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
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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. The cyano¬ bacterial strains were selected from the Culture Collection of Algae of the Swiss Fed¬
eral Institute for Water Resources and Water Pollution Control (EAWAG), Dübendorf,
Switzerland. The examined cyanobacteria are terrestrial and freshwater strains and
originate from different habitats, mainly from Switzerland. 19 of the 22 investigated
cyanobacteria induced a response in at least one of the test systems applied.
Based on the results of the biological screening, two cyanobacterial strains, Scy-
tonema spirulinoides (designated EAWAG strain number 161a) and Cylindrospermum
sp. (EAWAG 76), were chosen for large-scale cultivation and subsequent extraction
and fractionation by means of various chromatographic methods (VLC, CC, HPLC).
The investigation of the methanol extract resulting from the Amberlite elution of
the culture medium of Scytonema spirulinoides led to the isolation of four new extra¬
cellular naphthalenone derivatives. One of them showed a moderate antibacterial activ¬
ity against the Gram-positive bacterium Bacillus cereus.
The fractionation of the dichloromethane/methanol 2:1 extract from the lyophilized
cell material of Cylindrospermum sp. led to the isolation of the carotenoid caloxanthin and of three heterocyte glycolipids.
The structures of the isolates were established by spectroscopic and spectrometric
methods (NMR, UV, MS), including different ID and 2D NMR experiments.
The presented results prove that terrestrial and freshwater cyanobacteria are a
promising source of natural products with interesting chemical and biological proper¬
ties. XII
Zusammenfassung
Die Extrakte von zweiundzwanzig Cyanobakterienstämmen wurden im Rahmen eines biologischen Screenings auf ihre antibakterielle, antifungale und zytotoxische
Wirkung untersucht. Die Cyanobakterienstämme wurden aus der Algenkultursam- mlung der Eidgenössischen Anstalt für Wasserversorgung, Abwasserreinigung und Gewässerschutz (EAWAG), Dübendorf, Schweiz, ausgewählt. Die untersuchten
Cyanobakterien sind Frischwasser- und terrestrische Stämme und stammen aus veschiedenen Habitats, hauptsächlich aus der Schweiz. 19 der 22 untersuchten
Cyanobakterien waren aktiv in mindestens einem der angewandten Testsysteme. Aufgrund der Ergebnisse des biologischen Screenings wurden zwei Cyanobakte¬ rienstämme, Scytonema spirulinoides (EAWAG Stammnummer 161a) und Cylin¬ und drospermum sp. (EAWAG 76), für eine Kultivierung in grossem Massstab anschliessender Extraktion und Fraktionierung mittels verschiedener chromatographi¬ scher Methoden ausgewählt.
Die Untersuchung des Methanolextraktes aus der Amberlite-Elution des Kulturme¬ diums von Scytonema spirulinoides führte zur Isolierung von vier neuen extrazel¬ lulären Naphthalenonderivaten. Eine der vier isolierten Substanzen zeigte eine moder¬ ate Aktivität gegen das Gram-positive Bakterium Bacillus cereus.
Die Fraktionierung des Dichlormethan/Methanolextraktes aus dem lyophilisierten
Zellmaterial von Cylindrospermum sp. führte zur Isolierung des Carotenoids Caloxan¬ thin und von drei Heterozyt-Glykolipiden. Die Strukturaufklärung der isolierten Substanzen erfolgte mittels spektroskopis¬ cher und spektrometrischer Methoden (NMR, UV, MS), wobei insbesondere ver¬ schiedene ein- und zweidimensionale NMR-Experimente angewendet wurden. Die hier beschriebenen Ergebnisse beweisen, dass Frischwasser- und terrestrische
Cyanobakterien eine vielversprechende Quelle von Naturstoffen mit interessanten che¬ mischen und biologischen Eigenschaften sind. 1 INTRODUCTION 1
1 Introduction
1.1 Cyanobacteria
Cyanobacteria, also known as blue-green algae, Cyanophyta or cyanophyceae, are autotrophic prokaryotic micro-organisms. In evolutionary terms they represent a link between bacteria and higher plants. They have a prokaryotic cell organization charac¬ terized by the lack of membrane-bound organelles like a true nucleus, a chloroplast or a mitochondrion, resembling that found in bacteria: the genetic material, the photosyn¬ thetic apparatus, and the respiratory system are not segregated by means of internal membranes from the rest of the cell. However, they possess a photosynthetic apparatus similar in structure and function to that located in the chloroplast of eukaryotic algae and green plants.
The origins of cyanobacteria date back almost to the beginning of life on earth.
Their fossils are found in sediments from the Early Precambrian period, over three bil¬ lion years ago. At that time they were probably the primary producers of organic matter and the first organisms to release molecular oxygen into the former atmosphere, which was until then free from 02. Many strains of cyanobacteria have been shown to be capable of shifting between anoxygenic bacterial-type photosynthesis and oxygenic plant-type photosynthesis (Padan, 1979). Thus the cyanobacteria were most probably responsible for a major evolutionary transformation leading to the development of aer¬
obic metabolism and to the subsequent rise of higher plant and animal forms (Fay,
1983, p. 4). After flourishing in the oceans, they were probably the first organisms to
colonize the land 2.7 billion years ago (Watanabe et al., 2000).
Cyanobacteria are common in all kinds of natural habitats, both aquatic and terres¬
trial. In the aquatic environment they can be found in tropical, temperate and frigid
lakes, ponds and oceans, others are characteristic of rivers and hot springs. Among the
terrestrial forms, these can be found as hygropetric forms on rocks and in all types of
soils, both fertile and desert; they are known to be among the earliest colonizers of arid land, for example after volcanic eruptions.
The N2-fixing forms can convert atmospheric nitrogen into forms that plants and 2 1.2 REFERENCES
land animals can use in their own life processes. In this way, they fertilize agricultural throughout the world, most notably rice paddies, where they are often added to the soil (Carmichael, 1994).
Cyanobacteria are used also as source of pharmaceutical products and nutrients: Spirulina is particularly suitable because of the high protein content, the good digest¬
ibility, and the high vitamin content (Patterson, 1996). in Mass occurrences of cyanobacteria (water blooms) are frequently toxic fresh,
brackish and marine waters throughout the world. Such blooms have caused numerous
animal poisonings and constitute a health hazard to humans (Carmichael, 1994). In the metab¬ course of determining the toxic constituents in cyanobacteria, many secondary
olites with interesting chemical and biological features were discovered. These metab¬
olites are important pharmaceutical lead compounds and are considered as potential
medicines for the treatment of diseases.
1.2 References
Carmichael WW (1994): The toxins of cyanobacteria. Sei. Amer. 270: 64-72.
Fay P (1983): The blue-greens (Cyanophyta-cyanobacteria). Edward Arnold Ltd,
London.
Padan E (1979): Facultative anoxygenic photosynthesis in cyanobacteria. Ann.
Rev. Plant Physio. 30: 27-40.
Patterson GML (1996): Biotechnological applications of cyanobacteria. J. Sei. Ind.
Res. 55: 669-684.
Watanabe Y, Martini JEJ, Ohmoto H (2000): Geochemical evidence for terrestrial
ecosystems 2.6 billion years ago. Nature 408: 574-577. 1 INTRODUCTION 3
1.3 Aim of the Present Investigation
This study is part of a project between the Swiss Federal Institute of Technology
(ETH), Zurich (Switzerland) and the Swiss Federal Institute for Water Resources and
Water Pollution Control (EAWAG), Dübendorf (Switzerland), concerning the biolog¬ ical and phytochemical investigation of cyanobacteria.
Since cyanobacteria are a rich source of metabolites with a wide range of biological
activities and chemical structures, it is of growing interest to examine these organisms
in detail.
and The aim of this study was, therefore, to investigate 22 strains of terrestrial freshwater cyanobacteria derived from different habitats and countries, mainly Swit¬
zerland, in order to select the most active cyanobacterial strains for further investiga¬
tions. For this purpose, assays for antibacterial, antifungal and cytotoxic activities were performed.
Based on the results of these initial experiments, two strains, Cylindrospermum sp.
(EAWAG 76) and Scytonema spirulinoides (EAWAG 161a), were chosen for large metab¬ scale cultivation. The scope ofthis part of the work was to isolate the secondary olites from the cyanobacterial cell material and the culture media and to determine their
chemical structures and biological activities. 4 2.1 PHYLOGENY 2 Cyanobacterial Biology
2.1 Phylogeny
Microorganisms were divided into three groups by Woese and Fox (1977): archae- bacteria, eubacteria, and eukaryotes. This classification is the result of a phylogenetic analysis based upon ribosomal RNA sequence characterization.
According to the general scheme of prokaryotic phylogeny (fig. 2.1), the common ancestor of all extant organisms has given rise to three principal lines of development. The first lineage leads through the ancestral archaebacteria to methano-, hallophilic and thermoacidophilic bacteria. The second major evolutionary line is represented by the true bacteria (eubacteria). The third line of evolution proceeded through the hypo¬ thetical ancestral "urkaryote" group to eukaryotic organisms. The true eukaryotic cell is seen as a phylogenic chimera, which incorporates eubacterial components (mito¬ chondrion, chloroplasts) of endosymbiotic origin in the urkaryotic cytoplasm (Fay,
1983, pp. 74-76).
These three groups can be further categorized according to their principal mode of obtaining organic carbon and energy: Fenical and Jensen (1993) divided the eubacteria into autotrophs and heterotrophs. The first are primary producers, generating organic molecules from carbon dioxide. The energy required for carbon dioxide incorporation and for other cellular processes comes from either sunlight (photoautotrophy) or the oxidation of reduced inorganic substances (chemoautotrophy). Cyanobacteria
(together with the Prochlorales) belong to the photoautotrophic eubacteria, since they generate oxygen during the photosynthesis and, uniquely among prokaryotes, possess chlorophyll a. 2 CYANOBACTERIAL BIOLOGY 5
Animals Fungi
Slime moulds Protozoa Green plants
— Gram+ bacteria Thermo- acidophiles Halobacteria Spirochaetes
Green photosyn. bacteria
Archaebacteria
Figure 2.1 Evolutionary pathways among the prokaryotes and the origin of eukaryotic or¬ ganisms according to Fay (1983, p. 76)
2.2 Taxonomy
The cyanobacteria were traditionally treated as algae, defined by broad morpholog¬ ical, cytological, and chemical features and separated into distinct divisions, classes, orders, and lower categories on the basis of morphological distinctions. There was ample reason to proceed in this manner until the middle of the last century, since cyto¬ logical data were limited to impressions from light microscopy and only relative crude 6 2.2 TAXONOMY chemical data were available. Furthermore, the morphological characteristics of the blue-green algae and their ecological niches were so similar to those of many microal- gae that few investigators doubted their place as the base or a branch of a true algal phylogeny. When it had been realized that blue-green algae lacked the traditional nucleus and
chloroplasts of the green and other algae, and that their manner of cell division resem¬ bles bacteria, they were consequently recognized as oxygenic, photosynthetic, and Gram-negative eubacteria (Castenholz and Waterbury, 1989).
The capacity of the cyanobacteria to perform oxygenic photosynthesis, and their
morphology make the taxonomic treatment by the rules of the International Code of Botanical Nomenclature relevant. On the other side, their bacterial features make also
a classification based upon the principles of the International Code ofNomenclature of
Bacteria suitable.
2.2.1 Botanical Approach
Under the International Code of Botanical Nomenclature, the starting point for the
valid publication of names of nonheterocytous, filamentous blue-green algae is with Gomont (1892), and that for heterocytous forms is with Bornet and Flahault (1886-
1888). In other cases, i.e. the morphologically less complex forms, the starting point is
Linnaeus (1753), as for almost algal groups. A number of "floras" summarizing the
known species of blue-green algae in particular regions were published during the 20th
century. Several of these provide a lot of information about species occurring else¬
where in the world, and show that many species have a very wide distribution. The best
known flora is that of Geitler (1932), who produced a treatise, which recognized
approximately 1300 species, 145 genera, 20 families, and 3 orders. This treatise was
focussed on central Europe, but is in practice of worldwide use. A similarly compre¬
hensive treatise was published by Elenkin (1936, 1938, 1949) in Russian, but because
of the language, it was less used. Basing on these accounts, many other researchers
reorganized the freshwater blue-greens at the familial and generic level. All followed a
classification similar to that of Geitler: this has become known as the "Geitlerian" sys¬
tem (Whitton, 1992; Whitton and Potts, 2000).
A large number of species and genera had been described over the years on the 2 CYANOBACTERIAL BIOLOGY 7 basis of a single difference such as the presence or nature of sheath, and other features, which were later shown to have a great flexibility within clonal populations of some species, particularly when environmental conditions vary. This morphological vari¬ ability has prompted Drouet (1968) to revise the taxonomy profoundly. His basic idea was that there exist ecophenes, organisms sharing the same genotype but expressing distinct morphologies under the influence of environmental factors. He drastically reduced the number of species from over 2000 to 62 and the genera from over 140 to 24 by selecting certain morphological features, which he believed to be invariant with the environment (Wilmotte, 1994). However, the system of Drouet was not accepted by most scientists. Later, studies on DNA-DNA hybridization and DNA base composition showed that taxa placed in the same species by Drouet were genotypically different (Stam, 1980).
Recently, a number of papers by Anagnostidis and Komarek (1985, 1988, 1990) and Komarek and Anagnostidis (1986, 1989) made substantial improvements to the
"botanical approach". This revision is based on smaller, more coherent genera. The
authors made an extensive review of the literature and tried to integrate all the bio¬
chemical, ultrastructural, and molecular characters available. These papers provide the most detailed guides available to the morphological and ecological literature, consider¬
able new insight into morphology, and accounts not only of well-known forms, but also
ones which are little known. Perhaps half the accounts of plankton in European lakes made during the last decade have adopted the nomenclature recommended by Anag¬
nostidis and Komarek. However, its use is hindered by the lack of detailed explanation
of the principles underlying the changes, and the problem of dealing with species for
which the authors have not provided guidelines. The account of the Chroococcales
within a single volume (Komarek and Anagnostidis, 1999) overcomes the above prob¬
lems for this group. The organisms are arranged according to the various possible com¬
binations of pattern of cell division, cell shape and organization of cells within bound¬
ing sheaths and/or mucilage; generic status is assigned to most of the combinations, so
the number of species within genera tends to be low. This account is so detailed that it
is likely to remain the definitive text on botanical species in this group for a long time
(Whitton and Potts, 2000). Accounts for other orders are in preparation. 8 2.2 TAXONOMY
2.2.2 Bacteriological Approach
The bacterial approach was introduced by R.Y. Stanier in the early 1970s. This sys¬ tem aimed to treat the cyanobacteria in the same way as other prokaryotes, placing them under the International Code ofNomenclature of Bacteria, and is based on the use of axenic, clonal cultures only, and on some of their morphological, cytological, genet- ical, chemical and physiological characteristics.
The morphological and cytological characteristics include for example: the type of cellular division and the plane of subsequential divisions, the formation and structure of hormogonia, the presence and type of sheath, the shape and dimensions of cells and the presence of constrictions between adjacent cells of trichomes, the presence and
location of heterocytes, akinetes, and gas vacuoles. The chemical, genetical, and physiological characteristics include: motility and phototaxis, base composition of the DNA, chemoheterotrophic and photoheterotrophic
capability and substrate specificity, aerobic and anaerobic N2-fixation ability, types of phycobilin pigments, temperature and salt tolerance and requirement (Castenholz and Waterbury, 1989).
The basis of the bacteriological taxonomy of the cyanobacteria was published by
Rippka et al. (1979). This taxonomic system allows the identification of the strains of
the Pasteur Culture Collection of the Institut Pasteur in Paris at the generic level and
includes five major sections. A modified version of this system is given in the Bergey's
Manual of Systematic Bacteriology, in the sections written by Herdman et al. (2001),
Rippka et al. (2001), Castenholz et al. (2001), Hoffmann and Castenholz (2001). This
treatment is more global and includes taxonomic information on taxa which are not in
the Pasteur Culture Collection. A disadvantage of this system is that it gives a detailed
account of the strains in axenic, clonal cultures, but does not include genera not yet in
culture.
2.2.3 Recent Advances in Cyanobacterial Taxonomy
It is now recognized that modern approaches must be brought to solve the problems of cyanobacterial systematics, including ultrastructural studies and molecular biologi¬
cal methods. A number of new valuable techniques of taxonomic evaluation have been 2 CYANOBACTERIAL BIOLOGY 9 developed in recent years and give information on fine structure, DNA-DNA hybrid¬ ization, guanine-cytosine ratio, photopigment composition, presence of specialized secondary chemical components, etc.
The two molecular methods most frequently used today in bacterial systematics are the determination of sequences of small ribosomal subunit ribonucleic acids (SSU rRNAs) and DNA-DNA hybridization.
The use of SSU rRNAs to determine the evolutionary relationships among bacteria
of SSU rRNA was pioneered by CR. Woese and coworkers (1990). Through analysis data, prokaryotic organisms are seen to fall into two distinct "Domains", the Archaea and the Bacteria (Woese et al., 1990). Within the latter group, the oxygenic phototro- phs and derived organelles (cyanobacteria, prochlorophyta, and photosynthetic plas- tids) form a distinct assemblage, consistent with their unique phenotype of oxygenic photoautotrophy (Woese, 1987). SSU rRNA sequence analysis has been used to further
explore the evolutionary relationships within the cyanobacterial group (Reeves, 1996;
Turner, 1997).
Bacteria Archea Eucarya
nonsulfur Green Animals Ciliates bacteria Methano- c _ Green plants Extreme , . , microbiales Gram+ I , , ,., Fungi . halophiles Purple . / \ Methanobacteriales hacteria enal Flagellates Micro- sporidia Flavo bacteria
Thermotogales 1: Thermoproteus 2: Thermococcales 3: Methanococcales
Figure 2.2 Universal phylogenetic tree according to Woese et al. (1990)
Although SSU rRNA data are useful for determining differences among organisms
at the genus level, they generally are not reliable for lower levels of taxonomic assign¬
ment. DNA-DNA hybridization studies have been used with success to determine evo¬
lutionary relationships among strains of cyanobacteria both between and within genera 10 2.3 ANATOMY AND MORPHOLOGY
(Turner, 1997).
Conclusions on molecular data are still limited by the relatively small number of data available and there is a great need for molecular information to be integrated with other characteristics, leading to a taxonomy that will not only be of practical use but will also reflect as much as possible the evolutionary relationships of the organisms (Preisig, 2000).
2.3 Anatomy and Morphology
2.3.1 Cellular Organization
Cyanobacteria can be separated into unicellular, colonial, and filamentous forms.
The cells of unicellular algae are spherical (for example Chroococcus), ovoid or cylin¬ drical (e.g. Synechococcus). They occur singly if the daughter cells separate after cell
division, as in the case of Synechococcus, or they may aggregate into irregular loose
colonies, like those of Microcystis, which are held together by a slimy matrix secreted by the cells of the growing colony. Some genera, like Merismopedia or Gloeocapsa, produce more ordered colonies by means of a more or less regular sequence of cell divisions combined with sheath secretion. In Chamaesiphon the cells divide linearly to
give a pseudo-filamentous form.
Many blue-green species display a filamentous morphology formed by the daugh¬
ter cells remaining in close contact following repeated cell divisions which occur in a
single plane at right angles to the main axis of the filament. This process gives rise to a
simple multicellular structure which consist of a chain of cells and is called trichome.
The trichome may be straight, like that of Oscillatoria, or is coiled in a regular spi¬
ral as in Spirulina. Such variations in trichome morphology may occasionally appear
within one and the same species. In certain genera the cells are closely appressed, the
cross walls separating the cells are thin and scarcely visible under the light microscope,
and the trichome appears like a rod. In other groups the neighbouring cells are sepa¬
rated distinctly, conferring a moniliform appearance to the trichome. Frequently the tri¬
chome is covered by a mucilaginous sheath: trichome fragmentation within the sheath,
and subsequent protrusion of one or both trichome fragments through the sheath, gives 2 CYANOBACTERIAL BIOLOGY 11 the impression of a branched morphology (false branching). A true branch is formed by lateral or oblique division of a cell in a trichome. False branching is characteristic of, for example, Scytonema or Tolypothrix, true branching of the Stigonematales, such as
Westiella ox Fischerella (Fay, 1983, pp. 4-7; Fogg et al., 1973, pp. 17-18, 27-28).
2.3.2 Cellular Structure
The cyanobacterial cell measures from 1 urn to 100 urn in diameter (Castenholz and Waterbury, 1989), but is usually between 2 and 5 um. It has a typical prokaryotic organization (fig. 2.3), lacking the complex eukaryotic architecture of plant and animal cells. Nevertheless it is possible to make a rough distinction between two main regions of the cytoplasm: the peripheral and the central regions. The central (nucleoplasmic) region, often called "centroplasm", is lightly granulated due to the presence of ribo- somes, and contains the DNA. The peripheral (chromatoplasmic) region is traversed by thin sheets of the photosynthetic membrane system, incorporating the photosynthetic pigments. The cell is enclosed by a membrane, the plasmalemma, which is fortified by a multilayered cell wall. External to the wall, the cell may be surrounded by a gelati¬ nous sheath or a more firm envelope (Fay, 1983, p. 9). 12 2.3 ANATOMYAND MORPHOLOGY
Pore
Cytoplasmatic membrane
Cell wall layers
Sheath
Gas vacuole
Nucleoplasm
Pore
Polyphosphate granule
Thylakoid
Vacuole
Ribosomes
Cyanophycin granules
Cross wall
Figure 2.3 Structure of the cyanobacterial cell according to Wartenberg (1979)
2.3.2.1 Nuclear Apparatus
The DNA fibrils of cyanobacteria occur in a complex, folded arrangement, but each is circular when unfolded. The total molecular weight ranges between 1.6 and 8.6 x 109 daltons. Unicellular forms usually have genome sizes below 3.6 x 109 daltons, similar 2 CYANOBACTERIAL BIOLOGY 13 to those known for other prokaryotes. Cells of the filamentous cyanobacteria, however, contain larger genomes (Castenholz, 2001).
2.3.2.2 Ribosomes
Ribosomes are particularly concentrated in the nucleoplasmic region, but are dif¬ fusely distributed throughout the cytoplasm. Their size (10 to 15 nm) and their sedi¬ mentation properties (70 S) are typically prokaryotic (Fay, 1983, p. 9). Cleavage of the ribosomes to form 50 S and 30 S sub-units also follows the bacterial pattern (Fogg et al., 1973, p. 61).
2.3.2.3 Photosynthetic Membrane System
The most obvious internal feature distinguishing cyanobacteria from other eubac¬ teria is the type of pigment-bearing apparatus. In almost all cases, it consists of a series of thylakoid membranes, which appear mainly independent of the cytoplasmic mem¬ brane. In unicellular forms the membrane layers extend in concentric sheets beneath the cell membrane and surround the central nucleoplasmic region. The more elaborate membrane structure in the cells of filamentous species is formed by means of expan¬ sion and invagination of the membranes (Fay, 1983, p. 10). The thylakoids consist of two membranes of about 7 nm thickness, separated by an intrathylakoidal space of about 3-5 nm. However, they may be more closely appressed or separated by a greater gap. They are similar to the thylakoid membranes of chloroplasts, particularly those of the red algae (Castenholz and Waterbury, 1989). The lipid bilayer of the thylakoid membrane incorporate the lipophilic photosyn¬ thetic pigments, chlorophyll a and the various carotenoids. It also incorporates the components of the electron transport chain, like cytochromes, plastocyanin and ferre- doxin. Chlorophyll a, the only chlorophyll species in the cyanobacteria, is present in three different forms, which can be distinguished on the basis of their absorption char¬ acteristics (maximum light absorption, ^^ at 670, 680, and 700 nm, respectively).
Among the carotenoids, ß-carotene is universally present in all blue-greens while the presence of different xanthophylls (such as zeaxanthin or oscillaxanthin) varies from species to species (Fay, 1983, p. 10).
An important part of the photosynthetic pigment complement of the cyanobacteria 14 2.3 ANATOMYAND MORPHOLOGY is located in so called phycobilisomes, which are complex protein-pigment aggregates arranged in orderly rows on both surfaces of the thylakoids. They contain the chro- mophore-bearing water-soluble phycobiliproteins, i.e. the blue phycocyanin (maxi¬
red mum absorption, A^^ 620 nm) and allophycocyanin (^^ 650-670 nm), and the phycoerithrins (A^^ 550-570 nm). These accessory pigments are the primary light- harvesting pigments for Photosystem II in cyanobacteria. Light energy trapped by these pigments is transferred with high efficiency to chlorophyll a. The relative quan¬ tities of the phycobiliproteins may vary according to the species and the spectral com¬ position of light, and determine the coloured appearance of the cyanobacteria (Casten¬ holz, 2001; Fay, 1983, pp. 10-11).
2.3.2.4 Cytoplasmic Inclusions
In the cytoplasm of cyanobacteria there are many other components and "inclu¬ sions". They include (Castenholz, 2001):
Glycogen granules, usually between the thylakoids. They serve as a carbon and energy source.
Cyanophycin granules, which consist of a unique polypeptide composed only of arginine and aspartic acid. They are apparently unique to cyanobacteria, although some species lack them. They serve as reserves of nitrogen.
Carboxysomes, which contain a reserve form of the primary photosynthetic enzyme, ribulose-l,5-biphosphate carboxylase.
Polyphosphate bodies, called volutins, which serve as a phosphate store.
Lipid globules, which might represent lipid stores for use in membrane synthesis. Several other inclusions like microtubules, microfilaments, crystalline and trila- mellar bodies, and crystals have been observed, but their function is not known (Fay,
1983, p. 14).
2.3.2.5 Aerotopes
Aerotopes, formerly called gas vacuoles, are composed of many elongate cylindri¬
2 nm thick cal gas vesicles bearing a conical cap at each end. The vesicle is formed by a protein coat enclosing a gas-filled space (Castenholz, 2001). Gas vesicles are the buoy¬ ancy-regulating organelles and responsible for the diurnal vertical migration of certain 2 CYANOBACTERIAL BIOLOGY 15
planktonic cyanobacteria. This movement enables them to maintain their position
within a zone of the water column where their photosynthetic metabolism is optimal
and the supply of mineral nutrients adequate (Fay, 1983, p. 17).
2.3.2.6 Plasma Membrane
The physiological integrity of the cell is maintained by a thin plasma membrane or plasmalemma. It is about 7 nm thick and acts as a selective semipermeable membrane
(Fogg et al., 1973, p. 37).
2.3.2.7 Cell Wall
The cell wall of the cyanobacterial cell is of Gram-negative type. The wall is com¬ posed of two principal layers, the inner peptidoglycan and the outer lipoprotein layers. The peptidoglycan layer, also called murein, is primarily responsible for the mechani¬
cal strength of the wall; it confers the shape of the cell and protects it against osmotic
damage. Its thickness is usually 1-10 nm, but can reach 200 nm in some cases.
The lipoprotein layer probably controls the transport of solutes as in bacteria.
The space between the two layers, called periplasmic space, seams to have a similar
content of lipopolysaccharides and degradative enzymes.
Studies of thin sections of various filamentous cyanobacteria revealed the presence
of a single minute central pore or of numerous pores (microplasmodesmata) that
traverse the cross-wall between adjacent cells. These pores extend to the plasma mem¬
branes of the two cells and probably allow intercellular contact (Castenholz, 2001 ; Fay,
1983, p. 17).
2.3.2.8 Envelope
Numerous unicellular, colonial, and filamentous cyanobacteria possess an enve¬
lope outside of the outer membrane. This is called sheath, glycocalyx or capsule.
The sheaths are predominantly composed of polysaccharides, but in some strains
more than 20% of the weight consists of polypeptides (Castenholz, 2001). In the firm
sheaths of many cyanobacteria, yellow, red, or blue pigments may accumulate and
mask the colour of the cells. The typical yellow-brown pigment was characterized as 16 2.3 ANATOMY AND MORPHOLOGY scytonemin, a UV-absorbing, protective pigment (Garcia-Pichel and Castenholz, 1991 ; Castenholz and Garcia-Pichel, 2000). The function of the envelope is poorly understood, but it has been suggested that it may protect the cells from desiccation, or against invasion of pathogens or against phagocytosis from grazing animals. It may also render harmful substances ineffective and promote the attachment of the organisms to solid substrates (Fay, 1983, p. 18).
2.3.3 Heterocytes
In the process of nitrogen fixation atmospheric nitrogen, N2, is reduced to ammo¬ nia, NH3, in a reaction catalyzed by the nitrogenase, a complex enzyme system. An absolute prerequisite for this reaction is the absence of free O2, since nitrogenase is inactivated in presence of Oj (Fay, 1983, p. 19). Heterocytes are differentiated cells specialized for the fixation of N2 in an aerobic environment (Wölk et al., 1994).
Heterocytes are unique to cyanobacteria, and are produced only by members of the
Nostocales and Stigonematales; they are unknown in unicellular cyanobacteria (Cas¬ tenholz, 2001).
Main features of the heterocytes are the partial separation by a constriction from the vegetative cells, the presence of a massive cell envelope, reduced pigmentation, as compared with vegetative cells, a relatively homogeneous cell content, and the pres¬ ence of refractive structures, called polar nodules, near their attachment to the vegeta¬ tive cells (Fogg et al., 1973, p. 222).
The thick envelope consists of an inner laminated layer, a central homogeneous layer, and an outer fibrous layer. The inner layer comprises glycolipids; it is suggested that this glycolipid layer reduces the rate of diffusion of oxygen into the heterocytes to such an extant that most ofthat which does enter is reduced by oxidases present in the heterocytes. The central layer consists of polysaccharides, the outer layer probably of uncompacted fibres of the same polysaccharides. These two layers may protect the gly¬ colipid layer from degradation or dissolution (Adams, 1997).
Heterocytes retain significant quantities of chlorophyll a and carotenoids, but cyanophycin granules and phycobilisomes are usually degraded. While Photosystem I units are increased, much of the chlorophyll and phycobiliproteins associated with
Photosystem II units are lost during differentiation. They also lack the enzyme ribu- 2 CYANOBACTERIAL BIOLOGY 17 lose-l,5-biphosphate carboxylase, an essential component of Photosystem II. The con¬ sequence of these changes is a modified photosynthetic mechanism which no longer performs photosynthetic C02 fixation or 02 evolution (Fay, 1983, p. 21).
Heterocytes are produced at intervals in trichomes through the differentiation of vegetative cells, usually only after the concentration of combined inorganic nitrogen in the surrounding medium has been lowered (Castenholz, 2001). The transformation involves a whole range of structural and biochemical changes. During the initial period, proteases are activated: they degrade phycobiliproteins and numerous other proteins. Subsequently, new proteins, such as the nitrogenase enzymes, are synthe¬ sized. The thylakoids disintegrate, and a multilayered envelope is deposited on the external surface of the original cell-wall, which encloses the whole heterocyte, apart from the small areas of contact between heterocyte and vegetative cell (Fay, 1983, pp. 20-21). The intercellular connection between the vegetative cell and the heterocyte is restricted to a pore channel which may represent a pathway for the exchange of meta¬ bolic products; heterocytes are sustained by carbohydrates and organic acids from veg¬ etative cells, while combined nitrogen is supplied in organic form from heterocytes to vegetative cells (Castenholz, 2001).
2.3.4 Akinetes
Akinetes are specialized cells that occur in several groups of heterocytous cyano¬ bacteria, particularly in the Nostocaceae and Rivulariaceae. They are considered to be perennating structures, which are functionally, but not structurally, comparable to the bacterial endospore. They are resistant to adverse conditions, and remain viable for long periods (Adams and Carr, 1981). They tolerate drying, freezing, and long term storage in anaerobic sediments. Though the spatial distribution of akinetes within the trichome shows a variety of patterns, in all cases akinetes develop in a particular posi¬ tion with respect to the heterocyte. They always form adjacent to the terminal hetero¬ cyte in the genus Cylindrospermum, they develop from the vegetative cell 2-3 cells away from a heterocyte in most Anabaena species, and are approximately equidistant between two heterocytes in many Nostoe species. In some heterocytous genera aki¬ netes are never observed (Fay, 1983, p. 25).
The spacial relationship between heterocytes and akinetes suggests that heterocytes 18 2.4 PHYSIOLOGY may exert some degree of control over akinete formation, though it appears that some other external factors play apart in triggering the process of sporulation (Fay, 1983, pp.
25-26). These could be the presence or deficiency of some nutrients, the decrease of light penetration, or the release into the medium of a stimulating substance, but no clear relationship has yet been found (Humm and Wicks, 1980; Adams and Carr, 1981).
During the process of akinete differentiation the cell increases several fold in size, accumulates cyanophycin, glycogen, lipids, and carotenoid pigments, and acquire a thick extracellular capsule, which completely surrounds the akinete. Polyphosphate disappears, the RNA content increases, photosynthetic capacity decrease greatly or ceases completely (Castenholz, 2001).
The process of akinete germination begins with the gradual disappearance of reserve products, synthesis of DNA, RNA, pigments, and proteins, and the formation of new thylakoids. These are followed by one or two transverse divisions, and finally by the dissolution of the akinete envelope (Fay, 1983, p. 27). Little is known about the factors that induce akinete germination, but the process is probably light-dependent (Castenholz, 2001).
2.4 Physiology
2.4.1 Photosynthesis
Photosynthesis can be defined as the synthesis of organic compounds through the assimilation (fixation) of C02 with the use of light as energy source. During the pro¬ cess of carbon dioxide fixation, C02 is added to a 5-carbon substance, ribulose-1,5- biphosphate (RuBP) in a reaction catalyzed by the enzyme RuBP carboxylase. The product splits to yield two molecules of a 3-carbon substance, phosphoglyceric acid
(PGA). The reduction of PGA leads to the formation of a triose sugar phosphate and subsequently to a series of sugar phosphate intermediates, and finally to glucose. Dur¬ ing this process, known as Calvin cycle, the acceptor for C02, RuBP, is regenerated.
The C02 fixation is not directly dependent on light, but the energy (in form of
ATP) and reductant (in form of NADPH) required for the process are produced in the primary photochemical act which takes place in the thylakoids. In the photochemical 2 CYANOBACTERIAL BIOLOGY 19 act two distinct kinds of active centers (Photosystem I and II) are involved and act in series: Photosystem I receives the electron transferred from Photosystem II and raises it to a high enough energy level to reduce the electron carrier ferredoxin. Reduced ferredoxin in turn reduces NADP to NADPH, which can be used for carbon fixation.
Part of the energy released during this electron transport is incorporated in ATP in a process called photophosphorylation. The ultimate source of electrons for photosyn¬ thesis is water, which yields in the process of photolysis protons, electrons, and free 02
(Fogg et al., 1973, pp. 143-144; Fay, 1983, pp. 30-31).
This photosynthetic process is mechanistically identical with eukaryotic photosyn¬ thesis; there are, however, some aspects which are characteristic for cyanobacteria.
One concerns the spectral characteristics of light absorption, which are different from those of other photosynthetic organisms. High rates of photosynthetic activity are mea¬ sured in cyanobacteria not only in the spectral region between 665-680 nm, where light absorption by chlorophyll is greatest, but also around 620 nm or 560 nm, where phyco- cyanin and phycoerithrin, respectively, absorb light effectively. Energy is then trans¬ ferred to chlorophyll. This energy transfer proceeds from a pigment with an absorption maximum at a shorter wavelength of light to a pigment with an absorption maximum at a longer wavelength. Hence the sequence is: phycoerithrin -> phycocyanin -> allo- phycocyanin -> chlorophyll a (Fay, 1983, pp. 32-33).
Another particular aspect is that a group of cyanobacteria can use hydrogen sulfide
(H2S) or elemental hydrogen (H2) as an alternative electron donor to water. In some cyanobacteria Photosystem II is completely blocked, and they develop a sulfide-depen- dent anoxygenic photosynthesis. The Photosystem II of some other cyanobacteria shows resistance to sulfide toxicity: this allows the operation of oxygenic photosynthe¬ sis under sulfide in these organisms (Cohen et al., 1986).
2.4.2 Heterotrophy
Although cyanobacteria are basically oxygenic photosynthetic organisms, some can also utilize organic compounds, either by chemoheterotrophy or photoheterotro- phy. Photoheterotrophy is the growth in the light on an organic compound in the absence of C02 fixation, while chemoheterotrophy is the growth on an organic sub¬ strate in complete darkness. In the latter case, the organic compound provides the 20 2.4 PHYSIOLOGY organism with a source of carbon and energy, while in photoheterotrophy the organic compound is used only as a source of carbon, and light supplies the energy needs of the cell (Smith, 1982).
2.4.3 Nitrogen Metabolism
Cyanobacteria can assimilate nitrogen from nitrate, nitrite, ammonia, urea, amino acids, and N2. There is considerable variation among species as to which forms of nitrogen can be assimilated, but all cyanobacteria apparently can grow with nitrate as the sole nitrogen source. The reduction of nitrate proceeds through nitrite to ammonia.
Ferredoxin and NADH are required for nitrate reduction, while ferredoxin and
NADPH are required for nitrite reduction. As these reductants are available through the photosynthesis reactions, the reduction of nitrate appears to be light stimulated (Holm-
Hansen, 1968). Ammonia is the preferred nitrogen source, because no energy is needed, while the other nitrogen sources must be reduced using energy.
Many cyanobacteria are able to grow at the expense of atmospheric nitrogen, N2, as they can reduce elemental nitrogen to ammonia in a process called nitrogen fixation.
This process is essential for the maintenance of the nitrogen status of the whole bio¬ sphere. Nitrogenase, the enzymatic system for N2-fixation, is irreversibly inactivated by oxygen. Therefore, cyanobacteria able to perform N2-fixation in aerobiosis have evolved mechanisms for protecting their N2-fixing machinery from the effects of 02.
Because cyanobacteria perform oxygenic photosynthesis, they do not only have to pro¬ tect their N2-fixing machinery from atmospheric 02, but also from intracellularly gen¬ erated 02. Cyanobacteria probably had to develop mechanisms to protect nitrogenase from intracellular photosynthetically generated 02 before they had to handle high lev¬ els of atmospheric 02. In fact, the ability to fix N2 was probably already present in cyanobacteria living in the primitive anoxic atmosphere (Flores and Herrero, 1994).
The most effective adaptation for the protection of nitrogenase is the acquisition of the heterocyte, the highly specialized N2-fixation cell (see 2.3.3). Some non-heterocy- tous forms have succeeded in maintaining their ability to synthesize nitrogenase and to fix N2 in the absence or at low concentrations of exogenous 02, probably through a temporal or spatial segregation of photosynthetic and N2-fixing activities (Fay, 1983, pp. 41-44). 2 CYANOBACTERIAL BIOLOGY 21
The reduction of N2 to NH4+ catalyzed by the nitrogenase is a high endergonic reaction requiring metabolic energy in the form of ATP. The reduction equivalents derive from the photosystem I. The reduction of N2 by the nitrogenase always occurs concomitantly with the reduction of protons to H2, a reaction costing energy and reduc- tant that decreases the efficiency of the N2 fixation process. Cyanobacteria possess an enzyme, uptake hydrogenase, that returns electrons from at least a portion of the H2 produced by nitrogenase to the respiratory and photosynthetic electron transport chains for the production of ATP and reductant. Besides recovering ATP and reductant, the stimulated respiration promoted by the uptake hydrogenase contributes to the con¬ sumption of 02 and could represent a mechanism for protecting the nitrogenase from 02 (Flores and Herrero, 1994).
2.4.4 Reproduction
Most unicellular cyanobacteria reproduce by binary fission, which is followed by growth and expansion. An exception is seen in the genus Chamaesiphon where a new cell is produced in the process of an unequal fission or budding, which involves the for¬ mation of smaller cells (called exocytes) from larger ones. Some other genera repro¬ duce by multiple fission, a series of rapid divisions in three planes of the enlarged mother cell, not accompanied by cell growth. The small spherical endospores produced are released by rupture of the parental wall. These endospores are called baeocytes.
The major means of reproduction in filamentous cyanobacteria involves the forma¬ tion of hormogonia, short filaments of undifferentiated cells. In Cylindrospermum and
Oscillatoria, hormogoma are formed by the disintegration of whole filaments, while in branching forms such as Scytonema they form at the tips of branches. In some cases breakage of trichomes is aided by specialized cells (necridia) which become biconcave in shape due to lysis and dehydratation and permit fragmentation of the trichome and subsequent liberation of hormogonia. Hormogonia then glide or flow away, and grow into new individuals (Nichols and Adams, 1982). 22 2.4 PHYSIOLOGY
2.4.5 Motility
Many cyanobacteria show a sort of movement known as gliding, in which the cell
or filament exhibits a slow, smooth progression without signs of contraction or propul¬
sion. Gliding is seen most likely in the filamentous forms. Branched filaments do not glide, but the hormogonia, which differentiate from their ends, are motile once released. Gliding movement occurs only in cells which are in contact with a solid sur¬
face (Fogg et al., 1973, p. 111).
Motility may help the organism to adjust its position and settle in an area where conditions are favorable for growth, and is influenced by environmental factors, like temperature, light, and the chemical composition of the medium. Gliding is often accompanied by mucilage production, and early interpretations of the gliding movements were based on the assumption that the directional secretion of mucilage may result in a slow displacement of the trichome. It was shown, however, that mucilage secretion is a consequence and not the cause of the gliding movement.
More recent interpretations relate gliding with the presence of contractile organelles.
This theory is based on the discovery of many fine fibrous elements in the wall of the trichome. Synchronous waves propagated by these contractile fibrils could propel the
filament (Fay, 1983, p. 59).
2.4.6 Symbiosis
Cyanobacteria are unique in their capacity to form symbiotic associations with a remarkable range of eukaryotic hosts including plants, fungi, sponges, and protists. In most cases the profit to the host is the provision of combined nitrogen, or occasionally carbon. The benefit to the cyanobacterial symbiont (cyanobiont) is less clear. They
often receive carbon from photosynthetic hosts, but they are capable of carbon fixation themselves. The enclosed environment provided by the host may protect the cyanobac¬ teria from environmental extremes such as high light intensity, desiccation, and possi¬ bly prédation.
The infective agents in many cyanobacterial symbioses, particularly those involv¬ ing plants, are hormogonia, the specialized filaments produced by heterocytous cyano¬ bacteria in the Nostocaceae and Stigonemataceae. Hormogonia lack heterocytes but 2 CYANOBACTERIAL BIOLOGY 23 possess gliding motility, a crucial factor in their ability to establish symbioses, because the immotile parent filaments are unable to entry the plant structures that house the symbiotic colonies. Some plants enhance their chances of infection by producing chemical signals that induce hormogonia formation and serve as chemoattractants to guide the hormogonia into the plant tissue. Within the host additional plant signals repress further hormogonia formation and stimulate heterocyte development. The cyanobacteria then undergo considerable morphological and physiological changes, again in response to plant signals, usually resulting in reduced growth rate and carbon dioxide fixation, enhanced nitrogen fixation, and loss to the plant of much of the nitro¬ gen fixed (Adams, 2000).
2.4.6.1 Loose Associations with Plants and Bacteria
The presence of both free-living and symbiotic (y4zo//a-associated) cyanobacteria in rice fields improves growth and crop yield of the rice. Knowledge of the effects of cyanobacteria on crop plants other than rice is very limited, but investigations on wheat has shown that cyanobacteria associated with wheat roots can stimulate plant growth (Adams, 2000).
Many bacteria grow in the mucilaginous sheaths surrounding cyanobacteria. These bacteria are adapted to the micro-environment of the mucilage envelope, and use the extracellular organic compounds and 02 released from the cyanobacteria. Bacterial assimilation of organic substrates results in the production of C02 that can be immedi¬ ately re-assimilated from the cyanobacteria in the photosynthesis process (Fay, 1983, p. 60).
2.4.6.2 Associations with Angiosperm and Gymnosperms
Of all angiosperms only the genus Gunnera forms symbiotic associations with cyanobacteria. The cyanobiont, Nostoc sp., is found intracellularly, within specific mucus-secreting glands on the plant stem.
The cycads are the most primitive group of gymnosperms alive today. Their roots are frequently infected with nitrogen-fixing cyanobacteria, forming a dark band between the inner and the outer cortex of the root. In their limited natural habitats the cycads can make significant contributions to the local nitrogen economy (Adams, 24 2.4 PHYSIOLOGY
2000).
2.4.6.3 Associations with Bryophytes
The cyanobiont (usually Nostoc) occupies mucilage-filled intercellular cavities within the thalli. A colony of Nostoc filaments develop and this induces the host cells
surrounding the cavity to form protrusions into the Nostoc colony. These protuberances greatly increase the bryophyte-AWoc interface promoting the interchange of metabo¬
lites (Fay, 1983, p. 63).
2.4.6.4 Association with Azolla
The association between the floating water fern Azolla and its Anabaena endosym-
biont is the only known example of symbiosis between a fern and a cyanobacterium.
Anabaena inhabits cavities in the dorsal leaf lobes of the fern. Multicellular hairs pro¬
trude into the cavity space and allow the exchange of metabolites between fern and
cyanobacterium. As the leaf grows the cavities become colonized with Anabaena fila¬
ments, which begin to develop heterocytes and fix nitrogen. Both Photosystem I and II
of the Anabaena are suppressed. When isolated, however, Anabaena is capable of pho¬
tosynthesis and it performs light-dependent nitrogen-fixation but at a decreased rate.
The Azolla-Anabaena association is one of the most efficient nitrogen-fixing systems.
The exceptionally high rates of N2-fixation are almost certainly attributable to the
abundant supply of photosynthetic products from the host to the endosymbiont, which
permits N2-fixation to continue at night (Fay, 1983, pp. 64-66).
2.4.6.5 Association with Fungi
Lichens are associations between a fungus (usually an ascomycete) and an
autotroph (phycobiont) which is a green alga or a cyanobacterium. They are probably
the most common of all symbiotic associations. 8 to 15% of the 15000-20000 species
of lichens contain a cyanobacterium as phycobiont, usually Nostoc, although Calo-
thrix, Scytonema, and Fischerella are also common (Adams, 2000). The N2-fixing
activity of the cyanobacterial partner is clearly beneficial to the fungus and is counter¬
balanced by other factors like water supply, mineral nutrition, optimal light conditions, 2 CYANOBACTERIAL BIOLOGY 25 and reduced 02 tension. Bipartite lichens consist of two partners, a fungus and either a green alga or a cyanobacterium, whereas tripartite lichens contain a fungus, an alga, and a cyanobacterium.
In bipartite lichens with a cyanobacterial phycobiont only, the number of hetero¬ cytes is similar to that observed in free-living forms. In lichens incorporating both a green alga and a cyanobacterium, however, the number of heterocytes and the rates of
N2-fixation are much higher. Sugars released by the green alga may be taken up not only by the fungus but also by the cyanobacterium. This assimilation would enhance heterocyte development and nitrogenase synthesis (Fay, 1983, pp. 61-63).
2.4.6.6 Association with Invertebrates
Cyanobacteria are known to occur in several marine sponges, where they are able to fix N2 within the sponge system. This may be particularly beneficial to sponges in tropical seas, which are generally short of combined nitrogen (Fay, 1983, p. 66).
The presence of cyanobacteria in sponges makes reasonable that some sponge metabolites are produced by the symbiotic cyanobacteria. In fact, certain classes of compounds isolated from sponges are structurally identical or very similar to those known from cyanobacteria (Fusetani and Matsunaga, 1993).
2.4.6.7 Cyanelles and the Origin of Chloroplasts
Cyanelles are plastids found in the cells of photosynthetic eukaryotes. They possess many similarities with cyanobacteria, including a peptidoglycan wall, chlorophyll a, phycobiliproteins and carboxysome-like structures. However, their genome is similar in size to that of chloroplasts, but only about 5% of those of free-living cyanobacteria.
The association between host and cyanelle appears to be mutually obligate as neither of the partners can grow in isolation. Therefore, the cyanelle is considered as a photo¬ synthetic organelle analogous to the chloroplast.
The endosymbiont hypothesis of the evolution of chloroplasts assumes that chloro¬ plasts were derived from cyanobacteria which adopted an endosymbiotic existence within an ancestral eukaryotic cell. Cyanelles would then represent intermediate forms between these ancestral cyanobacteria and chloroplasts. However, in the last years this hypothesis was questioned and cyanelles are now thought to represent an evolutionary 26 2.5 ECOLOGY dead-end and not half-way stages to the development of a typical chloroplast (Whitton and Potts, 2000).
2.5 Ecology
2.5.1 Habitat
Cyanobacteria occur in a wide variety of habitats; they are ubiquitous in waters of a great range of salinity and temperature, they occur in and on the soil and on rocks. A number of cyanobacteria have even been recovered from the atmosphere. In freshwater and terrestrial environment they are absent below pH 4.0, and overall their abundance tends to increase with increasing pH value. Some species tolerate very high tempera¬ tures and can inhabit hot springs (up to 74 °C) and hot desert soils. Cyanobacteria also predominate at low and freezing temperatures in polar regions: extensive freshwater and terrestrial microbial mats of the Antarctic are composed mainly of cyanobacteria.
The ability to withstand high salinity allows cyanobacteria to dominate many hypersa¬ line marine lagoons and inland saline lakes. The predominance of cyanobacteria in hos¬ tile habitats is also due to the absence of competitors such as eukaryotic algae and of grazers, which are unable to withstand the adverse conditions (Castenholz, 2001).
A number of cyanobacteria grow in association with other organisms, as described in 2.4.6.
2.5.1.1 Freshwater Planktonic Forms
Cyanobacteria are widespread and abundant in freshwater lakes, where they fre¬ quently form dense populations, called water blooms, in eutrophic waters. In tropical regions the growth is usually continuous, but in temperate lakes there is a characteristic seasonal succession of the bloom-forming species. Usually the filamentous forms
(such as Anabaena species or Aphanizomenon flos-aquae) develop in late spring or
early summer, while the unicellular species (such as Microcystis species) bloom in mid-summer or autumn. The main factors which determinate the development of planktonic populations are light, temperature, nutrient concentrations, and the presence 2 CYANOBACTERIAL BIOLOGY 27
of organic solutes (Fay, 1983, p. 47).
Some planktonic forms can maintain their position in water at an optimum depth
for photosynthesis and growth due to the gas vesicles, which confer buoyancy to the
cyanobacterial cells. The gas vacuolation in the cells is dependent on light intensity: in
dim light the filaments become more buoyant due to an increased rate of gas vesicle
formation. In contrast, buoyancy is lost when the cells are exposed to intense radiation
(Fay, 1983, p. 49).
2.5.1.2 Attached Forms in Lakes
Attached forms can be classified in epilithic, growing on rocks and stones; epi¬ phytic, growing on larger plants; epipelic, growing on sediments. The occurrence of
different species is affected by a complex of factors, including the chemistry of the water and of the substratum, the physical nature of the substratum, light, temperature,
degree of exposure to waves, and variations in water level (Fogg et al., 1973, p. 272).
2.5.1.3 Cyanobacteria in Streams and Rivers
For obvious reasons streams and rivers can maintain only a limited planktonic pop¬
ulation since the floating forms are permanently swept downstream. But attached
forms may grow abundantly. Many forms are epiphytic on large algae and other river plants. The composition of the cyanobacterial population is influenced by light and the
rate of current flow, and by the chemistry of the water and substratum. For example,
calcareous rocks can support a larger and more complex community than non-calcare¬
ous rocks (Fay, 1983, p. 53).
2.5.1.4 Cyanobacteria in Thermal Waters
Cyanobacteria were found in hot thermal waters at temperatures up to 74 °C. The
hot source water contains most of the essential nutrients and permits a steady growth
throughout the year. Cyanobacteria are most abundant in the alkaline hot springs and
absent from the hot acid springs (Fay, 1983, p. 53). 28 2.5 ECOLOGY
2.5.1.5 Marine Planktonic Forms
The presence of cyanobacteria is, in general, less evident in the open sea than in
freshwaters, but they can occur in striking abundance in warmer seas. It was Trichodes- mium, a red-pigmented species, which gave the Red Sea its name (Fogg, 1982).
High rates of nitrogen-fixation were measured in Oscillatoria colonies collected
from subsurface layers, and rates may be even higher in the deeper water, where the physical conditions are more favourable for N2-fixation. Thus, the contribution of the marine Oscillatoria to the nitrogen level of the open sea is probably of enormous importance (Fay, 1983, p. 55).
2.5.1.6 Marine Littoral Forms
Cyanobacteria are widespread along maritime coasts, often forming visually con¬
spicuous growths on rocks and sediments. The colour of zones on limestone shores may, for instance, be due largely to the dominance of cyanobacteria (Whitton and
Potts, 1982). Their nitrogen-fixing activity contributes no doubt considerably to the productivity of the littoral zone. Filamentous cyanobacteria are abundant in the littoral region of coral reefs. They constitute the primary source of food for the associated her¬ bivores and play an important part in the high productivity of reef and tropical lagoon
communities (Fay, 1983, p. 56).
2.5.1.7 Terrestrial Cyanobacteria
Cyanobacteria are widely distributed in terrestrial habitats, occurring predomi¬ nantly on the surface of soils, stones, rocks, and trees, particularly in moist, neutral or
alkaline areas. They play a major role as primary colonizers, which help in the estab¬ lishment of other members of the soil flora and in the accumulation of humus. They do this in four main ways. They bind sand and soil particles, preventing erosion; they help to maintain moisture in the soil; they can fix nitrogen; they supply growth substances, helping the growth of higher plants (Fogg et al., 1973, p. 311).
Growth and propagation of terrestrial cyanobacteria are controlled by moisture,
soil temperature, irradiance, and pH. Soil desiccation is well tolerated probably because of the protection provided by the thick mucilaginous sheath. Heterocytous 2 CYANOBACTERIAL BIOLOGY 29
forms respond to adverse conditions by forming akinetes, which are able to withstand
long periods of drought and may readily germinate after rewetting of the soil (Fay,
1983, p. 57).
2.5.2 Cyanobacterial Toxins and Public Health
Cyanobacteria can form massive blooms in fresh and brackish waters worldwide.
These blooms do not only affect the water's taste, odour, and appearance, but more sig¬ nificantly, they are frequently high toxic to wildlife, domestic livestock, and humans.
The ecological and economic impacts of toxins seem to increase as blooms increase in many countries as a result of nutrient eutrophication (Dow and Swoboda, 2000).
At least 12 cyanobacterial genera have been documented as including toxin-form¬
ing species. Individual genera can include toxic and non-toxic species, and isolates of
a single species may, or may not, produce toxins, like hepatotoxins, neurotoxins, and cytotoxins (Codd and Poon, 1988).
Many Microcystis strains have been reported to be toxic to Zooplankton. In con¬
trast, some Microcystis strains proved to be a suitable food source for Daphnia. The
comparison of these results shows that not every Microcystis strain is toxic to Daphnia.
The toxicity ofMicrocystis strains and their ability to reduce the grazing pressure of the
Zooplankton have been suggested to be an algal defense that favors formation of algal blooms (Jungmann, 1992). Since the cyclic heptapeptides microcystins produced by
many Microcystis strains are toxic to vertebrates, it was first assumed that toxicity to
Zooplankton was caused by the same compounds. However, Jungmann and Benndorf
(1994) found no correlation between the concentrations of microcystins of different
Microcystis strains and the toxicity of extracts to Daphnia. Therefore, it must be con¬
cluded that the microcystins are not the compounds toxic to Daphnia.
Cyanobacterial toxins are accumulated by many aquatic organisms. It has been
shown, for example, that swan mussels (Anodonta cygnea) can accumulate remarkable
amounts of an Oscillatoria toxin. The total concentrations of toxin were 70-280 (xg per
mussel, which implies that mussels can concentrate low toxin concentrations over a
relatively short period of time. In the experiment, after 15 days in water contaminated
with the toxin, the mussels were transferred to clean water. After two months in clean
water the mussels still contained detectable amounts of toxin. The highest toxin con- 30 2.5 ECOLOGY centration was detected in the hepatopancreatic tissue, with about 40% of the total toxin content in the mussels. This high concentration of the toxin, however, had no considerable effect on the mussels, since they survived and remained motile during the whole experiment. Since the mussels do not metabolize the toxin, further investiga¬ tions are required to clarify the role of cyanobacterial toxin accumulation in aquatic food chains (Eriksson et al., 1989).
The effects of cyanobacterial toxins, especially microcystin-LR, on different fish species have been investigated. The lowest lethal dose of microcystin-LR in common carp (Cyprinus carpio) is 550 ug/kg. In fish injected with a lethal dose of microcystin-
LR a total loss of the liver parenchymal architecture and degeneration of the kidney tubuli were observed (Râberg et al., 1991). Acute toxicity is unlikely to occur in wild carp populations, but chronic poisoning may follow repeated sublethal exposure (Car- bis et al., 1996). Carps grown in a lake with Microcystis aeruginosa blooms showed higher serum aspartate aminotransferase activity and serum bilirubin concentrations than carps grown in a lake without cyanobacterial blooms, indicating an altered hepa- tocyte function. They also demonstrated difficulty in maintaining cation-anion homeo¬ stasis (Carbis et al., 1997). Similar results were obtained with the brown trout (Salmo trutta) and the rainbow trout (Oncorhynchus mykiss), which showed altered liver func¬ tion but, in contrast to the carp, lacked of kidney damage (Tencalla et al., 1994; Bury et al., 1997).
It is assumed that the toxicity to fish leads to a stronger growth of Zooplankton, which serves as an alternative nutrition source instead of the cyanobacteria.
Toxic cyanobacterial blooms have been reported for over a century, one of the first being a Nodularia bloom described by Francis in Nature in 1878. Numerous cases of wild and domestic animal poisonings have been reported, affecting birds, fish and mammals (Carmichael, 1992). For example, cyanobacterial toxins were responsible for numerous deaths of cattle on alpine pastures in the Canton of Grisons, south-eastern
Switzerland (Mez et al., 1997).
There are numerous reported cyanobacterial toxicoses in humans, but up to now there have been no definitive reports of human deaths due to cyanotoxins. However, world attention was drawn to this problem in 1996, when two Brazilian haemodialysis units, which had obtained water from a reservoir contaminated with cyanobacteria, caused toxic hepatitis in 126 patients, 60 of whom died (Pouria et al., 1998). 2 CYANOBACTERIAL BIOLOGY 31
Direct contact with cyanobacterial blooms during swimming has been associated with gastritis, acute dermatitis, rhinitis, conjunctivitis, and asthma. Poisoning is more often associated with drinking water; symptoms are fever, myalgia, hypotension, gas¬ troenteritis and acute hepatitis. Of even greater concern is the chronic, low-concentra¬ tion exposure to cyanotoxins. Continual oral exposure to low doses of microcystins have shown chronic liver injury, but more important is the possibility of carcinogenesis and tumour growth promotion. The incidence of human hepatocellular carcinoma in
China is one of the highest in the world; cancer mortality rates are lower when water is drawn from deep wells rather than from surface sources which sustain abundant cyano¬ bacterial populations (Dow and Swoboda, 2000).
To control toxic cyanobacterial blooms it is important to be able to determine quan¬ tity and quality of the toxins as well to understand the circumstances under which they occur.
The testing, monitoring, and quantification of the toxins has traditionally relied upon the intraperitoneal mouse bioassay. However, because of moral, economic, and practical considerations, additional assay techniques have been suggested and devel¬ oped (Campbell et al., 1994). Alternative bioassays are based on Zooplankton, like
Daphnia sp. and Artemia salina, but they are less reliable than the mouse bioassay
(Dow and Swoboda, 2000). Improved chromatographic techniques such as HPLC and
HPTLC have also been used (Codd et al., 1989). Other methods for the detection and quantification of cyanotoxins include enzyme-inhibition assays (like the protein phos¬ phatase inhibition assay, Ward et al., 1997), immunoassays with monoclonal antibod¬ ies (Dow and Swoboda, 2000), in vitro cytotoxicity assays (Codd et al., 1989), and the
Microtox bioluminescence assay (Campbell et al., 1994).
Some of the most important toxins isolated from cyanobacteria are described in Chapter 4.
2.6 Applications of Cyanobacteria
Although cyanobacteria constitute a vast potential resource, only a few relatively fast-growing strains have been studied intensively or commercially exploited. Agricul¬ tural uses are ancient and ever expanding. Many cyanobacteria produce high-value 32 2.6 APPLICATIONS OF CYANOBACTERIA chemicals or enzymes with unique properties that have been successfully commercial¬ ized, though in limited markets.
2.6.1 Biofertilizers
Biofertilizers are of great interest in many countries, especially in tropical Asia and
India, where rice is one of the major agricultural crops. Conditions typical of rice paddy are favourable for growth of both free-living and symbiotic cyanobacteria, many of which are capable of fixing atmospheric nitrogen and thus contribute to improve yields by 10 to 15%. Other potential benefits of cyanobacteria growing in rice paddy are the production and release of inhibitors of plant pathogens, and the amelioration of the negative effects of high salt concentration on soils, allowing the agricultural usage of saline and sodic soils (Patterson, 1996). Cyanobacterial genera commonly used in agriculture are Anabaena, Nostoc, Plectonema, Scytonema, and Tolypothrix.
Cyanobacteria are in general quite sensitive to herbicides and fungicides and this can reduce the beneficial effects of cyanobacteria, although it has been shown that some strains can develop herbicide-resistant mutants (Whitton, 2000).
Interesting is also the use of nitrogen-fixing cyanobacteria in land-based agricul¬ ture. Dried cyanobacterial preparations would be inoculated in the soil at the time of planting, so that they can proliferate and replace traditional chemical fertilizers.
Cyanobacterial exopolysaccharides would have a positive effect on soil stability and productivity, and it is suggested that this could be the only realistic hope for halting the spread of desert in sub-Saharan Africa (Whitton, 2000).
2.6.2 Food
The description of the consumption of a preparation of Spirulina by people in the vicinity of Mexico city in 1521 is the first record of the use of cyanobacteria as food. The consumption of Spirulina by native people in the vicinity of Lake Chad in Africa is also well documented. Other cyanobacteria, including Nostoc balls, have also been consumed as a staple or as delicacies.
The major success in large-scale, low-cost cultivation and marketing of cyanobac- 2 CYANOBACTERIAL BIOLOGY 33 teria was achieved with Spirulina. Worldwide commercial production oi Spirulina is in the order of 800-1000 tonnes/year. Spirulina is particularly suitable for food use because of the high protein content, which can constitute up to 70% of the dry weight, the good digestibility, and the high vitamin content (Patterson, 1996). Spirulina has been proposed to help in reducing appetite, as an aid in dieting, to prevent symptoms ascribed to y-linolenic acid deficiency, to decrease cholesterol levels, to activate the immune system (Allnutt, 1996).
The use of cyanobacterial proteins as a supplement for conventional protein sources (e.g. soybean meal or fish meal) in livestock feed has been widely tested, for example with poultry, pigs, and ruminants (Patterson, 1996).
2.6.3 Pollution Control
Pollution control through bioremediation is the most economical and ecofriendly approach. A number of toxic compounds such as phenolics, pesticides, and antibiotics can be degraded and detoxified by cyanobacteria fairly rapidly. Various metals, includ¬ ing heavy metals can be removed and even recovered from effluents by bioaccumula¬ tion and biosorption by cyanobacteria. Furthermore, cyanobacteria show high flexibil¬ ity and adapt themselves to varied environments.
However, cyanobacteria have to be screened extensively to select the right type of organism to perform the right function. With gene cloning and manipulative tech¬ niques, it would be even possible to develop suitable microorganisms to effectively treat and recycle wastes (Subramanian and Uma, 1996).
2.6.4 Commercial and Laboratory Chemicals
2.6.4.1 Phycobiliproteins
Phycobiliproteins are water soluble, coloured, highly fluorescent compounds, which occur naturally in cyanobacteria. The major markets for phycobiliproteins are the food, drug, and cosmetic industries (as natural colouring agents), and in clinical diagnostics (as fluorescent reagents) (Patterson, 1996). 34 2.6 APPLICATIONS OF CYANOBACTERIA
2.6.4.2 Isotopically-enriched Metabolites
Cyanobacteria can be used to produce, by biosynthesis, compounds that are isoto¬ pically-enriched through the process of growing the organisms on substrates such as
C02 enriched in a particular isotope, such as 13C or 14C. The resulting labelled com¬ pounds may be purified and utilized as tracers in biology and medicine. Some isotopi- cally labelled cyanobacterial metabolites are already commercially available: for example amino acids, sugars, lipids (Patterson, 1996).
2.6.4.3 Restriction Enzymes
Surveys indicate that cyanobacteria are a rich source of new restriction enzymes, with some strains reported to contain as many as five sequence-specific DNAses. Such enzymes with unique restriction sites are a useful tool in molecular biology. Many companies obtain the enzymes from cyanobacterial cultures, some others prepare them by fermentation of Escherichia coli strains that carry the cloned endonuclease genes from two Anabaena species (Allnutt, 1996).
2.6.4.4 Bioactive Compounds
Cyanobacteria are an important source of new pharmaceuticals, pharmaceutical lead compounds, and pharmacological tools. They produce many secondary metabo¬ lites with interesting chemical structures, like peptides, alkaloids, polyketides, and amides, and with important biological activities, such as cytotoxic, antifungal, antibac¬ terial, or antiviral activity.
Chapter 4 provides an overview of the most interesting secondary metabolites iso¬ lated from cyanobacteria.
2.6.5 Energy
Interest in alternatives to non-renewable fossil fuels has increased in the recent past due to both the finite nature of these resources as well as need in achieving non-pollut¬ ing alternative sources of energy.
One alternative is photosynthetically-generated biomass. It is renewable and car- 2 CYANOBACTERIAL BIOLOGY 35 bon utilization is a closed cycle: any carbon dioxide that is liberated by combustion was just recently incorporated by the photosynthetic organism, resulting in no net change in
C02 concentration. However, the costs associated with growth, harvesting, and drying make the cultivation of cyanobacteria solely for biomass production economically not competitive (Patterson, 1996).
It is possible to use cyanobacteria for the direct production of energy-rich fuels, especially hydrogen. Hydrogen can be produced using the nitrogenase or the hydroge¬ nase activity of cyanobacteria. The nitrogenase catalyzes the reduction of molecular nitrogen to ammonia, but also the production of hydrogen gas. Under the proper culture conditions it may be possible to exploit this enzymatic system and use solar energy for the generation of large volumes of hydrogen gas. Two problems prevent the application of this technique. First, the synthesis of ammonia and hydrogen gas is competitive, so the presence of nitrogen would reduce the hydrogen yield. The second problem is the presence of a hydrogen uptake enzyme which traps any H2 produced and reoxidizes it.
This is probably a means of recapturing the reducing power lost in the production of hydrogen. Of course, this activity results in a decrease of the hydrogen yield (Calvin and Taylor, 1989). Genetic manipulation of the hydrogen uptake enzyme could lead to the development of strains with high hydrogen yields. Another approach is the screen¬ ing of various cyanobacteria for strains with high rates of H2 productivity: Kumazawa and Mitsui (1994) found a Synechococcus sp. strain with little H2 oxidation activity.
Cyanobacteria possess also a hydrogenase capable of both H2 evolution and con¬ sumption, that is inhibited by 02 and C02. To overcome this limitation, hydrogen must be generated under conditions of low C02 concentration, and 02 removed (Kerby and Rowell, 1992).
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Wilmotte A (1994): Molecular evolution and taxonomy of the cyanobacteria. In: The molecular biology ofcyanobacteria (Bryant DA, ed.), Kluwer Academic Publish¬ ers, Dordrecht, pp. 1-25.
Woese CR, Fox GE (1977): Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc. Natl. Acad. Sei. USA 74: 5088-5090. Woese CR (1987): Bacterial evolution. Microbiol. Rev. 51: 221-271.
Woese CR, Kandier O, Wheelis ML (1990): Towards a natural system of organ¬ isms: Proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sei.
USA 87: 4576-4579.
Wölk CP, Ernst A, Elhai J (1994): Heterocyst metabolism and development. In: The molecular biology ofcyanobacteria (Bryant DA, ed.), Kluwer Academic Publish¬
ers, Dordrecht, pp. 769-823. 3 ISOLATION AND CULTIVATION 41
3 Isolation and Cultivation
Advances in the knowledge of cyanobacteria depends largely on the use of cul¬ in tures. Microorganisms used in research are grown generally in axenic cultures, i.e. artificial isolation from other species because it is difficult otherwise to attribute a par¬ The medium is ticular feature or response to the organism under investigation. syn¬ Such thetic and the physical conditions such as light and temperature are controlled. the maximum conditions are selected usually to give optimum growth and provide yield of algal material. This is important because often large quantities of material are
required in order to extract sufficient amounts of specific substances present in minute
amounts in the cells.
the course of in The physiological activities may vary considerably during growth is culture, and for most experimental purposes material in the active phase of growth
used.
Hence, purity, yield, and activity are the main criteria used in developing culture
techniques (Fogg et al., 1973, p. 129).
3.1 Isolation and Purification
Isolation is defined as the separation of individual clones of cyanobacterial species
from other photosynthetic organisms present in the crude material. As unicyanobacte-
rial isolates, they are subsequently purified to the axenic state.
3.1.1 Isolation by Liquid Enrichment
Many cyanobacteria presently in axenic culture have been isolated by the so-called of liquid enrichment technique, which imposes a positive selection of those members
the population which can better proliferate in the medium and under the physical con¬ ditions (light, temperature) provided by the investigator. In general, such liquid enrich- 42 3.1 ISOLA TION AND PURIFICA TION ments will contain only one or very few dominant cyanobacterial strains, and their iso¬ lation and purification are relatively easy, provided that they will grow equally well on solid media (Rippka, 1988).
3.1.2 Direct Isolation
The liquid enrichment method has the disadvantage that the cyanobacteria isolated are seldom representative of the total variety of species, and their relative number, encountered in the natural habitat. To obtain a more representative result, it is better to establish pure cultures via direct isolation.
With this method, aliquots of the sample are transferred directly to solid media.
After streaking the deposited crude material on solid media, many of the larger fila¬ mentous forms or colonial aggregates of unicellular cyanobacteria may be sufficiently
separated to attempt their isolation to the unicyanobacterial state without prior incuba¬ tion of the plates. If this is not the case, plates are incubated under the light and temper¬
ature conditions of choice, and examined daily under the dissection microscope to maximize the chances of isolating filaments and colonies before they are overgrown by
contaminants. Microscopic colonies are picked and restreaked on fresh solid media.
These steps are repeated until pure colonies have been obtained.
Motile filaments can be placed in the middle of a plate: as a result of gliding motil¬
ity, the filament will migrate from the center outwards, eventually freeing itself from
contaminants (Rippka, 1988; Rippka et al., 1981).
3.1.3 Purification Methods
It is often possible to obtain axenic cultures by performing isolation and the subse¬
quent purification steps on plates as described. Sometimes, however, this is not the
case, particularly if the original sample is highly contaminated by bacteria and the
cyanobacterial species are immotile and incapable of self-purification by gliding away
from their contaminants, or if the single filaments or microcolonies do not grow on
solid media.
Purification of such cyanobacteria can be attempted by filtration. The cyanobacte- 3 ISOLATION AND CULTIVATION 43 ria are partly purified by washing procedures using membrane filters for separation from their contaminants on the basis of differential cell sizes. Single filaments are then transferred to fresh plates and to liquid medium, unicellular species are incubated and grown on plates. If they do not grow on solid media, the washed sample should be used for serial dilutions in a sterile medium. After incubation and growth the individual dilu¬ tions are tested for purity. This dilution method works only if the cyanobacterial population is well superior to that of the contaminants. If this is not the case, the latter must be reduced by treat¬ ment with bactericidal agents or antibiotics. Aliquots of the treated culture are then removed, streaked on plates, grown, and tested for purity (Rippka, 1988).
3.2 Cultivation
3.2.1 Culture Media
There are many culture media, which differ mainly in the contents of combined nitrogen, phosphate, type of chelating agents, concentration and form of iron, complex¬ ity and concentration of trace metals. Of course, no medium is perfect for all cyanobac¬ teria, and modifications are needed depending on the purpose. For example, if the goal is to obtain the highest yield in the shortest time, a rich medium may be required. A deficiency of combined nitrogen is generally required to elicit heterocyte differentia¬ tion and synthesis of nitrogenase. Phosphorus deficiency is needed for akinete forma¬ tion in some species (Castenholz, 1988).
The quality of the water used for preparation of the media is of paramount impor¬ tance. The water should be deionized and the glassware used should be free of traces of high-phosphate detergents.
The great majority of cyanobacteria grow best in neutral to alkaline environments
(pH 7-10), and even those encountered in mildly acidic habitats (pH 4.5-5.0) are gen¬ erally acid tolerant rather than acidophilic. For this reason, practically all media employed for cyanobacteria are alkaline (Rippka et al., 1981). 44 3.2 CULTIVATION
3.2.2 Temperature
the The temperature used for cyanobacterial cultures will depend on the strain and in habitat from which it was isolated. Although many cyanobacteria are dominant very
for is much cold waters, a general rule is that the optimum temperature growth higher, above 30 °C 15-20 °C. Nevertheless, many cyanobacteria cannot tolerate temperatures (Castenholz, 1988).
3.2.3 Light Regimes
the use of fluores¬ The spectral range of light absorbed by cyanobacteria requires
because other sources cent light sources (i.e. cool-white, warm-white, daylight) light
near-infrared which are have a great proportion of the output in the far red and regions
not available to oxygenic phototrophs. cultivation is The light intensity can vary enormously, but, unless the attempted
under air enriched with C02 (a condition leading to a higher light tolerance), the light
in are intensities are kept low (less than 500 lux). Strains rich phycoerithrin particularly light sensitive (Rippka, 1988).
rather than under a Cyanobacteria are often kept under a continuous light regime light-dark cycle. However, it is possible that continuous light may favor the selection
of mutants defective in regulatory mechanisms that are under the control of light-dark
cycles in the natural habitat (Rippka, 1988).
3.2.4 Solid Media
medium with the solid¬ Solid media are prepared by mixing the mineral salts liquid of ifying agent, usually agar. Solid media should be prepared by separate autoclaving after have cooled to 50 the agar and the mineral solution, and subsequent mixing they sterilization of the min¬ °C. The correct preparation of agar media is important because toxic eral salts together with the agar can lead to the production of compounds (Allen, 1968). is If the solid media is used to isolate pure cyanobacterial strains and the sample 3 ISOLATION AND CULTIVATION 45 rich in eukaryotic contaminants, antibiotics like cycloheximide (actidione) can be added (Zehnder and Hughes, 1958).
3.2.5 Vessels
The type of vessels can vary greatly. For stock cultures in liquid medium, Erlen¬ meyer flasks are the most satisfactory. The intention of keeping stock cultures is not to and encourage high growth rates. For this reason, there is no need to add air or CO2, light intensity is kept at a low level. For cultures on solid media, Petri plates or test- tubes are used (Castenholz, 1988).
For experimental cultures, where growth on a relatively large scale should be achieved, bigger vessels are used. Various types of fermenter systems work well: long cylinders; Fernbach flasks, i.e. flasks with a broader base than Erlenmeyer flasks, in order to have a larger surface area; bottles with a 10-15 liter capacity and with inlets for addition of air, C02, and medium (Rippka, 1988).
3.3 References
Allen MM (1968): Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4: 1-4.
Castenholz RW (1988): Culturing methods for cyanobacteria. In: Methods in enzy- mology Vol. 167 (Packer L, Glazer AN, eds.), Academic Press, San Diego, pp. 68-93. Fogg GE, Stewart WDP, Fay P, Walsby AE (1973): The blue-green algae. Aca¬ demic Press, London.
Rippka R, Waterbury JB, Stanier RY (1981): Isolation and purification of cyano¬ bacteria: some general principles. In: The prokaryotes Vol. 1 (Starr MP, Stolp H,
Triiper HG, Balows A, Schlegel HG, eds.), Springer, Berlin, pp. 212-220.
Rippka R (1988): Isolation and purification of cyanobacteria. In: Methods in enzy- mology Vol. 167 (Packer L, Glazer AN, eds.), Academic Press, San Diego, pp. 3-27. Zehnder A, Hughes EO (1958): The antialgal activity of actidione. Can. J. Micro¬ biol. 4: 399-408. 46 4.1 CYANOBACTERIAL TOXINS 4 Biologically Active Compounds from Cyanobacteria
Cyanobacteria produce a wide variety of chemically unique substances derived from secondary biosynthesis. These compounds are called secondary metabolites because they are not used by the organism for its primary metabolism, such as cell divi¬ sion, photosynthesis or respiration (Carmichael, 1992). It was suggested that the sec¬ ondary metabolites are produced for protection against grazers, to control the growth of bacteria and green algae, as storage forms, or as detoxification products (Jones, 1988).
Systematic screening of cyanobacteria for bioactivity (Patterson et al., 1991; 1993;
1994; Honkanen et al., 1995; Falch et al., 1995; Jaki et al., 1999) showed that these microorganisms are a rich source of novel bioactive agents. The discovery rate for bio¬ active compounds is about 7%, about the same as that found for other microorganisms.
The rate of rediscovery of known bioactive compounds is, however, significantly lower among the cyanobacteria than among Actinomycetes (Carmichael, 1992). Thus, cyano¬ bacteria have a good potential for providing pharmaceutical substances with unusual or unknown chemical structures.
4.1 Cyanobacterial Toxins
Toxins are secondary metabolites that have a harmful effect on other cells, tissues, or organisms. The cyanotoxins are classified into two general groups based on the type ofbioassay used to screen for their activity. The cytotoxins are tested on cultured mam¬ malian cell lines, especially tumour cell lines; the biotoxins are assayed with small ani¬ mals. There are no reports that cytotoxins have been responsible for deaths of animals in cases of field poisoning. Biotoxins, on the contrary, have caused repeated cases of sickness and death in livestock, pets and wildlife after ingestion of water containing toxic algal cells (Carmichael, 1992). 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 47
4.1.1 Cytotoxins
Cytotoxins are not lethal to animals, but show antialgal, antimycotic, or antibacte¬ rial activity, or are active against cell lines. Their discovery was largely due to the search for new pharmaceutical and agrochemical compounds. For an overview of the most interesting cytotoxins see 4.2.1-4.2.8.
4.1.2 Biotoxins
Biotoxins with hepatotoxic, neurotoxic, dermatotoxic, and gastrointestinal effects are known.
4.1.2.1 Hepatotoxins
Acute hepatotoxicosis involving the hepatotoxins (liver toxins) is the most com¬ monly encountered toxicosis involving cyanobacteria. Clinical signs of hepatotoxico¬ sis have been observed in field poisonings involving livestock and domestic and wild animals. The signs of poisoning in these animals include weakness, anorexia, vomit¬
ing, diarrhoea. Death occurs within a few hours to a few days after initial exposure and Death from may be preceded by coma, muscle tremors and forced expiration of air. these hepatotoxins is the result of intrahepatic hemorrhage. The mechanism of action is
not completely clear. However, the toxins are absorbed into the blood and transported preferentially into the hepatocytes. Here, they induce changes in the actin microfila¬
ments which compose part of the cytoskeleton, leading to a loss of cellular support and
finally to cell destruction. With destruction of the parenchymal and sinusoid endothe¬
lial cells of the liver, lethal intrahepatic hemorrhage occur (Carmichael, 1992).
The major hepatotoxins are the microcystins, the nodularins, and cylindrospermop-
sin.
The microcystins are cyclic heptapeptides isolated from the genera Microcystis,
Nodularia, Anabaena, Nostoc, and Oscillatoria. They have the general structure cyclo(-D-Ala-L-X-erythro-ß-methyl-D-isoAsp-L-Z-Adda-D-isoGlu-N-methyldehy-
dro-Ala) where X and Z represent certain L-amino acids. The presence of Adda, (25',35',85',95)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, is 48 4.1 CYANOBACTERIAL TOXINS the most unusual structural feature. Adda plays an important role in hepatotoxicity, since hydrogénation or ozonolysis of the diene system in this unit gives an inactive product (Dow and Swoboda, 2000).
Variation in the chemical structure of microcystins is very common, most fre¬ quently in substitution of the L-amino acids at positions 2 and 4, and demethylation of the amino acids 3 and 7, but variations have been reported in each amino acid (Sivonen,
1996). Over 60 microcystins have been isolated and fully characterized to date (for example microcystin LR, fig. 4.1).
The microcystins are strong inhibitors of type 1 and 2A serine protein phos¬ and phatases. These enzymes are vital to various cellular processes such as cell growth tumour suppression and therefore these toxins are possible potent cancer promoters
(Carmichael, 1994). The occurrence of these toxins in potable water may therefore present a serious health hazard to humans if the peptide toxins are consumed over a long period of time, even at very low concentrations.
Certain chemicals have been used experimentally to prevent microcystin hepato¬ toxicity in laboratory animals. These include cyclosporin-A, rifampin, and Silymarin.
These antagonists have been most successful when given before or with the toxin. Their mechanism is unknown, but it is suggested that they inhibit the toxin uptake by
the hepatocytes (Carmichael, 1992).
(6) D-Glu (iso) (7) N-methyldehydroAla
COOH CH3 (3 1 I
„„/V/-^^ NH (5) Adda I 1 «- H3C*/,„ 1 I f^ OCH3 H3Cr V—q
\^ ^-^ ^^ ^y ^T CH3 CH3
CH3 CH3 ^^/^N^^N^^N ' < ^^ ^C I HN. N ^^ J f| (2)L"LeU Y^^^ ° *°0H ° U (4)L- Ar9 (3) D-erythro-ß-methylAsp ( so)
Figure 4.1 Microcystin-LR
Other hepatotoxins, the nodularins (fig. 4.2, nodularin), are produced by the genus 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 49
Nodularia. Nodularins are cyclic pentapeptides and contain the Adda unit or a deriva¬ tive. As the microcystins, they inhibit the protein phosphatases 1 and 2A, and are potent tumour promoters (Sivonen, 1996).
(4) D-Glu
COOH
CH3 /
(5) N-methyldehydro- butynne o
COOH
(1) D-erythro-ß-methylAsp
Figure 4.2 Nodularin
Another peptide hepatotoxin, cylindrospermopsin (fig. 4.3), has been isolated from
Cylindrospermopsis raciborskii, Umezakia natans, and Aphanizomenon ovalisporum. the Cylindrospermopsin is an alkaloid possessing a cyclic guanidin group and affects liver, the kidneys, the thymus, and the heart (Moore et al., 1993).
OH
H H O3SO,
IV. ,>NH HN. .NH H3Coçy ' NH
Figure 4.3 Cylindrospermopsin
4.1.2.2 Neurotoxins
Neurotoxins are produced by several cyanobacterial genera, such as Anabaena,
Aphanizomenon, Oscillatoria, and Trichodesmium. The major neurotoxins are ana¬ toxins, homoanatoxin-a (both fig. 4.4), anatoxin-a(s), saxitoxin, and neosaxitoxin. 50 4.1 CYANOBACTERIAL TOXINS
Anatoxin-a is a low molecular weight secondary amine, 2-acetyl-9-azabicyclo
[4.2.1]non-2-ene isolated from the genus Anabaena. It causes death via depolarizing blockage of neuromuscular transmission and subsequent respiratory paralysis. Ana¬
to the toxin-a is a highly potent and stereospecific agonist at nicotinic synapses; it binds nicotinic-acetylcholine receptors with higher affinity than acetylcholine itself, but is not degraded by acetylcholinesterase. The intraperitoneal LD50 for mice is 200 ug/kg body weight, with a survival time of 4-7 minutes (Sivonen, 1996). Clinical signs of tox¬ icosis in mice, rats and calves are muscle fasciculations, collapse, exaggerated abdom¬ inal breathing, cyanosis, convulsions and death. Death by respiratory failure occurs within minutes to a few hours depending on species and dose. The toxicity is so high that animals need to ingest only a few millilitres to a few litres of the toxic surface water bloom to receive a lethal bolus. No chemical antidote exists for anatoxin-a intox¬
ication (Carmichael, 1992). The anatoxin-a homologue homoanatoxin-a has been isolated and characterized
from a strain of Oscillatoriaformosa. It has an intraperitoneal LD50 for mice of 250 ug/ kg body weight (Skulberg, 1992).
o
Figure 4.4 Anatoxin-a (R=CH3) and homoanatoxin-a (R=CH2CH3)
Anatoxin-a(s) (fig. 4.5), the s denoting salivation in vertebrates, is produced by
strains ofAnabaena flos-aquae. Symptoms are similar to those of anatoxin-a, with the
addition of ataxia, hypersalivation, and tremors. However, this toxin is structurally and
physiologically different from anatoxin-a. It is an organophosphate and functions as an acetylcholinesterase inhibitor similarly to the organophosphate pesticides like malathion and parathion. The intraperitoneal LD50 for mice is 20 |xg/kg body weight,
ten times more lethal than anatoxin-a (Dow and Swoboda, 2000). 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 51
,CH3 F HN^ J\ CH3
,CH3 NH2 + , s\*
Figure 4.5 Anatoxin-a(s)
The neurotoxins saxitoxin and neosaxitoxin (fig. 4.6) were originally identified as from marine dinoflagellates, and are the cause of paralytic shellfish poisoning (PSP). They have then been isolated from the cyanobacterium Aphanizomenonflos-aquae and recently from Lyngbya wollei (Carmichael, 1997). The saxitoxins act by inhibiting nerve conduction by blocking sodium, but not potassium, transport across the axon membrane. Symptoms of acute poisoning in laboratory animals include loss of co-ordi¬ nation, twitching, irregular breathing, and death by respiratory failure. The intraperito¬ neal LD50 for mice is about 10 ug/kg body weight (Hunter, 1998).
Figure 4.6 Saxitoxin (R=H) and neosaxitoxin (R=OH)
4.1.2.3 Dermatotoxins and Gastrointestinal Toxins
Swimmers who have come in contact with the marine cyanobacterium Lyngbya majuscula have contracted acute dermatitis. The inflammatory activity is caused by aplysiatoxin (fig. 4.7) and debromoaplysiatoxin. These toxins are potent tumour pro¬ moter and protein kinase C activators (Sivonen, 1996). 52 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS
H3C
Figure 4.7 Aplysiatoxin
Lyngbyatoxin-a (fig. 4.8), found in another strain of Lyngbya majuscula, has caused dermatitis and severe oral and gastrointestinal inflammation (Sivonen, 1996).
Figure 4.8 Lyngbyatoxin-a
4.2 Cyanobacteria as a Source of Medicinal Agents
Microorganisms have historically proven to be an exceptionally rich source of bio¬ logically active metabolites. These metabolites have become important biomedically
as leads to new pharmaceutical compounds. The large effort expended in drug discov¬ fac¬ ery programs has greatly increased the risk of rediscovery of previously described
tors. One way to minimize this problem is to look at new and different sources of nat- 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 53 ural products, such as the cyanobacteria (Patterson et al., 1991).
Interest in investigating cyanobacteria has grown due to the recognition that their
human and animal presence in water reservoirs is often associated with the release of toxicants. During the studies on cyanobacterial toxins, many secondary metabolites with interesting chemical and biological features were discovered. These compounds include substances with antibacterial, antifungal, antiviral, cytotoxic, anti-inflamma¬ tory, and cardioactive properties. These secondary metabolites in their natural form will not necessarily lead directly to commercialization, but the activity induced by these natural compounds may be exploited to structure-function studies to develop ana¬ logues with stronger and more focused activities (Burja et al., 2001).
4.2.1 Antimicrobial Compounds
New antibiotics are continually needed because of the development of resistant pathogens, the evolution of new diseases, and the toxicity of some of the current com¬ pounds (Demain, 2000). Several cyclic peptides from cyanobacteria exhibit antimicrobial activity. The lax-
aphycins (like laxaphycin A, fig. 4.9), for example, are a large family of cyclic undeca-
and dodecapeptides with antifungal activity. The antifungal effect shown by these pep¬
tides is unusual in that the peptides act synergistically with each other to inhibit growth.
In order to achieve maximum biological potency, a member of each class of peptides
must be present (Frankmölle et al., 1992a; 1992b). The laxaphycins show also cyto¬ toxic activity against different human cell lines (Bonnard et al., 1997). 54 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS
Figure 4.9 Laxaphycin A
The laxaphycins closely resemble, both structurally and biologically, the hor- mothamnins, a group of cyclic peptides isolated from the marine cyanobacterium Hor- mothamnion enteromorphoides (Gerwick et al., 1989). Hormothamnin A, weak antimi¬
crobial to the Gram-positive bacterium Bacillus subtilis and the Gram-negative bacterium Pseudomonas aeruginosa, differs from laxaphycin A in the geometry of the double bond in the didehydrobutyrinyl unit.
Calophycin (fig. 4.10), a cyclic decapeptide containing a novel (2R,3R,4S)-3-
amino-2-hydroxy-4-methylpalmitic acid unit (Hamp), is a potent broad-spectrum fun¬ gicide from Calothrix fusca. The MIC against Candida albicans is 1.25 ug/ml (Moon
et al., 1992). 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 55
Pi 1H23
^ r*
NH2
Figure 4.10 Calophycin
Figure 4.11 Kawaguchipeptin A 56 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS
Kawaguchipeptins A (fig. 4.11) and B are two cyclic undecapeptides with antibac¬ terial activity isolated from Microcystis aeruginosa. They inhibit the growth of the
Gram-positive bacterium Staphylococcus aureus at a concentration of 1 ug/ml (Ishida etal., 1997a).
Many other cyclic peptides of cyanobacterial origin show antimicrobial properties: for example, majusculamide C from Lyngbya majuscula (Carter et al., 1984), schizot- from Nos¬ rin A from Schizothrix sp. (Pergament and Carmeli, 1994), nostofungicidine toc commune (Kajiyama et al., 1998). Not only peptides show antibiotic activity. For example the 5-lactone malyngolide (fig. 4.12) from the marine cyanobacterium Lyngbya majuscula is effective against
Mycobacterium smegmatis and Streptococcus pyogenes (Cardellina et al., 1979).
o N^o 0H
Figure 4.12 Malyngolide
The indolo[2,3-a]carbazoles named tjipanazoles (fig. 4.13, tjipanazole A), from
Tolypothrix tjipanasensis, are active against Candida albicans, Trichophyton menta¬ grophytes, and Aspergillus flavus (Bonjouklian, 1991).
Figure 4.13 Tjipanazole Al 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 57
Hierridin B (fig. 4.14) and 2,4-dimethoxy-6-heptadecyl-phenol from Phormidium ectocarpi are the first natural products from a marine cyanobacterium with activity against the malaria parasite Plasmodium falciparum. The IC50 of the mixture of the two compounds against chloroquine sensitive clones and chloroquine resistant clones are 5.2 ug/ml and 3.7 ug/ml, respectively (Papendorf et al., 1998).
OH
H3CO^ ^^^^ ^CisH3i V OCH3
Figure 4.14 Hierridin B
Also calothrixin A (fig. 4.15) and B, isolated from two Calothrix strains, inhibit the growth in vitro of a chloroquine resistant strain of Plasmodium falciparum. The IC5q
values of the two compounds were 58 nM and 180 nM, respectively. Calothrixin A and
B are pentacyclic metabolites with an indolo-phenanthridine ring system unique
among natural products (Rickards et al., 1999).
Figure 4.15 Calothrixin A
4.2.2 Antiviral Compounds
ini¬ Cyanobacteria appear to be also a rich source of new antiviral compounds. The
tial screening program conducted by Rinehart et al. (1981) indicated that a large per¬
centage of extracts of field-collected cyanophytes exhibited antiviral activity against 58 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS herpes simplex virus, type II (HSV-2). More recently, 900 strains of cultured cyano¬ bacteria were examined in vitro for their ability to inhibit the reverse transcriptases of avian myeloblastosis virus (AMV) and human immunodeficiency virus, type I (HIV-
1). Over 2% of these strains showed promising activities (Lau et al., 1993). Patterson and coworkers (1993) tested 600 strains of cultured cyanophytes, representing some
300 species. 10% demonstrated activity using test systems for inhibition of HSV-2 and
HIV-1, a smaller percentage (2.5%) of the extracts was active against respiratory syn¬ cytial virus (RSV).
Despite these promising results, only a few compounds with antiviral activity have been already isolated from cyanobacteria. One of the most important is cyanovirin-N,
from Nostoc ellipsosporum (Gustafson et al., 1997). Cyanovirin-N is a novel anti-HIV protein, consisting of a single chain of 101 amino acids. The protein was produced recombinantly in Escherichia coli by expression of a synthetic DNA coding sequence
corresponding to the amino acid sequence deduced for natural cyanovirin-N. The pro¬ tein potently inhibits the in vitro cytopathicity of diverse clinical isolates and labora¬
tory strains of HIV type 1, HIV type 2, and simian immunodeficiency virus. It also pre¬
vents cell-to-cell fusion and transmission of HIV from infected cells to uninfected host
cells. The inhibitory mechanism remains unclear. However, it has been shown that cyanovirin-N blocks the binding of the viral envelope glycoprotein gpl20 and cell-sur¬
face receptors. These interactions are required for successful virus fusion and entry into
the cell (Dey et al., 2000).
A group of known and new diacylated sulfoglycolipids (fig. 4.16) were isolated
from a strain of Scytonema sp., Oscillatoria raoi, O. trichoides, O. limnetica, andPhor-
midium tenue. Some of them are potent inhibitors of the enzymatic activity of the HIV-
1 reverse transcriptase (Reshef et al., 1997). It is suggested that the inhibitory activity
of these compounds results from their ability to inhibit the DNA polymerase activity of
the HIV-1 reverse transcriptase. It was shown that the sulfoglycolipids inhibit more
than 95% of the initial enzymatic activity at a final inhibitor concentration of 10 uM
(Loya et al., 1998). 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 59
S03Na+
Figure 4.16 Sulfoglycolipids: R^linoleoyl R2 = palmitoyl R3 = palmitoyl
Rj = linoleoyl R2 = palmitoyl R3 = H
Rl = palmitoyl R2 = palmitoyl R3 = H
Ry = oleoyl R2 = palmitoyl R3 = H
Compounds with anti-HSV-2 activity are the indolocarbazoles (fig. 4.17) from a strain of Nostoc sphaericum collected in Hawaii. These compounds reduce the virus titer in infected mink lung cells by 95% at 1 ug/ml (Knübel et al., 1990).
0CH3
Figure 4.17 6-cyano-5-methoxy-12-methylindolo[2,3-a]carbazole
Bauerins A-C (fig. 4.18) are three chlorine-containing ß-carbolines isolated from the terrestrial cyanobacterium Dichothrix baueriana. They show moderate anti-HSV-2 activity (Larsen et al., 1994).
Figure 4.18 Bauerine A 60 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS
Fractionation of an extract from Spirulina platensis led to the isolation of a polysaccharide named calcium spirulan (Ca-SP) with antiviral activity. Ca-SP is a sul¬ fated polysaccharide chelating calcium ion and mainly composed of rhamnose and fructose. It was found to have high activity against all enveloped viruses including HSV-1 (ED50 0.92 ug/ml), human cytomegalovirus (ED50 8.3 ug/ml), measles virus 9.4 (ED50 17.0 ug/ml), mumps virus (ED50 23.0 ug/ml), influenza A virus (ED50 ug/ ml), and HIV-1 (ED50 2.3 ug/ml). However, Ca-SP was inactive against nonenveloped of viruses, such as poliovirus. This compound is very promising for the treatment
HSV-1, HIV-1, and human cytomegalovirus infections, which is particularly advanta¬
infections. The geous for AIDS patients who are susceptible to these life-threatening h before antiviral activity was more potent in the cultures treated with Ca-SP from 3 infection compared with that in the cultures treated immediately after infection. These such results suggest that Ca-SP may interfere with a very early stage of viral replication as virus adsorption and penetration (Hayashi et al., 1996).
4.2.3 Compounds with Multidrug Resistance Reversing Activity
Tumour cells that survive initial chemotherapy in cancer patients often increase
their resistance not only to the original drug, but also to other unrelated drugs. This phenomenon is termed multidrug resistance (MDR) and is often associated with
increased expression of P-glycoprotein, which acts as an energy-dependent drug efflux
with a pump. Enhanced efflux results in a reduction of intracellular drug accumulation concomitant decrease in cytotoxicity and, consequently, ultimate failure of chemother¬
circumvent or apy. As a consequence, there is a growing interest to develop agents that
overcome MDR (Prinsep et al., 1992a).
Tolyporphins A-K are a group of porphinoids isolated from the terrestrial cyano¬ bacterium Tolypothrix nodosa. They all show multidrug resistance reversing activity
(Prinsep et al., 1992a; 1995; 1998; Minehan et al., 1999). Tolyporphins A (fig. 4.19), for example, potentiates the cytotoxicity of adriamycin and vinblastin in SK-VLB cells
at 1 ug/ml (Prinsep et al., 1992a). 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 61
Figure 4.19 Tolyporphin A
Hapalosin (fig. 4.20), a cyclic depsipeptide from Hapalosiphon welwitschii, is a
MDR-reversing agent that acts as antagonist for drug transport by P-glycoprotein
(Stratmann et al., 1994).
Figure 4.20 Hapalosin
MDR-reversing activity is also shown by dendroamide A (fig. 4.21), a cyclic dep¬ sipeptide from the terrestrial cyanobacterium Stigonema dendroideum. 10 uM den¬
droamide A fully reverse the resistance of MCF-7/VP cells to vincristine (Ogino et al.,
1996). 62 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS
o ^V"^
Figure 4.21 Dendroamide A
4.2.4 Cytotoxic Compounds
Cyanobacteria have been identified as one of the most promising groups of organ¬ isms from which new anticancer natural products can be isolated. Many of the isolated bioactive compounds possess unprecedented structures and therefore have the potential
for development of entirely new classes of drug agents (Gerwick et al., 1994a).
The nucleosides tubercidin and toyocamycin (fig. 4.22) and their glucosides are
among the first cytotoxic compounds isolated from cyanobacteria. Tubercidin was found in Tolypothrix byssoidea, its glucoside, tubercidin 5'-a-D-glucopyranose, in
Plectonema radiosum. Toyocamycin and toyocamycin 5'-a-D-glucopyranose were
isolated from Tolypothrix tenuis. In the KB cells assay, the MIC values are 0.07 ug/ml and 0.56 ug/ml for tubercidin and its glucoside respectively, and 0.06 ug/ml and 0.30
ug/ml for toyocamycin and its glucoside (Stewart et al., 1988).
HO OH
Figure 4.22 Tubercidin (R = H) and toyocamycin (R = CN) 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 63
The scytophycins are macrolides that inhibit the proliferation of a wide variety of mammalian cells. They were isolated from the genera Cylindrospermum, Scytonema, and Tolypothrix (Patterson et al., 1994). Tolytoxin (fig. 4.23), a potent cytotoxin iso¬ lated from a terrestrial strain of Tolypothrix conglutinata, is structurally related to the scytophycins. These compounds are also strongly antifungal and proved to be effective against some phytopathogenic fungi (Carmeli et al., 1990a).
N H
Figure 4.23 Scytophycin B (R{ = R2 = H) and tolytoxin (Rt = OH, R2 = CH3)
Cryptophycins are potent antitumour and antifungal peptolides that are found in
of Nostoc sp. The major naturally occurring representative of this class cyclic dep-
sipeptides, cryptophycin-1 (fig. 4.24), shows excellent activity against a broad spec¬ trum of solid tumours, including resistant ones, implanted in mice (Trimurtulu et al.,
1994). Over one hundred new cryptophycins were evaluated for their antitumour activ¬
ity. About twenty-five of these were obtained from the cyanobacteria (Trimurtulu et
al., 1994; Golakoti et al., 1995; Subbaraju et al., 1997). The remaining analogues were
either semi-synthesized or assembled by total synthesis. Cryptophycin-52, a new syn¬
thetic member of the cryptophycin group, is currently undergoing clinical evaluation. At high concentrations cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolimerizing spindle microtubules and disrupting chromosome organization. How¬
ever, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis
(IC50 = 11 pM) without significantly altering spindle microtubules mass or chromo¬ of some organization. Cryptophycin-52 appears to be the most potent suppressor
microtubule dynamics found so far (Panda et al., 2000). 64 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS
och3
Figure 4.24 Cryptophycin-1 (R = H) and cryptophycin-52 (R = CH3)
Tantazoles and mirabazoles (fig. 4.25, tantazole A and mirabazole A) are unusual alkaloids from the terrestrial cyanobacterium Scytonema mirabile which show murine and human solid tumour selective cytotoxicity (Carmeli et al., 1990b; 1991; 1993).
h3chn
Figure 4.25 Tantazole A and mirabazole A
and The lipid curacin A (fig. 4.26) was isolated from a strain ofLyngbya majuscula
= shows exceptional brine shrimp (LC50 = 3 ng/ml) and antiproliferative (IC50 6.8 ng/ ml in the Chinese hamster Aux Bl cell line) activities. Curacin A inhibits tubulin poly¬ merization and causes the accumulation of cells arrested in mitosis (Gerwick et al.,
1994b). 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 65
Curacins B-D, isolated also from Lyngbya majuscula, are structurally analogues of curacin A and possess antimitotic activity (Yoo and Gerwick, 1995; Marquez et al., 1998).
Figure 4.26 Curacin A
Westiellamide (fig. 4.27) is a cyclic hexapeptide from Westiellopsis prolifica. It is cytotoxic against KB and LoVo cell lines at 2 ug/ml (Prinsep et al., 1992b). Its struc¬ ture is identical with that of cyclazoline from the ascidian Lissoclinum bistratum and provides circumstantial evidence for algal symbionts playing a role in the biosynthesis of closely-related cyclic peptides found in marine tunicates (Moore, 1996).
h
Figure 4.27 Westiellamide
The dolastatins are a series of remarkable cytotoxic compounds isolated from the
Indian Ocean seahare Dolabella auricularia. The most important of these is dolastatin
10 (fig. 4.28), which is in clinical evaluation as potential new anticancer drug. The very low yields of dolastatins obtained from D. auricularia, however, imply that this mol- 66 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS lusk is not the true producer of these compounds. D. auricularia is a known generalist algal herbivore. Moreover, many metabolites that were originally isolated from seaha- res have been shown to be of dietary origin, and many analogues of dolastatin 10 were isolated from cyanobacteria. These findings demonstrate that some metabolites iso¬ lated from D. auricularia are of cyanobacterial origin (Harrigan et al., 1998).
Symplostatin 1 (fig. 4.28), a dolastatin 10 analogue from the marine cyanobacte¬ rium Symploca hydnoides, exhibited a cytotoxicity IC50 value of 0.3 ng/ml against KB cells (Harrigan et al., 1998). Symplostatin 2, a dolastatin 13 analogue, was isolated from the same algal strain (Harrigan et al., 1999). Symplostatin 3, another analogue of dolastatin 10 isolated from Symploca sp., possesses IC50 values of 3.9 and 10.3 nM against KB and LoVo cells, respectively (Luesch et al., 2002).
I o L s. OCH3 O OCH3 ^-^
^^
Figure 4.28 Dolastatin 10 (R = H) and symplostatin 1 (R = CH3)
Borophycin (fig. 4.29) is a boron-containing metabolite isolated from the marine
strains ofthe cyanobacteria Nostoc linckia and Nostoc spongiaeforme var. tenue (Hem-
scheidt et al., 1994; Banker and Carmeli, 1998). Borophycin, which is related both to
the boron-containing boromycins isolated from a terrestrial strain ofStreptomyces anti-
bioticus and to the aplasmomycins isolated from a marine strain of Streptomyces gri-
seus (actinomycetes), exhibits potent cytotoxicity against LoVo and KB cell lines, and
has recently been found to possess antimicrobial activity (Banker and Carmeli, 1998). 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 67
Figure 4.29 Borophycin
4.2.5 Enzyme Inhibitory Compounds
Several cyanobacterial cyclic depsipeptides show enzyme inhibitor activity. A90720A (fig. 4.30), for example, isolated from the terrestrial cyanobacterium Micro- chaete loktakensis, is a potent inhibitor of the serine proteases thrombin, trypsin, and plasmin. Since these enzymes are involved in the process of blood coagulation, their inhibitors could be useful therapeutic agents for cardiovascular diseases (Bonjouklian et al., 1996).
NH2
Figure 4.30 A90720A 68 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS
Micropeptins are a class of cyclic depsipeptides isolated from non-toxic Microcys¬ tis aeruginosa strains. They are known as protease inhibitors, like micropeptins A and
B (Okino et al., 1993a) and micropeptin 90 (Ishida et al., 1995), which inhibit plasmin and trypsin, but not other serine proteases such as thrombin, chymotrypsin, and elastase. Micropeptins 478-A (fig. 4.31) and B inhibit plasmin with IC50 values of 0.1 and 0.4 ug/ml, respectively. They don't inhibit trypsin, thrombin, chymotrypsin, and elastase (Ishida et al., 1997b). Micropeptins 88-A to 88-F are potent inhibitors of chy¬ motrypsin, with IC50 values between 0.4 and 10.0 ug/ml (Ishida et al., 1998). Many other micropeptins capable of protease inhibition have been isolated from Microcystis species (Murakami et al., 1997a; Banker and Carmeli, 1999; Okano et al., 1999;
Kodani et al., 1999; Reshef and Carmeli, 2001).
ho3so.
Figure 4.31 Micropeptin 478-A
A-D Other enzyme inhibitors from Microcystis aeruginosa are the cyanopeptolins
(Martin et al., 1993), cyanopeptolin S (Jakobi et al., 1995, fig. 4.32), and cyanopeptolin
SS (Jakobi et al., 1996). 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 69
NH2
HI\T NH
HO3SO
Figure 4.32 Cyanopeptolin S
Many protease inhibitors have been isolated from Oscillatoria agardhii, for exam¬ ple the oscillapeptins A-G (Itou et al., 1999; Sano and Kaya, 1996). Oscillapeptin G
(fig. 4.33) inhibits tyrosinase activity at 0.1 mM.
HO3S0 *PCH3
/> NH
\ /
HO OCH3
Figure 4.33 Oscillapeptin A 70 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS
Oscillatoria agardhii is also the source of the aeruginosas 205-A and -B, two serine protease inhibitory glycopeptides which inhibit trypsin (both with an IC50 of 0.07 ug/ml) and thrombin (IC50 of 1.5 and 0.17 ug/ml, respectively) (Shin et al.,
1997a). Many aeruginosins, for example aeruginosin 298-A (fig. 4.34), have been iso¬ lated also from Microcystis species; they inhibit trypsin, thrombin and plasmin (Kodani et al., 1998a; Ishida et al., 1999).
CH2OH
Figure 4.34 Aeruginosin 298-A
Agardhipeptin A and B (fig. 4.35) are cyclic hepta- and octapeptides isolated from
Oscillatoria agardhii. Agardhipeptin A is a plasmin inhibitor with an IC50 °f 65 ug/ml,
agardhipeptin B shows no activity (Shin et al., 1996a).
o\ J /V
V° o HN-'X
Figure 4.35 Agardhipeptin A 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 71
The microviridins (like microviridin B, fig. 4.36) are elastase, chymotrypsin, and tyrosinase inhibitors isolated from Microcystis viridis, M. aeruginosa, Oscillatoria agardhii, Nostoc minutum (Shin et al., 1996b; Murakami et al., 1997b). Microviridins are tricyclic depsipeptides, consisting of 14 amino acids (Ishitsuka et al., 1990; Okino et al., 1995).
HN
Figure 4.36 Microviridin B 72 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS
The elastase inhibitors nostopeptins A (fig. 4.37) and B have been isolated from a strain of Nostoc minutum. The have IC50 values of 1.3 and 11.0 ug/ml, respectively, and are also potent inhibitors of chymotrypsin (IC5q values of 1.4 and 1.6 pg/ml)
(Okino et al., 1997).
Figure 4.37 Nostopeptin A
Anabaenopeptins A (fig. 4.38) and B from Anabaenaflos-aquae are unusual cyclic peptides that possess a ureido linkage between two of the amino acids. They produce concentration-dependent relaxation of norepinephrine-induced contractions in rat aor¬ tic preparations (Harada et al., 1995). 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 73
OH
X -N NH
^N HN ^0 O^ >
Figure 4.38 Anabaenopeptin B
Circinamide (fig. 4.39) is the first papain inhibitor isolated from cyanobacteria. It is a linear peptide isolated from Anabaena circinalis, which inhibits papain selectively with an IC50 of 0.4 ug/ml, but have no inhibitory effect against thrombin, trypsin, chy¬ motrypsin, plasmin, and elastase (Shin et al., 1997b).
Figure 4.39 Circinamide
Radiosumin (fig. 4.40), a potent trypsin inhibitory peptide, has been isolated from the freshwater cyanobacterium Plectonema radiosum. The IC5q against trypsin is 0.14
ug/ml (Matsuda et al., 1996). Dehydroradiosumin, an analogue of radiosumin, has
been isolated from Anabaena cylindrica. It inhibits trypsin with an IC50 of 0.1 ug/ml
(Kodani et al., 1998b). 74 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS
COOH "*/, Figure 4.40 Radiosumin 4.2.6 Cardioactive Compounds Heart diseases are one of the most important afflictions of human beings and the interest in new antihypertensive or cardiotonic drugs is undiminished. A high percent¬ age of hydrophilic extracts of cyanobacteria exhibit cardiotonic activity in isolated mouse atria. Tyramine is frequently the agent responsible for increased chronotropic activity (Moore et al., 1989). The terrestrial cyanobacterium Anabaena sp. produces an unusual chlorine-con¬ taining cyclic decapeptide, puwainaphycin C (fig. 4.41), which elicits a strong inotro¬ pic effect without a concomitant chronotropic response. The ED50 of puwainaphycin C in mouse atria is 0.2 ppm, at 0.4 ppm (ED10o) the positive inotropic effect is 325-350% with only a slight increase in chronotropic activity to 128%. This cardioactive drug is accompanied by four structurally related but markedly less active cyclic decapeptides, puwainaphycins A, B, D, and E (Moore et al., 1989; Gregson et al., 1992). 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 75 Figure 4.41 Puwainaphycin C Scytonemin A (fig. 4.42) is a major metabolite of a Scytonema sp. strain and pos¬ sesses potent calcium antagonistic properties. On atria calcium antagonistic effects were observed at 5 ug/ml (by comparison diltiazem was active at 2.5 ug/ml). On rat portal vein calcium blocking was observed at 20 pg/ml (diltiazem showed activity at 0.5 ug/ml). Scytonemin A is weakly active against a wide spectrum of bacteria and fungi, and is mildly cytotoxic (Helms et al., 1988). 76 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS illOH OH Figure 4.42 Scytonemin A The angiotensin converting enzyme (ACE) participates in regulating blood pres¬ sure in the renin-angiotensin system; its inhibitors are used as antihypertensive drugs. Many human and animal studies have revealed that ingestion of the cyanobacterium Spirulina platensis, the most popular edible microalga in Japan since ancient times, results in decreased blood pressure. It has been demonstrated that the peptidic fractions of S. platensis decrease the blood pressure by regulating the renin-angiotensin system. Five ACE-inhibitory tri- and tetrapeptides could be isolated and identified; they have IC5o values ranging from 11.4 to 57.1 uM (Suetsuna and Chen, 2001). Microginin (fig. 4.43) is a linear peptide isolated from Microcystis aeruginosa with ACE-inhibitory activity. Microginin inhibits the ACE with an IC50 of 7.0 ug/ml, but doesn't inhibit papain, trypsin, chymotrypsin, and elastase (Okino et al., 1993b). Other microginin-type peptides have been isolated from Microcystis aeruginosa strains; 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 77 some of them exhibit ACE-inhibitory activity (Neumann et al., 1997; Ishida et al., 2000). OH 0 Figure 4.43 Microginin 4.2.7 Anti-inflammatory Compounds The first diterpenoid isolated from a cyanobacterium is tolypodiol (fig. 4.44), from Tolypothrix nodosa. Tolypodiol shows strong anti-inflammatory activity in the mouse ear edema assay, with an ED50 of 30 ug/ear (Prinsep et al., 1996). Figure 4.44 Tolypodiol The cyanobacterium Rivulariafirma contains seven brominated biindoles with sig¬ nificant anti-inflammatory activity. Five of these biindoles are optically active, with the chirality in each case being due to restricted rotation only. Therefore, these com¬ pounds are representative of a very small class of natural products which are chiral only 78 4.2 CYANOBACTERIA AS A SOURCE OF MEDICINAL AGENTS because of restricted rotation within the molecule (Norton and Wells, 1982; Hodder and Capon, 1991). Figure 4.45 (+)-2,3\5,5'-tetrabromo-7'-methoxy-3,4'-bi-l//-indole 4.2.8 Immunosuppressive Compounds Microcolins A (fig. 4.46) and B are two linear lipopeptides isolated from a speci¬ men of Lyngbya majuscula. They exhibit strong immunosuppressive activity and are more potent than cyclosporin A in suppressing lymphocyte proliferation induced by most stimuli (Koehn et al., 1992; Zhang et al., 1997). \ OH / OAc // Figure 4.46 Microcolin A 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 79 4.3 References Banker R, Carmeli S (1998): Tenuecyclamides A-D, cyclic hexapeptides from the cyanobacterium Nostoc spongiaeforme var. tenue. J. Nat. Prod. 61: 1248-1251. Banker R, Carmeli S (1999): Inhibitors of serine proteases from a waterbloom of the cyanobacterium Microcystis sp. Tetrahedron 55: 10835-10844. Bonjouklian R, Smitka TA, Doolin LE, Molloy RM, Debono M, Shaffer SA, Moore RE, Stewart JB, Patterson GML (1991): Tjipanazoles new antifungal agents from the blue-green alga Tolypothrix tjipanasensis. Tetrahedron 47: 7739-7750. Bonjouklian R, Smitka TA, Hunt AH, Occolowitz JL, Perun TJ, Doolin L, Steven¬ a son S, Knauss L, Wijayaratne R, Szewczyk S, Patterson GML (1996): A90720A, serine protease inhibitor isolated from a terrestrial blue-green alga Microchaete loktak¬ ensis. Tetrahedron 52: 395-404. Bonnard I, Rolland M, Francisco C, Banaigs B (1997): Total structure and biolog¬ ical properties of laxaphycins A and B, cyclic lipopeptides from the marine cyanobac¬ terium Lyngbya majuscula. Lett. Pept. Sei. 4: 289-292. Burja AM, Banaigs B, Abou-Mansour E, Burgess JG, Wright PC (2001): Marine - 57: 9347-9377. cyanobacteria a prolific source of natural products. 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Frankmölle WP, Knübel G, Moore RE, Patterson GML (1992b): Antifungal cyclic peptides from the terrestrial blue-green alga Anabaena laxa. II. Structures of laxaphy- cins A, B, D and E. J. Antibiot. 45: 1458-1466. Gerwick WH, Mrozek C, Moghaddam MF, Agarwal SK (1989): Novel cytotoxic peptides from the tropical marine cyanobacterium Hormothamnion enteromorphoides. 1. Discovery isolation and initial chemical and biological characterization of the hor- mothamnins from wild and cultured material. Experientia 45: 115-121. Gerwick WH, Roberts MA, Proteau PJ, Chen J-L (1994a): Screening cultured marine microalgae for anticancer-type activity. J. Appl. Phycol. 6: 143-149. 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 81 Gerwick WH, Proteau PJ, Nagle DG, Hamel E, Blokhin A, Slate DL (1994b): Structure of curacin A, a novel antimitotic, antiproliferative, and brine shrimp toxic natural product from the marine cyanobacterium Lyngbya majuscula. J. Org. Chem. 59: 1243-1245. 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Ishida K, Murakami M, Matsuda H, Yamaguchi K (1995): Micropeptin 90, a plas¬ min and trypsin inhibitor from the blue-green alga Microcystis aeruginosa (NIES-90). Tetrahedron Lett. 36: 3535-3538. Ishida K, Matsuda H, Murakami M, Yamaguchi K (1997a): Kawaguchipeptin B, an antibacterial cyclic undecapeptide from the cyanobacterium Microcystis aeruginosa. J. Nat. Prod. 60: 724-726. Ishida K, Matsuda H, Murakami M, Yamaguchi K (1997b): Micropeptins 478-a and -B, plasmin inhibitors from the cyanobacterium Microcystis aeruginosa. J. Nat. Prod. 60: 184-187. Ishida K, Matsuda H, Murakami M (1998): Micropeptins 88-a to 88-F, chymot¬ rypsin inhibitors from the cyanobacterium Microcystis aeruginosa (NIES-88). Tetra¬ hedron 54: 5545-5556. Ishida K, Okita Y, Matsuda H, Okino T, Murakami M (1999): Aeruginosins, pro¬ tease inhibitors from the cyanobacterium Microcystis aeruginosa. Tetrahedron 55: 10971-10988. Ishida K, Kato T, Murakami M, Watanabe M, Watanabe MF (2000): Microginins, zinc metalloproteases inhibitors from the cyanobacterium Microcystis aeruginosa. Tet¬ rahedron 56: 8643-8656. Ishitsuka M, Kusumi T, Kakisawa H, Kaya K, Watanabe MM (1990): Microviridin viridis. J. a novel tricyclic depsipeptide from the toxic cyanobacterium Microcystis Am. Chem. Soc. 112: 8180-8182. 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 83 Itou Y, Ishida K, Shin HJ, Murakami M (1999): Oscillapeptins A to F, serine pro¬ tease inhibitors from the three strains of Oscillatoria agardhii. Tetrahedron 55: 6871- 6882. Jaki B, Orjala J, Biirgi H-R, Sticher O (1999): Biological screening of cyanobacte¬ ria for antimicrobial and molluscicidal activity, brine shrimp lethality and cytotoxicity. Pharm. Biol. 37: 138-143. Jakobi C, Oberer L, Quiquerez C, Koenig WA, Weckesser J (1995): Cyanopeptolin S, a sulfate-containing depsipeptide from a water bloom of Microcystis sp. FEMS Microbiol. Lett. 129: 129-133. Jakobi C, Rinehart KL, Neuber R, Mez K, Weckesser J (1996): Cyanopeptolin SS, a disulphated depsipeptide from a water bloom: structural elucidation and biological activities. Phycologia 35: 111-116. Jones AK (1988): Algal extracellular products - antimicrobial substances. In: Bio¬ chemistry of the algae and cyanobacteria (Rogers LJ, Gallon JR, eds.), Clarendon Press, Oxford, pp. 257-281. Kajiyama S, Kanzaki H, Kawazu K, Kobayashi A (1998): Nostofungicidine, an antifungal lipopeptide from the field-grown terrestrial blue-green alga Nostoc com¬ mune. Tetrahedron Lett. 39: 3737-3740. Knübel G, Larsen LK, Moore RE, Levine IA, Patterson GML (1990): Cytotoxic antiviral indolocarbazoles from a blue-green alga belonging to the Nostocaceae. J. Antibiot. 43: 1236-1239. Kodani S, Ishida K, Murakami M (1998a): Aeruginosin 103-A, a thrombin inhibi¬ tor from the cyanobacterium Microcystis viridis. J. Nat. Prod. 61: 1046-1048. Kodani S, Ishida K, Murakami M (1998b): Dehydroradiosumin, a trypsin inhibitor from the cyanobacterium Anabaena cylindrica. J. Nat. Prod. 61: 854-856. Kodani S, Suzuki S, Ishida K, Murakami M (1999): Five new cyanobacterial pep¬ tides from water bloom materials of lake Teganuma (Japan). FEMS Microbiol. Lett. 178: 343-348. Koehn FE, Longley RE, Reed JK (1992): Microcolins A and B, new immunosup¬ pressive peptides from the blue-green alga Lyngbya majuscula. J. Nat. Prod. 55: 613- 619. Larsen LK, Moore RE, Patterson GML (1994): ß-carbolines from the blue-green alga Dichothrix baueriana. J. Nat. Prod. 57: 419-421. 84 4.3 REFERENCES Lau AF, Siedlecki J, Anleitner J, Patterson GML, Caplan FR, Moore RE (1993): Inhibition of reverse transcriptase activity by extracts of cultured blue-green algae (Cyanophyta). Planta Med. 59: 148-151. Loya S, Reshef V, Mizrachi E, Silberstein C, Rachamim Y, Carmeli S, Hizi A (1998): The inhibition of the reverse transcriptase of HIV-1 by the natural sulfogly¬ colipids from cyanobacteria: Contribution of different moieties to their high potency. J. Nat. Prod. 61: 891-895. Luesch H, Yoshida WY, Moore RE, Paul VJ, Mooberry SL, Corbett TH (2002): Symplostatin 3, a new dolastatin 10 analogue from the marine cyanobacterium Sym¬ ploca sp. VP452. J. Nat. Prod. 65: 16-20. Marquez B, Verdier-Pinard P, Hamel E, Gerwick WH (1998): Curacin D, an anti¬ mitotic agent from the marine cyanobacterium Lyngbya majuscula. Phytochemistry 49: 2387-2389. Martin C, Oberer L, Ino T, Koenig WA, Busch M, Weckesser J (1993): Cyanopep- tolins, new depsipeptides from the cyanobacterium Microcystis sp. PCC 7806. J. Anti¬ biot. 46: 1550-1556. Matsuda H, Okino T, Murakami M, Yamaguchi K (1996): Radiosumin, a trypsin inhibitor from the blue-green alga Plectonema radiosum. J. Org. Chem. 61: 8648- 8650. Minehan TG, Cook-Blumberg L, Kishi Y, Prinsep MR, Moore RE (1999): Revised structure of tolyporphin A. Angew. Chem. Int. Ed. 38: 926-928. Moon SS, Chen JL, Moore RE, Patterson GML (1992): Calophycin, a fungicidal cyclic decapeptide from the terrestrial blue-green alga Calothrix fusca. J. Org. Chem. 57: 1097-1103. Moore RE, Bornemann V, Niemczura WP, Gregson JM, Chen JL, Norton TR, Patterson GML, Helms GL (1989): Puwainaphycin C, a cardioactive cyclic peptide 1 'X 1 'X from the blue-green alga Anabaena BQ-16-1. Use of two-dimensional C- C and 1 ^ 1 ^ C- N correlation spectroscopy in sequencing the amino acid units. J. Am. Chem. Soc. Ill: 6128-6132. Moore RE, Ohtani I, Moore BS, de Koning CB, Yoshida WY, Runnegar MTC, Carmichael WW (1993): Cyanobacterial toxins. Gazz. Chim. Ital. 123: 329-336. Moore RE (1996): Cyclic peptides and depsipeptides from cyanobacteria: a review. J. Ind. Microbiol. 16: 134-143. 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 85 Murakami M, Kodani S, Ishida K, Matsuda H, Yamaguchi K (1997a): Micropeptin 103, a chymotrypsin inhibitor from the cyanobacterium Microcystis viridis (NIES- 103). Tetrahedron Lett. 38: 3035-3038. Murakami M, Sun Q, Ishida K, Matsuda H, Okino T, Yamaguchi K (1997b): Microviridins, elastase inhibitors from the cyanobacterium Nostoc minutum (NIES- 26). Phytochemistry 45: 1197-1202. Neumann U, Forchert A, Flury T, Weckesser J (1997): Microginin FR1, a linear peptide from a water bloom of Microcystis species. FEMS Microbiol. Lett. 153: 475- 478. Norton RS, Wells RJ (1982): A series of chiral polybrominated biindoles from the marine blue-green alga Rivularia firma. Application of C NMR spin-lattice relax¬ ation data and ^C^H coupling constants to structure elucidation. J. Am. Chem. Soc. 104: 3628-3635. Ogino J, Moore RE, Patterson GML, Smith CD (1996): Dendroamides, new cyclic hexapeptides from a blue-green alga. Multidrug-resistance reversing activity of den- droamide A. J. Nat. Prod. 59: 581-586. Okano T, Sano T, Kaya K (1999): Micropeptin T-20, a novel phosphate-containing cyclic depsipeptide from the cyanobacterium Microcystis aeruginosa. Tetrahedron Lett. 40: 2379-2382. Okino T, Murakami M, Haraguchi R, Munekata H, Matsuda H, Yamaguchi K (1993 a): Micropeptins A and B, plasmin and trypsin inhibitors from the blue-green alga Microcystis aeruginosa. Tetrahedron Lett. 34: 8131-8134. Okino T, Matsuda H, Murakami M, Yamaguchi K (1993b): Microginin, an angio- tensin-converting enzyme inhibitor from the blue-green alga Microcystis aeruginosa. Tetrahedron Lett. 34: 501-504. Okino T, Matsuda H, Murakami M, Yamaguchi K (1995): New microviridins, elastase inhibitors from the blue-green alga Microcystis aeruginosa. Tetrahedron 51: 10679-10686. Okino T, Qi S, Matsuda H, Murakami M, Yamaguchi K (1997): Nostopeptins A and B, elastase inhibitors from the cyanobacterium Nostoc minutum. J. Nat. Prod. 60: 158-161. Panda D, Ananthnarayan V, Larson G, Shih C, Jordan MA, Wilson L (2000): Inter¬ action of the antitumor compound cryptophycin-52 with tubulin. Biochemistry 39: 86 4.3 REFERENCES 14121-14127. Papendorf O, König GM, Wright AD (1998): Hierridin B and 2,4-dimethoxy-6- heptadecyl-phenol, secondary metabolites from the cyanobacterium Phormidium ecto- carpi with antiplasmodial activity. Phytochemistry 49: 2383-2386. Patterson GML, Baldwin CL, Bolis CM, Caplan FR, Karuso H, Larsen LK, Levine IA, Moore RE, Nelson CS, Tschappat KD, Twang GD, Furusawa E, Furusawa S, Norton TR, Raybourne RB (1991): Antineoplastic activity of cultured blue-green algae (Cyanophyta). J. Phycol. 27: 530-536. Patterson GML, Baker KK, Baldwin CL, Bolis CM, Caplan FR, Larsen LK, Levine IA, Moore RE, Nelson CS, Tschappat KD, Twang GD, Boyd MR, Cardellina JH, Col¬ lins RP, Gustafson KR, Snader KM, Weislow OS, Lewin RA (1993): Antiviral activity of cultured blue-green algae (Cyanophyta). J. Phycol. 29: 125-130. Patterson GML, Larsen LK, Moore RE (1994): Bioactive natural products from blue-green algae. J. Appl. Phycol. 6: 151-157. Pergament I, Carmeli S (1994): Schizotrin A; a novel antimicrobial cyclic peptide from a cyanobacterium. Tetrahedron Lett. 35: 8473-8476. Prinsep MR, Caplan FR, Moore RE, Patterson GML, Smith CD (1992a): Tolypor- phin, a novel multidrug resistance reversing agent from the blue-green alga Tolypothrix nodosa. J. Am. Chem. Soc. 114: 385-387. Prinsep MR, Moore RE, Levine IA, Patterson GML (1992b): Westiellamide, a bis- tratamide-related cyclic peptide from the blue-green alga Westiellopsis prolifica. J. Nat. Prod. 55: 140-142. Prinsep MR, Patterson GML, Larsen LK, Smith CD (1995): Further tolyporphins from the blue-green alga Tolypothrix nodosa. Tetrahedron 51: 10523-10530. Prinsep MR, Thomson RA, West ML, Wylie BL (1996): Tolypodiol, an antiinflam¬ matory diterpenoid from the cyanobacterium Tolypothrix nodosa. J. Nat. Prod. 59: 786-788. Prinsep MR, Patterson GML, Larsen LK, Smith CD (1998): Tolyporphin J and K, two further porphinoid metabolites from the cyanobacterium Tolypothrix nodosa. J. Nat. Prod. 61: 1133-1136. Reshef V, Mizrachi E, Maretzki T, Silberstein C, Loya S, Hizi A, Carmeli S (1997): New acylated sulfoglycolipids and digalactolipids and related known glycolipids from cyanobacteria with a potential to inhibit the reverse transcriptase of HIV-1. J. Nat. 4 BIOLOGICALLY ACTIVE COMPOUNDS FROM CYANOBACTERIA 87 Prod. 60: 1251-1260. Reshef V, Carmeli S (2001): Protease inhibitors from a water bloom of the cyano¬ bacterium Microcystis aeruginosa. Tetrahedron 57: 2885-2894. Rickards RW, Rothschild JM, Willis AC, de Chazal NM, Kirk J, Kirk K, Saliba KJ, Smith GD (1999): Calothrixins A and B, novel pentacyclic metabolites from Calothrix cyanobacteria with potent activity against malaria parasites and human cancer cells. Tetrahedron 55: 13513-13520. Rinehart KL, Shaw PD, Shield LS, Gloer JB, Harbour GC, Koker MES, Samain D, Schwartz RE, Tymiak AA, Weller DL, Carter GT, Munro MHG, Hughes RG, Renis HE, Swynenberg EB, Stringfellow DA, Vavra JJ, Coats JH, Zurenko GE, Kuentzel SL, Li LH, Bakus GJ, Brusca RC, Craft LL, Young DN, Connor JL (1981): Marine natural products as sources of antiviral, antimicrobial, and antineoplastic agents. Pure Appl. Chem. 53: 795-817. Sano T, Kaya K (1996): Oscillapeptin G, a tyrosinase inhibitor from toxic Oscilla¬ toria agardhii. J. Nat. Prod. 59: 90-92. Shin HJ, Matsuda H, Murakami M, Yamaguchi K (1996a): Agardhipeptins A and B, two new cyclic hepta- and octapeptide, from the cyanobacterium Oscillatoria agar¬ dhii (NIES-204). Tetrahedron 52: 13129-13136. Shin HJ, Murakami M, Matsuda H, Yamaguchi K (1996b): Microviridins D-F, serine protease inhibitors from the cyanobacterium Oscillatoria agardhii (NIES-204). Tetrahedron 52: 8159-8168. Shin HJ, Matsuda H, Murakami M, Yamaguchi K (1997a): Aeruginosins 205A and -B, serine protease inhibitory glycopeptides from the cyanobacterium Oscillatoria agardhii (NIES-205). J. Org. Chem. 62: 1810-1813. Shin HJ, Matsuda H, Murakami M, Yamaguchi K (1997b): Circinamide, a novel papain inhibitor from the cyanobacterium Anabaena circinalis (NIES-41). Tetrahe¬ dron 53: 5747-5754. Sivonen K (1996): Cyanobacterial toxins and toxin production. Phycologia 35: 12- 24. Skulberg OM, Carmichael WW, Andersen RA, Matsunaga S, Moore RE, Skulberg R (1992): Investigations of a neurotoxic oscillatorialean strain (Cyanophyceae) and its toxin. Isolation and characterization of homoanatoxin-a. Environ. Toxicol. Chem. 11: 321-329. 88 4.3 REFERENCES Stewart JB, Bornemann V, Chen JL, Moore RE, Caplan FR, Karuso H, Larsen LK, Patterson GML (1988): Cytotoxic fungicidal nucleosides from blue green algae belonging to the Scytonemataceae. J. Antibiot. 46: 1048-1056. Stratmann K, Burgoyne DL, Moore RE, Patterson GML, Smith CD (1994): Hapal¬ osin, a cyanobacterial cyclic depsipeptide with multidrug-resistance reversing activity. J. Org. Chem. 59: 7219-7226. Subbaraju GV, Golakoti T, Patterson GML, Moore RE (1997): Three new crypto¬ phycins from Nostoc sp. GSV 224. J. Nat. Prod. 60: 302-305. Suetsuna K, Chen J-R (2001): Identification of antihypertensive peptides from pep¬ tic digest of two microalgae, Chlorella vulgaris and Spirulina platensis. Mar. Biotech- nol. 3: 305-309. Trimurtulu G, Ohtani I, Patterson GML, Moore RE, Corbett TH, Valeriote FA, Demchik L (1994): Total structures of cryptophycins, potent antitumor depsipeptides from the blue-green alga Nostoc sp. strain GSV 224. J. Am. Chem. Soc. 116: 4729- 4737. Yoo HD, Gerwick WH (1995): Curacins B and C, new antimitotic natural products from the marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 58: 1961-1965. Zhang LH, Longley RE, Koehn FE (1997): Antiproliferative and immunosuppres¬ sive properties of microcolin A, a marine-derived lipopeptide. Life Sei. 60: 751-762. 5 THE GENERA SCYTONEMA AND CYLINDROSPERMUM 89 5 The Genera Scytonema and Cylin¬ drospermum 5.1 The Genus Scytonema The genus Scytonema belongs to the order Nostocales and to the family Scytone- mataceae (Komârek and Anagnostidis, 1989). The trichomes of the genus Scytonema are always isopolar and enveloped by a sheath. The apical cells are rounded or widened-rounded, sometimes with more or less spherical and vacuolized terminal cells. The false branching occurs obligatorily in Scy¬ tonema and usually originates after disintegration of a trichome between two vegeta¬ tive cells, sometimes after the death of one or several vegetative cells. From the divided trichome usually two branches arise aside, initially forming sometimes characteristic loops. The initiation of branching is without connection with a heterocyte. Cell division proceeds always perpendicularly to the long axis of the trichome, and only uniseriate trichomes develop. The heterocytes are always intercalar, akinetes are facultatively formed (Komarek and Anagnostidis, 1989). Figure 5.1 Scytonema sp. with filaments showing false branching and intercalary hetero¬ cytes (Humm and Wicks, 1980) 90 5.2 THE GENUS CYLINDROSPERMUM The genus Scytonema is usually subaerial or terrestrial; it forms coarse, felty masses on moist stones, wood, and soil (Bold and Wynne, 1985). 5.2 The Genus Cylindrospermum According to Komârek and Anagnostidis (1989), the genus Cylindrospermum belongs to the order Nostocales and to the family Nostocaceae, subfamily Ana- baenoideae. The trichomes are always isopolar symmetric and occur singly or embedded in an amorphous matrix. The trichomes of Cylindrospermum never form branches, neither true nor false. The heterocytes develop only from the terminal cells, usually at both ends of the trichome. Akinetes develop after fusion of several neighbouring cells just beside of heterocytes (paraheterocytic development); this process leads to the symmet¬ ric structure of the trichomes. Cell division proceeds, as in the genus Scytonema, always perpendicularly to the long axis of the trichome, and only uniseriate trichomes Both develop. The cells of the genus Cylindrospermum always lack of gas vesicles. aquatic and terrestrial species of Cylindrospermum are known (Bold and Wynne, 1985; Komârek and Anagnostidis, 1989). Figure 5.2 Cylindrospermum sp. with heterocyte (arrow) and akinete (stippled) at each ter¬ minus (Castenholz, 1994) 5 THE GENERA SCYTONEMA AND CYLINDROSPERMUM 91 5.3 References Bold HC, Wynne MJ (1985): Divisions Cyanophyta and Prochlorophyta. In: Intro¬ duction to the algae, Prentice-Hall, Englewood Cliffs, pp. 34-69. Castenholz RW (1994): Oxygenic phototrophic bacteria. In: Bergey's manual of determinative bacteriology (Holt JG, ed.), Williams & Wilkins, Baltimore, pp. 377- 425. Humm HJ, Wicks SR (1980): Introduction and guide to the marine bluegreen algae. John Wiley & Sons, New York, p. 87. Komârek J, Anagnostidis K (1989): Modern approach to the classification system of cyanophytes. 4-Nostocales. Arch. Hydrobiol. Suppl. 82: 247-345. 92 6.1 ANTIBACTERIAL ACTIVITY 6 Assays for Bioactivity A large number of bioassays are available for the bioactivity-directed screening and the bioactivity-guided fractionation leading to the isolation and characterization of used for these pure biologically active compounds from natural sources. Bioassays pur¬ and have statistical cor¬ poses should be simple to perform, fast, inexpensive, in-house relation with desired bioactivities (Anderson et al., 1991). In the crude extract, active principles are generally present at low concentrations only. The test systems have, therefore, to be sensitive enough to detect them reliably (Hamburger and Hostettmann, 1991). Both positive and negative controls should be incorporated into the bioassay design. The negative control ensures that it is not the procedure itself which is respon¬ a marker sible for any observed activity, while the positive control provides against which the potency of an active extract or compound can be measured (Cole, 1994). In this project, antibacterial, antifungal, and cytotoxicity assays were performed. 6.1 Antibacterial Activity Despite the wide availability of clinically useful antibiotics, a continuing search for have new anti-infective agents remains indispensable. Some of the major antibiotics indeed considerable drawbacks in terms of limited antimicrobial spectrum or serious side-effects. Moreover, the combination of the genetic versatility of microbes and widespread overuse of antibiotics has led to increasing clinical resistance of previously sensitive microorganisms and the emergence of previously uncommon infections. The search for new molecules exhibiting prominent activities against infectious microor¬ ganisms is therefore a task to be pursued. The choice of test organisms will depend greatly on the purpose of the investiga¬ tion. If the investigation is of general character, the test organisms selected should be as diverse as possible and representative of all important groups of pathogenic bacteria (Vanden Berghe and Vlietinck, 1991). Test organisms used in the present work are the gram-positive cocci Staphylococcus aureus and S. epidermidis, the gram-positive 6 ASSAYS FOR BIOACTIVITY 93 spore-forming rod Bacillus cereus, and the gram-negative rods Escherichia coli and Pseudomonas aeruginosa. 6.1.1 Agar Overlay Method TLC In the agar overlay method, an amount of an extract or a fraction is put on a plate, and the plate is covered with a suspension of bacteria in agar. Incubation permits growth of the bacteria. Zones of inhibition are then visualized by a dehydrogenase- activity-detecting agent, like methylthiazolyltetrazolium chloride (MTT) (Hamburger and Cordell, 1987). and the This assay has the advantage to be quick, easy to perform, relatively cheap, results are easy to interpret, since the inhibition of growth is frequently quite readily apparent. Moreover, before covering the plate with the suspension of bacteria in agar, the components of the extract or fraction can be separated using TLC in a suitable sol¬ vent. In this way, the antibacterial compounds in the mixture can be detected (Cole, 1994). In this work, the agar overlay method was employed for the antimicrobial screen¬ ing. 6.1.2 Minimal Inhibitory Concentration (MIC) For pure compounds, the minimal inhibitory concentration (MIC) is detected by means of a dilution method. The samples are mixed with a suitable medium, which has been previously incubated with the test organism. After incubation, growth of the microorganism may be determined by direct visual (i.e. aqueous solution of a tetrazo- lium salt) or turbidimetric comparison of the test culture with a control culture which did not receive an addition of the sample being tested. Usually a series of dilutions of the original sample in the culture medium is made and then inoculated with the test organism. After incubation, the endpoint of the test is taken as the highest dilution which will just prevent perceptible growth of the test organism (MIC-value) (Vanden Berghe and Vlietinck, 1991). 94 6.2 ANTIFUNGAL ACTIVITY 6.2 Antifungal Activity There is a growing interest in the search for antifungal drugs, mainly because of the increasing incidence of opportunistic systemic mycoses, associated primarily with patients with AIDS or those receiving treatment with immunosuppressive agents (Hadacek and Greger, 2000). There are a number of different types of antifungal assays. In vitro assays include examination of the effects of a compound or extract on fungal spore germination, on radial growth or increase in fungal mass. In vivo assays are also available for the exam¬ ination of the effects of test compounds and extracts on biotrophic fungi (Cole, 1994). In this work antifungal activity was tested against Candida albicans, using the same methods as for bacteria. 6.3 Brine Shrimp (Artemia salina) Lethality Assay Bioactive compounds are almost always toxic at high doses. Thus, in vivo lethality in a simple zoologie organism can be used as a convenient monitor for screening and fractionation in the discovery of new bioactive natural products. at low cost in The eggs of brine shrimp, Artemia salina Leach, are readily available pet shops as a food for tropical fish, and they remain viable for years in the dry state. Upon being placed in artificial sea water, the eggs hatch within 48 hours, providing large numbers of larvae (nauplii). Artemia salina has been proposed as a low cost sub¬ the stitute for cytotoxicity assays; because most antitumour compounds are cytotoxic, lethality of a simple organism like brine shrimp can be used as a prescreen method and allows the isolation of antitumour compounds (Meyer et al., 1982; McLaughlin et al., 1991). 6.4 KB-celI Activity The majority of research programmes dealing with the isolation and identification 6 ASSAYS FOR BIOACTIVITY 95 of potential antitumour compounds from natural sources have relied on cytotoxicity for bioassay-directed fractionation. Primarily, cultured KB or P388 cells have been employed. The procedure involves treating the cells with various concentrations of the KB P388 test substance or extract, and assessing cell growth after incubation with or cells. The number of cells surviving the treatment is determined and expressed relative to the negative controls (Swanson and Pezzuto, 1990). Although these procedures are that be of novel proven to be effective for the isolation of cytotoxic compounds may structure, it is often the case that compounds active with KB cells are not active with in Pez¬ vivo tumour models. In many cases, the isolates are simply toxic (Suffness and zuto, 1991). In the current study, KB cells have been utilized. The KB cell line is a HeLa cell line(ATCCCCL17). 6.5 References Anderson JE, Goetz CM, McLaughlin JL, Suffness M (1991): A blind comparison of simple bench-top bioassays and human tumour cell cytotoxicities as antitumor pre- screens. Phytochem. Anal. 2: 107-111. Cole MD (1994): Key antifungal, antibacterial and anti-insect assays - a critical review. Biochem. Syst. Ecol. 22: 837-856. Hadacek F, Greger H (2000): Testing of antifungal natural products: methodolo¬ gies, comparability of results and assay choice. Phytochem. Anal. 11: 137-147. Hamburger MO, Cordell GA (1987): A direct bioautographic TLC assay for com¬ pounds possessing antibacterial activity. J. Nat. Prod. 50: 19-22. Hamburger M, Hostettmann K (1991): Bioactivity in plants: the link between phy- tochemistry and medicine. Phytochemistry 30: 3864-3874. McLaughlin JL, Chang C.-J., Smith DL (1991): "Bench-top" bioassays for the dis¬ Products Chem¬ covery of bioactive natural products: an update. In: Studies in Natural istry, Vol 9 (Atta-ur-Rahman, ed.), Elsevier Science Publishers B.V., Amsterdam, pp. 383-409. Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL (1982): Brine shrimp: a convenient general bioassay for active plant constituents. 96 6.5 REFERENCES Planta Med. ¥5:31-34. Suffness M, Pezzuto JM (1991): Assays related to cancer drug discovery. In: Meth¬ ods in plant biochemistry Vol 6 (Hostettmann K, ed.), Academic Press, London, pp. 71-133. Swanson SM, Pezzuto JM (1990): Bioscreening technique for cytotoxic potential and ability to inhibit macromolecule biosynthesis. In: Drug Bioscreening. Drug Eval¬ uation Techniques in Pharmacology (Thompson EB, ed.), VCH, New York, pp. 273- 297. Vanden Berghe DA, Vlietinck AJ (1991): Screening methods for antibacterial and antiviral agents from higher plants. In: Methods in plant biochemistry Vol 6 (Hostett¬ mann K, ed.), Academic Press, London, pp. 47-69. 7 METHODOLOGY OF ISOLATION PROCEDURE 97 7 Methodology of Isolation Proce¬ dure 7.1 General Isolation Strategy 7.1.1 Isolation of Intracellular Compounds The isolation of a natural product can often be broken down into three main stages: extraction, fractionation, and purification. The first stage, the extraction, is the release of compounds from the cell mass and the removal of bulk of biomass. Most of the bulk of the biomass exists as fairly inert, insoluble, and often polymeric material, such as the microbial cell wall or the cellulose of plants. The first step of the extraction is to release and solubilize the smaller second¬ This can be ary metabolites by a thorough extraction with an organic solvent or water. which acts done by a series of stepwise extractions, using solvents of varying polarity, which as the first fractionation step, or by using a single solvent such as methanol, should dissolve most natural products. The initial extract is usually still a pretty complex mixture, and the aim of the sec¬ in ond step, the fractionation, is to remove a large portion of the unwanted material a fairly low-resolution step. Such a step may involve a vacuum liquid chromatography, The aim is to obtain a an open silica column, or a series of liquid-liquid extractions. mixture containing all the natural products of interest. The third general stage, the purification, is often a high-resolution separation to separate those components still remaining. This third step usually involves preliminary work modifying and altering conditions to achieve the desired separation before pre¬ parative work is carried out. This final stage is often achieved by HPLC (Cannell, 1998). 98 7.1 GENERAL ISOLATION STRATEGY 7.1.2 Isolation of Extracellular Compounds Microorganisms typically produce an extracellular product at low concentration (<3% w/v). Therefore, the primary goals of the initial capture stage are to concentrate the product, separate the product from the biomass, and purify it from impurities. The first step of isolation is the separation of the broth from the cell mass by means of fil¬ tration or separation. The second step is a solvent-extraction or, as in the present project, a solid phase extraction. The final purification is typically achieved via prepar¬ ative chromatography (Gailliot, 1998). 7.1.2.1 Solid Phase Extraction Solid phase extraction utilizes adsorbents for sample cleanup, trace enrichment, and fractionation of extracellular compounds in a crude fermentation broth. This method exploits the same product/sorbent interactions used in chromatography. Sepa¬ ration efficiency is largely a function of the sample application flow rate and the sam¬ ple/adsorbent volume ratio. As with any chromatographic method, there is a direct rela¬ tionship between flow rate and separation efficiency, but an inverse relationship between flow rate and time required for separation. Lower flow rates improve separa¬ tions but increase assay sample time. When flow rates are too high, the product may not have sufficient contact with the adsorbent. Overloading the sample can lead to variable recovery or loss of resolution. There are three general adsorbent classes used most often in solid phase extraction applications: polar, ion-exchange, and nonpolar. Each class of adsorbent-resins exhib¬ its unique properties of retention and selectivity based on interactive properties of the products and the adsorbent surface. In the present work, Amberlite XAD-2 was used. Amberlite XAD-2 is a polystyrene-divinylbenzene neutral resin with no ion-exchange groups, where the adsorption process occurs through hydrophobic forces. Resin parti- cles are typically 40 urn particles with either 60 or 300 A pores. The adsorbent's reten¬ tion capacity is approximately 5% of the adsorbent's weight. Prior to sample loading, the column should be prewashed with a strong eluent. After prewashing, the column must be equilibrated with an eluent of similar polarity to the sample solution. The sample should be loaded onto the column at less than 5 ml/ 7 METHODOLOGY OF ISOLATION PROCEDURE 99 min to achieve a narrow band of adsorbed product at the top of the column-bed. After loading, one or more suitable solvents for elution must be applied (Gailliot, 1998). 7.2 Chromatographic Methods 7.2.1 Thin Layer Chromatography Thin layer chromatography (TLC) is the simplest of all the widely used chromato¬ graphic methods to perform. A suitable closed vessel containing solvent and a coated plate are all that are required to carry out separations and qualitative and semiquantita¬ tive analysis. With optimization of techniques and materials, highly efficient separa¬ tions and accurate and precise quantification can be achieved (Sherma, 1996). TLC separations can also be used to select column chromatography conditions. TLC conditions that give a useful Rf value, i.e., compounds separate from the majority of other components without staying at the origin or with the solvent front, can be approximately transferred to column chromatography (Cannell, 1998). Detection is most simple when the compounds of interest are naturally colored or fluorescent or absorb ultraviolet light. However, application of a visualization reagent by spraying is usually required to produce color or fluorescence for most compounds (Sherma, 1996). 7.2.2 Vacuum Liquid Chromatography Vacuum liquid chromatography (VLC) is a simple and inexpensive chromato¬ graphic system, capable of producing good resolution in a short time. This technique involves the use of reduced pressure to increase the flow rate of the mobile phase through the stationary phase. The VLC column is a sintered glass funnel, which is vac¬ uum dry-packed with TLC grade sorbents. Due to the vacuum, stationary phases with a smaller particle size than in open column chromatography can be used, achieving a better separation. A preabsorbent layer of celite is also added, in order to protect the surface of the sorbent and to ensure that the sample moves onto the column with the 100 7.2 CHROMATOGRAPHIC METHODS solvent front as a uniform narrow band. Because the top of the column is at atmo¬ spheric pressure, solvent changes are very easy to perform (Targett et al., 1979). 7.2.3 Open Column Chromatography is still The conventional gravity-driven, open column chromatography method widely used in natural product chemistry, as it represents a rapid and efficient tech¬ nique for preliminary fractionation of crude extracts or fractions and in certain cases may yield even pure compounds. Silica gel is probably the most used stationary phase. The chemical nature of the surface of silica gel consists of exposed silanol groups. These hydroxyl groups are the active centers and potentially can form strong hydrogen bonds with compounds being chromatographed. Thus, in general, the stronger the hydrogen-bonding potential of a compound, the stronger it will be retained by silica gel, so that polar compounds are strongly adsorbed, while nonpolar molecules are poorly or nonretained on silica gel. of the How strongly a given compound is retained depends equally on the polarity mobile phase. The stronger the hydrogen-bonding potential of a solvent, the better it is as an eluant to elute polar compounds adsorbed on a silica gel column. Similarly, very nonpolar solvents would be used to Chromatograph nonpolar molecules. with differ¬ Silica gel may be chemically modified to give bonded phase silica gels ent chromatographic and physical properties. The most commonly employed bonded phase silica gels are the reverse phase class, where the silica particles are derivatized Since the with an alkylsilyl reagent to produce a nonpolar chromatographic support. degree of adsorption to reverse phase silica gel is proportional to the lipophilicity of the compounds being chromatographed, mobile phases used are usually aqueous. An organic modifier such as methanol, acetonitrile, or tetrahydrofuran is used and its con¬ centration increased during the development process (Salituro and Dufresne, 1998). 7.2.4 High Performance Liquid Chromatography Preparative high-performance liquid chromatography (HPLC) is a versatile, robust, and widely used technique for the isolation of natural products. The main difference 7 METHODOLOGY OF ISOLATION PROCEDURE 101 between HPLC and other modes of column chromatography is that the diameter of the stationary phase particles is comparatively low (3-10 urn), and these particles are tightly packed to give a very uniform column bed structure. The low particle diameter means that a high pressure is needed to drive the eluent through the bed. However, and because of the very high total surface area available for interactions with solutes the uniformity of the column bed structure, the resolving power of HPLC is very high. As for open column liquid chromatography, many different packing materials are available for HPLC; the most commonly applied to natural product isolation are nor¬ mal and reverse phase silica gels (Stead, 1998). 7.3 References Cannell RJP (1998). How to approach the isolation of a natural product. In: Natural products isolation (Cannell RJP, ed.), Humana Press, Totowa, New Jersey, pp. 1-51. Gailliot FP (1998). Initial extraction and product capture. In: Natural products iso¬ lation (Cannell RJP, ed.), Humana Press, Totowa, New Jersey, pp. 53-89. Salituro GM, Dufresne C (1998): Isolation by low-pressure column chromatogra¬ phy. In: Natural products isolation (Cannell RJP, ed.), Humana Press, Totowa, New Jersey, pp. 111-140. Sherma J (1996): Basic techniques, materials, and apparatus. In: Handbook ofthin- layer chromatography (Sherma J, Fried B, eds.), Dekker Inc., New York, pp. 3-47. Stead P (1998): Isolation by preparative HPLC. In: Natural products isolation (Cannell RJP, ed.), Humana Press, Totowa, New Jersey, pp. 165-208. Targett NM, Kilcoyne JP, Green B (1979): Vacuum liquid chromatography: an alternative to common chromatographic methods. J. Org. Chem. 44: 4962-4964. 102 8.1 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 8 Methodology of Structure Elucida¬ tion 8.1 Nuclear Magnetic Resonance Spectroscopy When placed in a magnetic field, nuclei that have a nonzero spin quantum number are able to absorb energy from the radio frequency range of the electromagnetic spec¬ trum. The specific environment of a nucleus slightly modifies the magnetic field it experiences: the nuclei are shielded from the applied magnetic field to differing extents, depending on the electron density about each nucleus. Thus, the frequency at which a nucleus is able to absorb energy is characteristic of the environment of the par¬ ticular nucleus, and this effect, the chemical shift, makes NMR spectroscopy very use¬ ful for structural determination. Almost all structural studies begin with the recording of an H spectrum. The num¬ ber of protons giving rise to each signal can be determined by comparing the areas under each signal (i.e. from the integrals). The C spectrum provides important structural information, since it arises from l the nuclei that form the framework of the molecule, in contrast to the H nuclei that are at the periphery. It also confirms the presence of carbonyl groups and other nonproto- 1 ^ nated carbon atoms whose presence in the molecule can be only inferred from the C spectrum. The normal 13C spectrum is acquired with full proton decoupling: in the i i o absence of coupling to H nuclei, all of the C signals in the spectrum appear as single lines, allowing the number of carbons in the molecule to be readily determined. The most commonly used method for determining the number of hydrogens bonded to each carbon atom is the DEPT (distortionless enhancement by polarization transfer) experiment. The DEPT-135° spectrum shows all protonated carbon signals, with CH3 and CH resonances being positive, while CH2 signals are negative. To distin¬ guish CH3 from CH signals, the DEPT-90° experiment is used: here only those reso¬ nances of carbons bearing one hydrogen can be seen. Two dimensional NMR spectra show the frequency on both axes, while the signal intensities correspond to the third dimension. These spectra are obtained by recording 8 METHODOLOGY OF STRUCTURE ELUCIDATION 103 a series of ID spectra differing only by a time increment. Homonuclear experiments contain frequencies of the same nuclei in both dimen¬ sions. DQF-COSY (double quantum filtered correlation spectroscopy) shows vicinal and geminal protons correlated via scalar coupling; TOCSY (total correlation spectros¬ copy) shows correlations of all protons within the same spin system via scalar cou¬ pling. The nuclear Overhauser effect (NOE) allows the identification of those nuclei within a molecule that are close in space. The use of two dimensional NOE experi¬ ments, like NOESY (nuclear Overhauser enhancement spectroscopy) or ROESY (rotating-frame Overhauser spectroscopy), helps identifying pairs of protons close molecule. enough to interact through space, hence the relative stereochemistry of the Heteronuclear correlation experiments contain frequencies of different nuclei (e.g. H- C correlations). The HSQC (heteronuclear single quantum coherence) experi¬ ment shows correlations of directly bonded protons and carbons; the HMBC (heteronu¬ clear multiple bond correlation) experiment allows to determine connectivities between H and C atoms separated by two or three bonds. These two- or three-bond correlations provides the means to span heteroatoms and quaternary carbons, thus giv¬ ing powerful structural information. In addition, long-range heteronuclear couplings also provide the means to link structural fragments through intervening quaternary car¬ bons (Byrne, 1993; Martin and Crouch, 1991). 8.2 Ultraviolet Spectroscopy UV/VIS spectroscopy deals with the interaction of electromagnetic radiation with a compound in a wavelength range of 200 to 800 nm (ultraviolet: 200-380 nm; visible: 380-800 nm). The frequency of the absorbed radiation correlates with the structure of the tested compound, and therefore absorption spectra (showing the amount of absorbed light for each wavelength) show maxima characteristic for a chemical struc¬ ture. Since the absorption depends on the electronic structure of the molecule, for many compounds the absorption does not occur in this portion of the spectral region. There¬ fore, UV spectroscopy is limited to compounds possessing a conjugated system (Har- borne, 1998). 104 8.3 MASS SPECTROMETRY 8.3 Mass Spectrometry Mass spectrometry consists of degrading trace amounts of a compound and record¬ ing the fragmentation pattern according to mass. The sample diffuses into the low pres¬ sure system of the mass spectrometer where it is ionized with sufficient energy to cause fragmentation of the chemical bonds. The resulting charged ions are accelerated in a magnetic field which disperses and permits relative abundance measurements of ions of given mass-to-charge ratio. The resulting record of ion abundance versus mass con¬ of stitutes the mass spectral graph, which thus consists of a series of lines varying a molecular intensity at different mass units. With this technique it is possible to obtain mass and often a molecular formula for an unknown compound and to gain structural is the information regarding functional groups or subunits. An additional feature high sensitivity, which makes mass spectrometry an attractive analytical technique (Har- borne, 1998). Many different methods of ionization are available. In the present work, EI and MALDI techniques were used. 8.3.1 Electron Impact (El) A compound is bombarded with electrons from a glowing filament at low source ion of internal pressure to produce a positively charged molecular radical high energy, which usually fragments to form positively charged fragment ions, neutral fragments, and radicals. The relative abundance of fragments and radicals together with their iso¬ molecule tope ions forms a characteristic fragmentation pattern for a given organic (Bloor and Porter, 1993). 8.3.2 Matrix Assisted Laser Desorption/Ionization (MALDI) is dis¬ The MALDI technique uses a laser for ionizing the molecules. The sample and transfers it to persed in a UV-chromophoric matrix which absorbs the laser energy the analyte, producing protonated molecular ions or [M+Na]+ or [M+K]+. This tech¬ a wide of nique is very sensitive, and has been used in particular to study variety pro¬ teins, peptides, oligosaccharides, and oligonucleotides (Baldwin, 1995). 8 METHODOLOGY OF STRUCTURE ELUCIDATION 105 8.4 Optical Rotation Chiral molecules possess an optical activity, i.e. the ability to rotate the plane of linear polarized light so that transmitted light emerges at a measurable angle to the plane of the incident light. Optical activity is usually expressed by the specific optical rotation [oc]D. The specific rotation is a characteristic physical constant of a compound. 8.5 References Baldwin MA (1995): Modern mass spectrometry in bioorganic analysis. Nat. Prod. Rep. 12: 33-44. Bloor SJ, Porter LJ (1993): Mass spectrometry. In: Bioactive natural products. Detection, isolation, and structural determination (Colegate SM, Molyneux RJ, eds.), CRC Press, Boca Raton, pp. 105-123. Byrne LT (1993): Nuclear magnetic resonance spectroscopy: strategies for struc¬ tural determination. In: Bioactive naturalproducts. Detection, isolation, and structural determination (Colegate SM, Molyneux RJ, eds.), CRC Press, Boca Raton, pp. 75-104. Harborne JB (1998): Phytochemical methods. A guide to modern techniques of plant analysis. Chapman and Hall, London, pp. 1-39. Martin GE, Crouch RC (1991): Inverse-detected two-dimensional methods: appli¬ cations in natural products chemistry. J. Nat. Prod. 54: 1-70. 106 9.1 COLLECTION 9 Collection and Cultivation 9.1 Collection The Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), Dübendorf, Switzerland, maintains a culture collection of about 200 differ¬ ent strains of terrestrial and freshwater cyanobacteria derived from all over the world. Twenty-two cyanobacterial species originating from this collection were selected for cultivation and biological and chemical investigation. The investigated species and their geographical origin are listed in Table 9.1. 9.2 Cultivation 9.2.1 Stock Cultures At the EAWAG, stock cultures of the cyanobacteria are cultivated under weak illu¬ mination with intervals of several months between reinoculations. The cultures are grown in different inorganic media as outlined in Table 9.1. Five different inorganic media were used during this project. Medium Z is the basic culture medium. Medium Z2 is poor in nitrogen, media Z4 and Z454 lack of nitrogen, media Z45 and Z454 lack of carbonate (the composition ofthe media is described in Tables 9.2 and 9.3). Aliquots of 5-10 ml from these stationary phase stock cultures were used to inoculate 100 ml of media. These samples were cultivated under constant illumination with fluorescent lamps (Philips TLM/33 Rs 40 W) at 2000 lx, at a temperature of 20±1 °C. The cyano¬ bacterial cultures were harvested after 4-6 weeks. The cells were separated from the medium by filtration and were employed directly for biological screening. 9 COLLECTION AND CULTIVATION 107 9.2.2 Large-Scale Cultures During this work two cyanobacterial strains, Scytonema spirulinoides (designated EAWAG strain number 161a) and Cylindrospermum sp. (EAWAG 76), were further scaled-up to 10-1 bottles. Each cultivation bottle is equipped with three inlets for addi¬ tion of air, C02, and culture medium, as well as an outlet for collecting the culture. At the beginning, a sterile glass bottle was inoculated with 200-400 ml of an actively growing culture, and 500 ml of sterile medium were added. Every week sterile medium was pumped in the bottle, approximately doubling the volume of the culture. The cul¬ tures were aerated with a mixture of 2% C02 in air, incubated at a temperature of 20±1 °C, and illuminated continuously with fluorescent lamps (Philips TLM/33 Rs 40 W) at 2000 lx. An even distribution of light in the bottle was achieved by permanent mixing with Teflon stirring bars. The cyanobacterial cultures were harvested after 30-40 days. The supernatant was separated from the cells by filtration and adsorbed on a column filled with 300 g Amberlite XAD-2 resin (non-polar, surface area 353 m2/g; Supelco). Subsequently, the column was washed with 1.5 1 methanol and 1.5 1 dichloromethane. The cell material was freeze-dried. The yields of freeze-dried cells were ranging from 0.1to0.5g/l. Table 9.1 Investigatedspeciesand their geographicalorigin. Strain3 Species,family Origin Medium 116d Anabaena flos-aquae(Lyng.)Bréb., Hallwilersee (CH), 1965 Z Nostocaceae 118b Chamaesiphonpolonicus(Rostaf.)Hans., Vallée de la Brévine (CH), 1958 Z Chamaesiphonaceae waterfall Z 214 Chroococcus sp., Chroococcaceae Davos (CH), sprinkledzone near Schiabachfall, 1975 stone 1951 Z 76 Cylindrospermumsp., Nostocaceae Yaounde (Cameroon),wet gneiss wall, 209a Dichothrix cf. orsiniana Born, et Flah., Davos (CH), sprinkledzone near waterfall Schiabachfall, Z2 Rivulariaceae 1975 1951 73 Gloeothece rupestris(Lyng.)Born., Yaounde (Cameroon),wet gneissstone wall, Z Synechococcaceae 172 Gloeotrichia echinulata (Smith)Rieht., Erkensee (S),1950; Culture Collection Algae and Proto- Z Rivulariaceae zoa, 1432/1,Cambridge (GB) 199b Hammatoidea normanni W. et G.S. West, Gotthard Pass (CH), water from melted snow, 1972 Z45 Homoeotrichaceae moist 1965 Z 119 Hydrocoleussp., Phormidiaceae Stansstaad (CH), rock, la from Z 48 Nostoc sp. ex Peltigeraaphthosavar. vario¬ Vallée de Brévine (CH), 1958; cyanobacterium losa,Nostocaceae the lichen Peltigeraaphthosavar. variolosa 138a Oscillatoria limosa Ag., Oscillatoriaceae River Rhine near Stein (CH), 1966 Table 9.1 (continued)Investigatedspeciesand their geographicalorigin. Strain2 Species,family Origin Medium 188b Petalonema alatum Berk.,Microchaetaceae Bürglen,Schächental (CH), wet stone wall,1969 Z4 197 Petalonema alatum Berk.,Microchaetaceae Berner Oberland (CH), waterfall Reichenbachfall,1969 Z4 Z 131a Phormidium sp., Phormidiaceae Berninapass(CH), on rock,1965 Z 134 Phorm idium sp., Phormidiaceae Berninapass(CH), on rock, 1965 Z 136 Phormidium sp., Phormidiaceae Berninapass(CH), on rock,1965 196a Rivularia haematites (DC) C. Ag., Rigi(CH), on rock, 1970 Z Rivulariaceae 196c Rivularia haematites (DC) C. Ag., Rigi (CH), on rock, 1970 Z Rivulariaceae 196f Rivularia haematites (DC) C. Ag., Rigi(CH), on rock, 1970 Z Rivulariaceae 161a Scytonema spirulinoidesGardner, Pokhara (Nepal),graniterock,1967 Z454 Scytonemataceae 8 SynechococcuselongatusNaeg., Cambridge (GB), gardenpond, 1940; Culture Collection Z Synechococcaceae Algae and Protozoa, 1479/1 A, Cambridge (GB) 1976 Z2 208b Tolypothrixsp., Microchaetaceae Ruin Stein,Baden (CH), limestone, Strain numbers accordingto the Culture Collection of Algae of the EAWAG. 110 9.2 CULTIVATION Table 9.2 Composition of culture mediaa Salt Medium Medium Medium Medium Medium Z Z2 (N-poor) Z4 (N-free) Z45 Z454 (N-free) CaCl22H20 0.00 0.00 37.00 0.00 37.00 NaN03 467.00 8.50 0.00 467.00 0.00 Ca(N03)4H20 59.00 59.00 0.00 59.00 0.00 KH2P04 0.00 0.00 0.00 41.00 41.00 K2HP04 31.00 31.00 31.00 17.00 17.00 MgS047H20 25.00 25.00 25.00 25.00 25.00 Na2C03 21.00 21.00 21.00 0.00 0.00 FeEDTAb 10.00 10.00 10.00 10.00 10.00 Gaffron 0.08 0.08 0.08 0.06 0.06 a in ml/1. Concentrations of salts are given in mg/1, FeEDTA solution and Gaffron solution are given b The FeEDTA solution consists of 10 ml of 0.1 M FeCl3 6H20 solution in 0.1 M HCl and 10 ml of 0.1 M Na2EDTA solution in 1 1. Table 9.3 Composition of the Gaffron solution Salt Concentration (mg/1) H3BO3 310.0 MnS044H20 223.0 Na2W042H20 3.3 (NH4)6Mo70244H20 8.8 KBr 11.9 KI 8.3 ZnS047H20 28.7 Cd(N03)24H20 15.4 Co(N03)26H20 14.6 CuS045H20 12.5 NiS04(NH4)2S04 6H20 19.8 Cr(N03)37H20 3.7 VOS042H20 2.0 A12(S04)3K2S04- 24H20 47.4 10 BIOLOGICAL SCREENING 111 10 Biological Screening During this project, 44 lipophilic and hydrophilic extracts obtained from 22 sam¬ ples of cultured terrestrial and freshwater cyanobacteria were investigated for their bio¬ logical activities. The cyanobacterial cells were cultivated and harvested as described in chapter 9.2.1. The lyophilized material (2-3 g) was extracted by maceration with dichloromethane/methanol 2:1 followed by methanol/water 7:3. The extracts were dried in vacuo. 10.1 Bioassays 10.1.1 Antimicrobial Assay Sta¬ Direct bioautographic assays were performed with the Gram-positive bacteria phylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), coli Bacillus cereus (ATCC 10702), and the Gram-negative bacteria Escherichia (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853), as well as the fungus Candida albicans (H29 ATCC 26790). An aliquot (30 pi) of the bacterial or fungal for stock suspension was transferred into 10 ml of broth (Nutrient Broth, OXOID, UK bacteria; Sabouraud Liquid Medium, OXOID, UK for Candida albicans) and incu¬ bated at 37 °C. 10 ml of nutrient agar (Müller-Hinton Agar, OXOID, UK for bacteria; Malt Extract Agar, OXOID, UK for Candida albicans) were inoculated with 50-100 pi of overnight culture of the test organisms. 500 pg of the extract were applied directly 24 h onto a TLC plate and the inoculated nutrient agar was poured over it. After incu¬ bation at 37 °C the plates were sprayed with MTT (methylthiazolyltetrazolium chlo¬ ride, 2.5 mg/ml, Fluka, Switzerland). Chloramphenicol (5 pg, Siegfried, Switzerland) and tetracycline hydrochloride (5 pg, Fluka, Switzerland) were used as antibacterial standards, and miconazole nitrate (5 pg, Sigma, USA) as antifungal standard. 112 10.2 RESULTS 10.1.2 Brine Shrimp (Artemia salina Leach) Lethality Assay The assay was performed at a concentration of 500 ppm extract as previously out¬ lined (Meyer et al., 1982; McLaughlin et al., 1991). Extracts showing a percent death value of more than 50% were considered as active. As reference standard, podophyllo- toxin at a concentration of 50 pg/ml (Fluka, Switzerland, batch 297667/1792) was used. 10.1.3 Cytotoxicity Assay The cytotoxicity of the extracts was assessed using the KB cell line (ATCC CCL 17) according to Swanson and Pezzuto (1990), and Orjala et al. (1994). Cultivation was done in 96-well plates with Minimum Essential Medium (21090-022; Gibco, Life Technologies, Switzerland) supplemented with 10% fetal calf serum (FCS), 1% nones¬ sential amino acids, 1 pg/ml fungizone (amphotericin B), 100 units/ml penicillin, 100 pg/ml streptomycin, 2 mM glutamine (all from Gibco, Life Technologies, Switzer¬ land). The extracts were tested at a concentration of 50 ppm. MTT (4 mg/ml) was used as detection reagent. Podophyllotoxin at a concentration of 0.05 pg/ml was used as positive control. 10.2 Results 10.2.1 Antimicrobial Activity The bioautographic assay allowed the detection of antibacterial and antifungal extracts on TLC plates. The extracts were considered active when exhibiting a zone of inhibition > 2 mm. 24 (54.5%; 11 DCM/MeOH 2:1 and 13 MeOH/H20 7:3 extracts) of the 44 extracts showed activity against Gram-positive bacteria, 4 (9.1%; 2 DCM/ MeOH 2:1 and 2 MeOH/H20 7:3 extracts) against Candida albicans (see Table 10.1). None of the extracts was active against Gram-negative bacteria. 10 BIOLOGICAL SCREENING 113 10.2.2 Brine Shrimp Lethality The brine shrimp Artemia salina Leach has been proposed as a low cost substitute for cytotoxicity assays. Literature data suggest a good correlation between the activity in the brine shrimp assay and the cytotoxicity against some tumor cell lines (Anderson et al., 1991) as well as hepatotoxic activity (Kiviranta et al., 1991). Only two of the 44 extracts exhibited a significant activity (lethality > 50%) at 500 ppm (see Table 10.2). 10.2.3 Cytotoxicity The KB cell assay was used to determine the cytotoxic activity. 17 extracts (38.6% of 44; 11 DCM/MeOH 2:1 and 6 MeOH/H20 7:3 extracts) exhibited cytotoxic effects. Stronger activities (inhibition > 60%) were shown only by the lipophilic extracts (see Table 10.2). Table 10.1 Antibacterial and antifungalactivitiesof the extracts.21 C. albicans Species Strain Extract B. cereus S. aureus S. epidermidis Anabaena flos-aquae 116d A + B Chamaesiphon polonicus 118b A B + ++ + Chroococcus sp. 214 A + B ++ Cylindrospermum sp. 76 A ++ B + ++ Dichothrix cf. orsiniana 209a A B + Gloeothece rupestris 73 A + B + + ++ Gloeotrichia echinulata 172 A + B Hammatoidea normanni 199b A B Table 10.1 (continued)Antibacterial and antifungalactivitiesof the extracts.3 C. albicans Species Strain Extract B. cereus S. aureus S. epidermidis ++ Hydrocoleussp. 119 A B Nostoc sp. ex Peltigera 48 A aphthosavar. variolosa B + + ++ Oscillatoria limosa 138a A B + ++ Petalonema alatum 188b A + + B + + + Petalonema alatum 197 A B + Phormidium sp. 131a A B Phormidium sp. 134 A B Phormidium sp. 136 A B ++ Table 10.1 (continued)Antibacterial and antifungalactivitiesof the extracts.3 Species Strain Extract B. cereus S. aureus S. epidermidis C. albicans Rivularia haematites 196a A + B ++ Rivularia haematites 196c A B ++ Rivularia haematites 196f A B ++ Scytonema spirulinoides 161a A ++ B ++ ++ Synechococcus elongatus 8 A ++ B Tolypothrixsp. 208b A B Chloramphenicol +++ +++ +++ Miconazole nitrate +++ a + ++ = +++ = for Zone of inhibition at 500 ng (-= no inhibition; = 2-3 mm; 3-5 mm; > 5 mm). Control: 5 ng chloramphenicol bacteria, 5 jxg miconazole nitrate for C. albicans. A = dichloromethane/methanol 2:1 extract; B = methanol/water 7:3 extract. 10 BIOLOGICAL SCREENING 117 Table 10.2 Brine shrimp lethality and KB-cell activity of the extracts.3 Species Strain Extractb Brine shrimpc KB cellsd Cylindrospermum sp. 76 A + (+) B Gloeotrichia echinulata 172 A + B (+) Hammatoidea normanni 199b A (+) B + Hydrocoleus sp. 119 A + B (+) Petalonema alatum 188b A (+) B Petalonema alatum 197 A + B Rivularia haematites 196a A + B (+) Rivularia haematites 196c A + B (+) Rivularia haematites 196f A (+) B (+) Scytonema spirulinoides 161a A (+) B (+) + Tolypothrix sp. 208b A B Podophyllotoxin + a Only active strains are shown. A = dichloromethane/methanol 2:1 extract; B = methanolAvater 7:3 extract. c Brine shrimp lethality: mortality at 500 ppm (+ = mortality > 50%); control: 50 ng/ml podophyllotoxin. d = > = 0.05 KB cells: inhibition at 50 ppm (+ inhibition 60%, (+) inhibition 10-60%); control: ng/ml podophyllotoxin. 118 10.3 REFERENCES 10.2.4 Conclusions No significant difference between the percentage of antibacterial active lipophilic and hydrophilic extracts could be found in the antimicrobial assays. 11 of the DCM/ MeOH 2:1 extracts and 13 of the MeOH/H20 7:3 extracts were active. 11 extracts showed activity against rods (B. cereus) and 20 against cocci (S. aureus and S. epider¬ midis), with 9 extracts active against both rods and cocci. Additionally, two lipophilic and two hydrophilic extracts were active against C. albicans. There was no correlation observable between cytotoxicity against KB cells and brine shrimp lethality: only two extracts were active against brine shrimps, whereas 17 with data were cytotoxic against KB cells. This result is in full accordance published by Jaki et al. (1999), dealing with the lacking correlation between the toxicity against KB cells and the brine shrimp lethality of further 43 samples of cyanobacteria. Regard¬ ing these studies, it does not seems suitable to monitor cytotoxicity by using the brine 19 of the 22 shrimp lethality assay instead of cellular cytotoxicity assays. investigated These cyanobacteria induce a response in at least one of the test systems applied. still a source results prove that terrestrial and freshwater cyanobacteria are promising of new bioactive natural products. Based on the results of the biological screening, two cyanobacterial strains, Scy¬ tonema spirulinoides (designated EAWAG strain number 161a) and Cylindrospermum 11 and sp. (EAWAG 76), were chosen for phytochemical investigations (see chapters 12). 10.3 References Anderson JE, Goetz CM, McLaughlin JL, Suffness M (1991): A blind comparison of simple bench-top bioassays and human tumour cell cytotoxicities as antitumor pre- screens. Phytochem. Anal. 2: 107-111. Jaki B, Orjala J, Bürgi H-R, Sticher O (1999): Biological screening of cyanobacte¬ ria for antimicrobial and molluscicidal activity, brine shrimp lethality, and cytotoxic¬ ity. Pharm. Biol. 37: 138-143. Kiviranta J, Sivonen K, Niemelä SI, Huovinen K (1991): Detection of toxicity of 10 BIOLOGICAL SCREENING 119 cyanobacteria by Artemia salina bioassay. Environ. Toxicol. Wat. Qual. 6:423-436. McLaughlin JL, Chang C.-J., Smith DL (1991): "Bench-top" bioassays for the dis¬ In: Studies in Natural Products Chem¬ covery of bioactive natural products: an update. istry, Vol 9 (Atta-ur-Rahman, ed.), Elsevier Science Publishers B.V., Amsterdam, pp. 383-409. Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL constituents. (1982): Brine shrimp: a convenient general bioassay for active plant Planta Med. 45: 31-34. Orjala J, Wright AD, Behrends H, Folkers G, Sticher O, Ruegger H, Rali T (1994): Cytotoxic and antibacterial dihydrochalcones from Piper aduncum. J. Nat. Prod. 57: 18-26. Swanson SM, Pezzuto JM (1990): Bioscreening technique for cytotoxic potential and ability to inhibit macromolecule biosynthesis. In: Drug Bioscreening. Drug Eval¬ uation Techniques in Pharmacology (Thompson EB, ed.), VCH, New York, pp. 273- 297. 120 11.1 FRACTIONA TION AND ISOLA TION 11 Phytochemical Investigation of Scytonema spirulinoides (EAWAG 161a) The investigation of the methanol extract resulting from the Amberlite elution of the culture medium of the terrestrial cyanobacterium Scytonema spirulinoides Gardner (EAWAG 161a) led to the isolation of four new extracellular naphthalenone deriva¬ tives (1-4). The structures of the isolates were elucidated by spectroscopic methods, mainly ID- and 2D-NMR and mass spectrometry. 11.1 Fractionation and Isolation The MeOH extract (470 mg) obtained from the Amberlite elution of 60 1 culture 90 reversed medium was subjected to a 3 x 10 cm VLC column filled with g phase material (C-18 HL RSil, 40-63 pm). Elution with a step gradient of 20% ACN to 100% ACN yielded 23 fractions each of 100-120 ml. The fractions were combined, based on TLC similarities, to yield 11 combined fractions (Ml-Ml 1). ACN as Fraction M6 (30 mg) was separated by reversed phase HPLC with 60% eluent to yield 8.9 mg of 1. 15% ACN as Fraction M4 (11 mg) was separated by reversed phase HPLC with eluent to yield 0.6 mg of 2 and 0.7 mg of 3. 15% ACN as Fraction M3 (10 mg) was separated by reversed phase HPLC with isolation eluent to yield 1.3 mg of 4. Figure 11.1 shows a flow chart with the procedure of the four isolated metabolites. MeOH extract (470 mg) RP-VLC H20->ACN, step gradient H20-ACN 6:4 H20-ACN1:1 H20-ACN 4:6 300 ml 300 ml 300 ml Ml M2 M3 M4 M5 M6 M7 M8 M9 MIO Mil (10 mg) (11mg) (30 mg) RP-HPLC RP-HPLC 15% ACN RP-HPLC 60% ACN 15% ACN 4 (1.3mg) rt= 10 min (8.9mg) rt = 9 min 2 3 (0.6mg) (0.7mg) rt = 13 min rt= 17 min Figure 11.1 Isolation procedurefor the MeOH extract of the culture medium of Scytonema spirulinoides161a. 122 11.2 STRUCTURE ELUCIDATION OF THE ISOLATES 11.2 Structure Elucidation of the Isolates The high resolution EI mass spectrum of 1 displayed a molecular ion at m/z 192.0786 corresponding to 0^,203 (calculated 192.0786). The *H NMR spectrum exhibited two vicinal aromatic protons (b\i 6.78, H-7, d, J= 8.5 Hz and b\i 7.38, H-6, d, J= 8.5 Hz), one methine proton at 5^ 5.10 (H-4, t, J- 3.0 Hz), two non equivalent ketomethylene proton multiplets (b\i 3.16 and 2.52, H-2a and b), one methylene proton multiplet at b\i 2.25 (H2-3), and one methyl group at d\i 2.37 (5-Me, s). The low-field shifted signal at ^ 12.48 (8-OH, s) suggested the presence of a phenolic OH that par¬ ticipates in an hydrogen bond (for !H NMR data see Table 11.1). The 13C NMR spectrum contained resonances consistent with the presence of an aromatic ring, besides additional resonances indicating the presence of a carbonyl group (Sc 206.7, C-1), a methine connected to a hydroxyl group (<5C 63.1, C-4), two methylenes at 8C 32.8 (C-2) and Sc 30.6 (C-3), and of one methyl group at 8C 17.7 (5- Me). (For 13C NMR data see Table 11.2). DQF-COSY correlations were observed between H-4 and H2-3, which in turn showed cross-peaks to H-2a and b. From the HMBC spectrum measured in MeOD, correlations were observed from H2-3 and H2-2 to C-1; the HMBC spectrum allowed also to establish the connectivity of this partial structure to the aromatic ring. Further¬ more, HMBC correlations showed the methyl group at b\i 2.37 to be bonded to C-5, and, in the HMBC spectrum measured in acetone, the hydroxyl group at d\i 12.48 to be bonded to C-8. 1 is therefore 3,4-dihydro-4,8-dihydroxy-5-methyl-l(2H)-naphthale- none. oh o CH3 OH Figure 11.2 Compound 1 11 PHYTOCHEMICAL INVESTIGATION OF SCYTONEMA SPIRULINOIDES (EAWAG 161 A) 123 a co CD I ^ LT) CO CVJ CO f> o s OD CM X o I CO 5-Me 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 ppm Figure 11.4 HSQC spectrum of 1 (<5ppm, 600 MHz, 295 K, MeOD) 5-Me I -< oH 2a 2b o UJL m Li ppm i o 5-Me. > 3, < m COH CD> OCO H-< 8a, \ o m 5 — > CO TJ 4a c zr; 8 -- g o m CO m > $ 1 - CD 03 Figure 11.5 HMBC spectrum of 1 (ô ppm, 500 MHz, 295 K, MeOD) no CXI 126 11.2 STRUCTURE ELUCIDATION OF THE ISOLATES The high resolution EI mass spectrum of 2 displayed a molecular ion at m/z 208.0731, consistent with the molecular formula Q iH1204 (calculated 208.0736). The 1 1 ^ H and C NMR spectra were very similar to those of 1, the only significant difference each J = being the presence of a hydroxymethylene group (c^ 4.76 and 4.68, 1H, d, 12.4 Hz, 8C 62.0; 5-CH2) instead of the methyl group. On the basis of these data and of 2D-NMR analysis, the structure of 2 was assigned as 3,4-dihydro-4,8-dihydroxy-5- hydroxymethylene-1 (2H)-naphthalenone. oh o Figure 11.6 Compound 2 The high resolution EI mass spectrum of 3 indicated the molecular formula ci 1H12°4 (molecular ion at m/z 208.0730, calculated 208.0736). The lH and 13C NMR of an data were very similar to those of 1. The main difference here is the presence additional hydroxyl group at C-3 (b\{ 4.07, H-3, m, <5C 68.3, C-3). 3 is therefore 3,4- dihydro-3,4,8-trihydroxy-5-methyl-l(2H)-naphthalenone. oh o ch3 oh Figure 11.7 Compound 3 11 PHYTOCHEMICAL INVESTIGATION OF SCYTONEMA SPIRULINOIDES (EAWAG 161 A) 127 The HREIMS (molecular ion at m/z 208.0724), lU and 13C NMR data of 4 sug¬ gested the molecular formula CiiH1204. In contrast to the previously described com¬ pounds 1-3, no low-field shifted signal at about ^12 ppm could be detected in the H NMR spectrum of 4, indicating the absence of a chelated phenolic OH. The H and C NMR spectra were consistent with the presence of an aromatic ring, and showed addi¬ tional signals for a carbonyl group (8C 202.2, C-1), two methines connected to a = hydroxyl group (^ 4.80, H-2, dd, J= 5.1, 13.1 Hz, 5C 70.4, C-2; b^ 5.35, H-4, t, J 3.1 Hz, 8C 62.6, C-4), two non equivalent methylene protons at position 3 (b\i 2.50, IH, m and 2.14, IH, td, J= 3.7, 13.1 Hz, 8C 40.1) and one methyl group ($h 2.50, s, <5C 21.6; 8-Me). DQF-COSY cross-peaks were observed between H-4 and H2-3 and between H2-3 and H-2. The HMBC spectrum allowed to establish the connectivity of this partial structure to the aromatic ring and to the carbonyl group. Furthermore, HMBC correla¬ tions and chemical shift data showed the methyl group at 8\i 2.50 to be bonded to C-8, and a phenolic OH group to be bonded to C-5. 4 is therefore 3,4-dihydro-2,4,5-trihy- droxy-8-mefhyl-1 (2H)-naphthalenone. ch3 o OH OH Figure 11.8 Compound 4 128 11.2 STRUCTURE ELUCIDATION OF THE ISOLATES Table 11.1 !H NMR data of compounds l-4a Compound Position 1 2 3 4 2a 3.16, m 3.12,m 3.12, dd (12.2, 16.9) 4.80, dd (5.1, 13.1) 2b 2.52, m 2.55, m 2.70, m 3a 2.50, m 2.25,m 2.28, m 4.07, m 3b 2.14, td (3.7, 13.1) 4 5.10, t (3.0) 5.23, t (3.1) 5.00, m 5.35, t (3.1) 6 7.38, d (8.5) 7.59, d (8.6) 7.41, d (8.5) 6.98, d (8.2) 7 6.78, d (8.5) 6.90, d (8.6) 6.83, d (8.5) 7.10, d (8.2) 5-Me 2.37, s 2.41, s 5-CH2 4.76, d (12.4) 4.68, d (12.4) 8-Me 2.50, s 8-OH 12.48, s 12.60, s 12.38, s a In acetone-c^, 500 MHz, <5ppm; m (/Hz), 295 K. 11 PHYTOCHEMICAL INVESTIGATION OF SCYTONEMA SPIRULINOIDES (EAWAG 161 A) 129 Table 11.2 13C NMR data of compounds l-4a. Compound Position lb 2e 3e 4e 1 206.7, s 206.8, s 205.4, s 202.2, s 2 32.8, t 33.2, t 41.1,t 70.4, d 3 30.6, t 30.4, t 68.3, d 40.1, t 4 63.1, d 63.2,d 67.9, d 62.6, d 4a 143.2, s 143.7, s 141.3, s 131.2,s 5 127.5, s 131.3, s 128.9, s 154.8, s 6 139.7, d 138.8, d 140.0, d 120.9, d 7 117.4, d 118.1,d 118.2, d 133.7, d 8 161.5, s 163.1, s 161.7, s 131.6, s 8a 116.2, s 116.5, s 116.6, s 129.9, s 5-Me 17.7, q 17.6, q 5-CH2 62.0, t 8-Me 21.6, q a S in ppm. All assignments are based on HSQC and HMBC measurements. b MeOD, 150 MHz, 295 K. c In acetone-d6, 125 MHz, 295 K. 11.2.1 Physical Data of the Isolates Compound 1. Amorphous solid; [a]25D +7° (c 0.1, MeOH); UV (MeOH) À^ax (log e) 261 (4.13), 344 (3.80) nm; EIMS m/z 192 (100) 174 (80) 164 (11) 146 (30) 135 (59) 107 (20) 91 (10) 77 (15); HREIMS m/z 192.0786 (calcd for CnH1203, 192.0786); *H and 13C NMR data see Tables 11.1 and 11.2. Compound 2. Amorphous solid; [cc]25D +14° (c 0.1, MeOH); UV (MeOH) A^ (log 8) 262 (4.02), 345 (3.44) nm; EIMS m/z 208 (23) 190 (100) 162 (20) 147 (14) 134 (39) 105 (15) 91 (10) 77 (14); HREIMS m/z 208.0731 (calcd for CnH1204,208.0736); *H and 13C NMR data see Tables 11.1 and 11.2. 130 11.3 BIOLOGICAL ACTIVITY Compound 3. Amorphous solid; [a]25D +11° (c 0.1, MeOH); UV (MeOH) À^ (log 8) 254 (3.92), 335 (3.34) nm; EIMS m/z 208 (43) 190 (100) 164 (45) 147 (20) 135 (93) 107 (39) 91 (11) 77 (30); HREIMS m/z 208.0730 (calcd for C{ iH1204, 208.0736); lR and 13C NMR data see Tables 11.1 and 11.2. Compound 4. Amorphous solid; [a]25D -5° (c 0.1, MeOH); UV (MeOH) À^ (log 8) 264 (3.73), 348 (3.33) nm; EIMS m/z 208 (6) 190 (100) 164 (4) 147 (21) 135 (11) 107 (6) 91 (7) 77 (10); HREIMS m/z 208.0724 (calcd for CnH1204,208.0736); *H and 13C NMR data see Tables 11.1 and 11.2. 11.2.2 General Experimental Procedures Optical rotations were recorded with a Perkin Elmer 241 Polarimeter using MeOH as a solvent. The UV spectra were recorded in MeOH using an Uvikon 930 spectropho¬ tometer. EIMS spectra were measured on a Hitachi-Perkin Elmer-RMUGM mass spec¬ trometer at 70 eV. NMR spectra were recorded at 295 K on a Bruker DRX-600 (oper¬ ating at 600.13 MHz for lH and 150.92 MHz for 13C) or a Bruker DRX-500 spectrometer (operating at 500.13 MHz for *H and 125.77 MHz for 13C). The spectra were measured in CD3OD or CD3COCD3 using the residual nondeuterated solvent (CH3OH, lH <53.31, 13C 549.0; CH3COCH3, lH 82.05, 13C 5206.0) as internal ref¬ erence. HPLC separations were performed with a Merck-Hitachi L-6200 intelligent pump connected to a Rheodyne 7125 injector, a Merck-Hitachi L-4250 UV7VIS detec¬ tor, a Merck D-2500 Chromato-Integrator, and a Knauer HPLC column (Spherisorb S5 ODSII, 5 pm, 250x8 mm). Silica gel for column chromatography, particle size 63-200 pm (Merck), was used for VLC. Silica gel 60 F254 precoated aluminium sheets (0.2 mm, Merck) and RP-18 F254 precoated sheets (0.25 mm, Merck) were used for TLC controls. All solvents were HPLC grade. 11.3 Biological Activity Compound 1 showed weak antibacterial activity against Bacillus cereus (MIC 64 ppm). A MIC value of 4 ppm was detected for reference compound chloramphenicol. 11 PHYTOCHEMICAL INVESTIGATION OF SCYTONEMA SPIRULINOIDES (EAWAG 161 A) 131 No inhibition of growth was observed against Staphylococcus aureus, Escherichia coli and Candida albicans. The MIC value was determined by the broth doubling dilution method (Frost, 1994), using a modified procedure as described below. Test organisms were Staphylo¬ coccus aureus (ATCC 25923), Bacillus cereus (ATCC 10702), Escherichia coli (ATCC 25922), and Candida albicans (H29 ATCC 26790). Bacterial and fungal sus¬ pensions were obtained from overnight cultures in broth (Nutrient Broth, OXOID, UK for bacteria; Sabouraud Liquid Medium, OXOID, UK for C. albicans) cultivated at 37 °C and diluted to ca. 105 cells/ml in fresh medium. The samples were dissolved in MeOH to 1 mg/ml as stock solutions. The required amount of stock solution was pipet¬ ted into the wells of the first column of a 96-well tissue culture plate and dried. The sample was redissolved in 50 pi DMSO, 50 pi sterile broth and 100 pi dilute culture suspension. Twofold dilutions were made in 100 pi volumes of dilute bacterial or fun¬ gal suspensions. The plates were kept in a moist atmosphere at 37 °C for 20 h. After incubation, 10 pi of a 0.25% aqueous solution of methylthiazolyltetrazolium chloride was added in each well and incubated for 4 h to detect living bacteria as violet turbid solutions. No cytotoxic activity was observed against KB cells. Due to the small yields, no bioassays were performed for compounds 2,3, and 4. 11.4 Reference Frost JA (1994): Testing for resistance to antimicrobial drugs. In: Methods in prac¬ tical laboratory bacteriology (Chart H, ed.), CRC Press, Boca Raton, pp. 73-82. 132 12.1 FRACTIONATION AND ISOLATION 12 Phytochemical Investigation of Cylindrospermum sp. (EAWAG 76) The investigation of the dichloromethane/methanol 2:1 extract from the lyophilized cell material of the terrestrial cyanobacterium Cylindrospermum sp. (EAWAG 76) led to the isolation of the carotenoid caloxanthin and of three heterocyte glycolipids (6-8). 12.1 Fractionation and Isolation The DCM/MeOH 2:1 extract (13 g) obtained from 110 g lyophilized cell material was subjected to a 6.5 x 21 cm VLC column filled with 210 g silica gel 60 (0.063-0.200 mm). Elution with a step gradient of hexane/EtOAc/MeOH yielded 50 fractions each of 100 ml. The fractions were combined, based on TLC similarities, to yield 9 com¬ bined fractions (D1-D9). Fraction D6 (160 mg) was applied to an open column chromatography (silica gel 60, 40 g, 0.040-0.063 mm) and eluted with a step gradient of hexane/EtOAc/MeOH. Caloxanthin (5, 10.0 mg) precipitated from fraction D6.3. Fraction D7 (4.0 g) was subjected to an open column chromatography (silica gel 60, 120 g, 0.040-0.063 mm) using a step gradient EtOAc/MeOH (from 100% EtOAc to 100% MeOH) as eluent to yield 10 fractions. A mixture (30.0 mg) of three glycolipids (6-8) precipitated from fraction D7.8. Unfortunately, the three compounds decom¬ posed during the attempt to separate them by preparative TLC. Figure 12.1 shows a flow chart with the isolation procedure of caloxanthin and the three glycolipids. DCM/MeOH 2:1 extract (13 g) VLC (silicagel) Hexane->EtOAc->MeOH, step gradient Hexane-EtOAc 6:4 Hexane-EtOAc 4:6 500 ml 300 ml D1-D5 D6 D7 D8-D9 (160 mg) (4.0g) Open column (silicagel) Open column (silicagel) Hexane-EtOAc 4:6 EtOAc-MeOH 9.5:0.5 D6.3 D7.8 (42.8mg) (564.8mg) Precipitation Precipitation Caloxanthin 5 Mixture of 6-8 (10.0mg) (30.0mg) Figure 12.1 Isolation procedurefor the DCM/MeOH 2:1 extract of the cell material of Cylindrospermum sp. 76. 134 12.2 STRUCTURE ELUCIDATION OF CALOXANTHIN 12.2 Structure Elucidation of Caloxanthin The MALDI mass spectrum of caloxanthin displayed a positive ion peak [M+H]+ at m/z 585. Together with NMR data, this suggested the molecular formula C40H56O3. A DEPT 135 experiment indicated the presence of 10 methyl groups, 3 methylene, 17 C- methine groups, and 10 quaternary carbons. Of the 17 methine groups, 3 (8q 65.1, 3'; Sc 67.7, C-3; 8C 80.1, C-2) are bonded to a hydroxyl group; the remaining 14 (chemical shifts between 125 and 140 ppm) form double bonds. This, together with the strong red colour of the substance, suggested the presence of a system of conjugated double bonds, characteristic for a carotenoid-type structure. The structure of the ole¬ finic part of the molecule was elucidated by means of HSQC, HSQC-TOCSY, and HMBC experiments. The comparison of the *H NMR data of the isolated compound with literature data of different /3-carotene isomers (see Table 12.3) indicated that the most probable conformation of the olefinic fragment is the all-trans-conformation. The !H NMR spectrum exhibited ten methyl groups at 8^ 1.98 (4 CH3), 1.74,1.72, at 1.98 1.14,1.08 (2 CH3), and 1.01 ppm, all as singlets. The four methyl groups <5pj (H- 19, H-20, H-19', H-20'; Sc 12.8) showed HMBC correlations to the olefinic fragment. NMR data indicated also the presence of two cyclic fragments. One fragment con¬ tains one double bond (Sc 125.2, C-5; 8C 131A, C-6) connected to the methyl group at position 18 (b\i 1.72; Sc 21.3), one methylene at position 4 (8\{ 2.48, H-4a, dd, J= 6.3, 17.2 Hz and ^ 2.15, H-4b, dd, J = 10.0, 17.2 Hz; 8C 40.1, C-4), two methines con¬ nected to a hydroxyl group (^ 3.33, H-2, d, J= 10.1 Hz, <5C 80.1, C-2; ^ 3.84, H-3, m, 8C 67.7, C-3), and a quaternary carbon at 8C 40.8 (C-1) with two methyl groups (<5^ 1.01, H3-I6, s, Sc 21.5, C-16; 8^ 1.14, H3-17, s, 8C 26.1, C-17). The coupling constant (J = 10.1 Hz) between the two protons at 2 and 3 suggested that the two hydroxyl differ¬ groups are oriented to trans. The other cyclic fragment is very similar, the only ence being the presence of only one hydroxyl group at position 3' (o\i 4.01, IH, m, 8C 65.1). The connectivities of these two rings to the olefinic fragment were established by means of a HMBC experiment, showing the isolated compound to be caloxanthin (2/2,3/2,3 '/?-/3,jß'-carotene-2,3,3 '-triol). 12 PHYTOCHEMICAL INVESTIGATION OF CYLINDROSPERMUM SP. (EAWAG 76) 135 Figure 12.2 Caloxanthin The H NMR data of caloxanthin, together with literature data for caloxanthin iso¬ lated from Anacystis nidulans (Buchecker et al., 1976) and Erythrobacter longus (Takaichi et al., 1990) are shown in Table 12.1. The 13C NMR data of caloxanthin, together with literature data for the similar car¬ otenoids zeaxanthin and /3,/3'-carotene (Palermo et al., 1991) are shown in Table 12.2. 12.2.1 Physical Data of Caloxanthin Red amorphous solid; UV (EtOAc) À^ (log e) 451 (4.80), 477 (4.80) nm; MALDI-MS m/z 585, 524, 503, 442, 273, 202; lU- and 13C-NMR data see Tables 12.1 and 12.2. 136 12.2 STRUCTURE ELUCIDATION OF CALOXANTHIN Table 12.1 lH NMR data3 of the isolated caloxanthin (5) compared to literature data of caloxanthin from Anacystis nidulans and Erythrobacter longus0. Position 5 Lit. values Lit. values0 2 (ax) 3.33, d (10.1) 3.31, d (10.5) 3.33, d (10.1) 3 (ax) 3.84, m -4.0, m 3.84, m 4a (eq) 2.48, dd (6.3, 17.2) - 2.48, dd 4b (ax) 2.15, dd (10.0, 17.2) - -2.20 7 6.10, m 6.10, s - 8 6.10, m 6.12, s - 10 6.17, d (15.0) - 11 6.65, m - 12 6.38, d (15.0) 6.15-6.9 - 14 6.27, m - 15 6.65, m - 16 1.01, s 1.00, s 1.00, s 17 1.14, s 1.12, s 1.13,s 18 1.72, s 1.72, s 1.72, s 19 1.98, s 1.97, s 1.97, s 20 1.98, s 1.97, s 1.97, s 2'a (eq) 1.77, m - -1.77 2'b (ax) 1.49, t (11.9) - 1.48, dd 3'(ax) 4.01, m -4.0, m 4.00, m 4'a(eq) 2.39, dd (4.6, 16.6) - 2.39, dd 4'b (ax) 2.05, m - 2.05, dd 7' 6.10, m 6.10, s - 8' 6.10, m 6.12, s - 10' 6.16, d (15.0) - 11' 6.65,m - 12' 6.37, d (14.9) 6.15-6.9 - 14' 7.38, d (8.5) - 15' 6.65,m - 16' 1.08, s 1.07, s 1.07, s 17' 1.08, s 1.07, s 1.07, s 18' 1.74, s 1.72, s 1.74, s 19' 1.98, s 1.97, s 1.97, s 1.98, s s 20' — — s 1.97, 7 1.97, a CDC13, 500 MHz, S ppm; m (/Hz), 295 K b Bucheckeretal., 1976. c Takaichi et al., 1990. 12 PHYTOCHEMICAL INVESTIGATION OF CYLINDROSPERMUM SP. (EAWAG 76) 137 Table 12.2 C NMR data of caloxanthin3 (5) compared to literature data of zeaxanthin and jö,/3'-caroteneb. Position 5 Lit.b Lit.b Position 5 Lit.b Lit.b 1 40.8, s 37.2 34.3 r 37.1, s 37.2 34.3 2 80.1, d 48.4 39.6 2' 48.4, t 48.4 39.6 3 67.7, d 65.0 19.3 3' 65.1, d 65.0 19.3 4 40.1, t 42.5 33.1 4' 42.5, t 42.5 33.1 5 125.2, s 126.0 129.2 5' 126.2, s 126.0 129.2 6 137.4, s 138.3 137.8 6' 137.8, s 138.3 137.8 7 125.2, d 125.4 126.5 7' 125.6, d 125.4 126.5 8 139.1, d 138.5 137.6 8' 138.5, d 138.5 137.6 9 135.5, s 135.5 135.8 9' 135.7, s 135.5 135.8 10 131.6, d 131.1 130.7 10' 131.3,d 131.1 130.7 11 124.8, d 124.7 124.8 11' 124.9, d 124.7 124.8 12 137.8, d 137.6 137.1 12' 137.5, d 137.6 137.1 13 136.5e, s 137.4 136.3 13' 136.4e, s 137.4 136.3 14 132.7, d 132.4 132.4 14' 132.6, d 132.4 132.4 15 130.1c,d 129.9 129.8 15' 130.0e, d 129.9 129.8 16 21.5, q 30.2 29.8 16' 30.2, q 30.2 29.8 17 26.1, q 28.7 28.9 17' 28.7, q 28.7 28.9 18 21.3, q 21.6 21.7 18' 21.6, q 21.6 21.7 19 12.8, q 12.8 12.8 19' 12.8, q 12.8 12.8 20 12.8, q 12.8 12.8 20' 12.8, q 12.8 12.8 a CDC13, 75 MHz, 295 K. Multiplicities determined by DEPT 135 experiment. b Palermo et al., 1991. c Values between C-13 and C-13' and between C-15 and C-15' may be interchanged. Table 12.3 H NMR data of caloxanthin3(5)compared to literature data of different /3-carotene isomers0. Position 5 ail-trans 1-cis 9-cis \3-cis 15-c/s 7 6.10 6.17 5.83 (-0.34) 6.17 6.17 6.18 T 6.10 6.17 6.17 6.17 6.17 6.18 8 6.10 6.12 6.09 (-0.03) 6.66 (+0.54) 6.13 6.13 8' 6.10 6.12 6.12 6.11 6.13 6.13 10 6.17 6.14 6.21 (+0.07) 6.04 (-0.10) 6.15 6.15 10' 6.16 6.14 6.14 6.14 6.15 6.15 11 6.65 6.64 6.56 (-0.08) 6.74 (+0.10) 6.63 6.68 (+0.04) 11' 6.65 6.64 6.63 6.64 6.64 6.68 (+0.04) 12 6.38 6.34 6.32 (-0.02) 6.28 (-0.06) 6.87 (+0.53) 6.41 (+0.07) 12' 6.37 6.34 6.34 6.34 6.34 6.41 (+0.07) 14 6.27 6.24 6.23 6.23 6.09 (-0.15) 6.65 (+0.41) 14' 6.27 6.24 6.23 6.23 6.22 (-0.02) 6.65 (+0.41) 15 6.65 6.62 6.61 6.61 6.79 (+0.17) 6.38 (-0.24) 15' 6.65 6.62 6.61 6.61 6.55 (-0.07) 6.38 (-0.24) a CDC13, 500 MHz, 5ppm;m(7Hz), 295 K. b Koyama et al.,1989. c ß-carotene. The numbers in parenthesisshow the chemical shiftdifference between the respectivecw-isomer and the all-trans 12 PHYTOCHEMICAL INVESTIGATION OF CYLINDROSPERMUM SP. (EAWAG 76) 139 12.3 Structure Elucidation of the Heterocyte Gly¬ colipids The 13C NMR signals near 8C 98 ppm (three anomeric carbons at Sc 98.6, 98.4, 98.8; C-1 ') and between 70 and 75 ppm (carbons of the sugar ring), and the *H NMR signals near 8\i 4.6 ppm (anomeric protons; H-l') and between 3.0 and 4.0 ppm (pro¬ tons of the sugar ring) indicated the presence of three sugar molecules. Furthermore, signals consistent with the presence of a long alkyl chain were detected (8^ between further 1.0 and 1.7 ppm, 8C between 20 and 42 ppm). In the 13C NMR spectrum, sig¬ nals were observed at 8C 208.7, suggesting the presence of a ketone carbonyl carbon, five and at 8C 61.4, 67.0, and 65.7 ppm in a ratio of 1:1:3, indicating the presence of methines connected to hydroxyl groups. The position of the carbonyl group and of the hydroxymethines was established by means of HSQC-TOCSY and HMBC spectra. Combining the information of the ID and 2D spectra, three very similar partial structures could be identified. Since there is no correlation between these structures, these must be three different substances. These could be determined as heterocyte gly¬ colipids, two with two hydroxyl groups at positions 3 and y (compounds 6 and 7), and one with a carbonyl group at position 3 and a hydroxyl group at position y (compound 8). Due to the strong overlapping of the signals and the decomposition of the com¬ pounds during an attempt to separate them, the stereochemistry of the sugar moieties and the length of the alkyl chain could not be determined. Compounds 6 and 7 Figure 12.3 Heterocyte glycolipids (6-8) Tables 12.4 and 12.5 show the !H and 13C NMR data of compounds 6-8 which could be assigned in spite of the overlapping signals. 140 12.3 STRUCTURE ELUCIDATION OF THE HETEROCYTE GLYCOLIPIDS Table 12.4 *H NMR data of compounds 6-8a. Compound Position 6 7 8 la 3.68, m 3.73, dt (6.9, 9.8) 3.82, dt (6.5, 10.2) lb 3.46, m 3.42, m 3.57, m 2a 1.63, m 1.63,m 2.67, t (6.4) 2b 1.53, m 1.53, m 2.66, t (6.4) 3 3.55, m 3.52, m - 4 1.32, m 1.32, m 2.43, t (7.3) 5 - - 1.45, m y 3.55, m 3.55, m 3.55, m z 1.02, d (6.1) 1.02, d (6.1) 1.02, d (6.1) 1' 4.62, m 4.62, m 4.62, m 2'-5' 3.1-3.4 3.1-3.4 3.1-3.4 6'a 3.60, m 3.60, m 3.60, m 6'b 3.47, m 3.47, m 3.47, m a DMSO-d6, 500 MHz, S ppm; m (JHz), 310 K. Only unambiguously assignable signals are shown. Table 12.5 13C NMR data of compounds 6-8a. Compound Position 6 7 8 1 64.6, t 64.7, t 62.6, t 2 36.9, t 36.8, t 42.0, t 3 67.1, d 67.4, d 208.7, s 4 37.3, t 37.5, t 42.1, t 5 - - 22.9, t X 39.0, t 39.0, t 39.0, t y 65.7,d 65.7, d 65.7, d z 23.5,q 23.5, q 23.5, q r 98.6, d 98.4, d 98.8, d 2'-5' 70 - 74, d 70 - 74,d 70 - 74, d 6' 60.9, t 60.9, t 60.9, t a DMSO-d6, 75 MHz, 310 K. Only unambiguously assignable signals are shown. 12 PHYTOCHEMICAL INVESTIGATION OF CYLINDROSPERMUM SP. (EAWAG 76) 141 12.4 References Buchecker R, Liaaen-Jensen S, Borch G, Siegelman W (1976): Carotenoids of Anacystis nidulans, structures of caloxanthin and nostoxanthin. Phytochemistry 15: 1015-1018. Koyama Y, Hosomi M, Hashimoto H, Shimamura T (1989): *H NMR spectra of the all-trans, 1-cis, 9-cis, 13-cis and \5-cis isomers of ß-carotene: elongation of the double bond and shortening of the single bond toward the center of the conjugated chain as revealed by vicinal coupling constants. J. Mol. Struct. 193: 185-201. Palermo JA, Gros EG, Seldes AM (1991): Carotenoids from the red algae of the Corallinaceae. Phytochemistry 30: 2983-2986. Takaichi S, Shimada K, Ishidsu J (1990): Carotenoids from the aerobic photosyn¬ thetic bacterium Erythrobacter longus: ß-carotene and its hydroxyl dérivâtes. Arch. Microbiol. 153: 118-122. 142 13.1 SELECTION OF CYANOBACTERIAL STRAINS 13 Discussion 13.1 Selection of Cyanobacterial Strains Seventeen cyanobacterial strains from Switzerland, two from Cameroon, and one from Great Britain, Sweden, and Nepal were chosen for this study to provide a broad spectrum of different cyanobacterial species. Many of the selected genera are known for producing biologically active compounds, however, they are not well documented in literature. 13.2 Cultivation The selected cyanobacterial strains were cultivated in suitable inorganic media. The different media provide a spectrum of nutrients that allows the growth of nearly every terrestrial and freshwater cyanobacterium. The large scale cultivation of cyano¬ bacteria has the goal to gain sufficient biomass in an easy and fast way. However, the cultivation of cyanobacteria has not yet reached the efficacy achieved with bacteria, as cyanobacteria grow slower and their metabolites give lower yields in comparison to bacteria or fungi. Since relative large amounts of biomass are needed for phytochemi- cal studies, it is of great importance to optimize the cultivation conditions and the pro¬ duction of metabolites. A further problem in the cultivation of cyanobacteria is to get axenic cultures. As most of the antibiotics active against bacteria would kill also cyanobacteria, it is very difficult to separate the cyanobacterial cells from accompanying bacteria. For this rea¬ son, it is necessary to check the cultures regularly, to make sure that the cyanobacterial mass is free from bacteria, fungi, and also from other cyanobacterial species. 13 DISCUSSION 143 13.3 Bioassays A large number of bioassays are available for the testing of crude extracts and iso¬ lated compounds, and the results obtained are profoundly influenced by the methods selected. Bioassays used for phytochemical purposes should be simple to perform, fast, economical, in-house, and have valid statistical correlation with the desired bioactivi- ties. During this work, the extracts of the cyanobacterial cells, as well as the extracts of the large scale cultivated strains and their culture media, were tested. Bioassays were performed also with the fractions obtained during the separation of these extracts and with the isolated compounds. Interestingly, 19 of the 22 cyanobacteria investigated during the biological screen¬ ing induced a response in at least one of the test systems applied, indicating the poten¬ tial of cyanobacteria to provide new biologically active compounds. 13.4 Extraction and Isolation The extraction of the cyanobacterial cells for the biological screening as well as for the phytochemical investigation was performed with only two solvent systems (dichlo- romethane/methanol 2:1 followed by methanol/water 7:3). Because of the small amounts of the starting cell material, the use of more than two solvent systems would have led to extracts too small to allow further investigation. Vacuum liquid chromatography (VLC) and open column chromatography were suitable methods for a preliminary fractionation of the extracts and prepurifications of the fractions prior to HPLC. Pure compounds were obtained by precipitation or by semipreparative HPLC. The isolation procedure was guided by bioassays, TLC and H NMR. Especially TLC and lU NMR showed to be suitable methods for identifying interesting fractions. The major problem during the isolation was the small amount of starting cell mate¬ rial: this often led to small fractions which could not be treated further. Most of the pure compounds were isolated in very small amounts, which caused problems in the struc¬ ture elucidation and made it impossible to perform bioassays. 144 13.5 STRUCTURE ELUCIDATION 13.5 Structure Elucidation The structure of the isolated compounds was elucidated mainly by means of mass spectrometry and NMR. For the four new naphthalenone derivatives isolated from Scytonema spirulinoides, mass spectrometry and *H and 13C NMR data provided the molecular weight and the molecular formula. By means of HSQC, HSQC-TOCSY and COSY spectra the struc¬ ture of fragments could be established. HMBC correlations allowed to determine the correlations between these fragments. The structure of the carotenoid caloxanthin from Cylindrospermum sp. was deter¬ mined by ID- and 2D-NMR, and mass spectrometry. The unambiguous assignment of all resonances was very difficult because of the overlapping of many signals, and was achieved also by comparison with similar carotenoids, as described in chapter 12.2. The complete structure of the heterocyte glycolipids isolated from Cylindrosper¬ the mum sp. could not be determined because the three compounds decomposed during attempt to separate them. However, partial structures could be elucidated by ID- and especially 2D-NMR. 13.6 Isolated Compounds The four naphthalenone derivatives from Cylindrospermum sp. are the first com¬ pounds of this class isolated from cyanobacteria. Similar compounds, like shinanolone, differing from the isolated naphthalenone 1 only in the position of the methyl group, were isolated from some Asian Diospyros species (family Ebenaceae) (Kuroyanagi et al., 1971; Zakaria et al., 1984). Furthermore, di- and trihydroxynaphthalenones like vermelone and scytalone have been postulated as precursors for melanin in fungi (Bell et al, 1976; Stipanovic and Bell, 1976). The isolated compound 1 showed a weak anti¬ bacterial activity against Bacillus cereus (MIC 64 ppm, the reference compound chloramphenicol had a MIC value of 4 ppm). No inhibition of growth was observed against Staphylococcus aureus, Escherichia coli and Candida albicans. Carotenoids are ubiquitous compounds in cyanobacteria, as they are involved in the photosynthetic process. Caloxanthin, the carotenoid isolated in this work from Cylin- 13 DISCUSSION 145 drospermum sp., has been isolated previously from the cyanobacterium Anacystis nid¬ ulans (Buchecker et al., 1976) and from the bacterium Erythrobacter longus (Takaichi et al., 1990). However, only incomplete lH NMR and no 13C NMR data were avail¬ able. The heterocyte glycolipids, isolated during this work from Cylindrospermum sp., occur only in nitrogen-fixing cyanobacteria (Soriente et al., 1992). They are localized in the cell wall, where they form a thick layer; it is suggested that this glycolipid layer reduces the rate of diffusion of oxygen into the heterocytes to such an extent that most ofthat which does enter is reduced by oxidases present in the heterocytes. Heterocyte glycolipids have been isolated for example from Nodularia harveyana (Soriente et al., 1992), Cyanospira rippkae (Soriente et al., 1993), Anabaena cylindrica (Soriente et al., 1995), Scytonema hofmanni, Calothrix desertica, Fischerella muscicola, Tolypothrix tenuis (Gambacorta et al., 1998), but they have not been reported from the genus Cylin¬ drospermum. Unfortunately, the three compounds decomposed during the attempt to separate them by preparative TLC. 13.7 Conclusions In this investigation, chemically different and interesting compounds were isolated from cyanobacteria, and therefore give promise that further chemical and biological investigations may provide interesting findings. In further studies following points should be included: - The influence of the composition of the culture media on the growth rate of the cyanobacteria and on the chemical production should be evaluated. Furthermore, the cultivation procedures have to be optimized, in order to improve the synthesis of inter¬ esting substances. - The hazardous effects and especially the ecological role of the cyanobacterial sec¬ ondary metabolites are poorly understood. Therefore, the ecological aspects should be also taken into consideration. - A comparison of the chemical and morphological characteristics of re-collected organisms from nature with the long-term cultivated strains may reveal the environ¬ mental influence on these organisms. 146 13.8 REFERENCES - As many compounds are isolated from the culture media, it would be interesting to study the mechanisms ofrelease ofthese compounds in the medium, in order to opti¬ mize the culture conditions and the release rate. - As cyanobacteria often produce substances with unusual structures, it is of great importance to investigate the biosynthetic pathways of these organisms. 13.8 References Bell AA, Stipanovic RD, Puhalla JE (1976): Pentaketide metabolites of Verticil- Hum dahliae. Identification of (+)-scytalone as a natural precursor to melanin. Tetrahe¬ dron 32: 1353-1356. Buchecker R, Liaaen-Jensen S, Borch G, Siegelman W (1976): Carotenoids of Anacystis nidulans, structures of caloxanthin and nostoxanthin. Phytochemistry 15: 1015-1018. Gambacorta A, Pagnotta E, Romano I, Sili C, Sodano G, Trincone A (1998): Het¬ erocyst glycolipids from nitrogen-fixing cyanobacteria other than Nostocaceae. Phy¬ tochemistry 48: 801-805. Kuroyanagi M, Yoshihira K, Natori S (1971): Naphthoquinone derivatives from the Ebenaceae. III. Shinanolone from Diospyros japonica Sieb. Chem. Pharm. Bull. 19:2314-2311. Soriente A, Sodano G, Gambacorta A, Trincone A (1992): Structure of the "hetero¬ cyst glycolipids" of the marine cyanobacterium Nodularia harveyana. Tetrahedron 48: 5375-5384. Soriente A, Gambacorta A, Trincone A, Sili C, Vincenzini M, Sodano G (1993): Heterocyst glycolipids of the cyanobacterium Cyanospira rippkae. Phytochemistry 33: 393-396. Soriente A, Bisogno T, Gambacorta A, Romano I, Sili C, Trincone A, Sodano G (1995): Reinvestigation of heterocyst glycolipids from the cyanobacterium Anabaena cylindrica. Phytochemistry 38: 641-645. Stipanovic RD, Bell AA (1976): Pentaketide metabolites of Verticillium dahliae. Identification of (-)-3,4-dihydro-3,8-dihydroxy-l(2H)-naphthalenone [(-)-vermelone] as a precursor to melanin. J. Org. Chem. 41: 2468-2469. 13 DISCUSSION 147 Takaichi S, Shimada K, Ishidsu J (1990): Carotenoids from the aerobic photosyn¬ thetic bacterium Erythrobacter longus: ß-carotene and its hydroxyl dérivâtes. Arch. Microbiol. 153: 118-122. Zakaria M, Jeffreys JAD, Waterman PG, Zhong S-M (1984): Naphthoquinone and triterpenes from some Asian Diospyros species. Phytochemistry 23: 1481-1484. 148 13.8 REFERENCES Publications and poster presentations Publications Orjala J, Mian P, Rali T, Sticher O (1998): Gibbilimbols A-D, cytotoxic and anti¬ bacterial alkenylphenols from Piper gibbilimbum. J. Nat. Prod. 61: 939-941. Mian P, Heilmann J, Biirgi H-R, Sticher O: Biological screening of terrestrial and freshwater cyanobacteria for antimicrobial activity, brine shrimp lethality, and cyto¬ toxicity. Pharm. Biol., accepted. Mian P, Heilmann J, Sticher O: Four new extracellular naphthalenone derivatives from the terrestrial cyanobacterium Scytonema spirulinoides. J. Nat. Prod., in prepara¬ tion. Poster presentations Mian P, Orjala J, Rali T, Sticher O: New biologically active alkenylphenols from Piper gibbilimbum. 44th Annual Congress of the Society for Medicinal Plant Research and a Joint Meeting with the Czech Biotechnology Society, Prague, September 3-7, 1996 Mian P, Heilmann J, Biirgi H-R, Sticher O: Evaluation of the biological activity of extracts from 22 cyanobacertia and isolation of a new diaryldecanoide from Scytonema spirulinoides. Traits, Tracks and Traces, International Congress and 49th Annual Meeting of the Society for Medicinal Plant Research, Friedrich-Alexander-Universität, Erlangen-Nürnberg, Germany, September 2-6, 2001. Curriculum vitae 1969 Born on 23rd March in Trieste (Italy) 1975 - 1980 Primary School in Trieste 1980 - 1983 Middle School in Trieste 1983-1989 High School in Trieste 1989 - 1995 Study in "Chimica e Tecnologia Farmaceutiche" at the Facoltà di Farmacia, Università degli Studi di Trieste Oct. 1995-Sept. 1996 Diploma work at the division of Pharmacognosy-Phytochem- istry, Departement of Pharmacy, ETH Zurich, Switzerland March 1997 Final examination (Laurea in Chimica e Tecnologia Farma¬ ceutiche) since April 1997 Ph. D. study under the supervision of Prof. Dr. Otto Sticher at the division of Pharmacognosy-Phytochemistry, Institute of Pharmaceutical Sciences, ETH Zurich, in collaboration with the Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), Dübendorf, Switzerland Teaching in practical courses "Phytochemisches Praktikum I und II", ETH Zurich October 2002 Final examination to obtain the degree of Doctor of Natural Sciences, ETH Zurich since June 2002 Floor Manager Solid Dosage Forms and Oral Contraceptives, Cilag AG, Schaffhausen, Switzerland