 
secondary metabolites (Figures 4.8 and 4.9). Further investigation of BUSUA2B-T 
was therefore abandoned. 
 
 
 
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Plate 4.4:Pure endophytic fungi BUSUA 2B-T isolated from the trunk of an 
indigenous mangrove plant sampled at Busia beach. 
 
 
Figure 4.8: HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of 
BUSUA2B-T liquid culture in malt extract. 
 
 
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Figure 4.9: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia 
of BUSUA2B-T cultured in malt extract. 
 
4.1.5 Dereplication of crude extracts of BUSUA2B-F 
The species BUSUA2B-F after purification was seen as a dark brown carpet on the 
malt extract agar plates (Plate4.5). Mass spectrometry analysis of the ethyl acetate and 
methanol extracts showed that, BUSUA2B-F produced predominantly the 
mycosporine amino acid N-methylpalythine-serine (Figures 4.10 and 4.16). This 
compound was previously isolated and characterized by Teai et. al.(1997) from a 
stony coral Pocillopora eydouxi. The article did not describe any form of bioactivity 
test for this compound or its derivatives but, mycosporine amino acids are widespread 
in marine chemistry and known to act as sun screens amongst other biological 
activities. Therefore, further chemical characterization of BUSUA2B-F was 
immediately abandoned.170 
 
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Plate 4.5: Pure endophytic fungi BUSUA 2B-F isolated from the fruit of an 
indigenous mangrove plant sampled at Busia beach. 
 
 
 
 
Figure 4.10: HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of 
BUSUA2B-F liquid culture in malt extract. 
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Figure 4.11: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia 
of BUSUA2B-F cultured in malt extract. 
 
4.1.6 Dereplication of crude extracts of BRS2A-AR2 
The marine derived specie BRS2A-AR2 was arguably the most interesting of the six 
(6) species studied in this project. On solid media, dark stretches of mycelia could be 
found growing within the agar and on top of the agar was a thick yellowish puss 
(Plate4.6). Coincidentally, these morphological features are very characteristic of the 
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genus Penicillium and BRS2A-AR2 was noted as one of such species of endophytic 
fungi although its full characterization is yet to be determined. 
 
  
 
Plate 4.6:Pure endophytic fungi BRS2A-AR2 isolated from the aerial root of an 
indigenous mangrove plant sampled at Butre River. 
 
Analysis of the mass spectrometry data for ethyl acetate and methanol extracts of 
BRS2A-AR2 revealed this species as very talented for two main reasons: 
 It makes quite a number of secondary metabolites as seen in the 
chromatograms shown in Figure4.12 and 4.13. 
 The structural skeletons of the metabolites seen in the chromatogram of 
Figures4.12 and 4.13 are not the same. This was detected by comparing the 
fragmentation patterns of all the compounds present in the mass spectrometry 
data. 
Interesting masses were detected at m/z values of 478.2562, 641.2723, 625.3962 
(681.4227), 293.1262, 563.2631. All these masses did not come up in the database 
searches with MarinLit, Antimarin and Antibase with the exception of 625.3962 
which came up as the novel pentacyclic alkaloid initially isolated and characterized by 
Tsuda et. al. from the marine-derived fungus Penicillium citrinum.171 
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Figure 4.12: HRESI/HPLC-DAD-MSn profile of the ethyl acetate extract of 
BRS2A-AR2 liquid culture in malt extract. 
 
Figure 4.13: HRESI/HPLC-DAD-MSn profile of the methanol extract of mycelia 
of BRS2A-AR2 cultured in malt extract. 
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Further fractionation and subsequent isolation of some of these metabolites are 
described in sections of the methodology outlined in Chapter 3 and the results are 
analysed here in this section.  
4.2 Structure determination of Citrinadin A and Butrecitrinadin 
Citrinadin A was isolated as a very deep yellow crystalline substance that was soluble 
in both dichloromethane and methanol. This compound showed prominent 
wavelength absorption maxima at λmax 221, 250, 270 and 332 nm in a 0.1% formic 
acid methanol solution. The HRESIMS of this compound gave m/z = 625.3962 for [M 
+ H]+ corresponding to a molecular formula of C35H52N4O6 ( = 0.2 ppm) with twelve 
(12) degrees of unsaturation. 
Due to the limited amount of the compound isolated from the crude extracts, it was 
difficult to get a good 13C NMR data for citrinadin A. Hence, the 13C chemical shifts 
were extracted from the gHSQC and HMBC data. Analysis of the 1H, 13C and 
multiplicity edited gHSQC spectra, suggested the presence of 11 quaternary carbons, 
9 methine, 5 methylene and 10 methyl carbons. 
The huge molecular weight of 625.3962 with about 31% of the carbon atoms being 
quaternary alongside the observation of only a few correlations in the 1H-1H-gCOSY 
spectrum was direct indication of the presence of several very short spin systems 
which is typical of the citrinadin A backbone. The only substructures obtained by 
analysis of 1H-1H-gCOSY data are as shown in Figure 4.14.  
The aromatic proton at H 7.20 (1H, ov., H5) showed correlations to protons H 7.76 
(1H, d, J = 7.2, H6) and 7.65 (1H, d, J = 7.2, H4) (substructure A). Correlations from 
the proton at H 5.28 (1H, m, H14) to protons at 1.94 (1H, m, H13)/1.91 (1H, m, 
H13') and 1.94 (1H, m, H15)/1.91 (1H, m, H15') confirmed substructure B with 3.75 
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(1H, ov., H12) also correlating to 1.94 (1H, m, H13)/1.91 (1H, m, H13') and 1.94 (1H, 
m, H15)/1.91 (1H, m, H15'). 
 
Figure 4.14: Substructures obtained solely from the analysis of 1H-1H-gCOSY 
data of citrinadin A. 
 
Evidence for substructure C involved correlations that were seen from H 2.04 (1H, 
ov., H-4') to 2.75 (1H, d, J = 10.8, H2'), 1.00 (3H, d, J = 6.4, H5') and 0.90 (3H, d, J = 
6.8, H6'). Also correlations such as 3.75 (1H, ov., H10) to 3.26 (1H, d, J = 11.6, H10') 
were rather confirmatory of the presence of diastereotopic protons at carbon positions 
C10, C8, C13, C15 and C17. 
 
Unlike the 1H-1H-gCOSY data, the HMBC was very detailed and provided a lot of 
correlations to complete a large part of the structure. The aromatic benzene ring 
portion of the indolinone ring (substructure A) was completed with the aid of HMBC 
correlations from C6-H4, C7-H5, C3a-H5, C7a-H6, C7a-H4 and C4-H6. There were 
no 1H-1H-gCOSY or HMBC data observed for the pentacyclic portion of the 
indolinone ring but, the observations of the carbons C2 (C 185.3) and C3 (C 64.4) 
provided concrete evidence of the existence of an indolinone ring. Substructure C 
which was meant to be part of the cyclopenta [b] quinolizidine moiety was also 
extended with the aid of crucial HMBC correlations C18-H29, C19-H28, C9-H29, 
C9-H28, C28-H29, C18-H28, C9-H8 and C18-H8. The full HMBC and 1H-1H-
gCOSY data are as summarized in Figures 4.15 and 4.16. 
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Figure 4.15:Full 1H-1H-gCOSY data of citrinadin A. 
 
 
 
 
Figure 4.16: Full HMBC correlation data of citrinadin A provides extension for 
the substructures obtained from 1H-1H-gCOSY data analysis. 
 
HMBC correlations C24-H23, C23-H24, C22-H23, C22-H24, C21-H23 and C23-H21 
provided proof of the presence of the epoxy propane ring and its substituents while 
HMBC correlations C2'-H7', C2'-H8', C2'-H6', C2'-H5', C5'-H6', C7'-H8', C8'-H7' and 
C4'-H5' made it obvious that, the N, N-dimethylvaline residue was present in this 
structure.  
 
In order to confirm the structural assignments made by 1H-1H-gCOSY and HMBC 
data, a detailed interpretation of the 2D-TOCSY of citrinadin A was made. Here 
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further confirmation of all the spin systems made it obvious that, the structure in 
question was citrinadin A. A detailed illustration of the data obtained from the 2D-
TOCSY is shown in Figure 4.17. All 1D and 2D NMR data are summarized in Table 
4.1 for citrinadin A and raw data can be found in the Appendix. 
 
 
Figure 4.17: Full 2D TOCSY correlations of citrinadin A. 
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Table 4.1:1H and 13C NMR data of citrinadins in CD3OD. 
ATOM δ 13C 13C δ 1H  Mult  1H-1H HMBC 2D-TOCSY 
(ppm) mult (ppm) (Hz) COSY 
1' 166.8 C      
20 194.8 C    H6, H5, H21  
2 185.3 C    H8  
7a 142.7 C    H6, H4  
3a 134.6 C    H5  
4 133.2 CH 7.65 d, 7.2 H5 H6 H6 
6 127.5 CH 7.76 d, 7.2 H5 H4 H4 
5 122.6 CH 7.20 ov. H6, H4   
7 117.6 C    H5  
18 82.3 C    H8, H29, H28  
2' 74.4 CH 2.75 d, H4' H7', H8', H5', H6', H5', H4' 
10.8 H6' 
14 65.7 CH 5.28 m H13, H15  H16, H12, H27, 
H17, H15, H13 
3 64.4 C      
21 64.1 CH 4.06 s  H23, H24  
9 63.2 C    H8', H29, H28  
22 61.6 C    H21, H23, H24  
12 56.5 CH 3.75 ov. H13, H15 H27 H14, H15, H13 
19 51.2 C    H29, H28  
10 50.8 CH2 3.75 ov. H10, H10'  11-NH 
3.26 d, 
11.6 
16 47.5 CH 4.02 ov.   11-NH, H14, 
H17, H15, H13 
8 41.7 CH2 2.21 ov.    
2.13 ov. 
7' 41.4 CH3 2.33 s  H8'  
8' 41.4 CH3 2.33 s  H7'  
15 34.3 CH2 1.94 m H14, H12  H14, H16, H12, 
1.91 m H27, H17 
13 33.0 CH2 1.94 m H14, H12 H27 H14, H16, H12, 
1.91 m H27, H17 
17 31.5 CH2 1.71 m   H14, H16, H15, 
1.67 m H13 
26 30.0 CH3 2.45 s    
29 28.1 CH3 1.39 s  H28 H28 
4' 27.5 CH 2.04 ov. H2', H5', H6' H5' H2', H6', H5' 
23 24.3 CH3 1.61 s  H24, H21 H24 
28 22.1 CH3 1.04 s  H29 H29 
6' 20.0 CH3 0.90 d, 6.8 H4' H5' H2', H4', H5' 
5' 19.2 CH3 1.00 d, 6.4 H4' H6' H2', H4', H6' 
24 18.5 CH3 1.25 s  H23 H23 
27 15.1 CH3 1.55 m   H14, H15', H13' 
1-NH   9.65 s, br.    
25-NH        
11-NH   11.23 s, br.   H10, H10', H16 
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Interestingly, some aspects of the spectrometric and spectroscopic data of BRS2A-
AR2 extracts cannot be overlooked. The mass spectrometry data of the crude extract 
of this strain (Figure 4.13) showed the presence of m/z 625.3962 and 681.4226 in a 
ratio of 1:1.5. The UV of the two compounds were virtually identical and their mass 
fragmentation patterns were also very similar. Also, the HPLC chromatogram of a 
mixture of the two compounds on preparative HPLC showed very poor resolution 
between the two compounds. Hence both compounds co-eluted with the same 
retention time and were collected together. The mass spectrometry analysis of the 
purified compounds gave the profile shown in Figure 4.24 below. 
 
 
Figure 4.18:HRESI/HPLC-DAD-MSn profile of citrinadin A and butrecitrinadin. 
 
Careful look at the data in Figure 4.18 showed peaks c, d, e and f as break down 
products of the two main compounds present and a reversal of the amounts of 
m/z625.3962 and 681.4226 present to give a ratio of 1:0.5. This reversal of the 
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amounts of these two compounds in the mass spectrometry data was indicative of the 
fact that: 
 Both compounds did degrade to some extent during the various purification 
procedures applied to the compound. 
 m/z 681.4226 suffered more from the effects of degradation than 625.3962 
hence a decrease in the percentage amount of the compound present. 
Fortunately, the main final degraded product of the two compounds was visible in 
Figure 4.18 and labelled as peak B. The mass fragmentation pattern of citrinadin A 
which has already been shown to be m/z 625.3962 was analysed into detail and 
chemical structures were proposed for the various fragments as shown in Figure 4.19 
and Scheme 4.4. 
 
 
Figure 4.19: Mass fragmentation pattern of citrinadin A m/z = 625.3962. 
 
 
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Scheme 4.4: Proposed structures for the mass fragments of citrinadin A = 
625.3962 
 
Mathematically, the difference between m/z 625.3962 and 681.4226 was found to be 
56.0264. Surprisingly, this consistent mass difference was seen throughout the 
fragmentation of m/z 681.4226 as compared to 625.3962 fragments and this is 
indicated in Figures 4.20 and Scheme 4.5. Clearly, the compound whose structure was 
responsible for the m/z 681.4226 has a structure as shown below and it was named 
butrecitrinadin after the river which produced the source organism.  
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Figure 4.20: Mass fragmentation pattern of butrecitrinadin m/z = 681.4226. 
 
 
 
 
 
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N N
OH H OH H
O O
O O
O HN N O O HN N O
O NH H O NH H
Chemical Formula: C +37H53N4O7
O O Exact Mass: 665.3909
Exact Mass: 681.4222
N
OH H
O
O
O HN N O
O NH H
Chemical Formula: C H N O •+36 50 4 7
H
O Exact Mass: 650.3674
O HN N
O NH H
N
H
O
O
O HN N O
O
O NH H
Chemical Formula: C H •+30 37N3O3
Exact Mass: 487.2829 Chemical Formula: C36H48N O
•+
4 6
O Exact Mass: 632.3568
H H
HO O
O HN N O HN N
O NH H O NH H
O O
Chemical Formula: C •+30H39N3O4 Chemical Formula: C31H40N3O
+
4
Exact Mass: 505.2935 Exact Mass: 518.3013
 
Scheme 4.5: Proposed structures for the mass fragments of butrecitrinadin = 
681.4246 
 
Furthermore, evidence from NMR spectroscopy was used to confirm the structure of 
butrecitrinadin as shown in Figure 4.21 and 4.22. The carbonyl carbon of the 
proposed 3-butanone side chain attached to the citrinadin backbone was observed at 
C206.2 ppm which is what is comparable to the values that were calculated 
theoretically for such a ketone. The methyl protons of the terminal carbon attached to 
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the ketone was also observed at H 2.00 (3H, s) and the corresponding carbon atom 
atC 33.0 ppm which is consistent with theoretical values. Hence, it appears that, the 
nature of the 3-butanone side chain makes it difficult to get correlations that link it to 
the rest of the citrinadin backbone but, butrecitrinadin undoubtedly is present with 
citrinadin A in the purified sample.  
 
 
Figure 4.21: HMBC spectrum of butrecitrinadin 
 
 
 
 
 
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Figure 4.22:1H NMR spectrum of butrecitrinadin 
 
4.3 Structure determination of Quinolactacins A1 and A2 
The quinolactacins A1 and A2 were isolated as a very light yellow substance which 
on drying turned into very white and flaky bits that easily became airborne. 
Apparently the light yellow portion was only remnants of impurities from the crude 
extracts. The compounds were soluble both in dichloromethane and methanol. Their 
UV profile was very interesting with wavelength absorption maxima at λmax221, 248, 
255, 315 and 328 nm0.1% formic acid methanol solution. The mass spectrometry data 
of quinolactacins A1 and A2 was very interesting and actually played a part to 
conceal the identity of these compounds until late in the structure elucidation process. 
Two peaks 293.1262 and 563.2631 were very consistent in the mass spectrometry 
data of the crude extracts as well as pure fractions containing quinolactacins A1 and 
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A2. Initially, during the dereplication stage, these masses were entered into the 
databases but they returned with no hits and rightfully so. It turned out after the 
analysis of 1D and 2D NMR data that, 293.1262 was [M + Na]+ and 563.2631 was 
[2M + Na]+. The molecular weight of the isolated quinolactacins was therefore 
270.1362 representing a molecular formula of C16H18N2O2 (( = 0.1 ppm) and nine 
(9) degrees of unsaturation.  
The 1H, 13C and multiplicity edited gHSQC spectra, suggested the presence of 6 
quaternary carbons, 6 methine, 1methylene and 3 methyl carbons. Analysis of the 1H-
1H COSY data gave the correlations 7.68 (1H, ddd, J = 8.0, 7.2, 4.0, H6) to 7.50 (1H, 
dd, J = 8.8, 4.4, H5), 7.68 (1H, ddd, J = 8.0, 7.2, 4.0, H6) to 7.40 (1H, ddd, J = 8.0, 
7.2, 6.8 H7), 8.39 (1H, ddd, J = 8.8, 6.4, 4.8 H8) to 7.40 (1H, ddd, J = 8.0, 7.2, 6.8 
H7), 7.40 (1H, ddd, J = 8.0, 7.2, 6.8 H7) to 8.39 (1H, ddd, J = 8.8, 6.4, 4.8 H8), 7.40 
(1H, ddd, J = 8.0, 7.2, 6.8 H7) to 7.68 (1H, ddd, J = 8.0, 7.2, 4.0, H6) and 7.50 (1H, 
dd, J = 8.8, 4.4, H5) to 7.68 (1H, ddd, J = 8.0, 7.2, 4.0, H6) confirmed the first spin 
system as indicated by substructure A.Also, 1H-1H COSY correlations 2.12 (1H, ov., 
H1') to 5.98 (1H, d, J = 45.2, NH), 4.76 (1H, s, H3), 0.53 (3H, J = 6.8, 1'-CH3) and 
additional correlations from 1.63 (1H, m, H2') to 4.76 (1H, s, H3), 4.76 (1H, s, H3) to 
1.63 (1H, m, H2')/1.49 (1H, m, H2''), 5.98 (1H, d, J = 45.2, NH) to 2.12 (1H, ov., H1') 
and 0.53 (3H, J = 6.8, 1'-CH3) to 2.12 (1H, ov., H1') confirmed the substructure B as 
indicated in Figure 4.23 below. 
 
 
 
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Figure 4.23: Substructures obtained solely from the analysis of 1H-1H-gCOSY 
data of quinolactacin A1 and A2. 
 
Subsequent extensions of substructure A and B were aided by several HMBC 
correlations including C9-H8, C3a-4-CH3, C4a-H8, H6 and 4-CH3, C6-H8, C8a-H5 
and H7, C8-H6, C7-H5, C5-H7 that helped to construct fully the unique N-methyl 
quinolone moiety that is fused to a lactam ring. HMBC correlations from C3-H2' and 
1'-CH3, C1'-H2', H2'', H3' and 1'-CH3 provided evidence for the sec-butyl group 
attached at C3 which is characteristic to quinolactacins A1 and A2. Illustrations of all 
COSY and HMBC data are shown in Figures 4.24 and 4.25. 
 
Figure 4.24: Full 1H-1H-gCOSY data of quinolactacin A1/A2. 
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Figure 4.25: Full HMBC correlation data of quinolactacin A1/A2 provides 
extension for the substructures obtained from 1H-1H-gCOSY data analysis. 
 
In order to confirm the structure obtained from all 1H-1H COSY and HMBC data, a 
2D-TOCSY experiment was conducted. Correlations from the 2D-TOCSY 
experiment were rather few probably due to the short acquisition time of five (5) 
hours. However, the few correlations obtained only served to further confirm those 
that were obtained previously from the HMBC and 1H-1H COSY. All 2D-TOCSY 
data are summarized in Figure 4.26 and detailed 1D and 2D NMR data can be found 
in Table 4.2 with raw data in the Appendix. All the NMR data obtained in the current 
project was consistent with those documented in the literature.172 
 
Figure 4.26:Full 2D TOCSY correlations of quinolactacin A1/A2. 
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Table 4.2:1H and 13C NMR data of quinolactacins in CD3OD. 
ATOM δ 13C 13C δ 1H  Mult  1H-1H HMBC 2D-
(ppm) mult (ppm) (Hz) COSY TOCSY 
9 173.5 C    H8  
1 170.6 C      
3a 164.1 C    4-CH3   
4a 141.3 C    H8, H6,   
4-CH3 
6 133.0 CH 7.68 ddd, 8.0, H5, H7 H8 H8 
7.2, 4.0 
8a 128.2 C    H5, H7  
8 127.1 CH 8.39 ddd, 8.8, H7 H6 H6 
6.4, 4.8 
7 125.0 CH 7.40 ddd, 8.0, H8, H6 H5  
7.2, 6.8 
5 115.7 CH 7.50 dd, 8.8, H6 H7  
4.4 
9a 110.9 C      
3 58.5 CH  4.76 s, broad  H2', 1'-CH3  
1' 36.9 CH 2.12 ov. NH, H3,  H2', H2'',  
1'-CH3 H3', 1'-CH3 
4-CH3 36.1 CH3 3.79 s    
2' 28.1 CH2 1.63 m H3', H3' H3', 1'-CH3  
1.49 m 
3' 12.2 CH3 1.05 t, 7.2 H2', H2'' H2', H2'' H2', H2'' 
1'-CH3 11.8 CH3 0.53 d, 6.8 H1'  H1', H2', 
H2'', H3' 
NH   5.98 d, 45.2 H1'   
 
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4.3.1 Acid-base rapid epimerization of quinolactacins 
Only thirteen (13) quinolactacins have been isolated and characterized to-date but, 
their structural novelty and biological activity has inspired a lot of research. 
Interestingly, out of the thirteen molecules described so far, only quinolactacins A2 
and B2 are genuine or native natural product molecules with the remaining eleven 
(11) described structures the result of very quick acid-base catalysed epimerization 
reactions.172 These reactions lead to the formation of quinolonimide and then 
eventually to quinolonic acid. Clark et. al. (2006) proposed mechanisms for these 
acid-base catalysed epimerization reactions, an example of which is shown in 
Figure4.27 for the transformation of quinolactacin A2 to A1 and vice versa.   
 
 
These reactions are the reason why it is very difficult to have pure solutions of one 
quinolactacin. While we are confident that the current research led to the isolation of 
quinolactacin A, we are not sure which of the epimers predominates and therefore we 
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assume an equilibrium mixture of both as can be seen from the very broad 1H NMR 
signals that were observed in this project.  
 
Figure 4.27: Acid-base epimerization interconversions of quinolactacin A2 to A1. 
 
4.4 Anti-proliferative activity of compounds 
The anti-proliferative activities of six compounds cholest-5-ene-3,25,26-triol (3,25) 
(SF2C5HG), 20-ethyl-5-pregnen-3-ol (SF2C5HH), -sitosterol (SF2C5HI), 
butrepyrazinone, quinolactacin A1/A2, citrinadin A/butrecitrinadin were tested on 
human cancer cell lines as shown in Figure 4.28A-F.  Quinolactacin A1 and A2 were 
tested as an intimate mixture of both compounds in the ratio of 1:1 while citrinadin A 
and butrecitrinadin were tested as an intimate mixture in the ratio of 1.5:1. The 
compounds showed some level of anti-proliferative activity against the cell lines 
compared to curcumin as a positive control (Figure 4.28A-F). The cell growth-
inhibitory potencies of compounds, expressed as IC50values, are shown in Table 4.3. 
Two of the steroids labelled SF2C5HH and SF2C5HI showed high inhibition (IC50 of 
5.58 and 5.28µM, respectively) against the human prostate cancer cells (LNCap) than 
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the positive control, curcumin (IC50 of 6.15 µM) used. However, SF2C5HG, 
quinolactacins, citranadins and butrepyrazinone showed moderate anti-proliferative 
activity against LNCap cells. In addition, only the SF2C5HH showed a comparable 
IC50 of 24.54µM to that of curcumin (20.68µM) on the Human breast cancer cell line 
MCF-7.  
Curcumin is known to be a potent inhibitor of cancer cell lines but, in this case, the 
inhibitory effect ofSF2C5HH and SF2C5HI was stronger than curcumin. SF2C5HH 
and SF2C5HI exerted a high anti-proliferative activity on the Human Prostate cancer 
cell line LNCap than the effect of SF2C5HG. SF2C5HG has hydroxyl groups on the 
C26 and C27 which is absent in the other compounds. Garry et al., (2002), published 
a report which indicated that, hydroxyl groups are required to decrease viability in 
LNCap cells.173 This could account for why SF2C5HG had a lowered inhibitory 
activity as compared to SF2C5HH and SF2C5HI. 
Naturally occurring steroids in the human body are sometimes used to treat prostate 
cancer that has spread and not responding to hormone therapy. Body steroids are used 
to manage cancers by shrinking and inhibiting growth. The ability of the steroids 
SF2C5HH and SF2C5HI to inhibit the human prostate cancer cell line supports the 
fact that steroids have anti-prostate cancer activity. The fact that these compounds 
were selective against LNCap as compared to PC-3 cell lines (Table 4.3) even though 
they are both Human Prostate Cancer cells showed some level of selectivity and 
hence their potential as leads that could be developed asanti-prostate cancer drugs. 
The compounds may be acting on the androgen receptors since the LNCap cell line is 
an androgen dependent prostate cancer cell and has an androgen receptor whiles PC-3 
cell line is an androgen independent prostate cancer cell. One of the causes of prostate 
cancer is an androgenic disorder which arises mainly by the ill effects of testosterone 
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metabolism and its conversion to dihydrotestosterone in the presence of 5-alpha 
reductase.174 
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A B
100 100
80
80
60
60
40
40 20
0
20
20 40 60 80 100
-20
0 [SF2C5HH] M
0 20 40 60 80 100
[SF2C5HG] M
C D
100 100
80 80
60 60
40 40
20 20
0 0
0 20 40 60 80 100 0 20 40 60 80 100
[SF2C5HI] M [Quinolactacin] M
E F
100 100
80 80
60 60
40 40
20 20
0 0
0 20 40 60 80 100 0 20 40 60 80 100
[Citrinadin] M [Butrepyrazinone] M
G
100
80
60
40
20
0
20 40 60 80 100
-20
[Curcumin] M
HepG2 HL60 Jurkat
MCF-7 LNCap PC-3
 
 
 
Figure 4.28A-F: Anti-proliferative activities of pure compounds and standard on 
human cancer cell lines. 
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% Cell viability % Cell viability % Cell viabilty
% Cell viabilty
% Cell viability
% Cell viabilty % Cell Viability
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Table 4.3: Cell growth-inhibitory potencies of pure compounds expressed as IC50values. 
 
Cell Origin Compound IC50 (µM) 
line SF2C5HG SF2C5HH SF2C5HI Quinolactacin Citrinadin, Butrepyrazinone  Curcumin 
         
HEPG2 Human        
 Hepatocellular ND 99.56 >100 96.54 82.51 84.74 61.38 
Carcinoma 
         
HL-60 Human        
 Promyelocytic 93.85 82.95 43.20 54.47 57.23 91.06 13.78 
Leukemia   
         
Jurkat Human T-        
 Lymphoblastic 92.79 78.74 64.08 ND ND ND 8.91 
Leukemia 
         
MCF-7 Human         
 Breast  72.46 24.54 57.79 94.49 66.07 66.62 20.68 
Cancer 
         
LNCap Human        
 Prostate 32.88 5.58 5.28 45.71 41.42 33.36  6.15 
Cancer 
         
PC-3 Human        
 Prostate 87.59 53.21 69.40 >100 >100 >100 20.72 
Cancer 
Data presented are mean of two experiments done in triplicates. ND: Not Determined 
IC50 (µM) = Concentration that inhibits cell growth by 50%
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4.5 Anti-Plasmodial Activity of Compounds 
4.5.1 Anti-Plasmodial activity ofcompounds to chloroquine sensitivePlasmodium 
falciparum 3D7strain 
The six compounds cholest-5-ene-3,25,26-triol (3,25) (SF2C5HG), 20-ethyl-5-
pregnen-3-ol (SF2C5HH), -sitosterol (SF2C5HI), butrepyrazinone, quinolactacin 
A1/A2, citrinadin A/butrecitrinadin were also tested against the chloroquine sensitive 
Plasmodium falciparum 3D7 strain for the anti-plasmodial activity as shown in 
Figure4.29. Only the quinolactacin showed anti-plasmodial activity within the 
concentration range tested. 
Table 4.4 shows the Inhibition Concentration (IC50) of the compounds on the 
plasmodial strain. Quinolactacins gave an IC50 of 24.80 µM, all the other compounds 
gave values greater than 25 µM. 
 
Table 4.4: Effective concentration of the six compounds on the 3D7 plasmodial 
strain. 
 Compounds EC50 (µM) 
 
Strain SF2C5HG SF2C5HH SF2C5HI Quinolactacins Citrinadins Butrepyrazinone Artesunate 
3D7 >25 >25 >25 24.80 >25 >25 0.074 
 
 
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100 SF2C5HG
SF2C5HH
80 SF2C5HI
QUINOLACTACIN
60
CITRINADIN
40 BUTREPYRAZINONE
20
0
0 5 10 15 20 25
[Concentration] M
 
Figure 4.29: Anti-plasmodial activity of compounds on 3D7 plasmodial strain. 
 
Quinolactacins A1/A2 exhibited anti-plasmodial activity in vitro with IC50 of 24.80 
µM. This represents the first study of the anti-plasmodial activity of quinolactacins in 
general and the results proved very encouraging. Interestingly, citrinadins also 
alkaloids isolated from the same endophytic fungi did not show anti-plasmodial 
activity. Structurally, quinolactacins have a backbone comprising of a quinolone 
moiety fused to a lactam ring. Compounds containing the quinolone skeleton have 
been found to exhibit a variety of biological activity including cytotoxicity,175anti-
HIV,176and ability to remedy certain conditions of Alzheimers.177 However, their 
inhibitory role against malaria parasites has not been fully explored except for a few 
known antibiotics such as ciprofloxacin, nalidixic acid and ofloxacin.178-180In one 
study, some4-quinolone compounds were shown to have moderate antiplasmodial 
activities against a chloroquine-sensitive Plasmodium falciparumstrain with IC50 
values in the micro molar range.181Studies by other research groups have shown that 
changing the substituents on the nitrogen atom present in the quinolone affects the 
anti-plasmodium activity of these compounds.182 
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The antimalarial activities of lactams have not been studied into detail. The few 
synthesized β-lactams tested for their antimalarial potential against chloroquine 
sensitive Plasmodium falciparum D10 strain did not show any considerable 
antimalarial activity.183 However, in some studies, introduction of suitable 
substituents on the nitrogen atom present in the lactams have helped to fine tune the 
antimalarial activity of lactams.183 
Results obtained for antimalarial studies of the quinolactacins suggest that these 
compounds could act as good scaffolds for the development to antimalarials if they 
are able to effectively induce apoptosis.  
4.6 Plasmodial Apoptotic Activity of Quinolactacins A1/A2 
The apoptotic activity of the quinolactacins was evaluated on the strain by studying 
the effect of the compound on the mitochondrion membrane potential of the parasite. 
The effect of the compound on the loss of mitochondrion membrane potential of the 
strain treated with 0 µM, 6.25 µM, 12.5 µM, 25 µM and 50 µM concentrations of the 
intimate mixture of quinolactacins A1 and A2 for 24 h were analysed by flow 
cytometry after staining with JC-1 as shown in Figure 4.30A-Fand 4.31 shows the 
percentage of apoptotic cells on the parasites strain after treatment with the 
compound. The Figures show a concentration dependent activity on the parasite. 
Apoptosis is a highly regulated programmed cell death in cells which occur widely in 
multicellular organisms as well as unicellular parasites and is essential for normal 
development and immune defences. The process of apoptosis is initiated by the 
activation of death receptors, or by intracellular stress conditions. This leads to a 
series of genetically controlled and ordered biochemical changes, resulting in 
morphological changes to the cell. The process of apoptosis is characterised by 
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condensing of chromatin, DNA breakdown, mitochondrial alterations, membrane 
changes, shrinkage of the cell and finally and the formation of apoptotic bodies. Picot 
et. al., in (1997) first highlighted the relationship that exists between Plasmodium 
drug resistance and apoptosis.184Quinolactacins used in this study exhibited histogram 
displacement (Figure 4.30A-F) indicating dissipation of the mitochondrial membrane 
potential(ΔΨm) and abolition of probe accumulation in a concentration dependent 
manner (Figure 4.31).Mitochondria play an essential role in apoptosis induction by a 
variety of death stimuli therefore, the ability of quinolactacins to compromise 
mitochondrial changes leading to the loss of the ΔΨm which result in cytochrome C 
release from the mitochondria to the cytosol.  
Literature provides evidence that one of the mechanisms of malaria drug resistance is 
mediated through a decrease in apoptosis susceptibility. However, quinolactacins may 
provide evidence of apoptotic death in the stages of Plasmodium falciparum 
development. 
 
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Figure 4.30A-F: Effect of Quinolactacin A1/A2 on the loss of mitochondrion 
membrane potential. 
 
 
 
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Figure 4.31: Apoptotic effect of quinolactacins on the plasmodial strain 
 
4.7 Anti-Buruli ulcer Activity of Compounds 
The six compounds cholest-5-ene-3,25,26-triol (3,25) (SF2C5HG), 20-ethyl-5-
pregnen-3-ol (SF2C5HH), -sitosterol (SF2C5HI), butrepyrazinone, quinolactacin 
A1/A2, citrinadin A/butrecitrinadin were tested for Anti-buruli ulcer activity on 
Mycobacterium ulcerans MN209 characterised isolates and the MICs are shown in 
Table 4.5. All the compounds showed no Minimum Inhibition Concentration (MIC) 
within the concentration range tested 
.
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Table 4.5 Minimum Inhibition Concentration of the compounds on 
Mycobacterium ulcerans MN209. 
 
 MIC (µM) 
Strain SF2C5H SF2C5H SF2C5H Quinolactaci Citrinadin Butrepyrazino Artesunat
G H I ns s ne e 
Mu     >10 >10 >10 
MN20 >10 >10 >10 >10 
9 
 
The compounds were tested for their anti-buruli activity but none of the compounds 
were able to inhibit the Mycobacterium ulcerans MN209 characterised isolates. This 
is the first time each of the compounds was tested for their activity against 
Mycobacterium ulcerans MN209. 
 
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CHAPTER FIVE 
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5.0 CONCLUSION AND RECOMMENDATIONS 
5.1 Conclusion 
The Ghanaian mangrove ecosystem is underexplored for the isolation of important 
natural products with key pharmacophore. This thesis proves the potential of isolating 
new and novel compounds from endophytic fungi from Ghanaian mangrove plants. 
The mangrove plants sampled along the banks of River Butre were investigated for 
new or novel secondary metabolites. The HRESI/HPLC-DAD-MSn was an 
appropriate and efficient dereplication technique and also very useful in identifying 
and isolating new secondary metabolites from the mangrove plants.   
With the aid of the MarinLit, AntiMarin and Antibase database, detailed information 
on already known compounds and prior information about the structures of all new 
compounds was known. 
The HRESI/HPLC-DAD-MSn analysis of the total crude extracts showed that the 
marine-derived fungi, Penicillium sp. BRS2A-AR2 produced interesting compounds 
and therefore, large scale fermentation was carried out to isolate these compounds. 
Quinolactacin A1/A2 and Citrinadin A and the new Butrecitrinadin were isolated and 
characterized using appropriate techniques. 
The compounds isolated as well as other compounds from the research group were 
studied for their biological activity against the proliferation of cancerous cells lines, 
their anti-plasmodial activity as well as their anti-buruli ulcer potential. 
In the search of new compounds with inhibitory abilities, 20-Ethyl-5-pregnen-3-ol 
and β-sitosterol proved strong inhibitory activity against human cancer prostate 
LNCap cells and quinolactacins also showed inhibitory activity against chloroquine 
sensitive Plasmodium falciparum 3D7 strain as its apoptotic activity via the loss of 
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mitochondrion membrane potential of the parasite. These compounds are therefore 
lead for anti-cancer and anti-malarial candidates respectively. 
 
5.2 Recommendations 
Since this is the first attempt to isolate metabolites from endophytic fungi from 
Ghanaian marine habitat, this work should serve as a baseline for the investigation of 
natural products from mangrove habitats in Ghana. 
The remaining four species isolated in the course of the study should be investigated 
for the isolation of new or novel secondary metabolites by culturing them in a 
different media. In time past, scientists have mainly focused on the identification of 
endophytic fungi and isolation of metabolites however, knowledge in genetic 
engineering, microbial fermentation amongst other techniques should be employed to 
increase yield in compounds generated from endophytic fungi with efficient activity. 
The mechanism of action of 20-Ethyl-5-pregnen-3-ol and β-sitosterol against human 
cancer prostate LNCap cells should be studied to enhance the possibility of being 
potential anti prostate cancer agents. The in vivo activity of quinolactacins should be 
studied to fully understand their mode of action. The compounds that showed some 
level of anti-proliferative activity against the cancerous cell line should be modified  
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REFERENCES 
1. Newman, D. J., Cragg G. M. (2012). Natural products as sources of new drugs 
over the 30 years. Journal of Natural Products, 75(3), 311-35. 
2. Cotter, C., Sturrock, H. J. W., Hsiang, M. S., Liu, J., Phillips, A. A., Hwang, J., 
Gueye, C. S., Fullman, N., Gosling, R. D., Feachem, R. G. A. (2013). The 
changing epidemiology of malaria elimination: new strategies for new 
challenges The Lancet,382(9895), 900-911 
3. Liu, J., Modrek, S., Gosling, R. D., Feachem, R. G. A. (2013). Malaria 
eradication: is it possible? Is it worth it? Should we do it? The Lancet Global 
Health, 1(1), 2-4. 
4. Gray, H., (2013). The malaria vaccine – Status quo. Travel Medicine and 
Infectious Disease, 11(1), 2-7.  
5. Renslo, A. R., McKerrow, J. H. (2006). Drug discovery and development for 
neglected parasitic diseases. Nature Chemical Biology, 2, 701 -710. 
6. Fernando, C. (2014). Imported Infectious Diseases, The Impact in Developed 
Countries. Current status of malaria, 61–90. 
7. Hay, S. I., Guerra, C. A., Tatem, A. J., Noor A. M., Snow R. W.  (2004). The 
global distribution and population at risk of malaria: past, present, and future. 
Lancet Infectious Diseases, 4(6), 327–336. 
8. Suswardany, D. L., Sibbritt, D. W., Supardi, S.,Chang, S.,Adams J. (2015). A 
critical review of traditional medicine and traditional healer use for malaria and 
among people in malaria-endemic areas: contemporary research in low to middle-
income Asia-Pacific countries. Malaria Journal,14, 98. 
125 
 
University of Ghana  http://ugspace.ug.edu.gh
9. del Prado, G. R. L., García C. H., Cea, L. M., Espinilla, V. F., Moreno, M. F. M., 
Márquez A. D., Polo, M. J. P., García, I. A. (2014). Malaria in developing 
countries. Journal of Infection in Development Countries,8(1), 1-4. 
10. Ihtesham, A. Q., Nimmathota A , Mohtashim A. Q. (2014).Prevalence of malaria 
and anemia among pregnant women residing in malaria-endemic forest villages in 
India.International Journal of Gynecology & Obstetrics, 127(1), 93. 
11. Chukwuocha, U. M., Dozie I. N., Chukwuocha A. N. (2012). Malaria and its 
burden among pregnant women in parts of the Niger Delta area of Nigeria. Asian 
Pacific Journal of Reproduction, 1(2), 147–151. 
12. Ameyaw, E., Dogbe, J., Owusu, M. (2015).  Knowledge and practice of malaria 
prevention among caregivers of children with malaria admitted to a teaching 
hospital in Ghana. Asian Pacific Journal of Tropical Disease,  5(8), 658–661.  
13. Eugene-Ezebilo, D. N., Ezebilo, E. E. (2014). Malaria infection in children in 
tropical rainforest: assessments by women of Ugbowo Community in Benin City, 
Nigeria.Asian Pacific Journal of Tropical Medicine, 7(1), 97–103. 
14. Schwartz, L., Brown, G. V., Genton, B., Moorthy, V. S. (2012). A review of 
malaria vaccine clinical projects based on the WHO rainbow table. Malaria 
Journal,11, 11 
15. Ho, M. M.,Baca-Estrada, M., Conrad C., Karikari-Boateng, E., Kang, H-N. 
(2014). Implementation workshop of WHO guidelines on evaluation of malaria 
vaccines: Current regulatory concepts and issues related to vaccine 
quality.Vaccine, Available online 10 July 2015. 
16. Vandana S.,Hoyun, L. (2015). Chloroquine-based hybrid molecules as promising 
novel chemotherapeutic agents.European Journal of Pharmacology. 762, 472–
486. 
126 
 
University of Ghana  http://ugspace.ug.edu.gh
17. Mushtaque,Md.,Shahjahan, (2015). Reemergence of chloroquine (CQ) analogs as 
multi-targeting antimalarial agents: A review.European Journal of Medicinal 
Chemistry,  90, 280–295. 
18. Lehane, A. M., McDevitt,C. A., Kirk, K., Fidock D. A. (2012). Degrees of 
chloroquine resistance in Plasmodium – Is the redox system involved? 
International Journal for Parasitology: Drugs and Drug Resistance, 2, 47–57. 
19. Saheli S. (2010).  Combating Evolving Pathogens, Malaria: An Evaluation of the 
Current State of Research on Pathogenesis and Antimalarial Drugs. Yale Journal 
of Biology and Medicine, 83(4), 185-191. 
20. Wells, T. N. C., Huijsduijnen R. H. V., Voorhis, W. C. V. (2015). Malaria 
medicines: a glass half full? Nature Reviews Drug Discovery, 14, 424–442. 
21. Al-Harthi, S. A. (2011). Malaria drug resistant: current situation with reference to 
Saudi Arabia. Journal of Egyptian Society of Parasitology.41(3) 553-64.  
22. Kirandeep, K., Meenakshi, J., Ravi, P. R., Rahul, J. (2010). Quinolines and 
structurally related heterocycles as antimalarials. European Journal of Medicinal 
Chemistry, 45(8), 3245-3264. 
23. Adebayo, J. O., Krettli, A. U. (2011). Potential antimalarials from Nigerian plants: 
A review, Journal of Ethnopharmacology, 133(2), 289-302. 
24. Foley, M., Tilley, L. (1998). Quinoline Antimalarials: Mechanisms of Action and 
Resistance and Prospects for New Agents. Pharmacology & Therapeutics, 79(1), 
55–87. 
25. Frausin, G., Hidalgo A. de F., Lima R. B. S., Kinupp, V. F., Ming, L. C., Pohlit, 
A. M., Milliken, W. (2015). An ethnobotanical study of anti-malarial plants 
among indigenous people on the upper Negro River in the Brazilian Amazon. 
Journal of Ethnopharmacology, In Press, accepted online. 
127 
 
University of Ghana  http://ugspace.ug.edu.gh
26. Kaur, K.,Jain, M., Kaur, T., Jain, R. (2009). Antimalarials from nature. 
Bioorganic Medicinal Chemistry,17(9), 3229-3256. 
27. Rutledge, P. J.,  Challis, G. L. (2015). Discovery of microbial natural products by 
activation of silent biosynthetic gene clusters. Nature Reviews Microbiology, 13, 
509–523. 
28. Zhi-Qiang, X., Jian-Feng, W., Yu-You, H., Yong, W. (2013). Recent Advances 
in the Discovery and Development of Marine Microbial Natural Products. 
Marine Drugs, 11(3), 700-717. 
29. Harvey, A. L., Edrada-Ebel, R. A.,  Quinn, R. J. (2015). The re-emergence of 
natural products for drug discovery in the genomics era. Nature Reviews Drug 
Discovery, 14, 111–129. 
30. Demain, A. L., Sanchez, S. (2009). Microbial drug discovery: 80 years of 
progress. The Journal of Antibiotics, 62, 5–16.  
31. Vicente, M. F., Basilio, A., Cabello, A., Peláez, F. (2003) Microbial natural 
products as a source of antifungals. Clinical Microbiology and Infection, 9(1), 15-
32.  
32. Buragohain, P., Surineni, N., Barua, N. C., Bhuyan, P. D., Boruah, P., Borah, J.C., 
Laisharm, S., Moirangthem, D. S. (2015). Synthesis of a novel series of 
fluoroarene derivatives of artemesinin as potent antifungal and anticancer agent. 
Bioorganic & Medicinal Chemistry Letters,25(16), 3338-3341 
33. Abad, M. J., Ansuategui, M., Bermejo, P. (2007). Active antifungal substances 
from natural sources. ARKIVOC, (vii) 116-145. 
34. Yu, Z., Stewart, D., Zhao, L-X., Jiang, Y., Xu, L-H., Andes, D., Shen, B., Klein, 
B. (2013). Identification of antifungal natural products via Saccharomyces 
128 
 
University of Ghana  http://ugspace.ug.edu.gh
cerevisiae bioassay: insights into macrotetrolide drug spectrum, potency and mode 
of action BRAD TEBBETS. Medical Mycology 51, 280–289.  
35. Roemer, T., Krysan, D. J. (2014). Antifungal Drug Development: Challenges, 
Unmet Clinical Needs, and New Approaches. Cold Spring Harbor Perspectives in 
Medicine, 4, 1-13. 
36. Rex, J. H., Walsh, T. J., Nettleman, M., Anaissie, E. J., Bennett, J. E., Bow, E. J., 
Carillo-Munoz, A. J., Chavanet, P., Cloud, G. A., Denning, D. W., de Pauw, B. E., 
Edwards, Jr. J. E., Hiemenz, J. W., Kauffman, C. A., Lopez-Berestein, 
G., Martino, P., Sobel, J. D., Stevens, D. A., Sylvester,  R., Tollemar, J., Viscoli, 
C., Viviani, M. A., Wu, T. (2001). Need for alternative trial designs and 
evaluation strategies for therapeutic studies of invasive mycoses. Clinical 
Infectious Diseases, 33(1), 95–106. 
37. Boucher, H. W., Talbot, G. H., Bradley, J. S., Edwards, J. E., Gilbert, D., Rice, L. 
B., Scheld, M., Spellberg, B., Bartlett, J. (2009). Bad bugs, no drugs: No 
ESKAPE! An update from the Infectious Diseases Society of America. Clinical 
Infectious Diseases, 48(1), 1–12. 
38. Phoebe, C. Jr., Combie, J., Albert, F. G., Van, T. K.,Cabrera, J., Correira, H. J., 
Guo, Y.,Lindermuth, J., Rauert, N., Galbraith, W., Selitrennikoff, C. P.  (2001). 
Extremophilic orgainisms as unexplored source for antifungal compounds. The 
Journal of Antibiotics (Tokyo). 54(1), 56-65. 
39. Leary, D. K., Nijhoff, M. (2007). International Law and the Genetic Resources of 
the Deep Sea. Martinus Nijhoff Publishers, Law, 297 pages. 
40. Giddings L-A., Newman, D. J. (2015). Bioactive Compounds from Marine 
Extremophiles. Springer, Science, 150 pages. 
129 
 
University of Ghana  http://ugspace.ug.edu.gh
41. Singh, O. V. (2012). Extremophiles: Sustainable Resources and Biotechnological 
Implications, John Wiley & Sons, Science, 456 pages 
42. Ghosh, S., Bakshi, M., Bhattacharyya, S., Nath, B., Chaudhuri, P. (2015). A 
Review of Threats and Vulnerabilities to Mangrove Habitats: With Special 
Emphasis on East Coast of India. Journal of Earth Science and Climate Change, 
6(4), 270.  
43. Kuenzer, C., Bluemel, A., Gebhard, S., Quoc, T. V., Dech, S., (2011). Remote 
Sensing of Mangrove Ecosystems: A Review. Remote Sensing, 3, 878-928.  
44. Hrudayanath, T., Bikash, C. B., Rashmi, R. M., Sushil, K. D. (2012). Biodiversity 
and biotechnological potential of microorganisms from mangrove ecosystems: 
a review.Annals of Microbiology, 63(1), 1-19.  
45. Revathi, P., Jeyaseelan, S., Thirumalaikolundusubramanian, P., Prabhu, N. (2014). 
An overview of antidiabetic profile of mangrove plants. International Journal of 
Pharmacy and Pharmaceutical Sciences, 6(3), 1-5 
46. Jayanta, K. P., Hrudayanth, N. T. (2010). Metabolic diversity and bioactivity 
screening of mangrove plants: a review.  Acta Physiologiae Plantarum 33(4), 
1051-1061. 
47. Dong-Bo, X., Wan-Wan, Y., Ying, H., Zi-Xin, D., Kui, H. (2014). Natural 
Products from Mangrove Actinomycetes.Marine Drugs. 12(5): 2590–2613. 
48. Blunt, J. W., Copp, B. R., Keyzers, R. A., Munro, M. H. G., Prinsep, M. R. 
(2014). Marine natural products, Natural Product Reports, 31, 160-258. 
49. Panchanathan, M., Kyong-Hwa, K., Kannan, S., Li-Chan, E. C.Y., Hyun-Myung, 
O., Se-Kwon, K. (2014). Marine actinobacteria: An important source of bioactive 
natural products. Environmental Toxicology and Pharmacology, 38(1), 172–188. 
130 
 
University of Ghana  http://ugspace.ug.edu.gh
50. Xue-Gong, L., Xiao-Min, T., Jing, X., Guang-Hui, M., Li, X., Shu-Jie, X., Min-
Juan, X., Xiang, X., Jun, X. (2013). Harnessing the Potential of Halogenated 
Natural Product Biosynthesis by Mangrove-Derived Actinomycetes. Marine 
Drugs, 11, 3875-3890. 
51. Gitishree, D., Sushanto, G., Yugal, K. M., Jayanta, K. Patra. (2015). Mangrove 
plants: a potential source of anticancer drugs. Indian Journal of Geo-Marine 
Sciences, 44 (5), 1-7.  
52. Oteng-YeboahA. A. (1999). Biodiversity studies in three Coastal Wetlands in 
Ghana, West Africa. Journal of the Ghana Science Association, 1(3), 147-149. 
53. Saenger, P., Bellan, M.F., (1995). The Mangrove Vegetation of the Atlantic 
Coast of Africa - A Review. Southern Cross University ePublications@SCU. 
54. Salif, D., Jean-Paul, B., Cyr, D. (2014). The Land/ocean Interactions in the 
Coastal Zone of West and Central Africa, Springer,  Business & Economics, 210 
pages. 
55. Friends of the Nation (2014), Assessment of flora and fauna of ecological and 
socioeconomic significance within the Anlo Beach Wetland Complex for 
improved management and livelihood outcomes, Parks and Gardens, Adiembra, 
60. 
56. Nataša, R., Borut, Š. (2012). Endophytic fungi-The treasure chest of antibacterial 
substances. Phytomedicine, 19(14), 1270–1284. 
57. Costa, I. P. M. W., Maia, L. C., Cavalcanti M. A. (2012). Diversity of leaf 
endophytic fungi in mangrove plants of northeast Brazil. Brazilian Journal of 
Microbiology, 43(3): 1165–1173. 
58. Liu, A. R., Wu, X. P., Xu, T. (2007). Research advances in endophytic fungi of 
mangrove. Ying Yong Sheng Tai Xue Bao, 18(4), 912-918. 
131 
 
University of Ghana  http://ugspace.ug.edu.gh
59. Monnanda, S. N., Ningaraju, S., Harischandra, S. P., (2014). Endophytic Fungal 
Diversity in Medicinal Plants of Western Ghats, India. International Journal of 
Biodiversity, 2014, 1-9. 
60. Hongsheng, Y., Lei, Z., Lin, Li., Chengjian, Z., Lei, G., Wenchao, L., Peixin, S., 
Luping, Q., (2010). Recent developments and future prospects of antimicrobial 
metabolites produced by endophytes. Microbiological Research, 165(6), 437–
449. 
61. Hrudayanath, T., Bikash, C. B., Rashmi, R. M., (2013) Ecological role and 
biotechnological potential of mangrove fungi: a review. Mycology, 4(1), 54-71. 
62. Alaganadham, E., Gnanaprakash, S. R., Murugaiyan, K. (2012). Taxol producing 
mangrove endophytic fungi Fusarium oxysporum from Rhizophora 
annamalayana, Asian Pacific Journal of Tropical Biomedicine, 2(2), 1081-1085.  
63. de Vita-Marques, A. M., Lira, S. P., Berlinck, R. G. S., Seleghim, M. H. R., 
Sponchiado, S. R. R., Tauk-Tornisielo, S. M. (2008). A multi-screening approach 
for marine-derived fungal metabolites and the isolation of cyclodepsipeptides 
from Beauveria felina. Quimica Nova, 31, 1099 - 1103. 
64. Kumar, D.S.S., Hyde, K.D. (2004). Biodiversity and tissue-recurrence of 
endophytic fungi in Tripterygium wilfordii. Fungal Diversity, 17, 69-90. 
65. Saikkonen, K. (2007). Forest structure and fungal endophytes. Fungal Biology 
Reviews, 21, 67-74.  
66. Suryanarayanan, T. S., Thirunavukkarasu, N., Govindarajulu, M. B., Sasse, F., 
Jansen, R., Murali T. S. (2009). Fungal endophytes and bioprospecting. Fungal 
Biology Reviews, 23, 9–1 9. 
132 
 
University of Ghana  http://ugspace.ug.edu.gh
67. Petrini, O. (1991). Fungal endophytes of tree leaves. In: Andrews, J.H., Hirano, 
S.S. (Eds.), Microbial Ecology of Leaves. Springer Verlag, New York, pp. 179–
197. 
68. Be´rdy, J. (2005). Bioactive microbial metabolites: a personal view. The Journal 
of Antibiotics, 58, 1–26. 
69. Quin, M. B., Schmidt-Dannert, C. (2014). Designer microbes for 
biosynthesis.Current Opinion in Biotechnology, 29, 55–61. 
70. Berdy, J. (1974). Recent developments of antibiotic research and classification of 
antibiotics according to chemical structure. Advances in Applied Microbiology. 
18, 309-406. 
71. Mayer, A. M. S., Glaser, K. B., Cuevas, C., Jacobs, R. S., Kem, W., Little R. D., 
McIntosh, J. M., Newman, D.J., Potts, B. C., Shuster, D. E. (2010). The odyssey 
of marine pharmaceuticals: a current pipeline perspective. Trends in 
Pharmacological Sciences, 31(6) 255-265. 
72. Hamilton-Miller, J. M. T. (2000). Sir Edward Abraham’s contribution to the 
development of the cephalosporins: a reassessment. International Journal of 
Antimicrobial Agents, 15(3), 179-184. 
73. Blunt, J. W., Copp, B. R., Hu, W.-P., Munro, M. H. G., Northcote,P.T., Prinsep, 
M. R. (2014). Marine natural products. Natural Product Reports, 31, 160-258. 
74. Bugni, T. S., Ireland, C. M. (2004). Natural Product Reports. 21 143. 
75. Klaus, B. (2012). Acremolin, a stable natural product with an antiaromatic 1H-
azirine moiety? A structural reorientation. Tetrahedron Letters, 53(47), 6443–
6445.  
133 
 
University of Ghana  http://ugspace.ug.edu.gh
76. Julianti, E., Oh, H., Lee, H-S., Oh, D-C., Oh, K.-B., Shin, J. (2012). Acremolin, a 
new 1H-azirine metabolite from the marine-derived fungus. Acremonium strictum. 
Tetrahedron Letters 53(23), 2885–2886. 
77. Yao, Y., Tian, L., Li. J., Cao, J., Pei, Y. (2009). Cytotoxic piperazine-2,5-dione 
derivatives from marine fungus Gliocladium sp. Pharmazie.  64(9), 616-618. 
78. Huang, Y.-F., Tian, L., Hua, H-M., Pei, Y-H. (2007). Two diketopiperazines 
from marine fungus Gliocladiumsp. YUP08. Journal of Asian Natural Product 
Research, 9(3), 197-201. 
79. Yao, Y., Tian, L., Cao, J-Q., Pei Y-H. (2007). A new piperazine-2,5-dione from 
the marine fungus Gliocladium sp. Pharmazie, 62(6), 478-479. 
80. Park, Y. C., Gunasekera, S. P., Lopez, J.V., McCarthy, P.J., Wright, A.E. (2006). 
Metabolites from the Marine-Derived Fungus Chromocleistasp. Isolated from a 
Deep-Water Sediment Sample Collected in the Gulf of Mexico. Journal of 
Natural Products, 69(4), 580. 
81. Almeida, C., Hemberger, Y., Schmitt, S. M., Bouhired, S., Natesan, L., Kehraus, 
S., Dimas, K., Gutschow, M., Bringmann, G., Koenig, G.M. (2012). Marilines 
A–C: Novel Phthalimidines from the Sponge-Derived Fungus Stachylidium sp. 
Chemistry - A European Journal, 18(28), 8827-8834. 
82. Ryuya, F.,Atsushi, M.,Katsuya, G.,Hideaki, O. (2013). Biosynthetic assembly of 
cytochalasin backbone. Tetrahedron Letters, 54(23), 2999–3002. 
83. Cooper, J. A. (1987). Effects of Cytochalasin and Phalloidin on Actin. The 
Journal of Cell Biology, 105, 1473-1478. 
84. Carter, S. B. (1967). Effects of cytochalasins on mammalian cells. Nature,213, 
261-264 
134 
 
University of Ghana  http://ugspace.ug.edu.gh
85. Krishan, A. J. (1972). Tetraploid Mouse Embryos produced by Cytochalasin B 
during Cleavage. The Journal of Cell Biology, 54, 657. 
86. Shepro, D., Belamarich, F.A., Robblee, L., Chao, F.C. (1970). Antimotility effect 
of Cytochalasin B observed in mammalian clot retraction. The Journal of Cell 
Biology 47, 544-547. 
87. Majno, G., Bouvier, C. A., Gabbiani, G., Ryan, C.B., Statkov, P. (1972). 
Kymographic recording of clot retraction: effects of papaverine, theophylline and 
cytochalasin B. Thrombosis et. Diathesis Haemorrhagica, 28(1), 49-53. 
88. Kletzien, R. F., Perdue, J. F., Springer, A., (1972). The Journal of Biological 
Chemistry, 247, 2964.  
89. Estensen, R. D., Plagemann, P. G. (1972). Cytochalasin B: Inhibition of Glucose 
and Glucosamine Transport. Proceedings of National Academy Sciences. 69(6) 
1430-1434. 
90. Williams, J. A., Wolff, J. (1971). Cytochalasin B inhibits thyroid secretion. 
Biochemical and Biophysical Research. Communications. 44(2) 422-425. 
91. Betina V., Micekova D. Z. (1972). Antimicrobial properties of fungal macrolide 
antibiotics. Journal of Basic Microbiology, 12(5) 355-364.  
92. Fu, J., Zhou, Y., Li, H. F., Ye Y. H., Guo J. H. (2011). Antifungal metabolites 
from Phomopsis sp. By254, an endophytic fungus in Gossypium hirsutum. 
African Journal of Microbiolial Research, 5 1231-1236. 
93. Katagiri, K., Matsuura, S. J. (1971). Antitumor activity of cytochalasin 
D.Journal of Antibiotics, 24(10), 722-723.  
94. Zhang Y., Tian, R., Liu, S., Chen, X., Liu, X., Che, Y. (2008). Alachalasins A–
G, new cytochalasins from the fungus Stachybotrys charatum. Bioorganic and 
Medicinal Chemistry, 16, 2627-2634.  
135 
 
University of Ghana  http://ugspace.ug.edu.gh
 
95. Rochfort, S., Ford, J., Ovenden, S., Wan, S. S., George, S., Wildman, H., Tait, 
R.M., Meurer-Grimes, B., Cox, S., Coates, J., Rhodes, D. (2005). A novel 
aspochalasin with HIV-1 integrase inhibitory activity from Aspergillus flavipes. 
Journal of Antibiotics, 58(4), 279-283.  
96. Liu, R., Gu, Q., Zhu, W., Cui, C., Fan, G., Fang, Y., Zhu, T., Liu, H. (2006). 10-
Phenyl-[12]-cytochalasins Z7, Z8, and Z9 from the marine-derived fungus 
Spicaria elegans. Journal of Natural Produts, 69(6), 871-875.  
97. Wagenaar, M. W., Corwin, J., Strobel, G., Clardy, J. (2000). Three new 
chytochalasins produced by an endophytic fungus in the genus Rhinocladiella. 
Journal of Natural Products, 63(12), 1692-1695.  
98. Scherlach, K., Boettger, D., Remme, N., Hertweck, C. (2010). The chemistry and 
biology of cytochalasans. Natural Product Report, 27(6), 869-886. 
99. Kazuyasu, S., Kazumasa, O., Kaori, S., Masato, O., Kensaku, M. (2012). 
Cytochalasin D acts as an inhibitor of the actin–cofilin interaction. Biochemical 
and Biophysical Research Communications, 424(1), 52–57. 
100. Heptinstall, J. A., May H., Ratan J. R., Glenn W. L. (1998). "GPIIb-IIIa 
antagonists cause rapid disaggregation of platelets pre-treated with cytochalasin 
D. Evidence that the stability of platelet aggregates depends on normal 
cytoskeletal assembly." Platelets 9(3), 227–232. 
101. Shao, C.-L., Wang, C.-Y., Gu, Y.-C., Wei, M.-Y., Pan, J.-H., Deng, D.-S., 
She, Z.-G., Lin, Y.-C. (2010). Penicinoline, a new pyrrolyl 4-quinolinone 
alkaloid with an unprecedented ring system from an endophytic fungus 
Penicillium sp. Bioorganic and Medicinal Chemistry Letters, 20(11), 3284-3286. 
136 
 
University of Ghana  http://ugspace.ug.edu.gh
102. Smetanina, O. F., Kalinovsky, A. I., Khudyakova, Y.V., Pivkin, M.V., 
Dmitrenok, P.S., Fedorov, S.N., Ji, H., Kwak, J.-Y., Kuznetsova, T.A. (2007). 
Indole Alkaloids Produced by a Marine Fungus Isolate of Penicillium 
janthinellum Biourge. Journal of Natural Products, 70(6), 906. 
103. Gu, W., Cueto, M., Jensen, P.R., Fenical, W., Silverman, R.B. (2007). 
Microsporins A and B: new histone deacetylase inhibitors from the marine-
derived fungus Microsporum cf. gypseum and the solid-phase synthesis of 
microsporin A. Tetrahedron 63, 6535. 
104. Oh, D.-C., Jensen, P.R., Fenical, W. (2006).  Zygosporamide, a cytotoxic 
cyclic depsipeptide from the marine-derived fungus Zygosporium 
masonii.Tetrahedron Letters, 47, 8625-8628. 
105. Luque-Ortega, J. R., Cruz, L.J., Albericio, F., Rivas L. (2010). The 
antitumoral depsipeptide IB-01212 kills Leishmania through an apoptosis-like 
process involving intracellular targets. Molecular Pharmaceutics, 7(5), 1608-
1617. 
106. Boot, C. M., Amagata, T., Tenney, K., Compton, J. E., Pietraszkiewicz, H., 
Valeriote, F.A., Crews, P. (2007). Four classes of structurally unusual peptides 
from two marine-derived fungi: structures and bioactivities. Tetrahedron 63, 
9903. 
107. Crawford, J.M., Townsend, C.A., (2010). New insights into the formation of 
fungal aromatic polyketides. Nature Reviews. Microbiology, 8(12), 879-889. 
108. Yamada, T., Muroga, Y., Jinno, M., Kajimoto, T., Usami, Y., Numata, A., 
Tanaka, R. (2011). New class of azaphilone produced b a marine fish-derived 
Chaetomium globosum. The stereochemistry and biological activites. Bioorganic 
and Medicinal Chemistry 19, 4106-4113. 
137 
 
University of Ghana  http://ugspace.ug.edu.gh
109. Mohamed, I.E., Gross, H., Pontius, A., Kehraus, S., Krick, A., Kelter, G., 
Maier, A., Fiebig, H.-H., Koenig, G.M. (2009). Epoxyphomalin A and B, 
prenylated polyketides with potent cytotoxicity from the marine-derived fungus 
Phoma sp. Organic Letters, 11(21), 5014-5017. 
110. Du. L., Zhu, T., Fang, Y., Liu, H., Gu, Q., Zhu, W. (2007). Aspergiolide A, 
A, a novel anthraquinone derivative with naphtho[1,2,3-de]chromene-2,7-dione 
skeleton isolated from a maine-derived fungus Aspergillus glaucus. Tetrahedron, 
63(5), 1085-1088. 
111. Pontius, A., Krick, A., Kehraus, S., Brun, R., Koenig, G.M. (2008). 
Antiprotozoal Activities of Heterocyclic-Substituted Xanthones from the Marine-
Derived Fungus Chaetomium sp.  Journal of Natural Products, 71, 1579-1584. 
112. Rajput S. B., Karuppayil, S. M., (2013). Small molecules inhibit growth, 
viability and ergosterol biosynthesis in Candida albicans. Springerplus 2, 26-37. 
113. Sun, Y., Tian, L., Huang, J., Li, W., Pei, Y.-H., (2006) Cytotoxic sterols from 
marine-derived fungus Penicillium sp. Natural Product Research, 20(4), 381-
384. 
114. Song, S., Wang, N., Gao, H., Liu, H., Zhang, Q., Namikoshi, M., Yao, X. 
(2006). Zhongguo Yaowu Huaxue Zazhi 16, 93. 
115. Huang, Y. F., Qiao, L., Lv, A. L., Pei, Y. H., Tian L., (2008) Eremophilane 
sesquiterenes from the marine fungus Penicillium sp. BL27-2. Chin. Chem. Lett. 
19, 562. 
116. Li, H.-J., Lan, W.-J., Lam, C.-K., Yang, F., Zhu, X.-F. (2011). Hirsutane 
Sesquiterpenoids from the Marine-Derived Fungus Chondrostereum sp. 
Chemistry & Biodiversity, 8(2), 317-324. 
138 
 
University of Ghana  http://ugspace.ug.edu.gh
117. Cheng, Y., Prusoff, W. H. (1973). Relationship between the inhibition 
constant (KI) and the concentration of inhibitor which causes 50 percent 
inhibition (I50) of an enzymatic reaction. Biochemical Pharmacology 22(23): 
3099–3108 
118. Lazareno, S., Birdsall, N. J. (1993). Estimation of competitive antagonist 
affinity from functional inhibition curves using the Gaddum, Schild and Cheng-
Prusoff equations. British Journal of Pharmacology, 109(4), 1110–1119. 
119. Stewart, M, J., Watson, I. D. (1983). Standard units for expressing drug 
concentrations in biological fluids. British Journal of Clinical Pharmacology, 
16(1), 3–7. 
120. Robinson, S. F., Marks, M. J. Collins, A. C. (1996). Inbred mouse strains 
vary in oral self-selection of nicotine. Psychopharmacology 124(4), 332–339. 
121. Nascarella, M. A., Calabrese. E. J. (2009). The relationship between the IC50, 
toxic threshold, and the magnitude of stimulatory response in biphasic (hormetic) 
dose–responses. Regulatory Toxicology and Pharmacology, 54(3), 229–233. 
122. Hevia, D., Rodriguez-Garcia, A.,Cimadevilla, H. M., Mayo, J. C. (2012).A 
New Protocol for Determining Intracellular Concentrations of Antitumor 
Compounds – How to Calculate a Real and Effective IC50. European Journal of 
Cancer, 48(5), 246. 
123. Sebaugh, J. L., (2011). Guidelines for accurate EC50/IC50 estimation. Pharm 
Stat.  2011, 10(2), 128-134.  
124. Chen, Z., Bertin, R., Froldi, G. (2013). EC50 estimation of antioxidant activity 
in DPPH assay using several statistical programs. Food Chemistry, 138(1), 414–
420. 
139 
 
University of Ghana  http://ugspace.ug.edu.gh
125. Alexander, B., Browse, D. J., Reading, S. J., Benjamin, I. S., (1999).A simple 
and accurate mathematical method for calculation of the EC50. Journal of 
Pharmacological and Toxicological Methods, 419(2–3), 55–58. 
126. Hongzong, S., Shuping, Y., Kejun, Z., Aiping. F., Yun-Bo, D., Zhide, H. 
(2008). Quantitative structure activity relationship study on EC50 of anti-HIV 
drugs. Chemometrics and Intelligent Laboratory Systems, 90(1), 15–24. 
127. Huber, W., Koella, J. C. (1993). A comparison of three methods of estimating 
EC50 in studies of drug resistance of malaria parasites. Acta Tropica, 55(4), 257-
261.  
128. Patron-Bizet, F., Mentré, F., Genton, M., Thomas-Haimez, Maccario, Jean. 
(1998). Assessment of the global two-stage method to 
EC50determination.Journal of Pharmacological and Toxicological Methods, 
39(2), 103–108. 
129. Garrido-Acosta, O., Meza-Toledo, S. E., Anguiano-Robledo, L., Valencia-
Hernández, I., Chamorro-Cevallos, G.,  (2014). Adaptation of Lorke's method to 
determine and compare ED50 values: The cases of two anticonvulsants drugs. 
Journal of Pharmacological and Toxicological Methods, 70(1), 66–69.  
130. Cioli, D., Botros, S. S., Wheatcroft-Francklow, K., Mbaye, A., Southgate, V., 
Tchuenté, L.-A. T., Pica-Mattoccia L., Troiani, A. R., el-Din, S. H. S., Sabra, A.-
N. A., Albin, J., Engels, D., Doenhoff, M. J. (2004).   Determination of 
ED50 values for praziquantel in praziquantel-resistant and -
susceptible Schistosoma mansoni isolates. International Journal for 
Parasitology, 34(8), 979–987. 
131. Kam P. F. (1989). A computer program in BASIC for estimation of ED50 and 
LD50. Computers in Biology and Medicine, 19(2). 131–135.  
140 
 
University of Ghana  http://ugspace.ug.edu.gh
132. Kopman, A. F. (1995). An ED50 is not two times an ED25. Journal of Clinical 
Anesthesia, 7(2), 173.  
133. Pöch, G., Pancheva, S. N. (1995). Calculating slope and ED50 of additive 
dose-response curves, and application of these tabulated parameter values. 
Journal of Pharmacological and Toxicological Methods, 33(3), 137–145 
134. Mehta, S. C., Jain, D. K., Gupta, C. K. (1991). Estimation of ED50 or 
LD50 using a programmable pocket calculator.Computers in Biology and 
Medicine,  21(3), 167–172.,  
135. Gad, S. C., (2014).  LD50/LC50 (Lethal Dosage 50/Lethal Concentration 50). 
Reference Module in Biomedical Sciences, Encyclopedia of Toxicology (Third 
Edition), Pages 58–60.  
136. DePass L. R. (1989). Alternative approaches in median lethality (LD50) and 
acute toxicity testing. Toxicology Letters, 49(2-3), 159-170. 
137. Cutler, D. (2001). Death of LD50.Trends in Pharmacological Sciences,  
22(2), Page 62. 
138. McGrath, P., Seng, W. L., Willett, C., Augustine, K. A. (2008). 
Determination of LD50 and assessment of drug induced developmental toxicity 
in zebrafish. Journal of Pharmacological and Toxicological Methods,  58(2), 
150.  
139. Hendriks, A. J., Awkerman, J. A., de Zwart, D., Huijbregts, M. A. J. (2013). 
Sensitivity of species to chemicals: Dose–response characteristics for various test 
types (LC50, LR50 and LD50) and modes of action. Ecotoxicology and 
Environmental Safety, 97, 10–16. 
140. Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. 
Journal of Antimicrobial Chemotherapy, 48(1), 5–16 
141 
 
University of Ghana  http://ugspace.ug.edu.gh
141. Davison, H. C., Low, J. C., Woolhouse, M. E. (2000). What is antibiotic 
resistance and how can we measure it? Trends in Microbiology, 8(12), 554–9. 
142. de Boer, M., Heuer, C., Hussein, H., McDougall, S. (2015). Minimum 
inhibitory concentrations of selected antimicrobials against Escherichia coli and 
Trueperella pyogenes of bovine uterine origin. Journal of Dairy Science, 98(7), 
4427–4438. 
143. Papich, M. G. (2013).  Clinical Pharmacology and Therapeutics, 
Antimicrobials, Susceptibility Testing, and Minimum Inhibitory Concentrations 
(MIC) in Veterinary Infection Treatment. Veterinary Clinics of North America: 
Small Animal Practice, 43(5), 1079–1089.  
144. Chakraborty, B., Nath, A., Saikia, H., Sengupta, M. (2014). Bactericidal 
activity of selected medicinal plants against multidrug resistant bacterial strains 
from clinical isolates. Asian Pacific Journal of Tropical Medicine, 7(1), 435–
441. 
145. Medical microbiology, Mims and Playfair, Mosby Europe, 1993, 35.31. 
146. French, G. L. (2006). Bactericidal agents in the treatment of MRSA 
infections--the potential role of daptomycin. Journal of Antimicrobial 
Chemotherapy, 58(6), 1107–1117. 
147. Cushnie, T. P. T., Hamilton, V. E. S., Chapman, D. G., Taylor, P. W., Lamb, 
A. J. (2007). Aggregation of Staphylococcus aureus following treatment with the 
antibacterial flavonol galangin. Journal of Applied Microbiology, 103 (5), 1562–
1567. 
148. Suarez, M., Haenni, M., Canarelli, S., Fisch, F., Chodanowski, P., Servis, C., 
Michielin, O., Freitag, R., Moreillon, P., Mermod, N. (2005). Structure-function 
142 
 
University of Ghana  http://ugspace.ug.edu.gh
characterization and optimization of a plant-derived antibacterial 
peptide. Antimicrobial Agents and Chemotherapy 49(9), 3847–3857. 
149. Voskoglou-NomikosT., Pater, J. L., Seymour, L. (2003). Clinical Predictive 
Value of the in Vitro Cell Line, Human Xenograft, and Mouse Allograft 
Preclinical Cancer Models.Clinical Cancer Research, 9,4227.  
150. Holbeck S. L.,Collins, J. M.,Doroshow, J. H. (2010). Analysis of FDA-
Approved Anti-Cancer Agents in the NCI60 Panel of Human Tumor Cell Lines. 
Molecular Cancer Therapeutics. 9(5), 1451–1460. 
151. ASTMH. American Society for Tropical Medicine and Hygiene. Tropical 
Diseases. http://www.astmh.org/tropical medicine_qanda/1591.htm 
152. Globalization and Infections Diseases: Areview to the Linkages. 2004. Social 
Economic and Behavorial Research. 
153. WHO Malaria. World Malaria Report 2014. World Health Organization 
Geneva, Switz [Internet]. 2014; Available from: 
http://apps.who.int/malaria/wmr2014. 
154. Ṧlapeta, J., Morin-Adeline, V. 2001. Apicomplexa, http://tolweb.org/ 
155. Center for Disease Control and Prevention. www.cdc.gov 
156. WHO Child Health. http://www.afro.who.int 
157. Europrean Alliance Against Malaria. Malaria and the G8 – Leading or 
Lagging?  2009. 
http://www.mmv.org/sites/default/files/uploads/docs/news/REPORT_ON_G8_M
ALARIA_V4.pdf 
158. Tinto, H., Rwagacondo, C., Karema, C., Mupfasoni, D., Vandoren, W., 
Rusanganwa, E., Erhart, A., Overmeir, C. V., Marck, E. V., D’Alessandro, U. 
(2006). In-vitro susceptibility of Plasmodium falciparum to 
143 
 
University of Ghana  http://ugspace.ug.edu.gh
monodesethylamodiaquine, dihydroartemsinin and quinine in an area of high 
chloroquine resistance in Rwanda. Transactions of the Royal Society of Tropical 
Medicine and Hygyiene, 100(6), 509-514. 
159. Agtmael, M. A. V., Cheng-Qi, S., Qing, J. X., Mull, R., Boxtel, C. J. V. 
(1999). Multiple dose pharmacokinetics of artemether in Chinese patients with 
uncomplicated falciparum malaria. International Journal of Antimicrobial 
Agents, 12, 151–158. 
160. Ridley, R. G. (2002). Medical need, scientific opportunity and the drive for 
antimalarial drugs. Nature. 415, 686–93. 
161. Elabbadi, N., Ancelin, M., Vial, H. (1992). Use of radioactive ethanolamine 
incorporation into phospholipids to assess in vitro antimalarial activity by the 
semiautomated microdilution technique. Antimicrobial Agents and 
Chemotherapy, 36(1), 50-55. 
162. Kupchan, M. S., Britton, R. W., Zeigler, M. F., Sigel, C. W. (1973). 
Bruceantin, a new potent antileukemic simaroubolide from Brucea 
antidysenterica. Journal of Organic Chemistry, 38(1), 178-179. 
163. Ayisi, N. K., Appiah-Opong, R., Gyan, B., Bugyei, K., Ekuban, F. (2011) 
Plasmodium falciparum: assessment of selectivity of action of chloroquine, 
Alchornea cordifolia, Ficus polita, and other drugs by a tetrazolium-based 
colorimetric assay. Malaria Research and Treatment, 2011, 1-7. 
164. Smilkstein, M., Sriwilaijaroen, N., Kelly, J. X., Wilairat, P., Riscoe, M. 
(2004). Simple and Inexpensive Fluorescence-Based Technique for High-
Throughput Antimalarial Drug Screening. Antimicrobial Agents and 
Chemotherapy, 48(5), 50-55. 
144 
 
University of Ghana  http://ugspace.ug.edu.gh
165. Yemoa, A., Gbenou, J., Affolabi, D., Moudachirou, M., Bigot, A., Anagonou, 
S., Portaels, F., Quetin-Leclercq, J., Martin, A. (2011). Buruli ulcer: a review of In 
vitro tests to screen natural products for activity against Mycobacterium ulcerans. 
Planta Medica, 77(6), 641–646. 
166. Ito, T., Masubuchi, M. (2014). Dereplication of microbial extracts and related 
analytical technologies. The Journal of Antibiotics, 67(5), 353-360. 
167. Lang, G., Mayhudin, N. A., Mitova, M. I., Sun, L., van der Sar, S., Blunt, J. 
W., Cole, A. L. J., Ellis, G., Laatsch, H., Munro, M. H. G. (2008). Evolving 
Trends in the Dereplication of Natural Product Extracts: New Methodology for 
Rapid, Small-Scale Investigation of Natural Product Extracts. Journal of Natural 
Products. 71(9), 1595–1599 
168. Smyrniotopoulos, V., Rahman, M. M., Gibbons, S., Roussis, V. (2008). 
Brominated Diterpenes with Antibacterial Activity from the Red Alga 
Sphaerococcuscoronopifolius. Journal of Natural Products. 71(8), 1386–1392 
169. Shaari, K., Ling, K. C., Rashid, Z. M., Jean, T. P., Abas, F., Raof, S. M., 
Zainal, Z., Lajis, N. H., Mohamad, H., Ali, A. M. (2009). Cytotoxic Aaptamines 
from Malaysian Aaptos aaptos. Marine Drugs, 7, 1-8. 
170. Teai, T. T., Raharivelomanana, P., Bianchini, J-P., Faure, R., Martin P. M. 
V., Cambon, A. (1997). Structure de deux nouvelles iminomycosporines isolées 
dePocillopora eydouxi. Tetrahedron Letters, 38(33), 5799–5800. 
171. Tsuda, M., Kasai, Y., Komatsu, K., Sone, T., Tanaka, M., Mikam, Y., 
Kobayashi, J. (2004). Citrinadin A, a Novel Pentacyclic Alkaloid from Marine-
Derived Fungus Penicillium citrinum, Organic Letters, 6(18),3087-3089.  
145 
 
University of Ghana  http://ugspace.ug.edu.gh
172. Clark, B., Capon, R. J., Lacey, E., Tennant, S., Gill, J. H. (2006). 
Quinolactacins revisited: from lactams to imide and beyond. Organic and 
Biomolecular Chemistry 4, 1512–1519. 
173. Morris, G. Z., Williams, R. L., Elliott, M. S., Beebe, S. J. (2002). Resveratrol 
induces apoptosis in LNCaP cells and requires hydroxyl groups to decrease 
viability in LNCaP and DU145 cells. The Prostate, 52, 319-329. 
174. Goldenberg, L., So, A., Fleshner, N., Rendon, R., Drachenberg, D., Elhilali, 
M. (2009), The role of 5-alpha reductase inhibitors in prostate pathophysiology: Is 
there an additional advantage to inhibition of type 1 isoenzyme? Canadian 
Urological Association, 3(3), S109-114. 
175. Pessina, A., Gribaldo, L., Mineo, E., Neri, M. G. (1994), In vitro short-term 
and long-term cytotoxicity of fluoroquinolones on murine cell lines. Indian 
Journal of Experimental Biology, 32(2), 113-118. 
176. He, Q. Q., Gu, S. X., Liu, J., Wu, H. Q., Zhang, X., Yang, L. M., Zheng, Y. T., 
Chen, F.E. (2011). Structural modifications of quinolone-3-carboxylic acids with 
anti-HIV activity. Bioorganic and Medicinal Chemistry, 19(16), 5039-5045. 
177. Kim, W-G., Song, N-K., and Yoo, I-D. (2001). Quinolactacins Al and A2, 
New Acetylcholinesterase inhibitors from Penicillium citrinum. The Journal of 
Antibiotics, 54 (10), 831-835. 
178. Dubar, F., Anquetin, G., Pradines, B., Dive, D., Khalife, J., Biot, C. (2009). 
Enhancement of the antimalarial activity of ciprofloxacin using a double 
prodrug/bioorganometallic approach. Journal of Medicinal Chemistry, 52(4), 
7954-7957. 
146 
 
University of Ghana  http://ugspace.ug.edu.gh
179. Pradines, B., Rogier, C., Fusai, T., Mosnier, J., Daries, W., Barret, E., Parzy, 
D. (2001). In vitro activities of antibiotics against Plasmodium falciparum are 
inhibited by iron. Antimicrobial Agents and Chemotherapy, 45(6), 1746–1750. 
180. Divo, A. A., Sartorelli, A. C., Patton, C. L., Bia, F. J. (1988). Activity of 
fluoroquinolone antibiotics against Plasmodium falciparum In vitro. 
Antimicrobial agents and chemotherapy, 32(8), 1182–1186. 
181. Winter, R. W., Kelly, J. X., Smilkstein, M. J., Dodean, R., Hinrichs, D., 
Riscoe, M. K. (2008). Antimalarial quinolones: Synthesis, potency, and 
mechanistic studies. Experimental Parasitology, 118, 487–497. 
182. Kurasawa, Y., Yoshida, K., Yamazaki, N., Kaji, E., Sasaki, K., Zamami, Y., 
Sakai, Y., Fujii, T., Ito, H. (2011). Quinolone Analogs 11: Synthesis of Novel 4-
Quinolone-3-carbohydrazide Derivatives with Antimalarial Activity Journal of 
Heterocyclic Chemistry, 49, 288-292. 
183. Singh, P., Sachdeva, S., Raj, R., Kumar, V., Mahajan, M. P., Nasser, S., 
Vivas, L., Gut, J., Rosenthal, P. J., Feng, T-S., Chibale, K. (2011). Antiplasmodial 
and cytotoxicity evaluation of 3-functionalized 2-azetidinone derivatives. 
Bioorganicand Medicinal Chemistry Letters, 21. 4561-4563. 
184. Picot, S., Burnod, J., Bracchi, V., Chumpitazi, B. F., Ambroise-Thomas, P. 
(1997). Apoptosis related to chloroquine sensitivity of the human malaria parasite 
Plasmodium falciparum. Transactions of the Royal Society of Tropical Medicine 
and Hygiene91(5), 590-591. 
147 
 
University of Ghana  http://ugspace.ug.edu.gh
 
APPENDICES 
Appendix 1a: 13CNMR spectrum of quinolactacins 
 
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Appendix 1b: HSQC spectrumof quinolactacins 
 
 
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Appendix 1c:  1H NMR spectrum of quinolactacins 
 
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Appendix 1d: 1H-1H COSY spectrum of quinolactacins 
 
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Appendix 1e: 1H-1H COSY spectrum of quinolactacins 
 
 
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Appendix 1f: 1H-1H COSY spectrum of quinolactacins 
 
 
 
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Appendix 1g: HMBC spectrum of quinolactacins 
 
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Appendix 1h: HMBC spectrum of quinolactacins 
 
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Appendix 1i: HMBC spectrum of quinolactacins 
 
 
 
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Appendix 1j: HMBC spectrum of quinolactacins 
 
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Appendix 1k: HMBC spectrum of quinolactacins 
 
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Appendix 1l: 1H-1H TOCSY spectrum quinolactacin 
 
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Appendix 1m: 1H-1H TOCSY spectrum quinolactacin 
 
 
 
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Appendix 2a:  HSQCspectrum of citrinadins 
 
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Appendix 2b:  HSQCspectrum of citrinadins 
 
 
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Appendix 2c: 1H NMR spectrum of citrinadins 
 
 
 
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Appendix 2d:  1H NMR spectrum of citrinadins 
 
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Appendix 2e: 1H-1H COSY spectrum of citrinadins 
 
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Appendix 2f: 1H-1H COSY spectrum of citrinadins 
 
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Appendix 2g: 1H-1H COSY spectrum of citrinadin 
 
 
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Appendix 2h: HMBC spectrum of quinolactacin 
 
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Appendix 2i: HMBC spectrum of citrinadins 
 
 
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Appendix 2j: HMBC spectrum of citrinadins 
 
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Appendix 2k: HMBC spectrum of citrinadins 
 
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Appendix 2l: HMBC spectrum of citrinadins 
 
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Appendix 2m: HMBC spectrum of citrinadins 
 
 
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Appendix 2n: HMBC spectrum of citrinadins 
 
 
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Appendix 2o: TOCSY spectrum of citrinadin 
AAGIIF F FFF 
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Appendix 2p: HMBC spectrum of citrinadins 
 
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Appendix 2q: TOCSY spectrum of citrinadins 
 
 
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Appendix 2r: TOCSY spectrum of citrinadins 
 
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Appendix 2s:  1H-1H TOCSY spectrum of citrinadin 
 
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Appendix 2t:  1H NMR spectrum of citrinadin 
 
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3. Preparation of reagents used for 3c. Preparation of Phosphate buffered 
MTT assay saline (PBS) 
3a. Preparation of DMEM 4 g of NaCl, 0.1 g of KCl, 0.72 g of 
Na2HPO4and 0.12 g of KH2PO4 were 
4.75 g of DMEM powder was weighed 
weighed and dissolved with 450 mL of 
and 300mL of sterile distilled water 
distilled water in a 500 mL beaker. The 
added and stirred for 30 mins. Sterile 
pH was adjusted to 7.4 using NaOH 
distilled water was added to top up to 
and HCl. The solution was transferred 
500 mL and transferred into an 
into a 500 mL measuring cylinder and 
autoclave flask for autoclaving. After 
topped up to the 500 mL mark. The 
autoclaving, the content was cooled to 
solution was then transferred into a 1L 
37oC and 5 mL of NaHCO3, 5 mL of 
autoclave bottle and sterilised. After 
PSG, and 5 mL of L-glutamate are 
cooling, it was labelled and stored at 
added under sterile conditions. The 
4oC. 
medium was stored at 4oC for later use. 
3d. Preparation of MTT solution 
3b. Preparation of Acidified 
Isopropanol 0.125 g of MTT was weighed into a 
tube covered with aluminium foil. A 
1.7 mL HCl was added to 500mL of 
volume of 50 µL of PBS was added 
isopropanol in a reagent bottle and 
and mixed to dissolve completely the 
mixed Five millilitre of the solution 
MTT salt. After that the solution was 
was pipetted off and replaced with 5 
filtered using a 50 mL syringe barrel 
mL of triton X and stored at room 
and 0.45µM Millipore filter. It was 
temperature. 
labelled and wrapped in a 50 mL 
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centrifuge tube container with 
aluminium foil and stored at 4oC. 
 
4. CELL CULTURE 
4a. Cell recovery 
Cryopreserved cells in -80 ºC 
refrigeratorwas thawed in a water bath 
at 37ºC for 5 min and suspended in 10 
mL of completemedia in a falcon tube. 
The falcon tube was spun at 1000 rpm 
for 5 min.Discard supernatant was 
resuspended pellets in 1 mL of 
completemedia and transfer cells into 
25cc culture flask containing 5 mL of 
complete DMEM and incubatein a 
humidified chamber at 37oC in the 
presence of 5% CO2. 
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5a. Breast Cancer cell Line (MCF-7) Passage No. 10 
Passage No. 6 Cell count: 83/73/60/57 
Cell count: 35/35/25/36 Cell density: X104 
Cell density: X104 
 
                      = 27.3 X 105cells/mL 
                     = 13.1 X 105cells/mL For MTT Assay, 1X105 cells/mL 
required; 
For MTT Assay, 1X105 cells/mL 
required; For 10 mL cell suspension;   = 0.37 
For 10 mL cell suspension;   = 0.76 mL 
mL = 0.37 mL cells + 9.63 mL RPMI 
= 0.76 mL cells + 9.24 mL DMEM   
  
 5d. Human promyelocytic leukemia 
cells 
5b. Prostate Cancer cell Line (PC-3) 
Passage No. 6 
Passage No. 7 
Cell count: 104/107/84/95 
Cell count: 22/13/17/12 
Cell density: X104 
Cell density: X104 
                     = 39.0 X 105cells/mL 
 
For MTT Assay, 1X105 cells/mL 
                    = 6.4 X 105cells/mL 
required; 
For MTT Assay, 1X105 cells/mL 
required; For 10 mL cell suspension;   = 0.26 
mL 
For 10 mL cell suspension;   = 0.76 
 = l0.26 mL cells + 9.74 mL RPMI 
mL 
 
= 1.56 mL cells + 8.44 mL DMEM 
 
 
5e. Human T-lymphoblastic leukemia 
5c. Human Prostate Cancer cell Line 
cells 
(LNCap) 
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Passage No. 4 
Cell count: 93/85/94/107 
Cell density: X104 
 
                     = 37.9 X 105cells/mL 
 
For MTT Assay, 1X105 cells/mL 
required; 
For 10 mL cell suspension;   = 0.26 
mL 
= 0.26 mL cells + 9.74 mL RPMI 
 
 
 
 
 
 
 
 
 
 
 
 
 
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