Chemical, biological and ethnopharmacological studies of two Malian medicinal plants: Terminalia macroptera and Biophytum umbraculum Anh Thu Pham Thesis for the degree of Philosophiae Doctor Department of Pharmaceutical Chemistry School of Pharmacy University of Oslo Oslo 2014 © Anh Thu Pham, 2014 Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo No. 1552 ISSN 1501-7710 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Printed in Norway: AIT Oslo AS. Produced in co-operation with Akademika Publishing. The thesis is produced by Akademika Publishing merely in connection with the thesis defence. Kindly direct all inquiries regarding the thesis to the copyright holder or the unit which grants the doctorate. Contents CONTENTS ............................................................................................................................. I ACKNOWLEDGEMENTS ................................................................................................... III LIST OF PAPERS .................................................................................................................. III ABBREVIATIONS ................................................................................................................. V ABSTRACT ......................................................................................................................... VII 1. INTRODUCTION .......................................................................................................... 1 1.1 MALI ........................................................................................................................... 1 1.2 TRADITIONAL MEDICINE ............................................................................................ 2 1.3 INVESTIGATED PLANTS............................................................................................... 3 1.3.1 Terminalia macroptera Guill. & Perr. (Combretaceae).................................... 3 1.3.2 Biophytum umbraculum Welw. (Oxalidaceae) ................................................. 4 1.4 GENERAL ASPECTS OF ISOLATED POLYPHENOLS ....................................................... 6 1.4.1 Flavonoids......................................................................................................... 6 1.4.2 Hydrolyzable tannins ........................................................................................ 9 1.5 BIOLOGICAL ACTIVITITES ........................................................................................ 11 1.5.1 Free radicals and antioxidant activity ............................................................. 11 1.5.2 Į-Glucosidase inhibitory activity .................................................................... 13 1.5.3 Antimalarial activity ....................................................................................... 14 1.5.4 Toxicity ........................................................................................................... 15 2. AIMS OF THE THESIS ............................................................................................... 16 3. SUMMARY OF PAPERS ............................................................................................ 17 4. RESULTS AND DISCUSSION ................................................................................... 21 4.1 ETHNOPHARMACOLOGICAL RESEARCH ................................................................... 21 4.2 EXTRACTION AND ISOLATION PROCEDURES ............................................................ 23 4.3 CHEMICAL CHARACTERIZATION .............................................................................. 25 4.4 BIOLOGICAL ACTIVITIES .......................................................................................... 30 4.4.1 Antioxidant activity ........................................................................................ 30 I 4.4.2 Į-Glucosidase inhibitory activity .................................................................... 33 4.4.3 Antimalarial activity ....................................................................................... 34 4.4.4 Toxicity ........................................................................................................... 36 5. CONCLUSIONS .......................................................................................................... 38 REFERENCES ....................................................................................................................... 39 PAPERS ................................................................................................................................. 51 II Acknowledgements The studies presented in this thesis were carried out at the Pharmacognosy section of the Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, during the years 2009-2014. Field work was performed in Bamako, Siby, Dioïla and Dogonland of Mali. Financial support was provided by Norwegian Pharmaceutical Society (NFS) and UNIFOR, which are gratefully acknowledged. Foremost, I would like to express my sincere gratitude to my supervisors, Associate Professor Helle Wangensteen and Professor Emeritus Karl Egil Malterud. I cannot thank you enough for your guidance and support that you have provided me over the last few years. Your enthusiasm, knowledge and patience inspired me to persevere and allowed me to reach my full potential, so thank you.. My gratefulness also extends to my co-supervisor, Berit Smestad Paulsen, for her insight and for the advices she provided me with. I would also like to thank all of my colleagues at the Pharmacognosy section. In addition to creating a nice and friendly working environment, they were able to provide some useful insights and feedback that contributed to the continuous improvement of my work. Special thanks goes to my roomie, Ingvild Austarheim, for being there as we shared in the ups and downs of a common journey, as well as Associate Professor Hilde Barsett for her positive attitude and words of encouragement at times when I needed them the most. Drissa Diallo, Adiaratou Togola and N’Golo Ballo in Bamako are greatly appreciated for giving me the opportunity to work with plant materials from Mali, and for making my time in Mali pleasant and memorable. Finally, I would like to thank everyone who has contributed directly or indirectly to this project. You know who you are! Last but certainly not least, I would like to thank all of my family and friends for their unconditional love, help and support, as well as for their enormous patience with me throughout this long but fulfilling journey. $QK7KX3KDP III List of Papers Paper I Anh Thu Pham, Christina Dvergsnes, Adiaratou Togola, Helle Wangensteen, Berit Smestad Paulsen, Drissa Diallo and Karl Egil Malterud Terminalia macroptera, its current medicinal use and future perspectives Journal of Ethnopharmacology 2011, 137, 1486-1491 Paper II Anh Thu Pham, Karl Egil Malterud, Berit Smestad Paulsen, Drissa Diallo and Helle Wangensteen DPPH Radical scavenging and xanthine oxidase inhibitory activity of Terminalia macroptera leaves Natural Product Communications 2011, 6, 1125-1128 Paper III Anh Thu Pham, Karl Egil Malterud, Berit Smestad Paulsen, Drissa Diallo and Helle Wangensteen Į-Glucosidase inhibition, 15-lipoxygenase inhibition and brine shrimp toxicity of extracts and isolated compounds from Terminalia macroptera leaves Pharmaceutical Biology 2014, 52, 1166-1169 Paper IV Anh Thu Pham, Celine Nguyen, Karl Egil Malterud, Drissa Diallo and Helle Wangensteen Bioactive flavone-C-glycosides of the African medicinal plant Biophytum umbraculum Molecules 2013, 18, 10312-10319, doi: 10.3390/molecules180910312 Paper V Ingvild Austarheim, Anh Thu Pham, Celine Nguyen, Yuan-Feng Zou, Sibylle Sax, Sergio Wittlin, Karl Egil Malterud, Drissa Diallo and Helle Wangensteen The Malian medicinal plant Biophytum umbraculum as adjuvant treatment of cerebral malaria Manuscript IV Abbreviations 2D-NMR Two-dimensional nuclear magnetic resonance BuOH Butanol 13C-NMR Carbon nuclear magnetic resonance CM Cerebral malaria COSY Correlation spectroscopy CVD Cardiovascular diseases DCM Dichloromethane DMT Department of Traditional Medicine DMSO Dimethyl sulfoxide DPPH 1,1-Diphenyl-2-picrylhydrazyl ET Electron transfer EtOAc Ethyl acetate GI Gastrointestinal 1H-NMR Proton nuclear magnetic resonance HAT Hydrogen atom transfer HHDP Hexahydroxydiphenic HMBC Heteronuclear multiple bond correlation spectroscopy HPLC High performance liquid chromatography HSQC Heteronuclear single quantum correlation spectroscopy IC50 Concentration to give 50 % inhibition ICH50 Concentration to give 50 % hemolysis ITM Improved Traditional Medicine LC50 Concentration to give 50 % lethality LDL Low density lipoprotein LO Lipoxygenase LPS Lipopolysaccharide MeOH Methanol NO Nitric oxide NP Normal phase NMR Nuclear magnetic resonance OH Hydroxyl group PUFA Polyunsaturated fatty acid RNS Reactive nitrogen species ROS Reactive oxygen species V RP Reverse phase RS Reactive species RT Room temperature SEC Size exclusion chromatography STD Sexually transmitted disease TLC Thin layer chromatography UV Ultraviolet WHO World Health Organization XO Xanthine oxidase VI Abstract The aim of this thesis was to investigate the chemical and biochemical properties of the Malian medicinal plants Terminalia macroptera and