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Biochemical and pharmacological investigations of selected cyanobacteria
International Journal of Hygiene and Environmental Health
Int. J. Hyg. Environ. Health 203, 327-334 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/intjhyg
Biochemical and pharmacological investigations of selected cyanobacteria Sabine Mundt1, 2, Susann Kreitlow1, Andrea Nowotny1, and Uta Effmert3 1 2 3
Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany Institute of Marine Biotechnology, Greifswald, Germany Department of Biology, University Rostock, Rostock, Germany
Received September 13, 2000 · Accepted February 15, 2001
Abstract Cyanobacteria are a very old group of prokaryotic organisms that produce a variety of secondary metabolites with antibiotic, algicide, cytotoxic, immunosuppressive and enzyme inhibiting activities. In the last decades structures of pure compounds have been determined as phenols, peptides, alkaloids or terpenoids (Falch, 1996). Screening of lipophilic and hydrophilic extracts from cultured cyanobacteria or waterbloom material, isolated from German lakes and the Baltic sea for antiviral, antibiotic, immunomodulating and enzyme inhibiting activity in different in vitro systems revealed strains with interesting effects. These strains were cultivated in 45 litre photobioreactors to produce enough biomass for bioassay-guided isolation of the active substances. First results characterising active substances are reported. Key words: Cyanobacteria – immunomodulating – antibacterial – antiviral – cultivation – bioassay-guided isolation – α-linolenic acid
Introduction Cyanobacteria (blue-green algae) are a diverse group of photosynthetic, prokaryotic organisms found in freshwater and marine environments. The origin of these organisms is dated back three or four billion years (Schopf and Packer, 1987). Their cell structure resembles that of Gram negative bacteria, but as a rule they live photoautotrophically. Like higher plants they possess chlorophyll a and the water soluble red and blue phycobiliproteins as well as photosystem I and II. Therefore they can use water for photosynthesis and produce oxygen, which is released to the atmosphere.
With about 2000 strains cyanobacteria are distributed all over the world. They show a remarkable ecological diversity. Because of widespread eutrophication of lakes, ponds and some parts of oceans cyanobacteria often form blooms, which lead to water hygienic problems (Henning and Kohl, 1981; Skulberg et al., 1984; Chorus and Bartram, 1999; Duy et al., 2000). They may cause unpleasant tastes and odours through excretion of volatile compounds (Jones and Korth, 1995). Furthermore animal poisonings and risks to human health are described. Several genera of cyanobacteria form toxic waterblooms and different cyanobacterial toxins have been characterised (Carmichael, 1992,
Corresponding author: PD Dr. Sabine Mundt, Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst-MoritzArndt-University Greifswald, F.-L.-Jahn-Straße 15a, D-17487 Greifswald, Germany, Phone: +49/3834/864 869, E-mail: [email protected] 1438-4639/01/203/4-327 $ 15.00/0
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1994; Rinehart et al., 1994; Hunter 1995). Possibly the synthesis of highly active toxins is a defence option of cyanobacteria against attack by other organisms like bacteria, fungi, zooplankton and eukaryotic microalgae. Carmichael (1994) found that cyanobacterial toxins can be extremely harmful to zooplankters that feed on cyanobacteria. They may be directly lethal or they may reduce their offspring. Besides the toxins a lot of active substances with antibacterial, antiviral, fungicide, enzyme inhibiting, immunosuppressive, cytotoxic and algicide activity has been isolated from cyanobacterial biomass or in some cases from the medium of laboratory cultures (Falch, 1996; Hayashi et al., 1996; Namikoshi and Rinehart, 1996; Banker and Carmeli, 1998; Harrigan et al., 1998 and 1999; Jaki et al., 1998 and 1999). Producing active biocide components could be an important selective advantage. In the 1970’s a pronounced reduction of Gram positive bacteria was found in lakes during the occurence of cyanobacterial blooms (Chrost, 1975). The production of antibacterial substances could be the reason for this phenomenon. In cyanobacterial blooms often only one species comes up to more than 95 % of the population. Though this has been interpreted as a result of competition between species, the dominance of one species could be a hint for the formation of metabolites with cyanobactericidal activity. In addition, antibacterial, fungicide and antiviral effective compounds formed by cyanobacteria could contribute to an improvement of the water quality in our aquatic environment. The aim of our work was to identify cyanobacterial strains from German lakes and the Baltic sea with relevant bioactivity in different in vitro test systems. The following optimisation of large scale culture conditions of these strains to produce enough biomass was the prerequisite for a bioassay-guided isolation of bioactive compounds and their structural elucidation.
Materials and methods Screening The following 35 cyanobacterial strains were screened for biochemical and pharmacological effects in different in vitro test systems: The Chroococcales : Gloeocapsa caldariorum RABENH. 127 H, Microcystis aeruginosa KÜTZ. HUB 042 B, HUB 063 B, HUB 018 B, HUB 5-2-4 B, HUB 5-3 B, PCC 7813, PCC 7820, 14.85 B, SAG 14.85, SPH 01 G, Microcystis firma (BREB. et LENORM.) SCHMIDLE 398/5 R, Synechocystis aquatilis SAUV. 428 R; The Oscillatoriales: Oscillatoria species 234 H, Oscillatoria agardhii GOM. (syn. Planktothrix agardhii GOM.) HUB 011 B, Oscillatoria redekei van GOOR (syn. Limnothrix redekei MEFFERT) HUB 010 B, HUB 051 B, HUB 022 B, Oscil-
latoria prolifica GOM. (syn. Planktothrix prolifica GOM.) SPH 02 G, Oscillatoria rubescens DC. Ex GOM. (syn. Planktothrix rubescens Dc. Ex GOM.) HUB 016 B, Oscillatoria tenuis AG. Ex GOM (syn. Phormidium tenue AG. Ex GOM) SPH 03 G, Phormidium species Hg 71-212 J, Pseudanabaena catenata LAUTERB. 154 H. The Nostocales: Anabaena flos-aquae (Lyngb.) BREB. AFA K, HUB 039B, Anabaena variabilis KÜTZ: AV K, 1 H, Aphanizomenon flos-aquae (L.) RALFS. HUB 046 B, Calothrix gracilis FRITSCH 96 H, Cylindrospermum majus KÜTZ. 95 H, Nodularia harveyana THUR. NH K, Nodularia spumigena MERT. HÜ 280 H, Nostoc species 104 H, Nostoc linckia (ROTH.) BORN. et. FLAH. 230 H, Scytonema bonerii SCHMIDLE Hg 73–2858 J. The strains were provided by Prof. Kohl, Humboldt University of Berlin B, Prof. Pohl, University of Kiel K, Dr. Hegewald, Institute of Biotechnology, Jülich J, Dr. Hübel, University of Greifswald, Hiddensee H, bought from culture collections or isolated by our own group G. Field material was used in only one case. Biomass for screening was produced by cultivating the strains in mineral salt solution under continuous light at temperatures between 18 °C and 22 °C. After harvesting by centrifugation biomass was freeze dried and extracted either with water or with n-hexane, methanol and water successively. The supernatants of the extractions were combined, the organic solvents were removed in vacuum and the aqueous residues were freeze dried. These dry extracts were used for screening in different in vitro test systems. In vitro test systems For the Lymphocyte Transformation Test (LTT) to investigate immunomodulating activity (Lindl and Bauer, 1989) freshly prepared human peripheral blood lymphocytes (HPBLs) were cultivated in vitro for three days in presence of the mitogen phythaemagglutinin and the aqueous extracts of cyanobacterial biomass. The influence of the extracts on mitogen-stimulated proliferation of the cells was quantified by measuring the incorporation of 3H-thymidine in the DNA. Cytotoxic effects of the aqueous extracts to HPBLs were determined with trypane blue (Lindl and Bauer 1989) and only non cytotoxic extract concentrations were used in LTT. Inhibiting or stimulating activities of the aqueous extracts on the enzymatic activity of cyclooxygenase and lipoxygenase were tested by measuring the consumption of oxygen in presence of a substrate. The activity of leucine aminopeptidase was estimated colorimetrically by using the substrate leucine hydrazid. Protease inhibitory effects tested by trypsine inhibition were monitored with BAPNA (α-N-benzoylDL-arginine-p-nitroanilid) (Pilgrim et al., 1992). The agar plate diffusion test for antibacterial and fungicide activity of cyanobacterial extracts was described by Kreitlow et al. (1999). The activity of aqueous, methanol and n-hexane extracts was tested against the Gram positive bacteria Bacillus subtilis SBUG 14, Staphylococcus aureus SBUG 11 and Micrococcus flavus SBUG 16, the Gram negative bacteria Escherichia coli SBUG 17 and the yeast Candida maltosa SBUG 700. Tests for antibacterial effects against multiresistant
Cyanobacteria Staphylococcus strains were performed in cooperation with the Landeshygieneinstitut Greifswald. Antiviral activity against influenza A virus /WSN 33/London and herpes simplex virus Type 1 was investigated in a dye uptake assay with neutral red (Nowotny et al., 1997). Antiviral effects against adenovirus type 2 were tested in a modified focus reduction test (Mentel et al., 1996). Virus protein synthesis was analysed by SDS polyacrylamide gel electrophoresis (Mentel et al., 1994). In all cases, the cytotoxicity of the extracts was tested and only non cytotoxic concentrations were used in the cellular test systems. Large scale batch cultivation Culture conditions were optimised for the filamentous cyanobacterium Limnothrix redekei HUB 051 and the unicellular cyanobacterial strain Microcystis aeruginosa SPH 01. The growth of the cultures was examined by measuring the optical density at 730 nm (UVICON 930, KONTRON Instruments). In small culture flasks of 200 ml the influence of the following parameters on production of biomass was tested: light intensity (500 to 5000 lux, light/dark rhythm of 12 hours), temperature (18 °C, 30 °C), pH (4 to 11) and salt concentration (phosphate, nitrate) of culture medium Z1/2 (Meffert 1971) or BG 11 (Waterbury and Stanier 1981). Different ways to agitate the cultures were also tested. The optimal conditions were used for scaling up to a 45 l batch culture. The cylindrical bioreactor was made of glass with a bottom of stainless steel. Aeration (air and carbon dioxide) and mild agitation were realised by nozzles in the bottom of the cultivation tank. The concentration of carbon dioxide was regulated to maintain a pH of 10. An external illumination with alternatively two, four or six fluorescent lamps realised a light intensity of 500 to 5000 lux (Teuscher et al. 1992). Biomass was harvested every 3 days by filtration 10 l of the culture using filter paper circles with a pore wide of 12– 25 µm (Schleicher and Schuell). The medium loss was compensated by addition of the same volume of fresh one. Bioassay-guided isolation For isolation of the antibacterial substances of Limnothrix redekei HUB 051 the n-hexane extract was fractionated by column chromatography with silica gel (Silicagel 60, MERCK) using petrolether/ethyl acetate 7 2, followed by ethyl acetate and methanol. Fractions with antibacterial activity were further fractionated on reversed phase material (Lichroprep RP-18, MERCK) with a step gradient acetonitrile/water. Three active fractions were separated and repeatedly fractionated on reversed phase material. Two pure fractions were received and investigated by different methods of MS (HRFAB-MS, ESI-MS and DCI-MS) and NMR (1H, 13C, COSY, HMBC) in cooperation with the GBF Braunschweig. Attempts to isolate compounds with antiviral activity from the Microcystis aeruginosa waterbloom were also performed by column chromatography (Nowotny et al. 1997). The aqueous extract prepared from the biomass was precipitated with ethanol. After the removal of ethanol in vacuum the aqueous phase was separated using ion exchange chrom-
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atography (DOWEX 50 W, SERVA, 10 % ammonia; DOWEX 1 1, SERVA, 2 N formic acid) in a basic fraction, an acid fraction and a neutral fraction (unbound). The basic compounds were further separated by gel chromatography on Sephadex G 25 (PHARMACIA) using water as eluent. Alternatively the fraction of basic compounds was separated by HPLC (HPLC system SMART, PHARMACIA) on a Superdex column 75 PC (PHARMACIA) with acetonitrile/water 2 8. The trypsine inhibiting fractions were further separated on a µRPC C2/C18 column (PHARMACIA) in a gradient of acetonitrile, water, trifluoroacetic acid. The active fractions were analysed by MS and NMR as described above or by MALDI-TOF-MS in the TU Berlin (Erhard et al. 1997).
Results Screening The influence of aqueous extracts prepared from the biomass of 35 cyanobacterial strains on human peripheral blood lymphocytes was tested using extract concentrations of 70, 14 and 1.4 µg/ml. In these concentrations 24 extracts showed a considerable cytotoxicity to the lymphocytes. Therefore only the 11 non cytotoxic aqueous extracts were tested in LTT (Fig. 1.) Three of them, the extracts of Oscillatoria tenuis SPH 03, Limnothrix redekei HUB 051 and Synechocystis aquatilis 428, inhibited the mitogen stimulated proliferation of HPBLs by more than 30 %. The effect of the aqueous extracts on the activity of the enzymes cyclooxygenase, lipoxygenase and leucine aminopeptidase was also determined. The activity of cyclooxygenase was not influenced. In contrast the activity of lipoxygenase was inhibited by 33 of 35 extracts. On the other hand, only the aqueous extracts of Oscillatoria tenuis SPH 03 and the Microcystis strain SAG 14.85 inhibited the leucine aminopeptidase by about 75 %. For the detection of antibacterial activity extracts prepared from the biomass with n-hexane, methanol and water were tested against the Gram positive bacteria Bacillus subtilis SBUG 14, Staphylococcus aureus SBUG 11 and Micrococcus flavus SBUG 16, the Gram negative bacteria Escherichia coli SBUG 17 and the yeast Candida maltosa SBUG 700. The n-hexane extracts of Oscillatoria species 234, Nostoc species 104, Cylindrospermum majus 95, Calothrix gracilis 96 and Limnothrix redekei HUB 051 and the methanol extracts of Anabaena variabilis 1, Gloeocapsa caldariorum 127, Pseudanabaena catenata and Limnothrix redekei HUB 051 inhibited the growth of Bacillus subtilis SBUG 14. The n-hexane extract of Limnothrix redekei HUB 051 was also active against Staphylococcus aureus SBUG 11 and Micrococcus flavus SBUG 16. The aqueous extracts were ineffective.
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Fig. 1. Influence of cyanobacterial extracts on the 3H-thymidine incorporation of humane peripheral blood lymphocytes Cyanobacterial dry mass in µg/ml (Strains with substantial inhibition are accentuated in ⊂⊃).
Tests for antiviral activity against adenovirus, herpesvirus Type 1 and influenza A virus were performed with methanolic and aqueous extracts. No extract showed an activity against adenovirus. Only the methanolic extracts of Nodularia spumigena HÜ 280 and Synechocystis aquatilis 428 were weakly effective against herpesvirus Type 1. 12,5 µg/ml of the aqueous extracts of the filamentous strains Calothrix gracilis 96 and Oscillatoria species 234 inhibited the replication of Influenza A virus in MDCK cells completely. For the Microcystis aeruginosa waterbloom SPH 01 an ED50 of 10.2 µg/ml was estimated. If 100 µg/ml Microcystis extract were available for the whole incubation period the synthesis of viral proteins analysed by gel electrophoresis was clearly reduced. Testing this extract in a trypsine inhibitor assay revealed a considerable enzymatic inhibition in concentrations of 5 µg/ml. According to the results of screening tests the strain Limnothrix redekei HUB 051 (because of its good antibacterial activity) and Microcystis aeruginosa water bloom SPH 01 (because of the antiviral activity against influenza A virus) were selected for optimizing culture conditions to produce biomass for the isolation of bioactive substances. Cultivation The best growth of Limnothrix redekei HUB 051 was achieved using the mineral salt medium Z1/2 with three times the amount of phosphate and nitrate as
compared to the original medium, continuous illumination with warm white fluorescent lamps with an intensity of 500 lux, pH 9 to 10 and a relatively low temperature of 18 °C to 20 °C, an increase to 30 °C resulted in a die off of the culture. In the 45 l fermenter a specific growth rate of µ 0.5 d–1 was reached compared with cultivation in 200 ml volumes under control conditions (pH 7.5, 500 lux, Z1/2 unmodified), where a specific growth rate was estimated with µ 0,22 5 d –1 (fig. 2). In contrast to the small culture flasks the light intensity in the fermenter was increased from 500 lux to 5000 lux with increasing optical density of the culture. Cultivation in the fermenter over 6 to 8 weeks yielded of 1.15 g cyanobacterial biomass per litre and day. The best yield of the n-hexane extract was obtained under the same conditions under which biomass production was optimal. The isolation of a laboratory culture strain from the Microcystis waterbloom, that showed the same antiviral activity against influenza A virus as the waterbloom material was not yet sucessful. Bioassay-guided isolation of active components Bioassay-guided isolation of the antibacterial substances of Limnothrix redekei HUB 051 by column chromatography led to three fractions with activity against the Gram positive bacteria Staphylococcus aureus SBUG 11 and Micrococcus flavus SBUG 16. One active
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fraction was identified by MS and NMR using reference substance as α-linolenic acid. The minimal inhibition concentrations (MICs) of the isolated α-linolenic acid were determined against Staphylococcus aureus SBUG 11 with 75 µg/ml and against Micrococcus flavus SBUG 16 with 25 µg/ml. A concentration of αlinolenic acid of 500 µg/filter disk was also effective against multiresistent Staphylococcus strains. Inhibition zones from 10 to 20 mm were measured. The other two active fractions are further separated and structural elucidation of the isolated active substances is in progress. The antiviral activity of the aqueous extract of Microcystis aeruginosa waterbloom material against influenza A virus was concentrated in a fraction of basic compounds after ion exchange chromatography. Further separation by gel chromatography on Sephadex G 25 resulted in five fractions G1 to G5. Analysis of fraction G4 and G5 by MS and NMR showed adenosine and adenine to be the major components (Nowotny et al. 1997). Alternatively the fraction of basic compounds was separated by HPLC on Superdex in six fractions, which were tested for trypsine inhibiting activity. The two trypsine inhibiting fractions 5 and 6 were further separated on reversed phase material in several fractions. The most active fractions were analysed by MALDI-TOF-MS. One fraction was identified as the cyclic hexapeptide anabaenopeptine B. This peptide was detected at m/z 837 and analysed in the PostSource-Decay (PSD) mode using anabaenopeptine B as the reference substance. Additional four linear peptides with trypsine inhibiting activity were identified to be new aeruginosins. They were detected at m/z 619, 633, 715 and 729. PSD spectra have revealed the unusual unit 2-carboxy-6hydroxy-octahydroindol (Choi) as the common amino acid component, leucine, a guanidine-containing unit deriving from arginine at the C-terminus and p-hydroxyphenyl lactic acid at the N-terminus. Another fraction with trypsine inhibiting effect was identified to be a new cyanopeptoline with m/z 979. The structural elucidation of the cyanopeptoline and the testing of the characterised substances for activity on the replication of influenza A virus is in progress.
Discussion In the last decades screening programs have revealed that cyanobacteria are a potential source of new active substances for medicine and pharmacy and numerous active compounds have been isolated (Moore et al., 1989; Patterson et al., 1994; Falch, 1996, Hayashi et al., 1996; Namikoshi and Rinehart, 1996; Papendorf
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Fig. 2. Growth curves of the cyanobacterium Limnothrix redekei HUB 051 in culture flasks and in the fermenter lnE optical density of the culture at 730 nm.
et al., 1998; Kajiyama et al., 1998; Jaki et al., 1999; Singh et al., 1999; Luesch et al., 2000; Horgen et al., 2000). These active compounds could influence the aquatic environment of cyanobacteria by reducing the number of viruses, bacteria and other micoorganisms. Cyanobacteria of local habitats have been rarely screened for biological effects. Therefore, our aim was the investigation of cyanobacterial strains especially from ponds, lakes and rivers of the northern areas of Germany. Only a few strains from culture collections were included in the tests. The extracts of cyanobacterial biomass were tested for immunomodulating, enzyme inhibiting, antibiotic and antiviral activity in different in vitro test systems. The determination of proliferation of lymphocytes is a very common assay in immunological research. The Lymphocyte Transformation Test (LTT) records the proliferation of T-lymphocytes in response to a mitogen as one step of the immune reaction. If an effect exceeds the value of 30 % it is either defined as stimulation or inhibition of the proliferation due to the considerable standard deviation of the values measured. Under these conditions the aqueous extracts of Limnothrix redekei HUB 051, Oscillatoria tenuis SPH 03 and Synechocystis aquatilis 428 inhibited the mitogenstimulated proliferation clearly. Cyclooxygenase and lipoxygenase catalyse the formation of prostaglandins, hydroxylated fatty acids, hydroperoxylated fatty acids and leucotrienes. These substances play an important role in allergic reactions, so that an inhibition of the enzymes could be one possibility to prevent overshooting immune reactions. In presence of the aqueous extracts no effects to cyclooxygenase have been observed. In contrast, the activity of lipoxygenase was inhibited by nearly all extracts. Probably unspecific effects by ubiquitous substances are the reason for this phenome-
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non. Leucine aminopeptidase, a membrane-bound proteinase may influence immune reaction by antigen processing and improving antigen presentation (Beninga et al., 1998). Maturing and differentiating of immune cells are connected with an enhanced activity of the enzyme (Becker, 1984; Rohrbach and Conrad, 1991; Van Hal et al., 1992). Two of 35 extracts inhibited the activity of leucine aminopeptidase. One of the active extracts was also effective in the LTT. Antibacterial activity was especially found in the more lipophilic extracts; the aqueous extracts were ineffective. Only the growth of Gram positive bacteria was inhibited, a phenomenon that has already been described (Chrost et al., 1975). A reason could be that cyanobacteria are also Gram negative bacteria, but recently it has been shown, that comnostins and noscomin isolated from Nostoc commune are also active against Gram negative bacteria (Jaki et al. 1998 and 1999). Activities against herpesvirus Type 1 and influenza A virus were found in the dye uptake assay using neutral red. The aqueous extract of the Microcystis aeruginosa waterbloom SPH 01 inhibited the replication of influenza A virus completely without any cytotoxic effects against the host cells. The ED50, the concentration of the extract that resulted in a 50 % survival of cells infected with virus, was estimated at 10.2 µg/ml. For amantadine-HCl, a drug which is used in treatment of influenza, an ED50 of 16.8 µg/ml was estimated. The inhibition of virus protein synthesis has been shown by gel electrophoresis (Nowotny et al. 1997) Additionally, the extract inhibited the protease trypsine in very low concentrations. This protease inhibition could be the reason for the inhibition of influenzavirus replication because the processing of virus proteins is an obligatory step in virus life cycle (Kido et al. 1992). In last years several protease inhibitors, cyclic and linear peptides, have been isolated from Microcystis aeruginosa strains (Namikoshi and Rinehart 1996), so that the antiviral effects may be caused by these peptides too. According to the results of screening tests the strains Limnothrix redekei HUB 051 with antibacterial activity and Microcystis aeruginosa waterbloom SPH 01 with antiviral activity against influenza A virus were selected for further investigations. The main conditions for practical use of the active strains is to establish a method for a large scale cultivation with stable production of the active compounds in culture. Optimisation of culture conditions resulted in a biomass production of 1.15 g/l d. The yield was in the order of magnitude described for plate-type photobioreactors (Puls, 1992) and higher than for helical tubular photobioreactors (Watanabe et al. 1995). Laboratory cultures isolated from the Microcystis aeruginosa waterbloom were able to grow but the anti-
viral activity of the laboratory grown biomass was considerably lower than in the biomass collected from the field. Attempts to optimise the culture conditions for production of antiviral compounds were not yet successful. Therefore, isolation of antiviral components was attempted with the waterbloom material. Bioassay guided isolation of effective substances is a common method in natural product chemistry. The antibacterial n-hexane extract of Limnothrix redekei HUB 051 was fractionated using different separation techniques. Two active fractions were received and one was identified by NMR and MS as α-linolenic acid. The antibacterial activity of this polyunsaturated fatty acid is well known. In 1981 MC DONALD and coworkers (Mc Donald et al., 1981) reported about antibacterial effects of α-linolenic acid against methicillinresistent Staphylococcus strains. A Japanese group isolated α-linolenic acid as the antibiotic principle from the biomass of the green alga Chlorococcum species HS-101 (Ohta et al., 1995). In the culture medium of the cyanobacterium Phormidium tenue a mixture of different fatty acids containing also linoleic and linolenic acid was responsible for autolysis of the axenic culture (Murakami et al., 1990; Yamada et al., 1993). The MICs for α-linolenic acid range from 75 µg/ml against Staphylococcus aureus SBUG 11 to 25 µg/ml against Micrococcus flavus SBUG 16, but ampicillin was more effective against these bacteria. The MICs were 0.05 µg/ml and 0.25 µg/ml respectively. Another antibacterial fraction, separated after column chromatography on reversed phase material seems to be a mixture of polyunsaturated and hydroxylated fatty acids. Characterisation of structures is in progress. The isolation of the antiviral components of Microcystis aeruginosa waterbloom resulted in two fractions with activity against influenza A virus. Surprisingly, adenosine and adenine were identified to be the main components. Besides the potential effect of these substances, which has not yet been clarified, the antiviral activity could be attributed to fractions showing protease inhibiting activity. One of these fractions that has not been tested for antiviral activity was identified as anabaenopeptine B. This cyclic hexapeptide has been isolated for the first time from the filamentous cyanobacterium Anabaena flos-aquae (Harada et al., 1995). Meanwhile, anabaenopeptins have been detected in several Anabaena, Oscillatoria and Microcystis aeruginosa strains and in the brackish-water cyanobacterium Nodularia spumigena (Namikoshi and Rinehart 1996, Erhard et al., 1997). Anabaenopeptine B caused relaxations in rat aortic preparations precontracted with 0.1 µM norepinephrine (Harada et al., 1995). Trypsine inhibiting activity or antiviral effects of this compound have not been described so far. In our trypsine inhibiting test it revealed an ID50 = 0.12 µM/ml, for trypsine
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inhibitor from soybean an ID50 0.02 µM/ml was estimated. The influence of anabaenopeptine B on the replication of influenza A virus will be investigated. Further fractions showing trypsine inhibitory activity were analysed by MALDI-TOF-MS and four new aeruginosins were detected. Several aeruginosins with protease inhibiting effects have already been isolated from Microcystis aeruginosa and Microcystis viridis (Murakami et al. 1995, Matsuda et al. 1996). All these compounds are characterised by the unusual amino acid unit 2-carboxy-6-hydroxy-octahydroindole (Choi), p-hydroxyphenyl lactic acid or its derivative at the Nterminus and a guanidine-containing unit at the Cterminus, deriving from arginine. There are no reports about antiviral effects of theses compounds. In another trypsine inhibiting fraction a new cyanopeptoline was found. These cyclic depsipeptides were described for the first time as inhibitors of serine proteases from Microcystis strains by Weckesser et al. (1996), but no hints of antiviral activity were given. Structural elucidation and testing of the biological activity are on progress. Cyanobacteria from local habitats seem to be a source of potential new active substances that could contribute to reduction of the number of bacteria, viruses and other micro-organisms in our waters. In that way cyanobacteria could contribute to a better water quality, but on the other hand toxic cyanobacterial blooms occur and cause water hygienic problems. Acknowledgement. The authors wish to thank Dr. Rolf Jansen, GBF Braunschweig and Marcel Erhard, TU Berlin for the help in structural elucidation of the cyanobacterial metabolites. Dr. Holger Hippe, TTB Greifswald, is thanked for his participation in separating the Microcystis extract. Prof. Renate Mentel, Institute of Medical Microbiology is thanked for the possibility to carry out the antiviral tests. Hannelore Bartrow and Petra Meyer are thanked for excellent technical assistence. The financial support by the government of Mecklenburg-Vorpommern within the TEAM-Programme is gratefully acknowledged.
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field-grown terrestrial blue-green alga Nostoc commune. Tetrahedron Lett. 39, 3737–3740 (1998). Kido, H., Yokogoshi, Y., Sakai, K.: Isolation and characterisation of a novel trypsine-like protease found in rat bronchiolar epithelial Clara cells. J. Biol. Chem. 267, 13 573– 13 579 (1992). Kreitlow, S., Mundt, S., Lindequist, U.: Cyanobacteria – a potential source of new biologically active substances. J. Biotechnol. 70, 61–63 (1999). Lindl, T., Bauer, J.: Zell- und Gewebekultur. 2th ed., Gustav Fischer Verlag. Stuttgart 1989. Luesch, H., Yoshida, W. Y., Moore, R. E., Paul, V. J., Mooberry, S. L.: Isolation, structure determination and biological activity of Lyngbyabellin A from the marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 63, 611–615 (2000). Matsuda, H., Okino, T., Murakami, M., Yamaguchi, K.: Aeruginosins 102-A and B, new thrombin inhibitors from the cyanobacterium Microcystis viridis NIES 102. Tetrahedron Lett. 52, 14 501–14 506 (1996). McDonald, M. I., Graham, I., Harvey K. J., Sinclair A.: Antibacterial activity of hydrolysed linseed oil and linolenic acid against methicillin-resistant Staphylococcus aureus. Lancet Nov 7, 10–56 (1981). Meffert, M. E.: Cultivation and growth of two planktonic Oscillatoria species. Mitt. Internat. Verein. Limnol. 19, 189–205 (1971). Mentel, R., Matthes, E., Janta-Lipinski, M., Wegner, U.: Fluorescent focus reduction assayfor the screening of antiadenoviral agents. J. Virol. Meth. 59, 99–104 (1996). Mentel, R., Meinsen, D., Pilgrim, H., Herrmann, B., Lindequist, U.: In vitro antiviral effect of extracts of Kuehneromyces mutabilis on influenza virus. Pharmazie 49, 859– 860 (1994). Moore, R. E., Banarjee, V., Bornemann, V., Caplyn, F. R., Chen, J. L., Corley, D. J., Larsen, L. K., Moore, B. S., Patterson, G. M. L.: Novel cytotoxins and fungicides from blue-green algae and marine animals possessing algal symbionts. Pure Appl. Chem. 61, 521–524 (1989) Murakami, M., Ishida, K., Okino, T., Okita, Y., Matsuda, H., Yamaguchi, K.: Aeruginosins 98-A and B, trypsine inhibitors from the blue-green alga Microcystis aeruginosa (NIES-98). Tetrahedron Lett. 36, 2785–2788 (1995). Murakami, N., Yamada, N., Sakakibara, J.: An autolytic substance in a freshwater cyanobacterium Phormidium tenue. Chem. Pharm. Bull. Tokyo 38, 812–814 (1990). Namikoshi, M., Rinehart, K. L.: Bioactive compounds produced by cyanobacteria. J. Ind. Microbiol. 17, 373–384 (1996). Nowotny, A., Mentel, R., Wegner, U., Mundt, S., Lindequist, U.: Antiviral activity of an aqueous extract of the cyanobacterium Microcystis aeruginosa. Phytother. Res. 11, 93–96 (1997). Ohta, S., Shiomo, Y., Kawashima, A., Aozasa, O., Teruyuki, N.: Antibiotic effect of linolenic acid from the Chlorococcum strain HS-101 and Dunaliella primolecta on methicillin-resistant Staphylococcus aureus. J. Appl. Phycol. 7, 121–127 (1995).
Papendorf, O., König, G. M., Wright, A. D.: Hierridin B and 2,4-dimethoxy-6-heptadecyl-phenol, secondary metabolites from the cyanobacterium Phormidium ectocarpi with antiplasmodial activity. Phytochem. 49, 2383–2386 (1998). Patterson, G. M. L., Larsen, L. K., Moore, R. E.: Bioactive natural products from blue-green algae. J. Appl. Phycol. 6, 151–157 (1994) Pilgrim, H., Haasmann, S., Schröder, K.: Trypsininhibitoraktivität in Basidiomyceten. Zentralbl. Mikrobiol. 147, 400–404 (1992). Pulz, O.: Cultivation Techniques for microalgae in open and closed systems. In: Symposium papers of the 1st European Workshop on Microalgal Biotechnology; pp. 61–65. Potsdam 1992. Rinehart, K. L., Namikoshi, M., Choi, B. W.: Structure and biosynthesis of toxins from blue-green algae (cyanobacteria). J. Appl. Phycol. 6, 159–176 (1994). Rohrbach, M. S., Conrad, A. K.: Comparison of the T lymphocyte-dependent induction of angiotensin-converting enzyme and leucine aminopeptidase in cultured human monocytes. Clin. Exper. Immunol. 83, 510–515 (1991). Schopf, W., Packer, B.: Early Archean (3.3 – Billion to 3.5 Billion Year Old) Microfossils from Warrawoona Group, Australia. Science 70–72 (1987). Skulberg, O. M., Codd, G. A., Carmichael, W. W.: Toxic blue-green algal blooms in Europe: a growing problem. Ambio13, 244–247 (1984). Singh, I. P., Milligan, K. E., Gerwick, W. H.: Tanikolide, a toxic and antifungal lactone from the marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 62, 1333–1335 (1999). Teuscher, E., Lindequist, U., Mundt, S.: Cyanobakterien, Quellen biogener Wirkstoffe. Pharm. Ztg. Wiss. 137: 57– 69 (1992). Van Hal, P. T, Hopstaken-Broos, J. P., Wijkhuijs, J. M., Te Velde, A. A., Figdor, C. G., Hoogsteden, H. C.: Regulation of aminopeptidase N (CD 13) and Fc epsilon RIIb (CD23) espression by IL 4 depends on the stage of maturation of monocytes/macrophages. J. Immunol. 149, 1395–1401 (1992). Watanabe, Y., Noüe J., Hall, D. O.: Photosynthetic performance of a helical tubular photobioreactor incorporating the cyanobacterium Spirulina platensis. Biotechnol. Bioeng. 47, 261–269 (1995). Waterbury, J. B., Stanier, R. Y.: Isolation and growth of cyanobacteria from marine and hypersaline environments. In: The Prokaryotes (M. P. Starr, H. Stolp, H. G. Trüper, A. Balows, H. G. Schlegel, eds. ). Vol 1: pp. 221–223. Springer Verlag, Berlin 1981. Weckesser, J., Martin, C., Jacobi, C.: Cyanopeptolines, Depsipeptides from cyanobacteria. System. Appl. Microbiol. 19, 133–138 (1996). Yamada, N., Murakami, N., Morimoto, T., Sakakibara, J.: Auto-growth inhibitory substance from the fresh-water cyanobacterium Phormidium tenue. Chem. Pharm. Bull. Tokyo 41, 1863–1865 (1993).
Int. J. Hyg. Environ. Health 203, 327-334 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/intjhyg
Biochemical and pharmacological investigations of selected cyanobacteria Sabine Mundt1, 2, Susann Kreitlow1, Andrea Nowotny1, and Uta Effmert3 1 2 3
Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany Institute of Marine Biotechnology, Greifswald, Germany Department of Biology, University Rostock, Rostock, Germany
Received September 13, 2000 · Accepted February 15, 2001
Abstract Cyanobacteria are a very old group of prokaryotic organisms that produce a variety of secondary metabolites with antibiotic, algicide, cytotoxic, immunosuppressive and enzyme inhibiting activities. In the last decades structures of pure compounds have been determined as phenols, peptides, alkaloids or terpenoids (Falch, 1996). Screening of lipophilic and hydrophilic extracts from cultured cyanobacteria or waterbloom material, isolated from German lakes and the Baltic sea for antiviral, antibiotic, immunomodulating and enzyme inhibiting activity in different in vitro systems revealed strains with interesting effects. These strains were cultivated in 45 litre photobioreactors to produce enough biomass for bioassay-guided isolation of the active substances. First results characterising active substances are reported. Key words: Cyanobacteria – immunomodulating – antibacterial – antiviral – cultivation – bioassay-guided isolation – α-linolenic acid
Introduction Cyanobacteria (blue-green algae) are a diverse group of photosynthetic, prokaryotic organisms found in freshwater and marine environments. The origin of these organisms is dated back three or four billion years (Schopf and Packer, 1987). Their cell structure resembles that of Gram negative bacteria, but as a rule they live photoautotrophically. Like higher plants they possess chlorophyll a and the water soluble red and blue phycobiliproteins as well as photosystem I and II. Therefore they can use water for photosynthesis and produce oxygen, which is released to the atmosphere.
With about 2000 strains cyanobacteria are distributed all over the world. They show a remarkable ecological diversity. Because of widespread eutrophication of lakes, ponds and some parts of oceans cyanobacteria often form blooms, which lead to water hygienic problems (Henning and Kohl, 1981; Skulberg et al., 1984; Chorus and Bartram, 1999; Duy et al., 2000). They may cause unpleasant tastes and odours through excretion of volatile compounds (Jones and Korth, 1995). Furthermore animal poisonings and risks to human health are described. Several genera of cyanobacteria form toxic waterblooms and different cyanobacterial toxins have been characterised (Carmichael, 1992,
Corresponding author: PD Dr. Sabine Mundt, Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst-MoritzArndt-University Greifswald, F.-L.-Jahn-Straße 15a, D-17487 Greifswald, Germany, Phone: +49/3834/864 869, E-mail: [email protected] 1438-4639/01/203/4-327 $ 15.00/0
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1994; Rinehart et al., 1994; Hunter 1995). Possibly the synthesis of highly active toxins is a defence option of cyanobacteria against attack by other organisms like bacteria, fungi, zooplankton and eukaryotic microalgae. Carmichael (1994) found that cyanobacterial toxins can be extremely harmful to zooplankters that feed on cyanobacteria. They may be directly lethal or they may reduce their offspring. Besides the toxins a lot of active substances with antibacterial, antiviral, fungicide, enzyme inhibiting, immunosuppressive, cytotoxic and algicide activity has been isolated from cyanobacterial biomass or in some cases from the medium of laboratory cultures (Falch, 1996; Hayashi et al., 1996; Namikoshi and Rinehart, 1996; Banker and Carmeli, 1998; Harrigan et al., 1998 and 1999; Jaki et al., 1998 and 1999). Producing active biocide components could be an important selective advantage. In the 1970’s a pronounced reduction of Gram positive bacteria was found in lakes during the occurence of cyanobacterial blooms (Chrost, 1975). The production of antibacterial substances could be the reason for this phenomenon. In cyanobacterial blooms often only one species comes up to more than 95 % of the population. Though this has been interpreted as a result of competition between species, the dominance of one species could be a hint for the formation of metabolites with cyanobactericidal activity. In addition, antibacterial, fungicide and antiviral effective compounds formed by cyanobacteria could contribute to an improvement of the water quality in our aquatic environment. The aim of our work was to identify cyanobacterial strains from German lakes and the Baltic sea with relevant bioactivity in different in vitro test systems. The following optimisation of large scale culture conditions of these strains to produce enough biomass was the prerequisite for a bioassay-guided isolation of bioactive compounds and their structural elucidation.
Materials and methods Screening The following 35 cyanobacterial strains were screened for biochemical and pharmacological effects in different in vitro test systems: The Chroococcales : Gloeocapsa caldariorum RABENH. 127 H, Microcystis aeruginosa KÜTZ. HUB 042 B, HUB 063 B, HUB 018 B, HUB 5-2-4 B, HUB 5-3 B, PCC 7813, PCC 7820, 14.85 B, SAG 14.85, SPH 01 G, Microcystis firma (BREB. et LENORM.) SCHMIDLE 398/5 R, Synechocystis aquatilis SAUV. 428 R; The Oscillatoriales: Oscillatoria species 234 H, Oscillatoria agardhii GOM. (syn. Planktothrix agardhii GOM.) HUB 011 B, Oscillatoria redekei van GOOR (syn. Limnothrix redekei MEFFERT) HUB 010 B, HUB 051 B, HUB 022 B, Oscil-
latoria prolifica GOM. (syn. Planktothrix prolifica GOM.) SPH 02 G, Oscillatoria rubescens DC. Ex GOM. (syn. Planktothrix rubescens Dc. Ex GOM.) HUB 016 B, Oscillatoria tenuis AG. Ex GOM (syn. Phormidium tenue AG. Ex GOM) SPH 03 G, Phormidium species Hg 71-212 J, Pseudanabaena catenata LAUTERB. 154 H. The Nostocales: Anabaena flos-aquae (Lyngb.) BREB. AFA K, HUB 039B, Anabaena variabilis KÜTZ: AV K, 1 H, Aphanizomenon flos-aquae (L.) RALFS. HUB 046 B, Calothrix gracilis FRITSCH 96 H, Cylindrospermum majus KÜTZ. 95 H, Nodularia harveyana THUR. NH K, Nodularia spumigena MERT. HÜ 280 H, Nostoc species 104 H, Nostoc linckia (ROTH.) BORN. et. FLAH. 230 H, Scytonema bonerii SCHMIDLE Hg 73–2858 J. The strains were provided by Prof. Kohl, Humboldt University of Berlin B, Prof. Pohl, University of Kiel K, Dr. Hegewald, Institute of Biotechnology, Jülich J, Dr. Hübel, University of Greifswald, Hiddensee H, bought from culture collections or isolated by our own group G. Field material was used in only one case. Biomass for screening was produced by cultivating the strains in mineral salt solution under continuous light at temperatures between 18 °C and 22 °C. After harvesting by centrifugation biomass was freeze dried and extracted either with water or with n-hexane, methanol and water successively. The supernatants of the extractions were combined, the organic solvents were removed in vacuum and the aqueous residues were freeze dried. These dry extracts were used for screening in different in vitro test systems. In vitro test systems For the Lymphocyte Transformation Test (LTT) to investigate immunomodulating activity (Lindl and Bauer, 1989) freshly prepared human peripheral blood lymphocytes (HPBLs) were cultivated in vitro for three days in presence of the mitogen phythaemagglutinin and the aqueous extracts of cyanobacterial biomass. The influence of the extracts on mitogen-stimulated proliferation of the cells was quantified by measuring the incorporation of 3H-thymidine in the DNA. Cytotoxic effects of the aqueous extracts to HPBLs were determined with trypane blue (Lindl and Bauer 1989) and only non cytotoxic extract concentrations were used in LTT. Inhibiting or stimulating activities of the aqueous extracts on the enzymatic activity of cyclooxygenase and lipoxygenase were tested by measuring the consumption of oxygen in presence of a substrate. The activity of leucine aminopeptidase was estimated colorimetrically by using the substrate leucine hydrazid. Protease inhibitory effects tested by trypsine inhibition were monitored with BAPNA (α-N-benzoylDL-arginine-p-nitroanilid) (Pilgrim et al., 1992). The agar plate diffusion test for antibacterial and fungicide activity of cyanobacterial extracts was described by Kreitlow et al. (1999). The activity of aqueous, methanol and n-hexane extracts was tested against the Gram positive bacteria Bacillus subtilis SBUG 14, Staphylococcus aureus SBUG 11 and Micrococcus flavus SBUG 16, the Gram negative bacteria Escherichia coli SBUG 17 and the yeast Candida maltosa SBUG 700. Tests for antibacterial effects against multiresistant
Cyanobacteria Staphylococcus strains were performed in cooperation with the Landeshygieneinstitut Greifswald. Antiviral activity against influenza A virus /WSN 33/London and herpes simplex virus Type 1 was investigated in a dye uptake assay with neutral red (Nowotny et al., 1997). Antiviral effects against adenovirus type 2 were tested in a modified focus reduction test (Mentel et al., 1996). Virus protein synthesis was analysed by SDS polyacrylamide gel electrophoresis (Mentel et al., 1994). In all cases, the cytotoxicity of the extracts was tested and only non cytotoxic concentrations were used in the cellular test systems. Large scale batch cultivation Culture conditions were optimised for the filamentous cyanobacterium Limnothrix redekei HUB 051 and the unicellular cyanobacterial strain Microcystis aeruginosa SPH 01. The growth of the cultures was examined by measuring the optical density at 730 nm (UVICON 930, KONTRON Instruments). In small culture flasks of 200 ml the influence of the following parameters on production of biomass was tested: light intensity (500 to 5000 lux, light/dark rhythm of 12 hours), temperature (18 °C, 30 °C), pH (4 to 11) and salt concentration (phosphate, nitrate) of culture medium Z1/2 (Meffert 1971) or BG 11 (Waterbury and Stanier 1981). Different ways to agitate the cultures were also tested. The optimal conditions were used for scaling up to a 45 l batch culture. The cylindrical bioreactor was made of glass with a bottom of stainless steel. Aeration (air and carbon dioxide) and mild agitation were realised by nozzles in the bottom of the cultivation tank. The concentration of carbon dioxide was regulated to maintain a pH of 10. An external illumination with alternatively two, four or six fluorescent lamps realised a light intensity of 500 to 5000 lux (Teuscher et al. 1992). Biomass was harvested every 3 days by filtration 10 l of the culture using filter paper circles with a pore wide of 12– 25 µm (Schleicher and Schuell). The medium loss was compensated by addition of the same volume of fresh one. Bioassay-guided isolation For isolation of the antibacterial substances of Limnothrix redekei HUB 051 the n-hexane extract was fractionated by column chromatography with silica gel (Silicagel 60, MERCK) using petrolether/ethyl acetate 7 2, followed by ethyl acetate and methanol. Fractions with antibacterial activity were further fractionated on reversed phase material (Lichroprep RP-18, MERCK) with a step gradient acetonitrile/water. Three active fractions were separated and repeatedly fractionated on reversed phase material. Two pure fractions were received and investigated by different methods of MS (HRFAB-MS, ESI-MS and DCI-MS) and NMR (1H, 13C, COSY, HMBC) in cooperation with the GBF Braunschweig. Attempts to isolate compounds with antiviral activity from the Microcystis aeruginosa waterbloom were also performed by column chromatography (Nowotny et al. 1997). The aqueous extract prepared from the biomass was precipitated with ethanol. After the removal of ethanol in vacuum the aqueous phase was separated using ion exchange chrom-
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atography (DOWEX 50 W, SERVA, 10 % ammonia; DOWEX 1 1, SERVA, 2 N formic acid) in a basic fraction, an acid fraction and a neutral fraction (unbound). The basic compounds were further separated by gel chromatography on Sephadex G 25 (PHARMACIA) using water as eluent. Alternatively the fraction of basic compounds was separated by HPLC (HPLC system SMART, PHARMACIA) on a Superdex column 75 PC (PHARMACIA) with acetonitrile/water 2 8. The trypsine inhibiting fractions were further separated on a µRPC C2/C18 column (PHARMACIA) in a gradient of acetonitrile, water, trifluoroacetic acid. The active fractions were analysed by MS and NMR as described above or by MALDI-TOF-MS in the TU Berlin (Erhard et al. 1997).
Results Screening The influence of aqueous extracts prepared from the biomass of 35 cyanobacterial strains on human peripheral blood lymphocytes was tested using extract concentrations of 70, 14 and 1.4 µg/ml. In these concentrations 24 extracts showed a considerable cytotoxicity to the lymphocytes. Therefore only the 11 non cytotoxic aqueous extracts were tested in LTT (Fig. 1.) Three of them, the extracts of Oscillatoria tenuis SPH 03, Limnothrix redekei HUB 051 and Synechocystis aquatilis 428, inhibited the mitogen stimulated proliferation of HPBLs by more than 30 %. The effect of the aqueous extracts on the activity of the enzymes cyclooxygenase, lipoxygenase and leucine aminopeptidase was also determined. The activity of cyclooxygenase was not influenced. In contrast the activity of lipoxygenase was inhibited by 33 of 35 extracts. On the other hand, only the aqueous extracts of Oscillatoria tenuis SPH 03 and the Microcystis strain SAG 14.85 inhibited the leucine aminopeptidase by about 75 %. For the detection of antibacterial activity extracts prepared from the biomass with n-hexane, methanol and water were tested against the Gram positive bacteria Bacillus subtilis SBUG 14, Staphylococcus aureus SBUG 11 and Micrococcus flavus SBUG 16, the Gram negative bacteria Escherichia coli SBUG 17 and the yeast Candida maltosa SBUG 700. The n-hexane extracts of Oscillatoria species 234, Nostoc species 104, Cylindrospermum majus 95, Calothrix gracilis 96 and Limnothrix redekei HUB 051 and the methanol extracts of Anabaena variabilis 1, Gloeocapsa caldariorum 127, Pseudanabaena catenata and Limnothrix redekei HUB 051 inhibited the growth of Bacillus subtilis SBUG 14. The n-hexane extract of Limnothrix redekei HUB 051 was also active against Staphylococcus aureus SBUG 11 and Micrococcus flavus SBUG 16. The aqueous extracts were ineffective.
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Fig. 1. Influence of cyanobacterial extracts on the 3H-thymidine incorporation of humane peripheral blood lymphocytes Cyanobacterial dry mass in µg/ml (Strains with substantial inhibition are accentuated in ⊂⊃).
Tests for antiviral activity against adenovirus, herpesvirus Type 1 and influenza A virus were performed with methanolic and aqueous extracts. No extract showed an activity against adenovirus. Only the methanolic extracts of Nodularia spumigena HÜ 280 and Synechocystis aquatilis 428 were weakly effective against herpesvirus Type 1. 12,5 µg/ml of the aqueous extracts of the filamentous strains Calothrix gracilis 96 and Oscillatoria species 234 inhibited the replication of Influenza A virus in MDCK cells completely. For the Microcystis aeruginosa waterbloom SPH 01 an ED50 of 10.2 µg/ml was estimated. If 100 µg/ml Microcystis extract were available for the whole incubation period the synthesis of viral proteins analysed by gel electrophoresis was clearly reduced. Testing this extract in a trypsine inhibitor assay revealed a considerable enzymatic inhibition in concentrations of 5 µg/ml. According to the results of screening tests the strain Limnothrix redekei HUB 051 (because of its good antibacterial activity) and Microcystis aeruginosa water bloom SPH 01 (because of the antiviral activity against influenza A virus) were selected for optimizing culture conditions to produce biomass for the isolation of bioactive substances. Cultivation The best growth of Limnothrix redekei HUB 051 was achieved using the mineral salt medium Z1/2 with three times the amount of phosphate and nitrate as
compared to the original medium, continuous illumination with warm white fluorescent lamps with an intensity of 500 lux, pH 9 to 10 and a relatively low temperature of 18 °C to 20 °C, an increase to 30 °C resulted in a die off of the culture. In the 45 l fermenter a specific growth rate of µ 0.5 d–1 was reached compared with cultivation in 200 ml volumes under control conditions (pH 7.5, 500 lux, Z1/2 unmodified), where a specific growth rate was estimated with µ 0,22 5 d –1 (fig. 2). In contrast to the small culture flasks the light intensity in the fermenter was increased from 500 lux to 5000 lux with increasing optical density of the culture. Cultivation in the fermenter over 6 to 8 weeks yielded of 1.15 g cyanobacterial biomass per litre and day. The best yield of the n-hexane extract was obtained under the same conditions under which biomass production was optimal. The isolation of a laboratory culture strain from the Microcystis waterbloom, that showed the same antiviral activity against influenza A virus as the waterbloom material was not yet sucessful. Bioassay-guided isolation of active components Bioassay-guided isolation of the antibacterial substances of Limnothrix redekei HUB 051 by column chromatography led to three fractions with activity against the Gram positive bacteria Staphylococcus aureus SBUG 11 and Micrococcus flavus SBUG 16. One active
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fraction was identified by MS and NMR using reference substance as α-linolenic acid. The minimal inhibition concentrations (MICs) of the isolated α-linolenic acid were determined against Staphylococcus aureus SBUG 11 with 75 µg/ml and against Micrococcus flavus SBUG 16 with 25 µg/ml. A concentration of αlinolenic acid of 500 µg/filter disk was also effective against multiresistent Staphylococcus strains. Inhibition zones from 10 to 20 mm were measured. The other two active fractions are further separated and structural elucidation of the isolated active substances is in progress. The antiviral activity of the aqueous extract of Microcystis aeruginosa waterbloom material against influenza A virus was concentrated in a fraction of basic compounds after ion exchange chromatography. Further separation by gel chromatography on Sephadex G 25 resulted in five fractions G1 to G5. Analysis of fraction G4 and G5 by MS and NMR showed adenosine and adenine to be the major components (Nowotny et al. 1997). Alternatively the fraction of basic compounds was separated by HPLC on Superdex in six fractions, which were tested for trypsine inhibiting activity. The two trypsine inhibiting fractions 5 and 6 were further separated on reversed phase material in several fractions. The most active fractions were analysed by MALDI-TOF-MS. One fraction was identified as the cyclic hexapeptide anabaenopeptine B. This peptide was detected at m/z 837 and analysed in the PostSource-Decay (PSD) mode using anabaenopeptine B as the reference substance. Additional four linear peptides with trypsine inhibiting activity were identified to be new aeruginosins. They were detected at m/z 619, 633, 715 and 729. PSD spectra have revealed the unusual unit 2-carboxy-6hydroxy-octahydroindol (Choi) as the common amino acid component, leucine, a guanidine-containing unit deriving from arginine at the C-terminus and p-hydroxyphenyl lactic acid at the N-terminus. Another fraction with trypsine inhibiting effect was identified to be a new cyanopeptoline with m/z 979. The structural elucidation of the cyanopeptoline and the testing of the characterised substances for activity on the replication of influenza A virus is in progress.
Discussion In the last decades screening programs have revealed that cyanobacteria are a potential source of new active substances for medicine and pharmacy and numerous active compounds have been isolated (Moore et al., 1989; Patterson et al., 1994; Falch, 1996, Hayashi et al., 1996; Namikoshi and Rinehart, 1996; Papendorf
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Fig. 2. Growth curves of the cyanobacterium Limnothrix redekei HUB 051 in culture flasks and in the fermenter lnE optical density of the culture at 730 nm.
et al., 1998; Kajiyama et al., 1998; Jaki et al., 1999; Singh et al., 1999; Luesch et al., 2000; Horgen et al., 2000). These active compounds could influence the aquatic environment of cyanobacteria by reducing the number of viruses, bacteria and other micoorganisms. Cyanobacteria of local habitats have been rarely screened for biological effects. Therefore, our aim was the investigation of cyanobacterial strains especially from ponds, lakes and rivers of the northern areas of Germany. Only a few strains from culture collections were included in the tests. The extracts of cyanobacterial biomass were tested for immunomodulating, enzyme inhibiting, antibiotic and antiviral activity in different in vitro test systems. The determination of proliferation of lymphocytes is a very common assay in immunological research. The Lymphocyte Transformation Test (LTT) records the proliferation of T-lymphocytes in response to a mitogen as one step of the immune reaction. If an effect exceeds the value of 30 % it is either defined as stimulation or inhibition of the proliferation due to the considerable standard deviation of the values measured. Under these conditions the aqueous extracts of Limnothrix redekei HUB 051, Oscillatoria tenuis SPH 03 and Synechocystis aquatilis 428 inhibited the mitogenstimulated proliferation clearly. Cyclooxygenase and lipoxygenase catalyse the formation of prostaglandins, hydroxylated fatty acids, hydroperoxylated fatty acids and leucotrienes. These substances play an important role in allergic reactions, so that an inhibition of the enzymes could be one possibility to prevent overshooting immune reactions. In presence of the aqueous extracts no effects to cyclooxygenase have been observed. In contrast, the activity of lipoxygenase was inhibited by nearly all extracts. Probably unspecific effects by ubiquitous substances are the reason for this phenome-
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non. Leucine aminopeptidase, a membrane-bound proteinase may influence immune reaction by antigen processing and improving antigen presentation (Beninga et al., 1998). Maturing and differentiating of immune cells are connected with an enhanced activity of the enzyme (Becker, 1984; Rohrbach and Conrad, 1991; Van Hal et al., 1992). Two of 35 extracts inhibited the activity of leucine aminopeptidase. One of the active extracts was also effective in the LTT. Antibacterial activity was especially found in the more lipophilic extracts; the aqueous extracts were ineffective. Only the growth of Gram positive bacteria was inhibited, a phenomenon that has already been described (Chrost et al., 1975). A reason could be that cyanobacteria are also Gram negative bacteria, but recently it has been shown, that comnostins and noscomin isolated from Nostoc commune are also active against Gram negative bacteria (Jaki et al. 1998 and 1999). Activities against herpesvirus Type 1 and influenza A virus were found in the dye uptake assay using neutral red. The aqueous extract of the Microcystis aeruginosa waterbloom SPH 01 inhibited the replication of influenza A virus completely without any cytotoxic effects against the host cells. The ED50, the concentration of the extract that resulted in a 50 % survival of cells infected with virus, was estimated at 10.2 µg/ml. For amantadine-HCl, a drug which is used in treatment of influenza, an ED50 of 16.8 µg/ml was estimated. The inhibition of virus protein synthesis has been shown by gel electrophoresis (Nowotny et al. 1997) Additionally, the extract inhibited the protease trypsine in very low concentrations. This protease inhibition could be the reason for the inhibition of influenzavirus replication because the processing of virus proteins is an obligatory step in virus life cycle (Kido et al. 1992). In last years several protease inhibitors, cyclic and linear peptides, have been isolated from Microcystis aeruginosa strains (Namikoshi and Rinehart 1996), so that the antiviral effects may be caused by these peptides too. According to the results of screening tests the strains Limnothrix redekei HUB 051 with antibacterial activity and Microcystis aeruginosa waterbloom SPH 01 with antiviral activity against influenza A virus were selected for further investigations. The main conditions for practical use of the active strains is to establish a method for a large scale cultivation with stable production of the active compounds in culture. Optimisation of culture conditions resulted in a biomass production of 1.15 g/l d. The yield was in the order of magnitude described for plate-type photobioreactors (Puls, 1992) and higher than for helical tubular photobioreactors (Watanabe et al. 1995). Laboratory cultures isolated from the Microcystis aeruginosa waterbloom were able to grow but the anti-
viral activity of the laboratory grown biomass was considerably lower than in the biomass collected from the field. Attempts to optimise the culture conditions for production of antiviral compounds were not yet successful. Therefore, isolation of antiviral components was attempted with the waterbloom material. Bioassay guided isolation of effective substances is a common method in natural product chemistry. The antibacterial n-hexane extract of Limnothrix redekei HUB 051 was fractionated using different separation techniques. Two active fractions were received and one was identified by NMR and MS as α-linolenic acid. The antibacterial activity of this polyunsaturated fatty acid is well known. In 1981 MC DONALD and coworkers (Mc Donald et al., 1981) reported about antibacterial effects of α-linolenic acid against methicillinresistent Staphylococcus strains. A Japanese group isolated α-linolenic acid as the antibiotic principle from the biomass of the green alga Chlorococcum species HS-101 (Ohta et al., 1995). In the culture medium of the cyanobacterium Phormidium tenue a mixture of different fatty acids containing also linoleic and linolenic acid was responsible for autolysis of the axenic culture (Murakami et al., 1990; Yamada et al., 1993). The MICs for α-linolenic acid range from 75 µg/ml against Staphylococcus aureus SBUG 11 to 25 µg/ml against Micrococcus flavus SBUG 16, but ampicillin was more effective against these bacteria. The MICs were 0.05 µg/ml and 0.25 µg/ml respectively. Another antibacterial fraction, separated after column chromatography on reversed phase material seems to be a mixture of polyunsaturated and hydroxylated fatty acids. Characterisation of structures is in progress. The isolation of the antiviral components of Microcystis aeruginosa waterbloom resulted in two fractions with activity against influenza A virus. Surprisingly, adenosine and adenine were identified to be the main components. Besides the potential effect of these substances, which has not yet been clarified, the antiviral activity could be attributed to fractions showing protease inhibiting activity. One of these fractions that has not been tested for antiviral activity was identified as anabaenopeptine B. This cyclic hexapeptide has been isolated for the first time from the filamentous cyanobacterium Anabaena flos-aquae (Harada et al., 1995). Meanwhile, anabaenopeptins have been detected in several Anabaena, Oscillatoria and Microcystis aeruginosa strains and in the brackish-water cyanobacterium Nodularia spumigena (Namikoshi and Rinehart 1996, Erhard et al., 1997). Anabaenopeptine B caused relaxations in rat aortic preparations precontracted with 0.1 µM norepinephrine (Harada et al., 1995). Trypsine inhibiting activity or antiviral effects of this compound have not been described so far. In our trypsine inhibiting test it revealed an ID50 = 0.12 µM/ml, for trypsine
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inhibitor from soybean an ID50 0.02 µM/ml was estimated. The influence of anabaenopeptine B on the replication of influenza A virus will be investigated. Further fractions showing trypsine inhibitory activity were analysed by MALDI-TOF-MS and four new aeruginosins were detected. Several aeruginosins with protease inhibiting effects have already been isolated from Microcystis aeruginosa and Microcystis viridis (Murakami et al. 1995, Matsuda et al. 1996). All these compounds are characterised by the unusual amino acid unit 2-carboxy-6-hydroxy-octahydroindole (Choi), p-hydroxyphenyl lactic acid or its derivative at the Nterminus and a guanidine-containing unit at the Cterminus, deriving from arginine. There are no reports about antiviral effects of theses compounds. In another trypsine inhibiting fraction a new cyanopeptoline was found. These cyclic depsipeptides were described for the first time as inhibitors of serine proteases from Microcystis strains by Weckesser et al. (1996), but no hints of antiviral activity were given. Structural elucidation and testing of the biological activity are on progress. Cyanobacteria from local habitats seem to be a source of potential new active substances that could contribute to reduction of the number of bacteria, viruses and other micro-organisms in our waters. In that way cyanobacteria could contribute to a better water quality, but on the other hand toxic cyanobacterial blooms occur and cause water hygienic problems. Acknowledgement. The authors wish to thank Dr. Rolf Jansen, GBF Braunschweig and Marcel Erhard, TU Berlin for the help in structural elucidation of the cyanobacterial metabolites. Dr. Holger Hippe, TTB Greifswald, is thanked for his participation in separating the Microcystis extract. Prof. Renate Mentel, Institute of Medical Microbiology is thanked for the possibility to carry out the antiviral tests. Hannelore Bartrow and Petra Meyer are thanked for excellent technical assistence. The financial support by the government of Mecklenburg-Vorpommern within the TEAM-Programme is gratefully acknowledged.
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