Effectiveness of Bulb Extracts of the Genus Allium
Total Page：16
File Type：pdf, Size：1020Kb
Effectiveness of Bulb Extracts of the Genus Allium Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Pharmazie der Philipps-Universität Marburg vorgelegt von M.Sc. Sahar Samadi aus Tehran, Iran Marburg/Lahn 2018 Erstgutachter: Prof. Dr. Michael Keusgen Zweitgutachter: Prof. Dr. Klaus Reuter Eingereicht am 24 May 2018 Tag der mündlichen Prüfung am 11 July 2018 Hochschulkennziffer 1180 III Preface This thesis is the result of a research project that I carried out at the institute of pharmacy at Philipps-Universität Marburg. During this research I received academic and practical supports from different persons whom I would like to acknowledge here. First of all, I would like to express my sincere gratitude to my advisor Prof. Dr. Michael Keusgen for his continuous support, patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. Next, I would like to thank my thesis committee: Prof. Dr. Maike Petersen, Prof. Dr. Moritz Bünemannand and Prof. Dr. Klaus Reuter for taking the time to review this work and also for their insightful comments. I would like also to thank my colleagues and fellow lab mates for the stimulating discussions, for working together before deadlines, and for all the funs we had together. I wish also to acknowledge the role of my parents and my sister for their spiritual supports and their blessings throughout this research. Finally, I would like to acknowledge the most important person in my life – my husband Meysam, who has been a constant source of strength and inspiration. I can honestly say that it was only his determination and constant encouragement that ultimately made it possible for me to see this project through to the end. IV Table of Contents Preface ......................................................................................................................... III List of abbreviation .................................................................................................... VII Abstract .................................................................................................................... VIII 1. .................................................................................................................. Introduction ........................................................................................................................................ 1 1.1 History of natural products as medicines ........................................................... 1 1.1.1 Natural products from plant sources.............................................................................. 2 1.1.2 Plant products as antimicrobial agents .......................................................................... 2 1.2 The Genus Allium ................................................................................................ 4 1.2.1 The genus Allium and its phylogenetic classification ..................................................... 4 1.2.2 Pharmaceutical relevance of the genus Allium............................................................... 5 1.2.3 Alliinase reaction ......................................................................................................... 8 1.3 Fungi as plant pathogenic ................................................................................. 12 1.3.1 Aspergillus flavus ....................................................................................................... 12 1.3.2 Aspergillus niger ........................................................................................................ 14 1.3.3 Mucor hiemalis .......................................................................................................... 15 1.3.4 Penicillium italicum ................................................................................................... 16 1.4 Fungi as Human Pathogens (Dermatophyte) ................................................... 17 1.4.1 Basidiobolus ranarum ................................................................................................ 17 1.4.2 Cryptococcus neoformans .......................................................................................... 18 1.4.3 Epidermophyton floccosum......................................................................................... 19 1.5 Bioassays ............................................................................................................ 20 1.6 Antimicrobial susceptibility testing .................................................................. 20 2. ...................................................................................................... Aims and objectives ...................................................................................................................................... 22 2.1 Objectives .......................................................................................................... 22 V 3. ................................................................................................. Materials and Methods ...................................................................................................................................... 23 3.1 List of reagents and instruments ...................................................................... 23 3.2 Plant material ................................................................................................... 24 3.2.1 List of Allium samples ................................................................................................ 25 3.3 Microorganisms ................................................................................................ 27 3.3.1 Inoculum preparation ................................................................................................. 27 3.4 Plant Processing ................................................................................................ 28 3.4.1 Plant processing for bioactivity tests ........................................................................... 28 3.4.2 Plant Processing for Analysis of Volatile Sulfur Compounds ...................................... 28 3.5 Bioassay methods .............................................................................................. 28 3.5.1 Broth microdilution method ....................................................................................... 28 3.5.2 Agar microdilution method ........................................................................................ 30 3.5.3 Disk diffusion method ................................................................................................ 32 3.5.4 Double-dish chamber ................................................................................................. 33 3.5.5 Antimicrobial assay of different fractions ................................................................... 34 3.6 Instrumental Setup for bioactivity tests ........................................................... 34 3.6.1 HPLC Separation of the Volatile Sulfur Compounds .................................................. 34 3.6.2 HPLC micro-fractionation .......................................................................................... 35 3.6.3 HPLC-MS Analysis of the effective fractions ............................................................. 35 3.6.4 Structure Elucidation .................................................................................................. 35 4. ........................................................................................................................... Results ...................................................................................................................................... 36 4.1 Selection and development of methods ............................................................ 36 4.2 Results regarding plant pathogenic fungi ........................................................ 36 4.2.1 Broth microdilution method ....................................................................................... 37 4.2.2 Agar microdilution method ........................................................................................ 37 4.2.3 Disk diffusion test ...................................................................................................... 45 4.2.4 Double-dish chamber ................................................................................................. 50 4.3 Results regarding human pathogenic fungi ..................................................... 52 4.3.1 Agar microdilution method ........................................................................................ 52 4.3.2 Disk diffusion test ...................................................................................................... 56 4.3.3 Double-dish chamber ................................................................................................. 59 4.3.4 Summarized results regarding human pathogenic fungi .............................................. 60 4.4 Allium stipitatum, Identification of active substances ...................................... 61 VI 4.5 Bioassay-guided fractionation and isolation of the active compounds (Prep HPLC). ......................................................................................................................... 62 4.6 LC-MS adducts general Interpretation of A. stipitatum................................... 63 4.7 Chemical