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P. H. Nden Phd Pure Coventry University DOCTOR OF PHILOSOPHY Could traditional African herbal remedies be a source of novel antimicrobial compounds? An analysis of the antimicrobial and antibiofilm properties of Uvaria chamae and Prosopis africana Nden, Henry Award date: 2019 Awarding institution: Coventry University Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of this thesis for personal non-commercial research or study • This thesis cannot be reproduced or quoted extensively from without first obtaining permission from the copyright holder(s) • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 07. Oct. 2021 COULD TRADITIONAL AFRICAN HERBAL REMEDIES BE A SOURCE OF NOVEL ANTIMICROBIAL COMPOUNDS? An analysis of the antimicrobial and antibiofilm properties of Uvaria chamae and Prosopis africana By NDEN PANTONG HENRY JULY 2019 A thesis submitted in partial fulfilment of the University’s requirements for the Degree of Doctor of Philosophy ABSTRACT With the steady increase in global antimicrobial resistance rates, concomitant with a recent paucity of novel antibiotic discovery, it is essential that new antimicrobial compounds be discovered to prevent a return to the pre-antibiotic era over coming decades. Knowledge of the medicinal properties of traditional plants has given rise to many of the modern day medicines. Consequently, the aim of this work was to assess the antimicrobial and antibiofilm activities of a number of traditional African herbal remedies against a range of clinically important bacterial species. After a pilot study was performed, Prosopis africana and Uvaria chamae were selected from a range of medicinal plants due to their greater antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli than the other remedies tested. Crude extracts from P. africana (PAM, PAS and PAU) and U. chamae (UCM, UCS and UCU) were prepared using maceration, Soxhlet and ultrasound extraction method respectively. Cultures of both aerobic and anaerobic bacterial species were exposed to the crude extracts in agar-well diffusion and broth micro-dilution assays. In addition, semi-quantitative and quantitative antibiofilm assays were performed using PAS, PAU, UCS and UCU extracts against preformed biofilms of P. aeruginosa, S. aureus, S. epidermidis, Propionibacterium acnes and Clostridium difficile strains. Fractionation of the crude extracts was then performed using column chromatography; with antimicrobial, simple phytochemical and spectrophotometric analyses being performed on the resultant fractions. It was found that all extracts had both bacteriostatic and bactericidal activity against all of the bacterial species tested, although the Soxhlet and ultrasound extracts demonstrated a significantly greater activity than the maceration extracts. The extracts i demonstrated antibiofilm activity against preformed biofilm of P. aeruginosa, S. aureus, S. epidermidis, P. acnes and C. difficile strains by reducing the density of biofilm biomass significantly by at least 11.5 % compared to the control. In the antimicrobial activity test, the fractions that were the most effective against P. aeruginosa and S. aureus with MIC and MBC value ranges of 0.195 % - 2.60 % and 0.391 % - 6.25 % respectively, contained the following phytochemical groups; saponins, quinones, flavonoids, and alkaloids which have been previously shown to be antimicrobial by other workers. Absorption spectrum analysis further suggests that the fractions of U. chamae and P. africana with the greatest antimicrobial activity might contain flavonoid-related compounds as their major active components. This study demonstrated that P. africana and U. chamae extracts are effective antimicrobial and antibiofilm agents and therefore the isolation and evaluation of their active ingredients could yield potentially new leads to fight antimicrobial resisance. ii ACKNOWLEDGEMENT I wish to express my profound gratitude to my parents and mentors Mr and Mrs John J. Nden for their unending love and support both financially and morally towards my upbringing and educational pursuit. You are the greatest gift any child can have. Words alone cannot express my heartfelt gratitude. I also want to acknowledge the support and love of my brothers and my extended family at large. Thank you. My appreciation goes to my supervisor, Dr Timothy Aldsworth for his advice, suggestions and immense contributions towards the success of this research work. I want to acknowledge the efforts of my co-supervisors, Dr Jamie Beddow, Dr Jess Rollason, Dr Lauren Acton and Dr Larysa Paniwnyk for the knowledge, training and feedback they gave me throughout the PhD research period. Your support and contributions have not only broadened my knowledge in diverse research fields but have also made me a better researcher as a whole. I want to acknowledge the efforts of Dr Omar Janneh for his advice, contributions and for collaborating with Dr Bakary Touray to supply the medicinal plant samples from The Gambia for this research work. I want to appreciate the efforts and support of Mrs Sue Thompson and the entire laboratory staff. I want to acknowledge the Centre for Sport, Exercise and Life Science (CSELS, formerly ABES) of the Faculty of Health and Life Sciences, Coventry University, for partly funding this PhD research as part of their PhD studentship scheme. I want to acknowledge the Society of Applied Microbiology (SFAM) for financially supporting this research through the SFAM Research hardship grant (2017), and for sponsoring my conference attendance through the SFAM conference studentship scheme and SFAM Presidents’ grant. I am grateful. To my RCC/JCC family, friends and colleagues, am grateful for the prayers, support and well wishes. Words alone cannot express my gratitude. Thank you. iii TABLE OF CONTENTS TITLE PAGE CU ETHICS CERTIFICATE CANDIDATE DECLARATION ABSTRACT - - - - - - - - - i ACKNOWLEDGEMENTS - - - - - - - iii TABLE OF CONTENTS - - - - - - - iv LIST OF TABLES - - - - - - - - xi LIST OF FIGURES - - - - - - - - xv CHAPTER ONE - - - - - - - - 1 1.0 INTRODUCTION AND LITERATURE REVIEW- - - 1 1.1.0 INTRODUCTION- - - - - - - - 1 1.2.0 ANTIMICROBIAL RESISTANCE- - - - - 2 1.2.1 Intrinsic resistance - - - - - - 3 1.2.2 Acquired resistance- - - - - - 4 1.2.3 Biofilm: a major contributor to antimicrobial resistance- 7 1.3.0 IMPORTANCE OF MEDICINAL PLANTS IN COMBATING ANTIMICROBIAL RESISTANCE - - 9 1.3.1 Uvaria chamae (Sambe)- - - - - 11 1.3.2 Prosopis africana (Kembo)- - - - - 12 1.3.3 Cassia occidentalis (Kassala)- - - - 14 1.3.4 Ficus carica (fig)- - - - - - 15 1.3.5 Pterocarpus erinaceus- - - - - 17 1.3.6 Dracaena mannii - - - - - 18 1.4.0 STUDIES ON ANTIMICROBIAL ACTIVITIES OF PLANT - 20 1.4.1 Selected plant antimicrobial studies from last decade - 22 1.5.0 MECHANISM OF ACTION OF PLANT COMPOUNDS AGAINST MICROORGANISMS - - - - - 25 1.5.1 Phenols- - - - - - - 25 1.5.2 Quinones - - - - - - 26 1.5.3 Flavones, Flavonoids and Flavonols- - - 27 1.5.4 Tannins- - - - - - - 28 iv 1.5.5 Alkaloids- - - - - - - 28 1.5.6 Terpenoids and Essential oils- - - - 29 1.5.7 Saponins- - - - - - - 30 1.6.0 THE ANTIBIOFILM ACTIVITY OF MEDICINAL PLANTS - 31 1.7.0 EXTRACTION AND IDENTIFICATION OF BIOACTIVE COMPOUNDS FROM PLANT MATERIALS - - - 34 1.7.1 Preparation of Sample for extraction- - - - 34 1.7.2 Solvent selection - - - - - 34 1.7.3 Selecting the appropriate extraction method - - 35 1.7.4 Isolation, purification and identification of bioactive compounds from plant extracts- - - - 38 1.8.0 AIMS AND OBJECTIVES - - - - - - 40 CHAPTER TWO - - - - - - - - 42 2.0 PRELIMINARY ANTIMICROBIAL STUDY ON A SELECTION OF AFRICAN MEDICINAL PLANTS - - - - 42 2.1.0 INTRODUCTION - - - - - - - 42 2.2.0 MATERIALS AND METHODS - - - - - 43 2.2.1 Extraction (maceration) - - - - 44 2.2.2 Bacterial Strains and Growth Conditions - - 45 2.2.3 Antimicrobial assays - - - - - 46 2.2.3.1 Antibiotic susceptibility assay (Kirby-Bauer assay) 46 2.2.3.2 Agar-well Diffusion Assay - - - 46 2.2.3.3 Broth microdilution assay - - - 47 Minimum inhibitory Concentration (MIC) 47 Minimum Bactericidal Concentration(MBC) 48 2.2.3.4 Statistical analyses- - - - - 49 2.3 RESULTS - - - - - - - - 49 2.3.1 Antibiotic Susceptibility assay- - - - 49 2.3.2 Agar well diffusion assay - - - - - 51 2.3.3 Broth microdilution assay - - - - - 52 2.3.3.1 Minimum Inhibitory Concentration (MIC) - 52 2.3.3.2 Minimum Bactericidal Concentration (MBC) - 54 2.4.0 DISCUSSION - - - - - - - 56 v 2.4.1 Antimicrobial assay - - - - - 56 2.4.1.1 Antibiotic susceptibility assay - - 56 2.4.1.2 Determination of the antimicrobial activities of a selection of African medicinal plants from the Gambia - - - - - 60 2.4.2 Conclusion- - - - - - - 67 CHAPTER THREE - - - - - - - - 69 3.0 EXTRACTION EFFECIENCY OF PROSOPIS AFRICANA AND UVARIA CHAMAE USING DIFFERENT EXTRACTION METHODS AND THE PHYTOCHEMICAL SCREENING OF YIELDED EXTRACTS - - - - - - 69 3.1.0 INTRODUCTION
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