British Journal of Pharmacology, 42, 392-402
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KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY, KUMASI COLLEGE OF HEALTH SCIENCES, DEPARTMENT OF PHARMACOGNOSY BIOACTIVE ANTI-LEISHMANIAL AGENTS: PROSPECTS OF THE GHANAIAN BIODIVERSITY BY FRANCIS ACKAH ARMAH MAY, 2016 BIOACTIVE ANTI-LEISHMANIAL AGENTS: PROSPECTS OF THE GHANAIAN BIODIVERSITY A thesis submitted in fulfilment of the requirements for the degree Of DOCTOR OF PHILOSOPHY In the Department of Pharmacognosy Faculty of Pharmacy and Pharmaceutical Sciences College of Health Sciences Kwame Nkrumah University of Science and Technology By Francis Ackah Armah MAY, 2016 ii DECLARATION The experimental work described in this thesis was carried out at the Department of Pharmacognosy, KNUST and the Department of Biomedical and Forensic Science, University of Cape Coast. This work has not been submitted for any other degree. Francis Ackah Armah…………………………………… …………………….. (Student) Signature Date Prof. Kofi Annan………………………………………… …………………….. (Supervisor) Signature Date Prof. Mrs. Rita Akosua Dickson………………………… …………………….. (Supervisor) Signature Date Prof. Abraham Yaboah Mensah…………………………… …………………….. (Head, Department of Pharmacognosy) Signature Date ii DEDICATION This work is dedicated to my late grandparents Nana Asafo IV alias Mr Thomas Ekeyeboe Cudjoe and Madam Hannah Aya Ekeyeboe Cudjoe. iv ACKNOWLEDGEMENT My sincere thanks go to my supervisors Profs. Kofi Annan and Rita Akosua Dickson, for supervising this research. Their continual advice and encouragement have been very helpful. May the Almighty God bless you. I am greatly indebted to Prof. Abraham Yeboah Mensah for his support as Head of the Pharmacognosy Department. I am most grateful to Dr Isaac Kingsley Amponsah for his advice and encouragement throughout this research. I express my sincere appreciations to the teaching staff and my colleague graduate students at the Department of Pharmacognosy, KNUST, for creating the much needed and pleasant working atmosphere during my stay. I thank you all for your warm companionship. I also express my gratitude to the technicians of the Departments of Pharmacognosy, Pharmacology and Pharmaceutical microbiology for their technical assistance. Many thanks to Dr. Solomon Habtemariam, Pharmacognosy Research Laboratories, Medway School of Science, University of Greenwich, for generously running and providing the NMR spectra of the isolates. The technical assistance of the staff at the Greenwich NMR Laboratory and their willingness to allow access to their state-of-the-art, Energy and Physical Science Research Council (EPSRC) National Mass Spectrometry Facilities at Swansea University (Singleton Park, Swansea, UK) are greatly appreciated. I would like to say a big thank you to my dear and lovely wife Anita for her understanding and never failing support through out this work. I am also very grateful to God for His mercies and protection. Finally, my deepest gratitude goes to my entire family; my mother, Mrs. Monique Assuah Cophie and my father, Mr. Alfred v Ndede Armah for their fervent prayers and continual financial support during my academic journey. vi ABSTRACT Leishmaniasis, one of the neglected tropical diseases, is currently a significant health problem in North Africa with a rising concern in West Africa because of co-infection with the Human Immunodeficiency Virus (HIV). The aim of this research is to evaluate the anti-leishmanial, anti-inflammatory and antioxidant activities of methanol extracts of four (4) indigenous plants namely root bark of Erythrophleum ivorense and the stem barks of Coelocaryon oxycarpum, Omphalocarpum ahia and Anthostema aubryanum and determine the chemical constituents which confer on the species their purported activities in folklore medicine. These plants are traditionally used in Ghana to treat parasitic infections as well as pain and inflammation. In vitro anti-leishmanial potential of the selected plants was evaluated using direct counting assay of promastigotes of Leishmania donovani. Amphotericin B was used as positive control. Anti-inflammatory activity of the extracts and compounds were evaluated using the chick carrageenan model with diclofenac and dexamethasone as standards. The antioxidant potential of the extracts and compounds were also evaluated using the DPPH scavenging assay. E. ivorense showed the highest anti-leishmanial activity (IC50 = 14.10 μg/mL), followed by A. aubryanum (IC50 = 23.90 μg/mL), C. oxycarpum (IC50 = 31.31 μg/mL) and O. ahia (IC50 = 124.04 μg/mL) respectively. E. ivorense thus showed considerable activity compared to amphotericin B (IC50 = 2.4 μg/mL). Fractionation of E. ivorense afforded a moderately active ethyl acetate fraction and a significantly methanol fraction active (IC50 = 2.97 μg/mL). E. ivorense also showing the highest anti-inflammatory (ED50 = 16.91 mg/kg vii body weight) and antioxidant (IC50 = 1.11 μg/mL) activities, followed by C. oxycarpum, O. ahia and A. aubryanum respectively. Investigation of the pet-ether, ethyl acetate and methanol fractions of E. ivorense revealed the ethyl acetate fraction to be the most active. Further fractionation of the ethyl acetate fraction led to an isolation of the novel cassane diterpene erythroivorensin as well as the flavanone eriodictyol and the triterpene betulinic acid. The rank order of leishmanicidal potency of the compounds was erythroivorensin (IC50 = 29.10 μg/mL) > eriodictyol (IC50 = 103.80 μg/mL) > betulinic acid (IC50 = 1000 μg/mL). These compcounds may be working synergistically to produce the observed anti- leishmanial activity of the ethyl acetate fraction. The anti-inflammatory activity of betulinic acid (ED50 = 4.367±1.333 mg/kg body weight), the most active anti- inflammatory compound, was comparable to that of diclofenac (ED50= 3.74±0.333 mg/kg body weight). This was followed by erythroivorensin (ED50 = 18.66±0.667 mg/kg body weight) and eriodictyol (ED50 = 56.25±0.133 mg/kg body weight). With respect to the antioxidant activity of the compounds, eriodictyol was the most active (IC50 = 21.06 μg/mL), followed by betulinic acid (IC50= 33.07 μg/mL) and erythroivorensin (IC50 = 35.78 μg/mL) respectively. The present studies have shown that extracts of E. ivorense have considerable anti-leishmanial activity and could be used for the treatment of leishmaniasis after the establishment of its toxicity profile. The novel compound erythroivorensin possesses anti-leishmanial effects and has considerable anti- inflammatory properties. To the best of my knowledge, eriodictyol and betulinic acid are isolated for the first time in Erythrophleum ivorense. viii TABLE OF CONTENTS DECLARATION ....................................................................................................... III DEDICATION............................................................................................................IV ACKNOWLEDGEMENT........................................................................................... V ABSTRACT.............................................................................................................. VII TABLE OF CONTENTS ...........................................................................................IX LIST OF TABLES ............................................................................................... XVIII LIST OF FIGURES ................................................................................................. XIX ABBREVIATIONS .............................................................................................. XXIV CHAPTER 1 INTRODUCTION ................................................................................. 1 1.1 GENERAL INTRODUCTION ............................................................................ 1 1.2 JUSTIFICATION OF RESEARCH ..................................................................... 3 1.3 AIMS AND OBJECTIVES ................................................................................. 5 CHAPTER 2 LITERATURE REVIEW ..................................................................... 7 2.1 LEISHMANIASIS .............................................................................................. 7 2.1.1 Historical background of leishmaniasis ........................................................ 8 2.1.2 Geographical distribution of leishmaniasis ................................................... 8 2.1.3 Leishmaniasis in West Africa .................................................................... 10 2.1.4 Leishmaniasis in Ghana ............................................................................. 11 2.1.5 Classification of human leishmaniasis ........................................................ 13 2.1.5.1 Cutaneous leishmaniasis ........................................................................ 13 ix 2.1.5.2 Diffused leishmaniasis ........................................................................... 16 2.1.5.3 Mucocutaneous leishmaniasis ................................................................ 17 2.1.5.4 Visceral leishmaniasis ........................................................................... 18 2.1.6 Treatment of leishmaniasis ........................................................................ 19 2.1.7 Life cycle of the Leishmania parasite ......................................................... 20 2.1.8 Leishmania infection and the human immune system ................................. 21 2.1.9 Intracellular survival of Leishmania parasite in the macrophages ................ 22 2.1.10 Natural products with anti-leishmanial activity ..........................................