Research Collection Doctoral Thesis Biological screening of cyanobacteria and phytochemical investigation of Scytonema spirulinoides and Cylindrospermum sp. Author(s): Mian, Paolo Publication Date: 2002 Permanent Link: https://doi.org/10.3929/ethz-a-004455867 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss.ETHNo. 14851 Biological Screening of Cyanobacteria and Phytochemical Investigation of Scytonema spirulinoîdes and Cylindrospermum sp. A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH for the degree of Doctor of Natural Sciences Presented by PAOLO MIAN Pharmacist Born March 23, 1969 Trieste, Italy Accepted on recommendation of Prof. Dr. Otto Sticher, examiner Prof. Dr. P. August Schubiger, co-examiner Dr. Jörg Heilmann, co-examiner Dr. Hans-Rudolf Biirgi, co-examiner Zürich 2002 Acknowledgements The present study was carried out at the Division of Pharmacognosy and Phy- tochemistry, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technol¬ ogy (ETH), Zurich, Switzerland. I wish to express my gratitude to my supervisor Prof. Dr. Otto Sticher for giving me the opportunity to join his group and for providing excellent working facilities. Great thanks are due to Dr. Hans-Rudolf Burgi for fruitful discussions, his support, and being a co-examiner. I am most grateful to my co-examiner Dr. Jörg Heilmann for his assistance, encour¬ agement, and support. I am grateful to Prof. Dr. August Schubiger for accepting at short notice to be my co-examiner. Special thanks are due to Dr. Jimmy Orjala for introducing me to this project, and to Dr. Birgit Jaki for her support at the beginning of this work. I am especially indebted to Frank Sunder for his help in the cultivation of the algal material. I also wish to express my gratitude to Dr. Oliver Zerbe for very helpful discussions and for performing NMR experiments, to Michael Wasescha for his technical support and for performing the cytotoxicity assays, and to Dr. Walter Amrein, Oswald Greter and Rolf Häfliger for recording mass spectra. I am also grateful to Ivo Fähnle for his support in informatics, and to the "Schalter- team" for the efficient and patient service. Special thanks go to Annemarie Suter for her assistance in literature search. I wish to thank all my colleagues at the Institute of Pharmaceutical Sciences for the pleasant working atmosphere and the fruitful discussions. Special thanks are due to my laboratory colleagues, Dr. Tulla Quadri-Spinelli, Dr. Karin Winkelmann, and Marco Leonti. Further thanks go to Diana Schäffer, Thomas Kuoni, Bettina Wyss, Stefan Raduner, and Karin Walser for their help during their diploma and semester works. Last but not least I express my gratitude to my wife Ruth and to my family for their encouragement, support and patience during this work. Table of Contents Abbreviations IX Summary XI Zusammenfassung XII 1. INTRODUCTION 1 1.1 Cyanobacteria 1 1.2 References 2 1.3 Aim of the Present Investigation 3 2. CYANOBACTERIAL BIOLOGY 4 2.1 Phylogeny 4 2.2 Taxonomy 5 2.2.1 Botanical Approach 6 2.2.2 Bacteriological Approach 8 2.2.3 Recent Advances in Cyanobacterial Taxonomy 8 2.3 Anatomy and Morphology 10 2.3.1 Cellular Organization 10 2.3.2 Cellular Structure 11 2.3.2.1 Nuclear Apparatus 12 2.3.2.2 Ribosomes 13 2.3.2.3 Photosynthetic Membrane System 13 2.3.2.4 Cytoplasmic Inclusions 14 2.3.2.5 Aerotopes 14 2.3.2.6 Plasma Membrane 15 2.3.2.7 Cell Wall 15 2.3.2.8 Envelope 15 2.3.3 Heterocytes 16 2.3.4 Akinetes 17 2.4 Physiology 18 2.4.1 Photosynthesis 18 2.4.2 Heterotrophy 19 2.4.3 Nitrogen Metabolism 20 2.4.4 Reproduction 21 2.4.5 Motility 22 2.4.6 Symbiosis 22 2.4.6.1 Loose Associations with Plants and Bacteria 23 2.4.6.2 Associations with Angiosperm and Gymnosperms 23 2.4.6.3 Associations with Bryophytes 24 2.4.6.4 Association with Azolla 24 2.4.6.5 Association with Fungi 24 2.4.6.6 Association with Invertebrates 25 2.4.6.7 Cyanelles and the Origin of Chloroplasts 25 2.5 Ecology 26 2.5.1 Habitat 26 2.5.1.1 Freshwater Planktonic Forms 2 6 2.5.1.2 Attached Forms in Lakes 27 2.5.1.3 Cyanobacteria in Streams and Rivers 27 2.5.1.4 Cyanobacteria in Thermal Waters 27 2.5.1.5 Marine Planktonic Forms 28 2.5.1.6 Marine Littoral Forms 28 2.5.1.7 Terrestrial Cyanobacteria 28 2.5.2 Cyanobacterial Toxins and Public Health 29 2.6 Applications of Cyanobacteria 31 2.6.1 Biofertilizers 32 2.6.2 Food 32 2.6.3 Pollution Control 33 2.6.4 Commercial and Laboratory Chemicals 33 2.6.4.1 Phycobiliproteins 33 2.6.4.2 Isotopically-enriched Metabolites 34 2.6.4.3 Restriction Enzymes 34 2.6.4.4 Bioactive Compounds 34 2.6.5 Energy 34 2.7 References 35 ISOLATION AND CULTIVATION 41 3.1 Isolation and Purification 41 3.1.1 Isolation by Liquid Enrichment 41 3.1.2 Direct Isolation 42 3.1.3 Purification Methods 42 3.2 Cultivation 43 3.2.1 Culture Media 43 3.2.2 Temperature 44 3.2.3 Light Regimes 44 3.2.4 Solid Media 44 3.2.5 Vessels 45 3.3 References 45 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 46 4.1 Cyanobacterial Toxins 46 4.1.1 Cytotoxins 47 4.1.2 Biotoxins 47 4.1.2.1 Hepatotoxins 47 4.1.2.2 Neurotoxins 49 4.1.2.3 Dermatotoxins and Gastrointestinal Toxins 51 4.2 Cyanobacteria as a Source of Medicinal Agents 52 IV 4.2.1 Antimicrobial Compounds 53 4.2.2 Antiviral Compounds 57 4.2.3 Compounds with Multidrug Resistance Reversing Activity 60 4.2.4 Cytotoxic Compounds 62 4.2.5 Enzyme Inhibitory Compounds 67 4.2.6 Cardioactive Compounds 74 4.2.7 Anti-inflammatory Compounds 77 4.2.8 Immunosuppressive Compounds 78 4.3 References 79 5. THE GENERA SCYTONEMA AND CYLINDROSPERMUM 89 5.1 The Genus Scytonema 89 5.2 The Genus Cylindrospermum 90 5.3 References 91 6. ASSAYS FOR BIOACTIVITY 92 6.1 Antibacterial Activity 92 6.1.1 Agar Overlay Method 93 6.1.2 Minimal Inhibitory Concentration (MIC) 93 6.2 Antifungal Activity 94 6.3 Brine Shrimp (Artemia salina) Lethality Assay 94 6.4 KB-cell Activity 94 6.5 References 95 7. METHODOLOGY OF ISOLATION PROCEDURE 97 7.1 General Isolation Strategy 97 7.1.1 Isolation of Intracellular Compounds 97 7.1.2 Isolation of Extracellular Compounds 98 7.1.2.1 Solid Phase Extraction 98 V 7.2 Chromatographie Methods 99 7.2.1 Thin Layer Chromatography 99 7.2.2 Vacuum Liquid Chromatography 99 7.2.3 Open Column Chromatography 100 7.2.4 High Performance Liquid Chromatography 100 7.3 References 101 8. METHODOLOGY OF STRUCTURE ELUCIDATION 102 8.1 Nuclear Magnetic Resonance Spectroscopy 102 8.2 Ultraviolet Spectroscopy 103 8.3 Mass Spectrometry 104 8.3.1 Electron Impact (EI) 104 8.3.2 Matrix Assisted Laser Desorption/Ionization (MALDI) 104 8.4 Optical Rotation 105 8.5 References 105 9. COLLECTION AND CULTIVATION 106 9.1 Collection 106 9.2 Cultivation 106 9.2.1 Stock Cultures 106 9.2.2 Large-Scale Cultures 107 10. BIOLOGICAL SCREENING 111 10.1 Bioassays 111 10.1.1 Antimicrobial Assay 111 10.1.2 Brine Shrimp (Artemia salina Leach) Lethality Assay 112 10.1.3 Cytotoxicity Assay 112 10.2 Results 112 10.2.1 Antimicrobial Activity 112 VI 10.2.2 Brine Shrimp Lethality 113 10.2.3 Cytotoxicity 113 10.2.4 Conclusions 118 10.3 References 118 11. PHYTOCHEMICAL INVESTIGATION OF SCYTONEMA SPIRULINOIDES (EAWAG 161a) 120 11.1 Fractionation and Isolation 120 11.2 Structure Elucidation of the Isolates 122 11.2.1 Physical Data of the Isolates 129 11.2.2 General Experimental Procedures 130 11.3 Biological Activity 130 11.4 Reference 131 12. PHYTOCHEMICAL INVESTIGATION OF CYLINDRO¬ SPERMUM SP. (EAWAG 76) 132 12.1 Fractionation and Isolation 132 12.2 Structure Elucidation of Caloxanthin 134 12.2.1 Physical Data of Caloxanthin 135 12.3 Structure Elucidation of the Heterocyte Glycolipids 139 12.4 References 141 13. DISCUSSION 142 13.1 Selection of Cyanobacterial Strains 142 13.2 Cultivation 142 13.3 Bioassays 143 13.4 Extraction and Isolation 143 13.5 Structure Elucidation 144 13.6 Isolated Compounds 144 VII 13.7 Conclusions 145 13.8 References 146 Publications and poster presentations Curriculum vitae VIII IX Abbreviations acetone-d6 deuterated acetone ACN acetonitrile Md specific optical rotation ATCC American type cultures collection 13C NMR carbon-13 NMR spectroscopy CC column chromatography CDCI3 deuterated chloroform CD3OD deuterated methanol CD3COCD3 deuterated acetone 5 chemical shift d doublet ID one-dimensional 2D two-dimensional DCM dichloromethane dd double doublet DEPT distorsionless enhancement by polarization transfer DMSO dimethylsulphoxide DMSO-d6 deuterated dimethylsulphoxide DQF-COSY double quantum filtered correlation spectroscopy ED50 effective dose 50 ED100 effective dose 100 EIMS electron impact mass spectrometry EtOAc ethyl acetate ^NMR proton NMR spectroscopy HMBC heteronuclear multiple bond correlation H20 water HPLC high pressure liquid chromatography HREIMS high resolution electron impact mass spectrometry HSQC heteronuclear single quantum correlation Hz hertz IC50 50% inhibition concentration X J coupling constant KB cells epidermoid carcinoma cells LD50 lethal dose 50 m multiplet MALDI matrix assisted laser desorption/ionization MeOH methanol MIC minimal inhibitory concentration microgram microliter urn micrometer MHz megahertz MS mass spectrometry MTT methylthiazolyltetrazolium chloride m/z mass-to-charge ratio NMR nuclear magnetic resonance NOE nuclear Overhauser effect NOESY nuclear Overhauser enhancement spectroscopy ppm parts per million ROESY rotating-frame Overhauser spectroscopy RP reversed phase s singlet t triplet TLC thin layer chromatography UV ultraviolet VIS visible VLC vacuum liquid chromatography XI Summary The extracts of twenty-two cyanobacterial species were investigated during a bio¬ logical screening for their antibacterial, antifungal, and cytotoxic activities.