KAUNAS UNIVERSITY OF TECHNOLOGY UNIVERSITY OF TOULOUSE LINA SMOLSKAITĖ RARE FOREST AND COASTAL-DUNE MUSHROOM: EVALUATION OF ANTIOXIDANT AND BIOLOGICAL PROPERTIES Doctoral dissertation Physical Sciences, Chemistry (03P) 2016, Kaunas UDK 582.28 (043.3) Doctoral research was carried out at Kaunas University of Technology, Faculty of Chemical Technology, Department of Food Science and Technology and in France at the Institut National Polytechnique de Toulouse, Ecole Nationale Supérieure des Ingénieurs en Arts Chimiques Et Technologiques de Toulouse. Part of the research was performed in Latvia University of Agriculture, Faculty of Food Technology, Department of Food Technology. This study was supported, by Midi-Pyrénées Regional Council and the SMI (International mobility support) program of INP Toulouse. Scientific supervisors: Prof. Dr. Petras Rimantas VENSKUTONIS (Kaunas University of Technology, Physical Sciences, Chemistry - 03P). Dr. Thierry TALOU (University of Toulouse, Physical Sciences, Chemistry - 03P). Doctoral dissertation has been published in: http://ktu.edu Editor: UAB “Synergium” ©L. Smolskaitė, 2016 ISBN 978-609-02-1262-2 KAUNO TECHNOLOGIJOS UNIVERSITETAS TULŪZOS UNIVERSITETAS LINA SMOLSKAITĖ RETIEJI MIŠKO IR PAJŪRIO GRYBAI: ANTIOKSIDACINIŲ IR BIOLOGINIŲ SAVYBIŲ ĮVERTINIMAS Daktaro disertacija Fiziniai mokslai, chemija (03P) 2016, Kaunas UDK 582.28 (043.3) Dvigubo laipsnio disertacija parengta 2010–2016 metais Kauno technologijos universitete, Cheminės technologijos fakultete, Maisto mokslo ir technologijos katedroje, ir Prancūzijoje, - Nacionaliniame Tulūzos politechnikos institute, Chemijos inžinerijos ir technologijų padalinyje. Papildomi tyrimai atlikti Latvijos Žemės ūkio universitete, Maisto technologijos fakulteto, Maisto technologijos katedroje. Mokslinius tyrimus rėmė Midi-Pyrénées (Pietų Pirėnų) regiono mokslo taryba ir Tulūzos politechnikos instituto tarptautinė mainų programa. Moksliniai vadovai: Prof. dr. Petras Rimantas VENSKUTONIS (Kauno technologijos universitetas, fiziniai mokslai, chemija – 03P). Dr. Thierry TALOU (Nacionalinis Tulūzos politechnikos institutas, fiziniai mokslai, chemija – 03P). Interneto svetainės, kurioje skelbiama disertacija, adresas: http://ktu.edu Redagavo: UAB “Synergium” ©L. Smolskaitė, 2016 ISBN 978-609-02-1262-2 ACKNOWLEDGEMENTS I would like to gratefully acknowledge all the people who have walked alongside with me in recent years as I have worked on this thesis. They have guided me, placed opportunities in front of me and showed me the doors that might be useful to open. I would like to thank each and every one of them. First, I owe an enormous debt of gratitude to my supervisors Prof. Ph.D. Rimantas Petras VENSKUTONIS and Dr Thierry TALOU, for their expertise, understanding, patience and generous guidance. They encouraged and challenged me to be faithful throughout the years. I am grateful to Prof. Eric DEHARO and Prof. Louis CHAVANT for their pleasant words, suggestions, new experiences and the skills obtained regarding the topic of my research. A huge thanks to Dr Anne-Cécille LE LAMER and Dr Cynthia GIRARDI for being an excellent guide throughout the time together and for encouraging me with your kind words when I was struggling. I would like to express my warmest thanks to Dr sc. ing. Zanda KRUMA for the helpful discussions and recommendations. I would also like to thank the Research Council of Lithuania, Midi-Pyrénées Regional Council and SMI (International mobility support) program of INP Toulouse for their financial support. I would like to thank all my colleagues at the Department of Food Science and Technology, either the laboratory of Agro-Industrial Chemistry in Toulouse for an inspiring scientific and social environment. A special thanks to my family and my parents who have always been ready to help me, for giving the right advice at the right time and for being a source of motivation. The heartfelt thanks to my husband Vytautas, for his comprehension and belief in me. I really appreciate his tolerance and all his encouragement during this time. CONTENT ABBREVIATIONS ................................................................................................. 9 INTRODUCTION ................................................................................................. 11 1. LITERATURE REVIEW ................................................................................ 15 1.1. HISTORY, DIVERSITY AND IMPORTANCE OF MUSHROOMS ............. 15 1.1.1. General characterisation and definition of mushrooms ............................... 15 1.1.2. History, traditions and importance of mushrooms ...................................... 15 1.1.3. Diversity, composition and characteristics of mushrooms .......................... 17 1.2. THE MUSHROOM AS A SOURCE OF NATURAL BIOACTIVE MOLECULES ..................................................................................................... 19 1.2.1. Brief review on the investigated wild mushrooms bioactive molecules ...... 21 1.2.1.1. Heterocyclic compounds in mushrooms ................................................ 23 1.2.1.2. Polyketides in mushrooms ..................................................................... 24 1.2.1.3. Sterols in wild mushrooms .................................................................... 26 1.2.1.4. Terpene compounds in mushrooms ....................................................... 27 1.2.1.5. Miscellaneous compounds in mushrooms ............................................. 28 1.3. BRIEF REVIEW OF COLLECTED MUSHROOM SPECIES AND THEIR SECONDARY METABOLITES ........................................................................ 29 1.4. THE POTENTIAL ACTIVITY OF MUSHROOM SUBSTANCES AND THEIR RECOVERY ........................................................................................... 32 1.4.1. Antioxidants in mushrooms and their mode of action ................................. 33 1.4.2. Antimicrobials in mushrooms and their mode of action ............................. 37 2. MATERIALS AND METHODS ..................................................................... 39 2.1. MATERIALS .................................................................................................. 39 2.1.1. Research objects ......................................................................................... 39 2.1.2. Reagents ..................................................................................................... 39 2.2. PREPARATION OF EXTRACTS .................................................................. 46 2.2.1. Isolation of active compounds from P. schweinitzii extract ........................ 47 2.3. RESEARCH METHODS ................................................................................ 47 2.3.1. ANTIOXIDANT ANALYSIS ...................................................................... 48 2.3.1.1. DPPH•-scavenging capacity ................................................................... 48 2.3.1.2 ABTS•+ decolourisation assay ................................................................ 49 2.3.1.3. Ferric-reducing antioxidant power assay ............................................... 49 2.3.1.4. Oxygen radical absorbance capacity ...................................................... 49 2.3.1.5. Determination of total phenolic content ................................................. 50 2.3.2. MICROBIAL ANALYSIS ............................................................................ 50 2.3.2.1. Disc diffusion assay ............................................................................... 50 2.3.2.2. Agar-overlay assay (TLC bioautography) ............................................. 51 2.3.3. BIOLOGICAL ANALYSIS .......................................................................... 52 2.3.3.1. Antileishmanial activity on amastigotes of wild mushrooms species .... 52 2.3.3.2. Antileishmanial activity on promastigotes of isolated compounds ........ 53 2.3.3.3. Cytotoxicity assay ................................................................................. 53 2.3.3.4. Larval exsheathment assay .................................................................... 54 2.3.3.5. Detailed biological activity assays of hispidin ....................................... 54 2.3.3.5.1. Cell culture and cytogenetic procedures .............................................. 54 2.3.3.5.2. Cytokinesis-block micronucleus assay ................................................ 55 2.3.3.5.3. Chromosome aberration and sister chromatid exchange assay ............ 55 2.3.3.5.4. Comet assay ........................................................................................ 55 2.3.3.5.5. Salmonella/microsome test ................................................................. 56 2.3.4. CHROMATOGRAPHIC ANALYSIS .......................................................... 57 2.3.4.1. Thin layer chromatography on selected mushroom extracts .................. 57 2.3.4.2. Bioautography using DPPH• as the detection reagent ............................ 57 2.3.4.3. HPLC-preparative chromatography ....................................................... 58 2.3.4.4. UHPLC- analytical chromatography ..................................................... 58 2.3.5. STRUCTURE ELUCIDATION OF ISOLATED COMPOUNDS ................ 58 2.4. STATISTICAL ANALYSIS ........................................................................... 58 3. RESULTS AND DISCUSSION ....................................................................... 60 3.1. YIELDS AND