Ernst Moritz Arndt-Universität Greifswald Mathematisch-Naturwissenschaftliche Fakultät
Chemical and Biological Investigations of Vietnamese Cyanobacteria
Inauguraldissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald
vorgelegt von Le Thi Anh Tuyet geboren am 19.05.1973 in Thanh Hoa, Vietnam
Greifswald, Juli 2010
Dekan: Prof. Dr. Klaus Fesser………………………………………………
1. Gutachter: Prof. Dr. Johannes F. Imhoff
2. Gutachter: PD. Dr. Sabine Mundt
Tag der Promotion:……16.09.2010……………………………………………………………
Hiermit erkläre ich, daß diese Arbeit bisher von mir weder an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald noch einer anderen wissenschaftlichen Einrichtung zum Zwecke der Promotion eingereicht wurde.
Ferner erkläre ich, daß ich diese Arbeit selbständig verfaßt und keine anderen als die darin angegebenen Hilfsmittel benutzt habe.
Greifswald, den 19 Juli 2007 Unterschrift
Le Thi Anh Tuyet
a
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1.1 Cyanobacteria...... 1 1.1.1 Cyanobacterial physiology and morphology ...... 1 1.1.2 Ecology of cyanobacteria ...... 2 1.1.3 Classification of cyanobacteria ...... 3
1.2 Cyanobacteria–a new and rich source of novel bioactive compounds with pharmaceutical potential ...... 6 1.2.1 Antimicrobials ...... 9 1.2.2 Cytotoxic and antitumoural activities ...... 12 1.2.3 Antiviral activity ...... 18 1.2.4 Toxins and other pharmacologically active compounds ...... 19
1.3 Aim of the work...... 22
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2.1 Biological materials...... 23 2.1.1 Soil cyanobacteria ...... 23 2.1.2 Marine cyanobacteria ...... 29 2.1.3 Bacteria, yeast, and cancer cell lines as test organisms ...... 31
2.2 Chemicals...... 31 2.2.1 Cultivation of cyanobacteria ...... 31 2.2.2 Cultivation of bacteria and yeast as test organisms ...... 32 2.2.3 General laboratory chemicals ...... 32 2.2.4 Chemical reagents ...... 32 2.2.5 Fatty acid analysis ...... 33
2.3 Solvents...... 33
2.4 Equipment in generally company, town, country ...... 34 2.4.1 Cultivation of cyanobacteria ...... 34 2.4.2 Extraction ...... 34 2.4.3 Isolation of secondary metabolites ...... 35 2.4.3.1 Thin layer chromatography (TLC) ...... 35 2.4.3.2 Preparative TLC ...... 35 2.4.3.3 Open column chromatography ...... 35 2.4.3.4 HPLC ...... 36 2.4.4 Agar plate diffusion test ...... 36 2.4.5 Bioautographic TLC assay ...... 37 2.4.6 Fatty acid analyses ...... 37
2. 5 Cultivation of cyanobacteria ...... 38 2.5.1 The stock culture ...... 38 2.5.2 The batch culture ...... 38 2.5.3 The large scale culture ...... 38
2.6 Extraction ...... 39 2.6.1 Extraction of intracellular compounds ...... 39 2.6.2 Extraction of extracellular compounds ...... 40
2.7 Bioassays...... 41 2.7.1 Assays for antimicrobial activity ...... 41 2.7.1.1 Agar diffusion assay ...... 41 2.7.1.2 Bioautographic TLC assay ...... 42 2.7. 2 Assays for cytotoxic activity ...... 43
2.8 Fractionation and isolation of the secondary metabolites of 6 cyanobacterial strains ...... 43 2.8.1 Fractionation and isolation of the secondary metabolites of Westiellopsis sp.VN ...... 43 2.8.2 Fractionation and isolation of the secondary metabolites of Calothrix javanica ...... 45 2.8.3. Fractionation and isolation of the secondary metabolites of Scytonema ocellatum ...... 47 2.8.4 Fractionation and isolation of the secondary metabolites of Anabaena sp...... 49 2.8.5 Fractionation and isolation of the secondary metabolites of Nostoc sp...... 50 2.8.6 Fractionation and isolation of the secondary metabolites of Lyngbya majuscula ...... 51 2.8.6.1 Method 1 ...... 51 2.8.6.2 Method 2 ...... 53
2.9 Structure elucidation of the isolated secondary metabolites...... 55 2.9.1 Structure elucidation of compounds isolated from Westiellopsis sp.VN and Lyngbya majuscula ...... 55 2.9.2 Structure elucidation of compounds isolated from Calothrix javanica and Scytonema ocellatum ...... 56 2.9.3 Structure elucidation of compounds isolated from Anabaena sp...... 56 2.9.4 Structure elucidation of compounds isolated from Nostoc sp...... 56
2.10 Investigation of the active ethyl acetate extract of Westiellopsis sp. VN growth medium ...... 56
2.11 Gas chromatography- mass spectrometry...... 57
2.12 Culture optimization of Westiellopsis sp .VN...... 59 2.12.1 The effects of nitrogen deficiency ...... 59 2.12.2 The effects of cultivation time (culture age) ...... 59
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3.1 Screening of antibacterial activity ...... 61 3.1. 1 Extract preparation ...... 61 3.1.2 Screening of crude extracts ...... 61
3.2 Chemical investigation and culture optimization of Westiellopsis sp. VN...... 65 3.2.1 Chemical investigation of methanol extract obtained from biomass ...... 65 3.2.1.1 Fractionation of methanol extract by silica gel column chromatography ...... 65 3.2.1.2 Seperation of the combined fractions (FI, FII, and FIII) by sephadex LH-20 column .... 66 3.2.1.3 Purification of fraction WF1 using reversed-phase HPLC ...... 67 3.2.1.4 Structure elucidation of isolated compounds of Westiellopsis sp.VN ...... 68 3.2.1.4.1 Structure elucidation of fraction WF 1-3 (compound 1) ...... 68 3.2.1.4.2 Structure elucidation of fraction WF 1-5...... 69 3.2.1.4.3 Structure elucidation of fraction WF 1-6 (compound 4) ...... 73 3.2.1.4.4 Identification of compounds in fraction WF 1-8 ...... 74 3.2.2 Chemical investigation of the active ethyl acetate extract resulting from cultivation medium of Westiellopsis sp. VN ...... 76 3.2.3 Culture optimization of Westiellopsis sp.VN ...... 79 3.2.3.1 Nitrogen deficiency ...... 79 3.2.3.2 The effect of incubation time on biomass production and antibacterial production ...... 81
3.3 Chemical investigation of Calothrix javanica ...... 82 3.3.1 Fractionation of methanol extract from biomass by RPC 18 chromatography ...... 82 3.3.2 Purification of fraction CJF II by semi-preparative reversed-phase HPLC ...... 83
3.3.3 Structure elucidation of fraction CJF II-4 (compound 7) ...... 85
3.4 Chemical investigation of Scytonema ocellatum ...... 90 3.4.1 Fractionation of methanol extract obtained from biomass by RP C 18 column chromatography ...... 90 3.4.2 Separation of fraction SOF II using silica gel column ...... 91 3.4.3 Purification of the pooled fractions using reversed-phase RP HPLC ...... 91 3.4.4 Structure elucidation of fraction FSO 3 ...... 93
3.5 Chemical investigation of Anabaena sp...... 94 3.5.1 Purification of EtOAc extract using semi-preparative reversed-phase HPLC ...... 94 3.5.2 Structure elucidation of fraction AF 6 (compound 8) ...... 96
3. 6 Chemical investigation of Nostoc sp...... 98 3.6.1 Fractionation of methanol extract obtained from biomass using silica gel column ...... 98 3.6.2 Separation of the acitve fraction NF IV using RP C 18 column chromatography ...... 98 3.6.3 Purification of the active fraction NF IV-1 using semi-preparative reversed-phase HPLC ...... 99 3.6.4 Structure elucidation of fraction NsF 2 ...... 100
3.7 Chemical investigation of Lyngbya majuscula ...... 100 3.7.1 Isolation of cytotoxic compounds of methanol extract obtained from biomass according to method 1 ...... 101 3.7.1.1 Fractionation of methanol extract by silica gel column chromatography ...... 101 3.7.1.2 Separation of fraction F 8 by silica gel column chromatography ...... 102 3.7.1.3 Purification of fraction F 8-3 by preparative TLC ...... 103 3.7.1.4 Structure elucidation of fraction F 8-3-2 (compound 9) ...... 103 3.7.2 Isolation of cytotoxic compounds of methanol extract obtained from biomass according to method 2 ...... 105 3.7.2.1 Separation of methanol extract by silica gel column chromatography ...... 105 3.7.2.2 Purification of fraction F 10 using semi-preparative reversed-phase HPLC ...... 107 3.7.2.3 Structure elucidation of fractions F 10-3 and F 10-5 ...... 108 3.7.2.3.1 Structure elucidation of fraction F10-3 (compound 10) ...... 108 3.7.2.3.2 Structure elucidation of fraction F 10-5 (compound 11) ...... 110 3.7.3 Fatty acid analysis ...... 112
...... 113
4.1 Screening of crude extracts for antibacterial activity ...... 113 4.1.1 Selection of antibiotic screening and cyanobacterial strains ...... 113 4.1.2 Cultivation and extraction ...... 115 4.1.3 Antibacterial activity ...... 116
4.2 Chemical investigation and culture optimization of Westiellopsis sp. VN...... 117 4.2.1 Selection of Westiellopsis sp.VN ...... 117 4.2.2 Active intracellular metabolites of Westiellopsis sp.VN strain ...... 118 4.2.3 Chemical composition of volatile extracellular compounds of Westiellopsis sp.VN strain .. 122 4.2.4 Cultivation optimization of Westiellopsis sp.VN strain ...... 123 4.2.5 Effect of incubation time on biomass production and antimicrobial compound accumulation of Westiellopsis sp.VN strain ...... 128
4.3 Chemical investigation of Calothrix javanica and Scytonema ocellatum ...... 130 4.3.1 Selection of Calothrix javanica ...... 130 4.3.2 Selection of Scytonema ocellatum ...... 130 4.3.3 New cyclic peptide of Calothrix javanica and Scytonema ocellatum ...... 130
4.4 Chemical investigation of Anabaena sp...... 136 4.4.1 Selection of Anabaena sp...... 136 4.4.2 Active compound of Anabaena sp...... 137
4.5 Chemical investigation of Nostoc sp...... 138 4.5.1 Selection of Nostoc sp...... 138 4.5.2 Active compounds of Nostoc sp...... 139
4.6 Chemical investigation of the marine Lyngbya majuscula ...... 139 4.6.1 Selection of the marine cyanobacterium L. majuscula collected in Vietnam ...... 139 4.6.2 Cytotoxic compounds of the marine cyanobacterium Lyngbya majuscula collected in Vietnam ...... 140
4.7 Conclusion ...... 142
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Crytophycin 1 and crytophycin 8...... 11 Norharmane from cyanobacteria...... 12 Cryptophycin 1 and 52, potent antitumor agents from cyanobacteria...... 14 Prominent anticancer marine cyanobacterial secondary metabolites and synthetic analogues ...... 15 Borophycin from cyanobacteria...... 17 Nostocarboline from Nostoc 78-12A...... 21 Map of Vietnam with the locality of 12 cyanobacterial strains in Dak Lak province ...... 23 Morphology of 12 cyanobacterial strains ...... 28 Map of Vietnam with the collection area and collection place of Lyngbya majuscula in Khanh Hoa province indicated ...... 29 collection place of Lyngbya majuscula in Khanh Hoa province indicated ...... 29 Natural habit of Lyngbya majuscula and microscopic view of filament of Lyngbya majuscula ...... 31 Agilent 6890N gas chromatograph and mass selective detector ...... 37 Cultivation of cyanobacteria ...... 39 Antibacterial activity of cyanobacterial extracts against the Gram positive bacterium Bacillus subtilis ATCC 6501...... 62 Antibacterial activity of cyanobacterial extracts against the Gram positive bacterium Staphylococcus aureus ATCC 6538...... 62 Antibacterial activity of cyanobacterial extracts against the Gram negative bacterium Escherichia coli ATCC 11229 ...... 63 Antibacterial activity of cyanobacterial extracts against the Gram negative bacterium Pseudomonas aeruginosa ATCC 22853...... 64 Semi-preparative RP HPLC chromatogram of WF 1 ...... 67 Ambiguine D isonitrile ...... 68 UV and ESI-MS of fraction WF 1-3 ...... 68 UV and ESI-MS of compounds of fraction WF 1- 5...... 69
MS data of peak 1 of fraction WF 1-5 ...... 70 Ambiguine C isonitrile...... 70 MS data of peak 2 of fraction WF 1-5 ...... 71 Fischerellin A...... 71 MS data of peak 3 of fraction WF 1-5 ...... 72 Ambiguine B isonitrile...... 73 UV and ESI-MS of fraction WF 1-6 ...... 73 ESI-MS of compounds of fraction WF 1- 8 ...... 74 ESI-MS of peak 2 of fraction WF 1- 8 ...... 75
ESI-MS of peak 3 of fraction WF 1- 8...... 76 Bioautographic assay of ethyl acetate extract from cultivation medium of Westiellopsis sp.VN against S.aureu ...... 77 Analytical HPLC of MeOH fraction of EtOAc extract of cultivation medium of Westiellopsis sp. VN ...... 77 Fermenters for cultivation of Westiellopsis sp.VN ...... 79 Agar diffusion test of extracts prepared from biomass and cultivation medium of Westiellopsis sp.VN grown in BG-11 media ...... 80
a The effect of incubation time on dry weight and antibiotic production of Westiellopsis sp.VN...... 81 Agar diffusion test of MeOH extracts of Westiellopsis s p.VN biomass ...... 81 Analytical HPLC chromatogram of fraction CJF II ...... 83 Analytical HPLC chromatogram of fraction CJF II-4 ...... 84 Daklakapeptin ...... 85 Semi-preparative RP HPLC chromatogram of the pooled fractions SOF II-5 and SOF II-6 ...... 92 Analytical HPLC chromatogram of fraction FSO3 ...... 93 Semi-preparative RP HPLC chromatogram of the crude EtOAc extract from culture medium ...... 95 Agar diffusion test of 7 fractions obtained from ethyl acetate extract of culture medium of Anabaena sp...... 96 Fluourensadiol...... 96 Semi-preparative RP HPLC chromatogram of fraction NF IV-1 ...... 99 Thin layer chromatogram of 15 fractions obtained from silica gel column ...... 102 17-debromo-3, 4didehydro-3-deoxy –aplysiatoxin ...... 103 ESI-MS spectrum of fraction F 8-3-2 ...... 104 Thin layer chromatogram of 22 fractions obtained from silica gel column ...... 105 Semi-preparative RP HPLC chromatogram of F 10 ...... 107 Debromoaplysiatoxin...... 108 UV and ESI-MS spectrum of compound 10 ...... 110 3,4-didehydro-3-deoxy-aplysiatoxin...... 110 UV and ESI-MS spectrum of compound 11...... 111 Fischerellins from cyanobacteria ...... 121 Sequence of daklakapeptin ...... 131 Analytical HPLC chromatogram of methanol extract of Calothrix javanica ...... 132 Analytical HPLC chromatogram of methanol extract of Scytonema ocellatum ...... 133 The phylogenetic relationships of cyanobacteria inferred from 16S rRNA nucleotide sequence ...... 134
a a The orders of cyanobacteria according to the botanical classification and their correspondence to the subsections of the bacteriological code ...... 4 a Cyanobacterial strains ...... 24 a Step gradient used in purification of fraction WF 1 by semi-preparative HPLC ...... 44 a Step gradient used in purification of fraction CJF II by semi-preparative HPLC ...... 46 a Step gradient used in purification the pooled fractions (SOF II-5, SOF II-6) .... 48 a Step gradient used for purification of the EtOAc extract by semi- preparative HPLC ...... 49 a Step gradient used for purification of fraction NF IV-1 by semi-preparative HPLC ...... 51 a Step gradient used in purification of fraction F 10 by semi-preparative HPLC ...... 54 a Dry weight of extracts from biomass (1g) and culture media (1L) of 12 cyanobacterial strains...... 61 a Fractionation of methanol extract from Westiellopsis sp. VN biomass by silica gel chromatography and antibacterial activity of fractions to S. aureus ...... 65 a Fractionation of combined fractions F I, F II , and F III from Westiellopsis sp. VN biomass by LH-20 chromatography and antibacterial activity of fractions to S. aureus ...... 66 a Fractionation of WF 1 from Westiellopsis sp. VN biomass by semi- preparative reversed-phase HPLC and antibacterial activity of fractions to S. aureus .... 67 a 1H NMR data of compound 1 compared with literature data of ambiguine D isonitrile ...... 69 a 1H NMR data of compound 3 compared with literature data* of fischerellin A...... 72 a Comparison of 1H NMR data of compound 4 with reported data*...... 74 a Compounds of MeOH fraction analyzed as methyl ester in n-hexane after hydrolysis /derivatization...... 78 a Compounds of MeOH fraction analyzed as methyl ester in MeOH after hydrolysis/derivatization...... 78 a Dry biomass and antibacterial activity against S.aureus of extracts in BG-11 medium...... 80 a Fractionation of methanol extract from Calothrix javanica biomass by RP-18 chromatography and antibacterial activity of fractions to S. aureus ...... 82 a Step gradient used in HPLC analysis of CJF II by analytical HPLC ...... 83 a Separation of CJF II from Calothrix javanica biomass by semi- preparative reversed-phase HPLC and activity of the fractions to S. aureus ...... 84 a Step gradient used in HPLC analysis of seven fractions by analytical HPLC ...... 84 a NMR data of CJF II-4...... 86 a Sequence information deduced from the NOE’s found in the 2D NOESY spectrum of CJF II-4 ...... 88 a Calculation of the molecular mass from the structure of the residues deduced from the NMR data...... 88
a Comparison of the sequence from the high-resolution ESI-MS data with that from the NMR data...... 89 a Fractionation of methanol extract from Scytonema ocellatum biomass by RP-18 chromatography and antibacterial activity of fractions to S. aureus ...... 90 a Separation of SOF II from Scytonema ocellatum biomass by silical gel chromatography and antibacterial activity of the fraction to S. aureus ...... 91 a Separation of SOF II-5 and SOF II-6 from Scytonema ocellatum biomass by semi-preparative reversed-phase HPLC and antibacterial activity of the fractions to S. aureus ...... 92 a Step gradient used in HPLC analysis of FSO 3 by analytical HPLC...... 93 a Antibacterial activity of extracts from Anabaena sp. cultivated in large scale...... 94 a Separation of EtOAc extract from Anabaena sp.culture medium by semi-preparative reversed-phase HPLC and antibacterial activity of the fractions to E. coli ...... 95 a NMR data of AF 6 (Fluourensadiol) ...... 97 a Fractionation of methanol extract from Nostoc sp. biomass by silical gel chromatography and antibacterial activity of fractions to S. aureus ...... 98 a Fractionation of NF IV from Nostoc sp. biomass by RP C 18 chromatography and antibacterial activity of fractions to S. aureus ...... 99 a Fractionation of NF IV-1 from Nostoc sp. biomass by semi-preparative RP HPLC and antibacterial activity of fractions to S. aureus ...... 100 a Fractionation of methanol extract from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions against cell line 5637..... 101 a Separation of fraction F 8 from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions to 5637 cell line ...... 102 a Separation of fraction F 8-3 from Lyngbya majuscula biomass by preparative TLC...... 103 a Comparison of 1H NMR data of compound 9 with reported data *...... 104 a Fractionation of methanol extract from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions against cell line 5637..... 106 a Separation of fraction F 10 from Lyngbya majuscula biomass by semi- preparative RP HPLC and cytotoxic activity of fractions against cell line 5637 ...... 108 a Comparison of 13 C NMR and 1H NMR data of compound 10 with literature values of *Debromoaplysiatoxin...... 109 a Comparison of 1H NMR data of compound 11 with reported data *...... 111 a Fatty acids analyzed as methyl ester in n-hexane extract...... 112
Scheme of extraction...... 40 Extraction, fractionation, and isolation of the secondary metabolites of Westiellopsis sp.VN...... 45 Extraction, fractionation, and isolation of the secondary metabolites of Calothrix javanica ...... 46 Extraction, fractionation, and isolation of the secondary metabolites of Scytonema ocellatum ...... 48 Extraction, fractionation, and isolation of the secondary metabolites of Anabaena sp...... 49 Extraction, fractionation, and isolation of the secondary metabolites of Nostoc sp...... 51 Extraction, fractionation, and isolation of the secondary metabolites of Lyngbya majuscula (method 1)...... 53 Extraction, fractionation, and isolation of the secondary metabolites of Lyngbya majuscula (method2)...... 55
a
1D one-dimensional 2D two-dimensional AS anisaldehyd- sulphuric acid br broad
C18 octadecyl CC column chromatography COSY correlation spectroscopy d doublet DAD diot array detector DCM dichloromethane dd double of double ddd doublet of doublet of doublets DEPT distortionless excitation by polarization transfer dq doublet of quartets dt doublet of triplets ESI electrospray ionization EtOAc ethyl acetate EtOH ethanol GC-MS gas chromatography-mass spectrometry Gln glutamine HMBC heteronuclear multiple bond correlation HMQC heteronuclear multiple quantum coherence HPLC high performance liquid chromatography HR-ESI-MS high resolution-ESI-MS Hz hertz
IC 50 50% inhibitory concentration Ile isoleucine INT iodonitrotetrazolium chloride IR infrared IZ inhibition zone Leu leucine
m multiplet m/z mass/charge M+ molecular ion Me methyl MeCN acetonitrile MeOH methanol MHz megahertz MS mass spectrometry NMR nuclear magnetic resonance NOE nuclear Overhauser effect NOESY nuclear Overhauser effect spectroscopy ppm part per million Pro proline q quartet qd quartet of doublets
Rf retention factor ROESY rotating frame nuclear Overhauser effect spectroscopy rRNA ribosomal ribonucleic acid s singlet sp. species (singular) SS solvent system t triplet TFA trifluroacetic acid Thr threonine TLC thin layer chromatography TOCSY total correlation spectroscopy TOF time of flight tR retention time Tyr tyrocine UV ultraviolet vis visible δ chemical shift (in ppm) PTLC preparative- TLC
Chapter I Introduction
a a a a a a a Cyanobacteria, also known as blue-green algae, blue-green bacteria, cyanoprokaryots, and cyanophytes, are oxygenic photosynthetic prokaryotes that possess features familiar to both bacteria (prokaryota) and algae (eukaryota). Their cell structure and composition are similar to those of prokaryotic cell in that they lack the cell nucleus and distinctive organelles of eukaryotic cell, and their special structure and chemical composition of the cell wall are basically the same as those of gram-negative bacteria (Stanier & Cohen-Bazire, 1977; Van den Hoek et al., 1995; Sakamoto et al., 1997; Castenholz, 2001; Kalaitzis et al., 2009). However, in contrast to typical prokaryotes, they contain chlorophyll a and several accessory pigments providing them with oxygenic photosynthetic ability like other algae, and their blue- green color. They have two photosystems (PSII and PSI) and use water as an electron donor during photosynthesis, leading to the production of oxygen. Several cyanobacteria can also perform anoxygenic photosynthesis using only photosystem I if electron donors such as hydrogen sulphide are present (Madigan et al., 2003). All the known cyanobacteria are photoautotrophic, using primarily CO 2 as carbon source (Castenholz, 2001). Cyanobacteria show considerable morphological diversity (Whitton & Potts, 2000). They may either be unicellular, be aggregated into flat, spherical, regular or irregular colonies, or form single filaments without branches or filaments with false or true branching. Some cyanobacteria have the ability to produce two types of specialized cells: (1) heterocysts, which provide the site for nitrogen fixation and thereby counteract nitrogen demand under conditions of nitrogen deficiency, and (2) akinetes, which are resting cells that allow the species to survive unfavourable growth conditions. Many species of cyanobacteria possess gas vesicles, enabling them to regulate their buoyancy and to maintain a certain vertical position in the water column in response to physical and chemical factors (Reynolds, 1987; Walsby, 1994). Asexual reproduction of cyanobacteria occurs by the formation of hormogonia or endospores (exospores are modified endospores) or by fragmentation of colonies (Lee, 1999)
1
Chapter I Introduction
a a a Cyanobacteria have a long evolutionary history and documented fossil records date back to about 3500 million years ago (Schopf, 2000). However, the earliest DNA-biomarker evidence suggests that cyanobacteria appeared about 2600 million years ago (Hedges et al., 2001). It is widely accepted that ancient cyanobacteria evolved oxygenic photosynthesis and played a major role in the change of the oxygenless atmosphere to an oxygenic one (Schopf, 2000). Additionally, cyanobacteria are believed to have had a considerable effect on the formation of oxygen rich gas composition of Earth`s atmosphere (Dismukes, 2001; Paul, 2008), and today the production of oxygen by cyanobacterial photosynthesis continues to contribute to maintaining the balance of our atmosphere (Sielaft et al., 2006). Their long evolutionary history is considered as a reason for the successful survival of cyanobacteria in many habitats and their wide ecological tolerance (Whitton & Potts, 2000). In addition, cyanobacteria have developed a wide ecological tolerance to temperature, light, salinity, moisture, alkalinity, and possess many characteristics and adaptations that explain their world wide distribution and success. The distribution of cyanobacteria is expanded widely on the earth in diverse ecosystems of marine, freshwater, and terrestrial environments. They are most abundant in aquatic habitats as part of the plankton, some can be found tightly or loosely attached to surfaces of plants, rocks and sediments, and some can be found in hot and acidic springs, in salt lakes, in deserts, ice shelves, and the arctic (Mur et al., 1999; Rastogi & Sinha, 2009 ). Cyanobacteria are also important in many terrestrial environments and they can live in soils or rocks and form symbiotic associations with plants, fungi and animals (Whitton & Potts, 2000; Oren, 2000; Baracaldo et al., 2005; Thajuddin & Subramanian, 2005). The immense diversity within this group of microorganisms, apart from the variability of morphology and range of habitats, is also reflected in the extent of their synthesis of natural products. Cyanobacteria have evolved to produce a diverse array of secondary metabolites that have aided species survival in these varied and highly competitive ecological niches (Kalaitzis et al., 2009). Cyanobacteria are commonly associated with the toxic blooms encountered in many eutrophic fresh and brackish waters and are widely known for their potential to produce a range of neurotoxic, hepatotoxic, and tumour promoting-secondary metablites (Codd et al., 1999; Sivonen & Börner, 2008). Cyanobacteria are unique phyla that grow in competitive niches and, as a result, are promising sources of bioactive compounds (Clardy & Walsh, 2004; Lin et al., 2008).
2
Chapter I Introduction
a a a a a Taxonomic classification is a method for registration of the biodiversity and the arrangement of individual into taxonomic groups. Therefore it should reflect evolution, ecology, and phenotypic variations (Hoffmann et al., 2005). Taxonomic classification of cyanobacteria is quite complex. There are presently two main classification systems available (1) the botanical classification system (Komárek & Anagnostidis, 1989; 1999; 2005 and Anagnostidis & Komárek, 1990) and (2) the bacteriological classification system (Castenholz, 2001). Cyanobacteria were traditionally classified on the basis of their morphology only, according to the International Code of Botanical Nomenclature, ICBN (Greuter et al., 2000). The taxonomy based on morphological characteristics alone does not necessarily result in a phylogenetically reliable taxonomy despite the fact that the cyanobacterial morphology is complex compared to most other prokaryotic microbes (Giovanni et al., 1988; Wilmotte, 1994). Moreover, the problem of using only morphological criteria is that some characters may vary considerable in response to different environmental conditions making species delimitation difficult (Wilmotte & Golubic 1991; Barker et al., 1999; Otsuka, 1999). Cyanobacteria are also classified according to the International Code of Nomenclature of Prokaryots, ICNP (Oren & Tindall, 2005). The bacteriological classification is today widely based on phenotypic, chemotypic and genotypic characteristics, the so called polyphasic approach, of pure culture of cyanobacteria. It is a challenge to combine the traditional morphological classification and the classification based on molecular methods, however, effort is made to unify these two systems (Hoffmann, 2005; Oren & Tindall, 2005). In the current botanical classification system, Komárek & Anagnostidis (1989; 1999; and 2005) and Anagnostidis & Komárek, 1990) revised the taxonomy of cyanobacteria and also included phenotypic and genotypic features. The botanical approach distinguishes four orders of cyanobacteria (Komárek & Anagnostidis, 1989; 1999; 2005 and Anagnostidis & Komárek, 1990). The bacteriological taxonomic system created for cyanobacteria is divided in five subsections (Rippka et al., 1979; Castenholz, 2001) and they are to a large extent in agreement with the orders in the botanical system (see table 1-1). Ideally, taxonomy reflects evolutionary relationships of the classified organisms, and the taxa are monophyletic groups of organisms (e.g. Wilmotte & 3
Chapter I Introduction
Golubic, 1991; Wilmotte, 1994). DNA sequences make it possible to infer phylogenies of organisms (e.g. Moritz & Hillis, 1996) and DNA is not affected by environmental factors in the same manner as many morphological traits are. The 16S rRNA gene is universally present in bacteria and cyanobacteria and has a conserved function. Woese and coworkers (Woese et al., 1976; Woese, 1987) established the modern bacterial phylogenetic classification mainly based on the 16S rRNA gene sequence. The widely used 16S rRNA region has been useful in several phylogenetic analyses of cyanobacteria (e.g. Wilmotte& Golubic, 1991; Ben-Porath & Zehr, 1994; Nelissen et al., 1996; Fergusson & Saint, 2000; Wilmotte & Herdman, 2001). Some of the molecular methods have been also used for taxonomic studies of cyanobacteria including DNA-DNA hybridization (Stam, 1980; Stam & Stulp, 1988; Wilmotte et al., 1997), fingerprinting based on PCR with primers from short and long tandemly repeated repetitive sequences (Rasmussen & Svenning 1998), restriction fragment length polymorphism (RFLP) (Mazel et al., 1990; Asayama et al., 1996; Lehtimäki et al., 2000), DNA amplification methods (AFLP, ARDRA, REP-PCR, RAPD) (Neilan, 1995; Satish et al., 2001; Lyra et al ., 2001), sequencing of marker genes, e.g. rpoC1 (Fergusson & Staint, 2000), nifH (Ben-Porath & Zehr, 1994), cpcB and cpcA (Manen & Falquet, 2002; Ballot et al., 2004; Teneva et al., 2005), ITS region sequencing (the internal transcribed spacer between the 16S rDNA and 23S rDNA) (Gugger et al., 2002; Orcutt et al., 2002), PC-IGS region sequencing (phycocyanin operon intergenic spacer) (Neilan et al., 1995; Bolch et al., 1996; Laamanen et al., 2001; Dyble et al., 2002; Rohrlack et al., 2008).
a The orders of cyanobacteria according to the botanical classification and their correspondence to the subsections of the bacteriological code
a a a a a a a a a a a a a Subsection I Unicellular cyanobacteria that reproduce by binary cell division in one, two or three plane; or Synechocystis Order budding, either single cells or in Microcystis Chroococcales 1 colonies held together by
4
Chapter I Introduction
mucilage or laminated sheaths. Many species are planktonic and contain gas vesicles. They are widespread in freshwater, brackish water and marine environment. Subsection II Unicellular or non-filamentous Pleurocapsa aggregates of cells held together by outer wall or gel-like matrix. Some species can sometimes or always reproduce by small spherical cells (baeocytes) which are produced by multiple divisions of the mother cells. They generally grow in aquatic environments attached to substrata. Order Subsection III Organisms form trichomes of Oscillatoriales 2 vegetative cells, mostly uniseriate, without differentiated Oscillatoria cells such as heterocytes and Spirulina akinetes (resting cells, spores). Lyngbya The trichomes show not true Planktothrix branches but in some genera false Limnothrix ramifications may occur. The trichomes usually have a sheath and many species have gas vesicles. Cell division occurs always in one plane perpendicular to the longitudinal axis of trichome. The group is ecologically diverse and they live in plankton, benthic and periphytic in freshwater and in marine environments.
5
Chapter I Introduction
Order Subsection IV Binary division in one plane Nostocales 3 giving rise to 1-seriate trichomes, Anabaena though sometimes with "false" Nodularia branches; one or more cells per Nostoc trichome differentiate into Scytonema heterocysts, at least when Calothrix concentration of nitrogen is low; some also produce akinetes; some may form hormogonia (formation of motile trichomes that give rise to young filaments).They occur in plankton, benthic and periphytic in freshwater, marine, and terrestrial environments. Order Subsection V Binary division periodically or Fischerella Stigonematales 4 commonly in more than one Hapalosiphon plane, giving rise to multiseriate Westiellopsis trichomes, or trichomes with true branches or both; apparently always possess ability to form heterocysts; some also form akinetes; in some genera, there is a differentiation into main filament and branches. They occur in terrestrial and aquatic environments but usually not in the plankton. 1. Komárek & Anagnostidis, 1999; 2. Komárek & Anagnostidis, 2005; 3. Komárek & Anagnostidis, 1989; 4. Anagnostidis & Komárek, 1990; 5. Rippka et al., 1979, Castenholz, 2001
a a a a a a a a a a Natural products (secondary metabolites) are an important source of new pharmaceuticals and pharmaceutical 'lead' compounds. Natural products serve not only as drugs in their own right, but they may also serve as structural models for the
6
Chapter I Introduction creation of synthetic analogues, and as models in structure-activity studies (e.g. Quinn et al., 1993; Harvey, 2008; Gademann and Kobylinska, 2009). Of the 974 small molecule new chemical entities introduced as drugs worldwide during 1981-2006, 63% were inspired by natural products (Newman & Cragg, 2007). These include natural products (6%), natural product derivatives (28%), synthetic compounds with natural product derived pharmacophores (5%) and synthetic compounds based on knowledge gained from a natural product (natural product mimic, 24%). In certain therapeutic areas the productivity was higher: 77.8% of anticancer drugs are either natural products or derived from natural products. The overwhelming majority of active compounds have been derived from streptomycetes, fungi, bacteria and plants. A major problem in focusing on these sources in the search for novel, biologically active molecules, is the rediscovery of previously known natural products. One way to minimize this problem is to develop selective bioassays and investigate new therapeutic areas. Another approach is to look at new and different sources of natural products. Natural products have been isolated from a wide variety of taxa and tested for various biological activities. Among these taxa, cyanobacteria represent such a source. They have been identified as a new and rich source of bioactive compounds (Abarzua et al., 1999; Burja et al., 2001; Shimizu, 2003; Bhadury et al., 2004; Wiegand & Pflugmacher, 2005; Dahms et al., 2006; Tan, 2007; Jaiswal et al., 2008; Smith et al., 2008; Sivonen & Börner, 2008; Gademan & Portmann, 2008). Numerous bioactive compounds isolated from different cyanobacterial strains exhibited novel and a diverse range of biological activities and chemical structures including novel peptides (e.g. cyclic depsipeptides, cyclic peptides, lipopeptides), fatty acids, polyketides, alkaloids, amides, terpenes, carbohydrates, and other organic chemicals (Patterson et al., 1994; Namikoshi & Rinehart, 1996; Abarzua et al., 1999; Kreitlow et al., 1999; Burja et al., 2001; Singh et al., 2005;Welker &VonDöhren, 2006; Sielaff et al., 2006; Spolaore et al., 2006; Ramaswamy et al., 2006; Wagoner et al., 2007 ; Tan, 2007; Baumann, 2007; Blunt et al., 2008, 2010; Portmann et al., 2008; Medina et al., 2008;Gademann & Portmann, 2008 ; Van Wagoner et al., 2007; Tripathi et al., 2009; Tidgewel et al., 2009). Many of these are regarded as good candidates for drug discovery, with applications in agriculture (Biondi et al., 2004), industry (De Philippis et al., 1998; Spolaore et al., 2006; Jaiswal et al., 2008; Rastogi &Sinha, 2009), and especially, in pharmacy (Mundt et al., 2001; Singh et al., 2005; Sielaff et al., 2006; Dunlap et al., 7
Chapter I Introduction
2007; Tan, 2007; Gademann & Portmann, 2008; Gerwick et al., 2008; Rastogi & Sinha, 2009). The cyanobacterial bioactive compounds provide novel and useful pharmaceuticals that are difficult to produce synthetically because of their structural complexity (Kaushik & Chauhan, 2009). The chemical diversity and novelty seen in cyanobacteria are comparable to those of Actinomycetes, which have turned out many important drugs. It is not unusual that a single species produces many different chemotypes. Lyngbya majuscula is a good example. The diversity of structures found in this ubiquitous filamentous cyanobacterium is just incredible. Compounds isolated from this strain are lipopeptides (cyclic or linear), amino acids, lactones, fatty acids, amides, alkaloids, pyrroles , depsipeptides and many others (Burja et al., 2001; Shimizu, 2003; McPhai et al., 2007; Tripathi et al., 2009; Gutiérrez et al., 2008; Tan, et al., 2008; Jones et al., 2009, 2010; Blunt et al., 2010 ). Most of them possess characteristic biological activity. For example, curacin A isolated from a Cuasao strain by Gerwick's group (Gerwick et al., 1994b) was recognized as a new pharmacophore to perturb microtubule assembly and acts as potent antimitotic agent. With a rather simple structure, it is an important lead compound for new types of anticancer drugs. The relative disregard in the past of cyanobacteria compared with other microbial sources of natural products, and the microbial diversity of cyanobacteria, as well as the huge chemical and biologically active diversity of their products recommend them as an attractive source of novel drugs for use in diverse therapeutic areas and should imply opportunity for a most significant progress in the generation of novel bioactive substances. Furthermore, cyanobacteria also have the advantage over many other organisms as they can be cultured, thus providing an alternative to chemical synthesis for the production of their bioactive compounds. Cyanobacterial metabolites show an interesting and exciting range of biological activities ranging from antimicrobial, anticancer, antiviral, immunosuppressant, insecticidal, anti-inflammatory to proteinase-inhibiting activities which are striking targets of biomedical research (Borowitzka, 1995; Kulik, 1995; Luesch et al., 2002 ; Soltani et al., 2005; Tan, 2006; Dunlap et al., 2007; Wase & Wright, 2008; Gerwick et al. , 2008; Abed et al., 2009, Gademann & Kobylinska, 2009; Villa et al., 2010). 8
Chapter I Introduction
a Cyanobacteria have been shown to be a source of antibiotic compounds (e.g.Harvey, 2000; Jaki et al., 1999b; Burja et al., 2001; Volk &Furkert, 2006; Chlipala et al., 2009). Most of the antibiotic metabolites isolated until now were accumulated in the cyanobacterial biomass, but cyanobacteria are also known to excrete various antibiotic compounds into their environments (Moore et al., 1984a; De Caire et al., 1993; Jaki et al., 1999a and 2000a; Volk 2006; Jaiswal et al., 2008). In spite of the studies carried out so far, many cyanobacterial compounds are still largely unexplored and the chemicals involved are mostly unidentified, thus giving a rich opportunity for discovery of new bioactive compounds. The antibiotic activities include antibacterial, antifungal, and algicidal activity etc. Secondary metabolites with antibacterial activity are widely produced by cyanobacteria. These compounds are effective against Gram-positive and/or Gram – negative bacteria, however, it has been found that the antibacterial activity of cyanobacteria is mainly directed against Gram-positive bacteria since most Gram- negative bacteria are resistant to toxic agents in the environment due to the barrier of lipopolysaccharides on their outer membrane (Dixon et al., 2004). Both toxic and nontoxic strains of cyanobacteria are producers of antibacterial compounds that are distinct from cyanotoxins (Østensvik et al., 1998). Antibacterial effects of extracts from Fischerella sp. (Asthana et al., 2006), Spirulina platensis (Kaushik &Chauhan, 2008; Abedin &Taha, 2008), Anabaena variabilis (Kaushik et al., 2009), Nostoc sp. CCC537 (Asthana et al., 2009), Oscillatoria (Shanab, 2007), Anabaena and Nostoc (Svircev et al., 2008), Synechocystis and Synechococcus (Martins et al., 2008) and other species belonging to the orders of Chroococales, Pleurocapsales, Oscilatoriales, Nostocales, Stigonematales (Soltani et al., 2005; Taton et al., 2006; Chlipala et al., 2009; Patil et al., 2009) have been reported. Up to now, there have been published several reports of antibacterial compounds isolated from cyanobacteria, such examples as ambiguine isonitriles, aoscomin, comnostins A-E, norharmane, lyngbyazothrins, carbamidocyclophanes (Smitka et al., 1992; Jaki at el., 1999a and 2000a,b; Volk & Furkert, 2006; Bui et al., 2007; Raveh & Carmeli, 2007; Zainuddin et al., 2009; Mo et al., 2009). Several extracts of cyanobacteria belong to Stigonematales, Nostocales and Oscillatoriales have shown antifungal activity in in-vitro test systems (Moore et al., 9
Chapter I Introduction
1987; Parket et al., 1992; Smitka et al., 1992; Stratmann et al., 1994; Soltani et al., 2005; Pawar &Puranik, 2007; Abedin & Taha, 2008; Svircev et al., 2008). Antifungal compounds isolated from these extracts include hapalindoles, tolytoxin, scytophycins, toyocamycin, tjipanazoles, hassallidin A, nostocyclamide and nostodione (Abed et al., 2009). Antibacterial and antifungal substances identified also include fatty acids (Gerwick et al.,1987; Mundt et al., 2003; Asthana et al., 2006), phenolics (DeCano et al.,1990), bromophenols (Pedersén & DaSilva, 1973), terpenoids (Jaki et al., 2000a,b), N-glycosides (Bonjouklian et al., 1991), cyclic depsipeptides (Carter et al .,1984), lipopeptides (MacMillan et al., 2002; Neuhof et al., 2005), cyclic peptides (Pergament &Carmeli, 1994), cylic undecapeptides (Zainuddin et al ., 2009) and isonitrile-containing indole alkaloids (Moore et al.,1987; Smitka et al.,1992; Raveh & Carmeli, 2007, Mo et al., 2009). Relatively little has been known on the antimicrobial activity of cyanobacterial compounds from Chroococcales group (e.g., Synechoccystis and Synechoccocus ), however, Martins et al ., (2008) emphasise the potential of these genera as a source of antibiotic compounds that produce substances with inhibitory effects on prokaryotic cells and with apoptotic activity in eukaryotic cell lines, which highlights the importance of these organisms as potential pharmacological agents. Unfortunately, almost all of the studies have used only in vitro assays, it is likely that most of compounds responsible for antibiotic activity will have little or no application in medicine as they are either too toxic or are inactive in vivo ( Reichelt & Borowitzka, 1984; Borowitzka, 1995). They may, however serve as useful leads to create new synthetic antibiotics or may find application in agriculture. For example, cryptophycin 1 was first isolated from Nostoc sp. ATCC 53789 by the Merck's group as a fungicide (Schwartz et al ., 1990). However, it was found to be too toxic to use as an antifungal agent and no further studies were carried out with this compound. Subsequently, Moore and co-workers isolated this same compound from Nostoc sp. GSV 224, which exhibited powerful cytotoxicity against human tumor cell lines and good activity against a broad spectrum of drug-sensitive and drug-resistant murine and human solid tumors. Nevertheless, cryptophycin 1 again appeared to be too toxic to become a clinical candidate. This led to a detailed structure-function study by Moore in collaboration with Carmichael, and resulted in creation of cryptophycin 8 (see fig.1-1) a semisynthetic analogous which proved to have a greater therapeutic 10
Chapter I Introduction efficiency and lower toxicity than cryptophycin 1 in vivo (Moore et al., 1996; Liang et al., 2005).
Crytophycin 1 and crytophycin 8
It has also been shown that cyanobacteria produce a broad spectrum of antialgal compounds, which may be used to control cyanobacteria and algal blooms. Cyanobacteria probably use these compounds in order to out-compete other micro- organisms, to gain dominance over other organisms, or influence the type of conspecifics and successors (Gross, 2003; Dahms et al., 2006). A growing number of studies have identified cyanobacterial metabolites that act as algaecides (Mason et al., 1982; Vepritskii et al., 1991; Gromov et al ., 1991; Schlegel et al ., 1999; Smith & Doan, 1999; Volk & Furkert, 2006; Shanab, 2007; Berry et al ., 2008). Mason et al ., 1982 reported the identification and characterization of cyanobacterin (chlorinated γ- lactone) from the filamentous freshwater cyanobacterium Scytonema hofmanni that specifically inhibited a range of algae, including other cyanobacteria and green algae, at micromolar concentrations, but had little effect on non-photosynthetic microbes. It was later found that cyanobacterin specifically inhibits photosystem II (Gleason &Case, 1986). Compounds such as cyanobacterins LU-1 and LU-2 (Gromov et al ., 1991; Vepritskii et al. ,1991; Ishibashi et al ., 2005) were reported from Nostoc linckia that are structurally different from cyanobacterin but specifically inhibit transport of electrons in photosystem II. It was found that LU-1 inhibited cyanobacteria as well as algae but not photosynthetic microbes, whereas LU-2 inhibited cyanobacteria only. Flores & Wolk, 1986 and Schlegal et al., 1999 independently screened 65 and approximately 200 isolated of cyanobacteria, respectively, for algaecidal activity. Interestingly, it was found in these studies that antialgal activity was largely restricted to several genera, namely Fischerella, Nostoc, Anabaena, Calothrix and Scytonema . Fischerella produces the fischerellins and hapalindoles, which both inhibit photosynthesis and RNA polymerization (Srivastava et al. , 1999; Gantar et al., 2008).
11
Chapter I Introduction
The bioactive compounds of Oscillatoria species also showed antibiotic activity against green algae and cyanobacteria, including a toxic Microcystis aeruginosa species (Smith & Doan, 1999; Shanab, 2007). More recently, Berry et al ., 2008 screened 76 isolates of cyanobacteria from the Everglades for antialgal activity against two sympatric representatives of green algae ( Selanstrum 34-4 and Chlamydomonas Ev-29) and cyanobacteria ( Anabaena 66-2 and Synechococcus 40- 4). Of these, 40 isolates (53% of those tested) inhibited one or more of the representative strains. It has been described that the pentacyclic calothrixins, isolated from Calothrix (Rickards et al. 1999), inhibit RNA polymerase and DNA synthesis, and hence act as allelopathic compounds (Doan et al., 2000). In addition to cyanobacterins LU-1 and LU-2, the genus Nostoc produces several other metabolites that have been associated with algaecidal activity e.g., nostocyclamide, nostocine A as well as nostocarboline (Todorova et al., 1995; Hirata et al. , 2003; Blom et al., 2006). It has been reported that the microcystins (e.g. microcystin-LR) isolated from M. aeruginosa inhibit the growth of cyanobacteria, including Nostoc, Synechococcus and Anabaena species (Singh et al., 2001). Recently, an interesting compound, norharmane (see fig.1-2) from Nodularia harveyana exhibited anticyanobacterial activity against both filamentous and unicellular cyanobacteria and may be used for the control of toxic algal blooms (Volk, 2006).
Norharmane from cyanobacteria a a a a Blue-green algae have been found to be excellent sources of new anticancer agents (Moore et al., 1988; Patterson et al. , 1991; Gerwick et al., 1994a; Burja et al ., 2001; Simmons et al., 2005; Tan, 2007; Sivonen & Börner, 2008; Gerwick et al., 2008). Freshwater and marine cyanobacteria are well recognized for producing
12
Chapter I Introduction numerous and structurally diverse bioactive and cytotoxic secondary metabolites suited to drug discovery (Dunlap et al., 2007). Researchers found that a relatively high percentage of extracts from cultured cyanobacteria showed cytotoxicity, and was active in vivo. 6% of extracts of over 1000 cultured cyanobacterial strains showed cytotoxicity against the KB cell line
(a human epidermoid carcinoma of the nasopharynx) with MIC s< 30µg/ml) (Moore et al.,1988). According to Patterson et al., 1991, in their large-scale screening program initiated to evaluate laboratory-cultured blue-green algae (cyanobacteria) as a source of novel antineoplastic agents, approximately 1000 cyanophyte strains from diverse habitats were cultured to provide extracts for testing. This screening program showed approximately 7% of extracts tested inhibited proliferation of the KB cell line, the families Scytonemataceae and Stigonemataceae as prolific producers of novel cytotoxic compounds, and rates of rediscovery of known compounds were relatively low. Research of Gerwick et al., 1994b at Oregon State University had focused on marine cyanobacteria and primarily screening of compounds for anticancer activity. This work had clearly shown that marine cyanobacteria are an exciting source of novel bioactive compounds, with a high number of purified metabolites demonstrating activity. Interestingly, approximately 40% of the marine cyanobacterial compounds possess anticancer/antitumor activity, making them invaluable as potential therapeutic leads (Jaspars & Lawton, 1998; Tan, 2007). Marine cyanobacteria are also an exceptionally rich source of novel peptides and integrated peptide-polyketide type natural products. Many of these natural products are potently cytotoxic to mammalian cells, and this has furthered their exploration as a source of new anticancer lead compounds (Gerwick et al., 2008). To date, many researches have focused on screening for anticancer compounds (e.g. Martin et al ., 2008; Sivonen et al ., 2008) and a variety of cytotoxic compounds have been isolated from cyanobacteria, many of them possess unprecedented structures and therefore have the potential for development of entirely new classes of drug agents. Perhaps the cryptophycins are among the earliest and most prominent candidates for new anticancer drugs. When the cryptophycins, a group of >25 cyanobacterial metabolites with strong tubulin-destabilizing activities (Smith et al ., 1994; Panda et al., 1997; Corbett et al ., 1997; Eggen & Georg, 2002), were 13
Chapter I Introduction discovered, hopes were great that one of these natural products could be developed into a useful anticancer drug. In fact, the prototype cryptophycin 1, mentioned in 1.2.1, of this class of natural anticancer drugs from the cyanobacterium Nostoc sp. ATCC 53789, is one of the most potent tubulin-destabilizing agents ever found (Smith et al., 1994). In addition, the cryptophycins, like the epothilones, were not substrates of P-glycoprotein, an efflux pump that makes multidrug-resistant cancer cell lines immune against a multitude of anticancer drugs (Smith et al ., 1994; Fojo et al., 2005; Breier et al ., 2005). Consequently, cryptophycin 52 (see fig. 1-3), a synthetic analogue, was developed and reached phase II of clinical trials (Sessa et al., 2002; Edelman et al ., 2003; D'Agostino, 2006). The synthetic analogue 52 was chosen because no large-scale biotechnological production method existed for the cryptophycins. Eventually, the high production costs and toxic side effects of cryptophycin 52 stopped its development and that of any other analogues of the cryptophycin family. Nobody wanted to restart all of the trials with a different analogue, although preclinical studies showed that other analogues would have been a better choice (Liang et al., 2005). Nonetheless, studies to find new cryptophycin analogues or to develop semisynthetic/biotechnological methods for the generation of promising cryptophycin analogues continued (Liang et al., 2005; Beck et al., 2005). To date, the research group of David H. Sherman of the University of Michigan in collaboration with Richard E. Moore of the University of Hawaii (Magarvey et al., 2006) has studied in a very comprehensive way the biosynthesis of the cryptophycins and cryptophycin 52 is now biotechnologically producible, which may make this drug more easily accessible and less costly to produce if further pursued (Rohr, 2006).
Cryptophycin 1 and 52, potent antitumor agents from cyanobacteria
14
Chapter I Introduction
In addition, an increasing number of marine cyanobacteria are found to target tubulin or actin filaments in eukaryotic cells, making them an attractive source of natural products as anticancer agents (Jordan &Wilson, 1998). Prominent compounds isolated from marine cyanobacteria such as the anti-microtubuli agents, curacin A (1), dolastatin 10(2), and dolastatin 15 have been in preclinical and /or clinical trials as potential anticancer drugs (Gerwick et al., 2001; Newman & Cragg, 2004; Simmons et al ., 2005; Tan, 2007; Butler, 2008). Many of marine cyanobacterial molecules with potent biological activities are also lead compounds for the development of synthetic analogs having increased potency and decreased toxicity. As shown in figure 1-4. Curacin A , dolastatin 10 , and dolastatin 15 served as lead structures for the development of synthetic analogues. e.g. compound , TZT-1027 , ILX-651 , and LU-103793 , usually with improved pharmacokinetic properties (Wipf et al., 2004; Mita et al., 2006; Wantanabe et al., 2006; Tan, 2007; Butler, 2008).
Prominent anticancer marine cyanobacterial secondary metabolites and synthetic analogues (Tan 2007)
Curacin A (1) a metabolite isolated from a Curaçao strain of the marine cyanobacterium Lyngbya majuscula (Gerwick et al .,1994b) exhibits potent anti- proliferative and cytotoxic activity against colon, renal, and breast cancer derived cell
15
Chapter I Introduction lines but is effectively insoluble in any formulation and thus has not been reported to produce activity in in vivo animal models. However, as a lead molecule, it has inspired the synthetic production of numerous analogs, some of which show potent cytotoxic effects but with increased stability and water solubility (Wift et al., 2004), e.g. compound a synthetic analog of curacinA, is undergoing evaluation ( Tan, 2007; Gerwick et al., 2008; Jones et al., 2009). Dolastatin 10 (2) was first isolated in 1987 from the Indian Ocean sea hare, Dolabella auricularia (Pettit et al., 1987), and then isolated from the marine cyanobacterium Symploca (Luesch et al., 2001). It possesses potent antiproliferative activity in vitro against a variety of human leukemias, lymphomas, and solid tumor cell lines (Pezer et al., 2005; Simmons et al., 2005). Indeed, while dolastatin 10 is no longer a clinical trial agent derivatives such as TZT-1027 (soblidotin) are still being evaluated (http://www.clinicaltrials.gov/ct2/results?term=soblidotin ; Patel et al., 2006; Gerwick et al., 2008; Butler, 2008). Dolastatin 15 ( ) was initially isolated from extracts of the Indian Ocean sea hare D. auricularia and numerous dolastatin 15–related peptides have been also isolated from diverse marine cyanobacteria (Beckwith et al., 1993; Gerwick et al., 2001), Dolastatin 15 inhibits proliferation of human malignant cell lines in vitro and is active in a broad range of animal tumor models (Hamel et al., 2002; Ray et al., 2007). Obstacles to further clinical evaluation of dolastatin 15 include the complexity and low yield of its chemical synthesis and its poor water solubility. However, these impediments have prompted the development of various synthetic analogue compounds with enhanced chemical properties, including ILX-651 and LU103793 (Simmons et al., 2005; Ray et al., 2007). ILX-651(tasidotin or synthadotin) is an orally active third generation synthetic dolastatin 15 ( ) analogue, ILX-651 is currently undergoing Phase II trials after its successful run in Phase I trials (Simmons et al., 2005 ; Mita et al., 2006; Ray et al ., 2007; Dunlap et al ., 2007; Butler, 2008; http://www.clinicaltrials.gov/ct2/results?term=synthadotin) Belamide A, a highly methylated linear tetrapeptide related to the dolastatin family of potent anticancer agents, was purified from a Panamanian marine cyanobacterium Symploca sp. This compound contains two characteric residues, the N-terminal N, N-dimethylvaline and C-terminal benzyl (methoxy) pyrrolinone moieties (Simmons et al., 2006). Belamide A demonstrated cytotoxicities against the MCF7 breast cancer-and the HCT-116 colon cancer cell lines, and displayed classic 16
Chapter I Introduction microtubule-depolymerizing effects in A-10 cells, including concentration-dependent interphase microtubule loss, micronucleation and abnormal mitotic spindle formation. Therefore, belamide A may provide a valuable starting point for structure- activity relationship (SAR) studies (Baker et al., 2007). Other noteworthy marine cyanobacterial molecules reported in the literature having significant cytotoxic activity include borophycin and apratoxin A. Borophycin (see fig.1-5) is a complex boron containing polyketide isolated from marine strains of Nostoc linckia and Nostoc spongiaeforme var. tenue (Banker and Carmeli, 1998). Borophycin, which is related both to the boron-containing boromycins isolated from a terrestrial strain of Streptomyces antibioticus and to the aplasmomycins isolated from a marine strain of Streptomyces griseus (actinomycetes), exhibits promising antitumor activity against standard cancer cell lines (MIC 0.066mg/mL, LoVo and 3.3mg/mL KB), and human epidermoid carcinoma and human colorectal adenocarcinoma cell lines (Davidson, 1995; Banker et al., 1998; Gademann &Portmann, 2008).
Borophycin from cyanobacteria
Apratoxin A first isolated from Lyngbya majuscula found at Apra Harbor, Guam, is a potent cytotoxin with a unique carbon skeleton. It possesses an impressive biological profile in in vitro cytotoxicity assays against various human tumor cell lines with IC 50 values ranging from 0.36 to 0.52 nM. Total synthesis of apratoxin A has been accomplished leading the way to synthetic analogs for the purpose of new therapeutics (Chen & Forsyth, 2004). Recently, several of potent anticancer marine cyanobacterial metabolites have been published including grassypeptolide, aerucyclamide A and B, hantupeptin A, and Apratoxins D and E (Kwan et al., 2008; Portmann et al ., 2008; Gutiérrez et al., 2008; Matthew et al ., 2008; Tripathi et al., 2009).
17
Chapter I Introduction
a a Cyanobacteria also appear to be a rich source of new antiviral compounds. The initial screening program by Rinehart et al., 1981 indicated that a large percentage of extracts of field- collected cyanophytes exhibited antiviral activity when assayed against herpes simplex virus, type II (HSV-2). Then, screening programs of the University of Hawaii and the U.S. National Cancer Institute have demonstrated antiviral activity in approximately 10% of extracts tested (some 600 cyanophyte strains) using live virus test systems for inhibition of HSV-2 and human immunodeficiency virus, type 1 (HIV-1) whereas a smaller percentage (2.5%) of the extracts were active against respiratory syncytial virus (Patterson et al. , 1993). Lau et al., 1993 have also screened extracts of over 900 strains of cultured cyanobacteria for inhibition of the reverse transcriptases (RT) of avian myeloblastosis virus (AMV) and human immunodeficiency virus, type 1 (HIV-1), and they found that over 2% of extracts showed activity against AMV and HIV RTs. Few of the antiviral compounds were isolated from cyanobacteria so far. Bioassay-directed fractionation of the National Cancer Institute of cyanophytes grown at the University of Hawaii led to the isolation of a family of anti–HIV sulfolipids (Gustafson et al., 1989; Patterson et al., 1994). Other compounds were anti-HSV-2 indolocarbazoles from Nostoc sphaericum (Knübel et al., 1990). Ambiguol A from Fischerella ambigua has been shown to inhibit HIV-1 reverse transcriptase and cyclooxigenase (Falch et al., 1993). A sulphated polysaccharide isolated from Spirulina platensis (Hayashi et al., 1993 and 1996) named calcium spirulan, inhibits replication in enveloped viruses such as HSV-1, HIV-1, human cytomegalovirus, etc. This polysaccharide is composed of rhamnose, ribose, mannose, fructose, galactose, xylose, glucose, glucuronic acid, sulphate and calcium, and the calcium appears to be essential for maintaining the replication-inhibiting activity. Nostoflan isolated from Nostoc flagelliforme (Kanekiyo et al. , 2005) and ichthyopeptins A and B isolated from Microcystis ichthyoblabe (Zainuddin et al., 2007) exhibited antiviral activity. Two interesting antiviral compounds also have been isolated from cyanobacteria: cyanovirin-N from Nostoc ellipsosporum (Boyd et al., 1997) and scytovirin from Scytonema vatium (Bokesch et al., 2003). Cyanovirin-N, a novel 11 kDa protein, inactivates the human immunodeficiency virus (HIV) and has high potency against most strains of influenza A and B viruses (O’Keefe et al., 2003; 18
Chapter I Introduction
Sivonen & Börner, 2008; Xiong et al ., 2010). Cyanovirin –N is under development as an antiviral agent, thanks to its effectiveness against HIV, its non-toxicity to human cells, and its persistence (Bewley et al ., 1998). Currently, cyanovirin-N is available as a vaginal gel for local protection to HIV infection (http://www.aidsinfo.nih.gov). In addition, a recent ex vivo test showed that the antiviral effect of cyanovirin-N is stronger than that of PRO 2000 (Fischetti et al ., 2009; Huskens et al ., 2009), a nonspecific polyanion microbicide. Scytovirin is a protein that acts similarly to cyanovirin –N, but is less efficient in inactivation of viruses (Bokesch et al ., 2003, Xiong et al ., 2006). The potential of these drug candidates to achieve clinical success holds great strategic promise for exploiting the structural complexity of cyanobacterial metabolites across diverse therapeutic areas in future drug discovery (Sielaff et al ., 2006).
a a a a a Cyanobacteria are better known as producers of highly toxic compounds (cyanotoxins) (Dow & Swoboda, 2000; Codd et al., 2005; Sivonen & Börner, 2008; Jaiswal et al., 2008; Stewart et al ., 2009). Cyanotoxins are bioactive secondary metabolites produced by cyanobacteria and the majority are commonly grouped according to their physiological effects either as cytotoxins ( e.g. cryptophycins, dolastatins, symplostatins), neurotoxins ( e.g. anatoxins, saxitoxins), hepatotoxins ( e.g. microcystins, nodularins), or as irritants and gastrointestinal toxins ( e.g. aplysiatoxins and lyngbyatoxin). Up to now some of cyanobacterial toxins are known as allelochemicals with potential applications as algaecides, herbicides and insecticides (Berry et al ., 2008), and many of cytotoxins isolated from cyanobacteria have potential as anticancer drugs (Gerwick et al., 1986 and 1989; Moore et al., 1988; Borowitzka, 1995; Van Wagoner et al ., 2007; Gademann & Portmann, 2008), some of them are discussed in 1.2.2. The cyanobacteria have also showed to be a rich source of highly effective inhibitors of proteases (Itou et al ., 1999; Borowitzka, 1999; Hee et al., 2008). Since proteases are involved in a variety of biological processes, and many proteases are validated drug targets (Turk, 2006), the discovery of new protease modulators is important to the development of pharmacological tools as well as potential therapeutics. For example, cancer cells are more sentitive to the proapoptotic effects 19
Chapter I Introduction of proteasome inhibition than normal cells. Thus, proteasome inhibitors can be potential anticancer agents. Protease inhibitors can be produced by both toxic cyanobacterial strains (e.g., those that produce hepatotoxins or neurotoxins) and non- toxic cyanobacterial strains of Microcystic, Anabaena, Planktothrix/Oscillatoria and Nostoc (Smith et al., 2008). Several of protease inhibitors isolated from cyanaobacteria have been published including aeruginopeptins, anabaenopeptilides, cyanopeptins, micropeptins, nostopeptins, oscillapeptins, miroviridins, aeruginosins, microcins, anabaenopeptins, oscillamides (Welker & van Döhren, 2006; Smith et al., 2008), banyasin A (Plouno et al., 2005), largamides A-H (Plaza & Bewley, 2006), lyngbyastatins 4-7 (Matthew et al ., 2007; Taori et al ., 2007), planktocyclin (Baumann et al ., 2007), kempopeptins A and B (Taori et al., 2008), nostodione A (Hee et al ., 2008) and others. In addition, some protease inhibitors may also find application in medicine for treatment of stroke, coronary artery occlusions and pulmonary emphysema. For example, inhibitors of the serine protease, thrombin, could be used to control blood clot formation in these diseases. Thrombin acts by cleaving a peptide fragment from fibrinogen which then leads to the formation of fibrin, a major component of blood clots. Inhibition of this protease would thus also inhibit clot formation. Similarly, angiotensin-converting enzyme inhibitors are being developed as anti-hypertensive agents. Protease inhibitors are also used in the treatment of HIV infections (Richman, 1996). These inhibitors include linear and cyclic peptides as well as depsipeptides and have been isolated mainly from Microcystis and Oscillatoria strains (Borowitzka, 1999). Some of cyanobacterial metabolites have promising therapeutic applications showing anti-inflammatory activities, as for example, malyngamide F acetate isolated from Lyngbya majuscula exhibited strong concentration-dependent anti-inflammatory activity in the nitric oxide (NO) assay with an IC 50 of 7.1 µM and with no cytotoxicity at the concentrations tested (Villa et al., 2010). Haploindolone A and B, isolated from the cyanobacterium Fischerella (ATCC 53558), have been patented as vassopressin antagonists with possible application in the treatment of congestive heart failure, hypertension, oedema and hyponatriaemia (Schwartz et al., 1989). There are also several reports of cyanobacterial extracts acting on the immune system. For example, extracts of Spirulina platensis have been shown to
20
Chapter I Introduction enhance chicken macrophage function in vitro as well as cell-mediated immune function in chickens and cats (Qureshi et al ., 1995 and 1996; Qureshi & Ali, 1996). Other interesting activities such as antimalarial (McPhail et al ., 2007; Gademann & Kobylinska, 2009), immunosuppressant (Koehn et al. , 1992; Zang et al ., 1997), insecticidal activity (Beche et al., 2007), and antiplasmodial (Papendorf et al., 1998; Barbaras et al., 2008) isolated from cyanobacteria have been also reported. Recently, it has also been found that the carbolinium alkaloid, nostocarboline (see fig.1-6) isolated from Nostoc 78-12A (Becher et al., 2005) acts as cholinesterase inhibitor, an enzyme targeted in the treatment of Alzheimer'disease (Blom et al ., 2006). The effects of this compound were comparable to galanthamine, an approved drug for Alzheimer' disease. This discovery could lead to development of drugs for neurological disorders and shows, that cyanobacteria are possible sources of pharmaceuticals also for these diseases.
Nostocarboline from Nostoc 78-12A
During the last few years several novel and diverse metabolites combined with relevant pharmaceutical activities (e.g. antibiotic, enzymes, antiviral, anticancer, antifungal, and antiinflammatory agents as well as protease inhibitors) have been discovered from cyanobacteria which clearly indicates that cyanobacteria have a valuable potential for providing novel and diverse bioactive substances for drug discovery and can be considered a prime source for leads for drugs; this has stimulated researcher’s efforts to find novel and pharmacologically active cyanobacterial metabolites (Jaspars & Lawton, 1998; Burja et al., 2001; Singh et al., 2005; Sielff et al., 2006; Sivonen & Börner, 2008; Gademann & Portmann, 2008).
21
Chapter I Introduction
Isolation and screening of cyanobacteria for antibiotics and other pharmacologically active compounds have recently received considerable attention. During the last decade a large number of novel bioactive molecules have been isolated from cyanobacteria, but cyanobacteria are still viewed as unexplored source of potential drugs (Sielaff et al., 2006). Especially the collections of cyanobacterial strains from South East Asia where biodiversity is high (Tan, 2007) are still largely unexplored. In our going efforts toward finding novel and pharmacologically active marine and terrestrial cyanobacterial metabolites, we have investigated 12 strains of terrestrial cyanobacteria collected in Daklak province and one marine cyanobacterium Lyngbya majuscula from Khanh Hoa province of Vietnam were studied with following goals 1. Screening for antibacterial activity of the extracts prepared with organic solvents of different polarity and water from 12 terrestrial cyanobacterial strains; 2. Isolation, identification and structure elucidation of the antimicrobial compounds from extracts with prominent activity; 3. Isolation, identification and structure elucidation of the cytotoxic compounds from Lyngbya majuscula; 4. Culture optimization of selected strains showing strong antibacterial activity to enhance biomass production and synthesis of active compounds.
22
Chapter II Materials and methods
a a a a a a a a a
The cyanobacterial strains were isolated from acidic soil samples collected from rice, cotton and coffee fields in the Dak Lak province, Vietnam during April 2002-2003 and September 2002- 2003 (see fig. 2-1).
Map of Vietnam with the locality of 12 cyanobacterial strains in Dak Lak province
Dak Lak is a Central Highland province of Vietnam located at 11 °44’- 13 °25’N, 107 °23’- 109 °06’E (Ho, 2007). The isolates were established as laboratory cultures at the Department for Algal Biotechnology, Institute of Biotechnology (IBT), Hanoi, Vietnam. These strains are maintained in the culture collection of the Institute of Pharmacy, Ernst-Moritz- Arndt- University Greifswald, Germany as stock cultures. Firstly, according to morphology and classification of Komárek & Anagnostidis, 1989; 1999; 2005 and Anagnostidis & Komárek, 1990; Desikachary,
23
Chapter II Materials and methods
1959, and http://www.algaebase.org most of these strains were classified belonging to the genera Anabaena, Nostoc, Calothrix, Oscillatoria, Scytonema and Westiellopsis . Addtionally, genus Westiellopsis has been identified by molecular characterization of 16S rDNA sequence (Ho et al . 2005a) and four cyanobacterial strains which belong to genus Calothrix were explored for the genetic relationships by using Randomly Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) technique (Ho et al., 2006).
a Cyanobacterial strains
a a a a a a Oscillatoria sp. Trichomes long small without any Oscillatoriaceae branching, not constricted at the cross- TVN16 walls, sheath absent; cells discoid, no heterocysts in filament. Oscillatoriales Anabaena sp. Trichomes constricted at the cross- Nostocaceae walls, look like string of beads; cells Nostocales barrel-shaped, mostly longer than TVN40 broad; no heterocysts in filament; no branches. Nostoc Filaments flexuous, loosely entangled; spongiaeforme sheath thin; cells cylindrical or oblong; Agardh ex TVN7 heterocysts subspherical; spores in long Born. et Flah. chains.
Nostoc Filaments densely entangled, flexuous,
coeruleum sheath mostly indistinct; trichomes 5.1- Nostocaceae Lyngbye ex 6.8 µm broad; cells short, barrel-shaped; Nostocales Born. et Flah. TVN14 heterocysts subspherical; spores not known. Nostoc sp. Filaments single, flexuous, entangled; cells nearly spherical, no heterocysts in TVN9 filament, no branches. Calothrix Filaments single, pale blue-green to javanica de olive green, lamellated sheath,
24
Chapter II Materials and methods
Wilde gradually attenuated to a pointed apex; trichomes 5.1-6 µm broad; heterocysts basal, 4-5.1 µm broad, 4.8-6 µm in TVN1 length; spores single or two together, about 8.5-9 µm broad, 6-10 µm long Calothrix Filaments 80-250 µm, united in tuff, elenkinii swollen at the base, 6-9 µm broad; Kossinsk. sheath close to the trichome, thin, not TVN202 lamellated, open at the ends; trichomes at the base 5.1-6.8 µm broad, apical hair not formed; cells quadratic or somewhat Rivulariaceae shorter; heterocysts single, basal, 4.1- 6.8 µm broad Nostocales Calothrix sp. TVN20 Filaments single, unbranched; heterocysts basal. Calothrix TVN201 Filaments in groups, irregularly bent marchica var. and closely entangled, 8.5-13.6 µm crassa Rao, broad; sheath thin, firm; trichomes 8.5- C.B. 11.9 µm broad, constricted at the septa, ends tapering but without a hair, end cell with a rounded apex, sometimes pointed cells quadratic, as well as shorter or longer than broader; heterocysts single, basal, spherical or subspherical, 8.5 µm broad, 5.1 µm long. Scytonema TVN12 Filaments interwoven, false branches millei Bornet erect; sheath firm brownish; heterocysts ex Born. et discoid Flah. Scytonemataceae Scytonema TVN10 Filaments up to 3 mm long, 10.2-16.3 Nostocales ocellatum µm; false branched; sheath firm, often Lyngbye ex. lamellated; trichomes 6.8-8.5 µm broad; Born. et Flah. cells shorter than broad or quadratic; heterocysts subquadratic, size 6.8-6.8 µm
25
Chapter II Materials and methods
Westiellopsis TVN22 Filaments with true branching Stigonemataceae sp.VN filaments of two kinds, primary filaments and secondary filaments. Stigonematales Main filaments torulose, constricted at the cross-walls, with short barrel-shaped cells or longer than broad, 8.5 µm broad, 10.2 µm long. Branch filaments growing erect, generally thinner, not constricted at the cross-walls, with cylindrical cells, 5.1 µm broad, 10.2-12 µm long. Heterocysts rounded- cylindrical in main and branch filaments, 5.1-8.5 µm or 10.2 µm broad, 1.9-13.6 µm long. Pseudohormocysts formed on terminal portions of secondary filaments. Endospores single in each cell of the pseudohormocysts, 8.5- 11.9 µm diameter
Oscillatoria sp. (x 100) Anabaena sp. (x 100)
26
Chapter II Materials and methods
Nostoc spongiaeforme Nostoc coeruleum Agardh ex Born. et Flah. (x 600) Lyngbye ex Born. et Flah.(x 600)
Nostoc sp. (x 100) Calothrix javanica de Wilde (x 600)
Calothrix elenkinii Kossinsk (x 100) Calothrix sp. (x 600)
27
Chapter II Materials and methods
Calothrix marchica var. crassa Rao,C.B. a. Root part (x 600); b. Trichom(x 120)
Scytomema millei Scytonema ocellatum Bornet ex Born. et Flah. (x 600) Lyngbye ex. Born. et Flah. (x 600)
Westiellopsis sp .VN (x 600), (x 100), respectively
Morphology of 12 cyanobacterial strains
28
Chapter II Materials and methods
a a a a Living specimens of the marine cyanobacterium Lyngbya majuscula were collected at Hon Khoi locality in Khanh Hoa province, Vietnam on August 20, 2007 (see fig. 2-3). Khanh Hoa province is located at the South Central Coast of Vietnam. Its geographical coordinates are 108°40’33" to 109°27’55" E and 11°42’50" to 12°52’15" N, the length of the coast lines about 300 km. Hon Khoi is an area of Ninh Hoa district of Khanh Hoa, this area is situated at 12° 34′ 44″ N, 109° 13′ 49″ E
1 B
1 A
1A. Map of Vietnam with the collection area (the arrow indicates the collection site) 1B. The collection place of Lyngbya majuscula in Khanh Hoa province indicated (Geological Map of the sea waters of the Institute of Oceanography, Nha Trang, Vietnam)
29
Chapter II Materials and methods
Samples of the marine cyanobacterium Lyngbya majuscula Harvey ex Gomont (Oscillatoriaceae), growing on rocks, dead corals, and gravel in the lower intertidal to subtidal zone of shores and exposed to calm to moderate wave action were collected by hand from a water depth of 0.1-1m, placed into sample containers and shipped in the laboratory within the day. In the laboratory, the samples were air-dried in low natural light, lyophilized later, and stored at -200C until use. A voucher specimen is available in the Department for Algal Biotechnology, Institute of Biotechnology (IBT) of the Vietnam Academy of Science and Technology (VAST), Hanoi and the Ernst-Moritz-Arndt- University Greifswald, Germany under the strain number LMVN. The sample (see fig. 2-4) was identified morphologically as Lyngbya majuscula Harvey ex Gomont (Oscillatoriaceae) by algologist Pham Huu Tri, the Oceanography Institute, Nha Trang and Dr. Dang Diem Hong, the Department for Algal Biotechnology, Institute of Biotechnology (IBT) of the Vietnam Academy of Science and Technology (VAST), Hanoi. The thallus of marine cyanobacterium Lyngbya majuscula Harvey ex Gomont (Oscillatoriaceae) from Hon Khoi locality in Khanh Hoa province expanded, up to 3 cm long, dull blue-green to brown or yellowish-brown in color, with very long and curved filaments, seldom only slightly coiled, the sheath colorless, lamellated, the trichomes blue-green, not constricted at the cross-wall, not attenuated at both ends, the calyptra absent.
a
30
Chapter II Materials and methods
b c a and b Natural habit (took photograph during low tide) of Lyngbya majuscula c Microscopic view of filament (x 40) of Lyngbya majuscula
a a a a a a a • Gram positive bacteria Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6051 • Gram negative bacteria Escherichia coli ATCC 11229 , Pseudomonas aeruginosa ATCC 27853 • Yeast Candida maltosa SBUG 700 • Cancer cell lines: 5637 cell line: bladder cancer cell line (German Collection of Microorganisms and Cell Cultures (DSMZ Braunschweig, Germany) a a a a a All media were prepared and autoclaved at 121 0C for 20 min before use, but preparation of BG 11 medium citric acid combined with ferric ammonium citrate had to be autoclaved separately and after that added to the autoclaved medium. (Rippka et al., 1979)
Ingredient Stock solution Nutrient solution (g/100 mL) (mL/L) NaNO 3 15.0 10 K2HPO 4. x 3 H 2O 0.4 10 MgSO 4. x 7 H 2O 0.75 10 CaCl 2 x 2 H 2O 0.36 10 Citric acid 0.06 10 Ferric ammonium citrate 0.06 10 EDTA (disodium magnesium salt) 0.01 10 Na 2CO 3 0.2 10 *[Trace metal mix A5+Co] 1 Distilled water 919 31
Chapter II Materials and methods
*[Trace metal mix A5+Co]
Ingredient Stock solution Nutrient solution (g/L) (mL/L) H3BO 3 2.86 MnCl 2 x 4H 2O 1.81 ZnSO 4. x 7H 2O 0.222 CuSO 4. x 5H 2O 0.079 1 Na 2MoO 4. x 2H 2O 0.390 Co (NO 3)2. x 6H 2O 0.0494 Distilled water 1.0 L
(BG-11 without nitrate)
a a a a a a a Standard II nutrient agar for microbiology (Merck, Darmstadt, Germany) Composition: -Peptone from meat 3.45 g -Peptone from casein 3.45 g -Sodium chloride 5.1 g -Agar- agar 13.0 g Suspend 25 g standard II nutrient agar in 1 liter of demineralized water by heating in a boiling water bath or in a current of steam; autoclave (15 min at 121 0C), pH: 7.5 ±0.2 at 25 0C
a a a a Ampicillin Merck, Darmstadt, Germany Gentamycinsulfat Biochrom AG, Berlin, Germany Nystatin- Dihydrat Carl Roth GmbH & Co.KG, Karlsruhe,Germany NaCl Carl Roth GmbH & Co.KG, Karlsruhe,Germany Trifluroacetic acid (TFA) for spectroscopy Merck, Darmstadt, Germany
a a • Anisaldehyd- sulphuric acid (AS) reagent (Merck, Darmstadt, Germany):
32
Chapter II Materials and methods
Solution of 10 mL acetic acid containing 0.5 mL anisaldehyde was added to a mixture of 5 mL sulphuric acid and 85 mL methanol (Houghton and Raman, 1998).
• a (Sigma, Steinheim, Germany): 5 mg of the INT is dissolved in 1 mL of ethanol 50%.
a a a a Ethanol Merck, Darmstadt, Germany KOH VWR, Darmstadt, Germany HCl Bayer, Leverkusen, Germany
Na 2SO 4 VWR, Darmstadt, Germany Methoxyamine (MeOX) Sigma- Aldrich, Munich, Germany N-methyl-N- CS Chromatography Service, trimethylsilyltrifluoroacetamide (MSTFA) Langerwehe, Germany) Acetone Carl Roth GmbH & Co.KG, Karlsruhe, Germany Acetic acid Carl Roth GmbH & Co.KG, Karlsruhe, Germany
Acetonitrile (HPLC gradient Prolabo VWR International company, made in EC grade) or Carl Roth GmbH & Co.KG, Karlsruhe, Germany or Fisher Scientific, Loughborough, UK Dichloromethane Carl Roth GmbH & Co.KG, Karlsruhe, Germany Deionized water Ethanol Carl Roth GmbH & Co.KG, Karlsruhe, Germany Ethyl acetate Carl Roth GmbH & Co.KG, Karlsruhe, Germany Methanol Carl Roth GmbH & Co.KG, Karlsruhe, Germany
Methanol (HPLC gradient Prolabo VWR International company, made in EC grade) or AppliChem, AppliChem GmbH, Damstadt, Germany n-Hexane Carl Roth GmbH & Co.KG, Karlsruhe, Germany
All solvents were distilled prior to use except for HPLC and spectral grade solvents were used for spectroscopic measurements.
33
Chapter II Materials and methods
a a a a a a - For stock culture: 100-150 mL Erlenmeyer flasks (VWR GmbH, Darmstadt, Germany) - For batch culture: 1.5 L Fehrnbach flasks (Merck, Darmstadt, Germany) - Large scale cultivation: 45 liter-glass fermentor Self-construction,Ernst-Moritz-Arndt-university, Greifwald, Germany pH Redox- transducer (Ecoline Wissenschaftlich – Technische Werkstätten GmbH, pH 170) Weilheim, Germany Heater Tetra GmbH, Melle, Germany Centrifuge (Rotanta 460R) Hettich Zentrifugen, Tuttlingen; Germany Centrifuge Stratos D37520 Heraeus Instruments , Osterode, Germany Lyophylizer Alpha 1-4 Christ GmbH, Berlin, Germany Filter paper Ø 185 mm with 12- Schleicher & Schüll MicroscienceGmbH, Dassel, 25 µm pore size Germany Deep Freezer GTL 2811WS Bauknecht, Berlin Germany Autoclave Varioklav, H+P Labortechnik, Germany Sterilizer Memmert, Schwabach, Germany a Laboratory sea sand Merck, Darmstadt, Germany Porcelain mortar and pestle 250 mL and 500 mL Merck, Darmstadt, Germany Erlenmeyer flasks System of rotatory vacuum Büchi Labortechnik AG, Flawil, CH, Büchi & Co, evaporator: pump B-178, Berlin, Germany vacuum controller B-721, Rota vapor R-114, water bath B-480 Stirrer, Heidolph MR 3000 Schwabach, Germany Shaker Kika Labortecknik Staufen, Janke & Kunkel GmbH & Co.KG, Germany Banderlin Sonorex RK103H Bandelin electronic, Berlin, Germany
34
Chapter II Materials and methods
Ultrasonic cleaner Centrifuge (Rotanta 460R) Hettich Zentrifugen, Tuttlingen; Germany 100 mL centrifuge tubes, round Carl Roth GmbH & Co.KG, Karlsruhe, Germany bottom Lyophylizer Alpha 1-4 Christ Gefriertrocknungsanlagen, Osterode, Germany Filter paper Ø 185 mm with 12- Schleicher & Schüll MicroscienceGmbH, Dassel, 25 µm pore size Germany Banderlin Sonorex RK103H Banderlin electronic, Berlin, Germany Ultrasonic cleaner a a a a a a
TLC sheets: Silica gel 60, F 254, Merck, Darmstadt, Germany 20x20 cm
TLC tank Desaga GmbH, Heidelberg, Germany
Capillaries Hirschmann Laborgeräte GmbH & Co. KG, Eberstadt, Germany
UV lamp Desaga SARSTEDT- Desaga GmbH, Heidelberg, Germany
GRUPPE HP-UVIS UVPMultiDoc-It, Digital Cambridge, UK Imaging System
Thermoplate Desaga Desaga GmbH, Heidelberg, Germany SARSTEDT-GRUPPE
a a
Preparative TLC plate: PLC Silica gel F254 , 2mm, 20 x 20 cm (Merck, Darmstadt, Germany)
a a Glass column: 60 x 1.2 cm, Schott Duran, Mainz, Germany 50 x 4.0 cm; 50 x 2.0 cm;
35
Chapter II Materials and methods
35 x 2.5 cm; 35 x 1.2 cm (h x i.d.) 50 mL, 100 mL, 250 mL, 500 Merck, Darmstadt, Germany mL, and 1000 mL Erlenmeyer flasks Test tubes (10 mL) Schott Duran, Mainz, Germany Fra ction Collector Model 2110 Bio-Rad Laboratories, Richmond, USA Silica gel 60 ( 0.015-0.040 mm; Merck, Darmstadt,Germany 0.040-0.063 mm) Sephadex LH-20 Amersham Biosciences AB, Uppsala, Sweden Silica gel 60RP-18 ( 0.040-0.063 Merck, Darmstadt,Germany mm) Pasteur pipettes, 150 mm, 230 mm VWR GmbH, Darmstadt, Germany
Synergi Polar RP 4 µm (80 Å) column (Phenomenex Ltd, Aschaffenburg,
Germany)
+ Analytical column (250 x 4.6 mm) + Semi-preparative column (250x10 mm) HPLC system (Kontron Instruments, Italy) + Diode array detector (DAD 440) + Autosampler 360 (SA360) + Pump 422 & 422 S Water-system Clear UV plus SG Water Preparation and Recycling GmbH, Germany HPLC vials (1.5 mL) (VWR GmbH, Darmstadt,Germany)
a a Petri dishes plastic or glass (Ø 90 mm) Merck, Darmstadt, Germany Burner, beaker, variable Eppendorf pipettes with sterile tips, tweezers Sharp nails, polystyrene board, inoculating loops, lineal Steril antibiotic test paper discs Ø6mm Schleicher & Schuell Microscience
36
Chapter II Materials and methods
GmbH, Dassel, Germany 20-50 mL standard glasses Schott Duran, Mainz, Germany Incubator Mytron BS 120 Memmert, Schwabach, Germany Laminar flow box Heraeus Instruments, Hanau, Germany a a a a Thin layer chromatography sheets Merck KgaA, Darmstadt, Germany silica gel 60 F 254, 20x20 cm
Petri dishes 20- 50 mL (Ø 90 mm) Merck, Darmstadt. Germany
Incubator Mytron BS 120 Memmert, Schwabach, Germany
Sterile standard glass Merck, Darmstadt, Germany
Laminar flow box Heraeus Instruments, Hanau, Germany
a a a a
GC/MS Firma Agilent (USA) Gaschromatograph G1530N MSD G2588A Software G1701 CA Syringe G2613 A
syringe sample rack
column
Figure 2-5: Agilent 6890N gas chromatograph and mass selective detector (Agilent®5973 Network MSD)
37
Chapter II Materials and methods
a a a a For maintenance of laboratory culture, 2- 3 mL of a 3 weeks old cyanobacterial stock culture was used as inoculum in 50 mL of autoclaved BG 11 medium in 150 mL Erlenmeyer flasks. The cultivation was carried out at 20 ±2 0C, under continuous illumination of 8µmol/m2 by cool fluorescence lamps. The stock cultures were maintained for 20-30 days.
a During this work 12 cyanobacterial strains (see 2.1.1) were cultured in batch cultures. Aliquots of 50 mL from the stationary phase stock cultures were used to inoculate 500 mL of autoclaved BG 11 medium in 1.5 liter Fehrnbach flasks. These samples were cultivated at 20 ±2 0C, under continuous illumination of 8µmol/m2 by cool fluorescence lamps. The cyanobacterial cultures were harvested after 4-6 weeks. The cells were separated from the medium by centrifugation (4000 rpm/ 10 min/ 10 0C) followed by filtration with filter paper. The biomasses were lyophilized and stored at -20°C until use while cultivation media were concentrated to 1/10 (v/v) by rotary evaporation in vacuum at 40 0C and extracted immediately with EtOAc solvent.
a a During this work the large scale cultivation was applied for 5 cyanobacterial strains, Westiellopsis sp. VN, Anabaena sp., Nostoc sp., Scytonema millei , and Calothrix elenkinii . The large scale cultivation was carried out in a 45 liter-glass fermentor (Mundt et al., 2001). The fermentor was cleaned by distilled water and 70% isopropanol before use. At the beginning, the fermentor was filled with 15 L of medium and after 1- 2 hours 1.5 L of growing culture (after 20 days of cultivation in three Fehrnbach flasks) was added. Afterwards, every day 5 L of medium were added into the fermentor until 35 L of medium were reached. The cultures were illuminated continuously with banks of cool white fluorescent tubes of 8µmol/m2 and incubated at temperature of 26°C to 28°C adjusted using a heater. The pH-value of the large- scale culture was adjusted to 7.4-8.5 using CO 2 supplementation. Strain Westiellopsis sp. VN was grown for 8 weeks at 28°C and pH of 7.4 Strain Anabaena sp. was grown for 6 weeks at 26°C and pH of 8.5
38
Chapter II Materials and methods
Strain Nostoc sp. was grown for 4 weeks at 26°C and pH of 8.5 Strain Scytonema millei was grown for 4 weeks at 26°C and pH of 8.5 Strain Calothrix elenkinii was grown for 7 weeks at 28°C and pH of 7.4 The biomass was collected by centrifugation at 6500 rpm in a refrigerated 0 continuous-flow centrifuge and lyophilized, then stored at -20 C.
a
b c
Cultivation of cyanobactria a. Stock culture;
b. Batch culture; c. Large scale culture a a a a The harvested cells were freeze-dried and then successively extracted with three different organic solvents which increasing polarity starting with n-hexane, methanol, water in three steps (Mundt et al., 2001). Firstly, 5 g freeze-dried biomass was crushed with 1g sea sand and 5 mL of the first organic solvent (n-hexane) using porcelain mortar and pestle to get homogenous suspension. The remaining 245 mL n-hexane was added to the cell suspension followed by homogenization for 7 minutes in ultrasonic bath. After this, the biomass was extracted for 1 hour under stirring at room temperature. The supernatant was separated from the residue by centrifugation (Centrifuge 96R, 4500 39
Chapter II Materials and methods rpm, 10 minutes, and 4°C). The residue was extracted with n-hexane three times in all. After filtration the supernatants were pooled and evaporated to dryness with a rotary evaporator. The residue of the biomass was dried at room temperature over night. Subsequently, the dried residue was further extracted 3 times with the next solvents (methanol followed by water). The organic solvents were removed by rotary evaporation in vacuo at 40 0C to get dry extracts. The water was reduced by rotary evaporation at 40 0C and removed completely by lyophilization (lyophylizer Alpha 1-4). The dried extracts were weighed and stored at -20 0C until use.
a a a For media extraction, 3L of cyanobacterial culture medium were concentrated to 250-300 mL by rotary evaporation in vacuum at 40 0C. The media were then shaken for 24 h with equal volume of ethyl acetate then separated by separating funnel. Extraction of the lower phase (media) was repeated three times. The upper phase was collected to get ethyl acetate extract. All ethyl acetate extracts obtained from the three extractions were combined, dried with sodium sulfate and reduced to dryness in vacuum. The dried extract was weighed and stored at -20 0C until use.
Cyanobacteria
5g freeze-dried biomass Media - Crushed with n-hexane and see sand in mortar -Ultrasonicated for 7min 250ml ethyl acetate/ 3times/ shaking Suspension
250ml n-hexane/ 3times/ stirring Ethyl acetate extract
n-hexane extract Residue
250ml methanol/ 3times/ stirring
Methanol extract Residue
250ml water/ 3times/ stirring
Water extract Residue
Bioassay
Scheme of extraction
40
Chapter II Materials and methods
a a a a a a a a a An agar diffusion assay according to the Pharmacopoea Europaea was used to determine antibacterial and antifungal activity in screening the extracts (see 2.6) and for evaluation the activity of fractions during separation process and isolated compounds. As test organisms were used the gram positive bacteria Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6051, the gram negative bacteria Escherichia coli ATCC 11229 and Pseudomonas aeruginosa ATCC 27853 and yeast Candida maltosa SBUG 700. All experiments were repeated 3 times. Inhibition zone was measured including 6 mm paper disc. Ampicillin (10 µg for Staphylococcus aureus and Bacillus subtilis ; 50 µg for Escherichia coli), Gentamycin (25 µg for Pseudomonas aeruginosa ) or Nystatin (5 µg for Candida maltosa ) were used as positive control. The solvent was used as negative control. A sterile paper disc with a diameter of 6 mm was loaded with 50 µL test solution. For screening 2 mg of cyanobacterial extracts dissolved in the extraction solvent were applied on the paper disc. During bioassay-guided fractionation of cyanobacterial extracts, the extract was tested in a concentration of 2.0 mg/disc, fractions after first separation were tested in a concentration of 500 µg/disc and fractions of second separation and pure compounds were tested in a concentration between 100 and 200 µg/disc. One paper disc was loaded with only solvent as solvent control (50 µL) and another was loaded with ampicillin, gentamycin or nystatin as positive control. The paper discs were fixed by pins on a polystyrene plate and dried for 2 hours under sterile conditions to eliminate solvents completely. The bacteria were cultured on nutrient agar plate at 37 0C for 24 h (bacteria) and at 28°C for 48 h ( Candida maltosa ) then maintained at 40C. For antibiotic test, from the bacterial stock culture an inoculum was spread on a nutrient agar plate and incubated at 37 0C for 24 h (bacteria) and at 28°C for 48 h ( Candida maltosa ) before use. From this culture a pin-head size inoculum was suspended in 2 mL of sterile 0.9% NaCl and mixed thoroughly. 200 µL of this suspension was diluted with 20 mL sterile warm agar medium and poured immediately into the Petri dish. Temperature of
41
Chapter II Materials and methods nutrient agar should be below 40 0C. After solidification of the agar (about 15 minutes under sterile conditions) the paper discs containing test samples were placed on the surface of the agar and the Petri dishes were kept at 4°C over 4 h for prediffusion. After this, the plates were incubated for 24 h at 37°C for bacteria and for 48 h at 28°C for Candida maltosa in an inverseposition. At the end of the incubation period, the inhibition zones were measured and expressed as the diameter of the clear zone including the diameter of the paper disc ( ∅ 6 mm). For better detection of inhibition zones the agar plates were sprayed with INT solution. Inhibition zones were visible as clear zones around the paper discs against a dark red background.
a a a a For bioautographic TLC, an amount of an extract or a fraction is put on a TLC 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 using spray reagents (Hamburger and Cordell, 1987). 500 µg of extract or fraction were applied on analytical TLC sheet (6,5x3 cm) in duplicate and developed in the same solvent system. The control chromatogram was detected at 254 nm and 356 nm UV light and by spraying with anisaldehyde/sulfuric acid reagent, R f values were calculated. The test chromatogram was dried under sterile conditions for 3 hours. Approximately 15 mL of nutrient medium was poured into a Petri disc (Ø 90 mm) as basic layer. After solidification of the agar, the test chromatogram was applied free of air bubbles on the surface of nutrient agar. 20 mL of nutrient medium inoculated with 200 µL of bacterial suspension containing S. aureus as described in 2.7.1.1 was poured over the test chromatogram as top layer. The Petri dish was incubated at 4°C for 3 h and afterwards incubated at 37°C as described for agar diffusion assay. The inhibition zones were detected with INT reagent. After spraying with INT, the inhibition zones appear as clear sports against the red background. The inhibition zones were compared with the R f values of the control chromatogram so that the active compounds were located on TLC.
42
Chapter II Materials and methods
a a Cytotoxicity data of extracts, fractions, and pure compounds of Lyngbya majuscula towards 5637 cell line [bladder cancer cell line {(German Collection of Micro organisms and Cell Cultures (DSMZ Braunschweig, Germany)} were provided by Dr. Wende, Department of Phamaceutical Biology, Institute of Pharmacy, Ernst- Moritz-Arndt-University of Greifswald, Germany. Method was modified according to Bracht and Bednarski (Bracht et al ., 2006). Test was performed as previously published (Bui et al., 2007).
a a a a a a a a a a a a a a a a Westiellopsis The active methanol extract obtained from extraction of lyophilized biomass from large scale culture of this strain was separated first by silica gel chromatography, followed by gel filtration, and finally reversed-phase HPLC (see scheme 2-2). The methanol extract (260 mg) obtained from 2 g of the lyophilized biomass was fractionated using silica gel column chromatography. For preparation of the column 30 g of silica gel 60 (0.015-0.040 mm) was mixed and saturated with the first solvent system for 1 hour at room temperature. This silica gel solution was then poured into the column (35 x 2.5 cm, h x i.d.) The column was rinsed with the first solvent system for 30 minutes and a thin layer (1cm) of sea sand was applied on the top of column. The methanol extract was dissolved in a small amount of the first solvent system and applied on the surface of silica gel bed. The column was then eluted with 300 mL of each mobile phase, first DCM, followed by DCM/EtOAc (95:5), DCM/EtOAc (90:10), DCM/EtOAc (50:50), EtOAc, EtOAc/MeOH (75:25), EtOAc/MeOH (50:50), and finally MeOH. Each eluting solvent was collected in a different flask at flow rate 0.4 mL/min. After that, eight major fractions (F I to F VIII ) were obtained, and solvents were removed in vacuo using a rotary evaporator. All fractions were tested for their antibacterial activity . The fractions F I, FII, F III eluting with 100% DCM, 95% DCM/EtOAc, and 90% DCM/EtOAc were pooled since they all exhibited approximately the same antibacterial activity.
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Chapter II Materials and methods
The pooled fractions (68.5 mg) were further seperated using sephadex LH- 20 column chromatography. An amount of 18 g LH-20 gel was swollen in 100 mL
H2O/MeOH (10:90) for 1 hour at room temperature. The suspension was then poured slowly into the column (60 x 1.2 cm, h x i.d.) and rinsed with H 2O/MeOH (10:90) until the stationary was stable and reached a height of 50 cm. Then, sample dissolved in a small amount of the first solvent system was softly applied onto the column eluting first with 200 mL H2O/MeOH (10:90) and followed by 150mL MeOH, and finally washing with 100mL H 2O/Aceton (50:50). The outflow of the column was collected in sub-fractions of 5mL automatically by fraction collector with a flow rate 0.3 mL/min. The sub-fractions were analyzed by TLC using n-hexane/EtOAc/MeOH (75:25:5). TLC plate was visualized under UV light at 254 and 366 nm and by use of anisaldehyde/sulfuric acid reagent. Sub-fractions with the same spots on TLC were combined to main fractions. After TLC analyzing, four major fractions (WF 1 to WF 4) were collected and tested for antibacterial activity . Because fraction WF 1 which eluted with 10% H 2O/MeOH exhibited the highest antibacterial activity, fraction WF 1 was further purified.
The fraction WF 1 was further purified by using a semi-preparative HPLC column Synergi POLAR-RP 80A (250×10mm, 4 micron ) with a flow rate of 3.0 mL/min and detection at 210, 220, 238, 254 and 366 nm. A concentration of 500 µg/50 µL was injected per run. Altogether, 50.0 mg were purified using the step gradient described in table 2-2. Nine fractions (WF 1-1-WF 1-9) were collected under
UV of 238nm and then tested for the activity against S .aureus. Fractions WF 1-3, WF 1-
5, WF 1-6, and WF 1-8 exhibited significant antibacterial activity were therefore used for structural elucidation.
a Step gradient used in purification of fraction WF 1 by semi-preparative HPLC 0.50 3.50 40.50 42.50 50.50 52.50 80 50 15 0.0 0.0 80.0 20 50 85 100 100 20
Solvent A: H 2O and solvent B: CH 3CN; Flow rate: 3.0 mL/min; HPLC column: Synergi polar- RP80A/250×10mm, 4 micron
44
Chapter II Materials and methods
Lyophilized biomass
3 x n-Hexane Residue n-Hexane ext. 3 x EtOAc
EtOAc ext. Residue 3 x MeOH
MeOH ext. Silica gel column DCM/ EtOAc/ MeOH gradient
FI FII FIII FIV FV FVI FVII FVIII
LH-20 column
H2O/MeOH (1:9), MeOH, H 2O/Aceton (1:1)
WF 1 WF 2 WF 3 WF 4 -Semi-preparative HPLC Synergi-Polar RP -CH 3CH/H 2O gradient
WF 1-1 WF 1-2 WF 1-3 WF 1-4 WF 1-5 WF 1-6 WF 1-7 WF 1-8 WF 1-9
Structure elucidation
Extraction, fractionation, and isolation of the secondary metabolites of Westiellopsis sp.VN
a a a a a a Calothrix javanica The active methanol extract obtained from extraction of lyophilized biomass from batch culture of this strain was subjected to RP C 18 column chromatography, followed by reversed-phase HPLC (see scheme 2-3). 210 mg of MeOH extract obtained from 1.25 g lyophilized biomass was separated by reverse-phase C 18 column chromatography. For preparation of the column 15 g reverse-phase C 18 silica gel was swollen in 50mL MeOH/EtOH/H 2O (45:45:10) for 3 hours at room temperature. The slurry was poured into the column (35 x 1.2 cm, h x i.d.) and column was then rinsed with initial mobile phase for 30 minutes. After that, the sample was dissolved in a 1mL of initial mobile phase and applied onto the surface of the gel bed. The column was initially eluted with 200 mL
MeOH/EtOH/H 2O (45: 45:10), followed by 200 mL MeOH/H 2O (90:10), 200 mL MeOH, 200 mL MeOH/Acetone (50:50), and finally 200 mL DCM. The outflow of the column was collected in sub-fractions of 4 mL automatically by fraction collector at flow rate 0.3 mL/min. The received sub-fractions were analyzed by TLC in the first solvent system, and then detected under UV light at 254 nm and 366 nm and by spraying with AS reagent and heating. Sub-fractions with the same spots on TLC were combined to main fractions. After TLC analyzing ten major fractions (CJF I to 45
Chapter II Materials and methods
CJF X) were received and tested for the antibacterial activity . The fraction CJF II eluted with MeOH/EtOH/H 2O (45:45:10) was further purified because this fraction showed antibacterial activity.
The purification of fraction CJF II was carried out using a semi-preparative HPLC column Synergi POLAR-RP 80A (250×10mm, 4 micron ) with a flow rate of 3.0 mL/min and detection at 210, 220, 238, 246, 254 and 366 nm. A concentration of
2 mg/50 µL was injected per run. Altogether, 86.0 mg of CJF II were purified using the step gradient described in table 2-3. Seven peaks were collected separately at 220 nm to yield 7 fractions. Among of them, the fraction CJF II-4 was the most pure this fraction was therefore used for structural elucidation.
a Step gradient used in purification of fraction CJF II by semi-preparative HPLC 0.50 16.50 24.50 30.50 31.50 30 15 0 0 30 70 85 100 100 70
Solvent A: H 2O+0.05%TFA and solvent B: MeOH; Flow rate: 3.0 mL/min; HPLC column: Synergi polar-RP80A/250×10 mm, 4 micron
Lyophilized biomass
3 x n-Hexane Residue n-Hexane ext . 3 x MeOH
MeOH ext. Residue 3 x H 2 O
H2O ext.
Silica gel 60 RP 18 column - MeOH/ EtOH/H 2O= 45:45:10 - MeOH/ H 2O= 90:10 - MeOH - MeOH/ Acetone= 1:1 - DCM
CJF I CJF II CJF III CJF IV CJF V CJF VI CJF VII CJF VIII CJF IX CJF X
- Semi-preparative HPLC Synergi Polar RP - MeOH/(H 2O+0.05%TFA) gradient
CJF II-1 CJF II-2 CJF II-3 CJF II-4 CJF II-5 CJF II-6 CJF II-7
Structure elucidation
Scheme 2-3: Extraction, fractionation, and isolation of the secondary metabolites of Calothrix javanica
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Chapter II Materials and methods
a a a a a a Scytonema ocellatum The active methanol extract obtained from extraction of lyophilized biomass from batch culture of this strain was first fractionated using RP C 18 column chromatography, followed by silica gel column chromatography, and finally reversed- phase HPLC (see scheme 2-4). 230 mg of MeOH extract obtained from 1.26 g lyophilized biomass were fractionated by RP C 18 column chromatography. An amount of 15 g reverse-phase C 18 silica gel was swollen in 50mL MeOH/H 2O (1:1) for 3 hours at room temperature. The slurry was poured into the column (35 x 1.2 cm, h x i.d.). Column was then rinsed with initial mobile phase for 30 minutes. After that, the sample was dissolved in 1 mL of initial mobile phase and applied onto the surface of the gel bed. The elution was carried out first with 200 mL MeOH/EtOH/H 2O (45:45:10), 200 mL MeOH, and 200 mL MeOH/Acetone (10:10). The outflow of the column was collected in sub-fractions of 5 mL automatically by fraction collector at flow rate 0.2 mL/min. The received sub-fractions were analyzed by TLC in the first solvent system, and then detected under UV light at 254 and 366 nm and by spraying with of AS reagent and heating. Sub-fractions with the same spots on TLC were combined to main fractions. After
TLC analyzing 8 major fractions (SOF I to SOF VIII ) were obtained and tested for the antibacterial activity . Because only fraction SOF II which eluted with
MeOH/EtOH/H 2O (45:45:10) exhibited strong antibacterial activity, this fraction was further separated.
74.3 mg of fraction SOF II were separated using silica gel column. 10 g silica gel (0.040-063 mm) was mixed with the first solvent system, n-hexane/EtOAc (25:75), to form loose slurry for 30 minutes at room temperature. This slurry was then poured into the column (35 x 1.2 cm, h x i.d.) and column equilibration was carried out with the first solvent system for 30 minutes. The sample was dissolved in initial mobile phase and applied onto the surface of the gel bed protected by a thin layer (1 cm) of sea sand. The column was eluted with 100 mL n-hexane/EtOAc (25:75), 60 mL EtOAc, 60 mL EtOAc/MeOH (50:50), and 100 mL MeOH. The outflow of the column was collected in sub-fractions of 4 mL automatically by fraction collector at flow rate 0.2 mL/min. The sub-fractions were analyzed by TLC in mobile phase
MeOH/EtOH/H 2O (45:45:10). The thin layer chromatogram was detected under UV light at 254 and 366 nm and by spraying with AS reagent and heating. Sub-fractions 47
Chapter II Materials and methods with the same spots on TLC were combined to main fraction. After TLC analyzing ten major fractions (SOF II-1 to SOF II-10 ) were collected and tested for the antibacterial activity. The fractions SOF II-5 and SOF II-6 eluting with 50% EtOAc/MeOH exhibited approximately the same antibacterial activity. Thus, these two fractions were pooled and further purified. The purification of pooled fractions was carried out using semi-preparative HPLC column Synergi POLAR-RP 80A (250×10mm, 4 micron ) with a flow rate of 3.0 mL/min and detection at 210, 220, 238, 246, 254 and 366 nm. A concentration of 500 µg/50 µL was injected per run. Altogether, 26.0 mg of the mixture were purified using the step gradient described in table 2-4. Three peaks were collected separately under UV of 220 nm to give three fractions. All these fractions were tested for antibacterial activity. Because the fraction FSO 3 exhibited antibacterial activity, this fraction was used for structural elucidation.
a Step gradient used in purification the pooled fractions (SOF II-5, SOF II-6) by semi-preparative HPLC 0.50 18.50 21.50 24.50 25.50 95 5 0 0 95 5 95 100 100 5
Solvent A: H 2O and solvent B: CH 3CN; Flow rate: 3.0 mL/min; HPLC column: Synergi Polar- RP80A/250×10mm, 4 micron
Lyophilized biomass
3 x n-Hexane Residue n-Hexane ext. 3 x MeOH
MeOH ext. Residue 3 x H 2O
H2O ext.
Silica gel 60 RP 18 column - MeOH/ EtOH/H 2O= 45:45:10 - MeOH - MeOH/Acetone= 1:1
SOF I SOF II SOF III SOF IV SOF V SOF VI SOF VII SOF VIII Silica gel column - n-hexane/EtOAc =25:75 - EtOAc - EtOAc/MeOH =50:50 - MeOH
SOF II-1 SOF II-2 SOF II-3 SOF II-4 SOF II-5 SOF II-6 SOF II-7 SOF II-8 SOF II-9 SOF II-10
- Semi-preparative HPLC Synergi Polar RP
- CH 3CN/H 2O gradient
FSO 1 FSO 2 FSO 3 Structure elucidation
Extraction, fractionation, and isolation of the secondary metabolites of Scytonema ocellatum
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Chapter II Materials and methods
a a a a a a Anabaena The ethyl acetate extract obtained from the microscopically cell-free growth medium of Anabaena sp. of the large scale culture exhibiting very strong antibacterial activity against Gram-positive and Gram-negative bacteria, as well as yeast Candida maltosa was analyzed by HPLC. Based on the results of the HPLC analysis, the ethyl acetate extract was separated by semi-preparative HPLC. Procedure of this separation was carried out by using a semi-preparative HPLC column Synergi POLAR-RP 80A (250×10mm, 4 micron) with a flow rate of 3.0 mL/min and detection at 210, 220, 238, 254 and 366 nm. A concentration of 1mg/50 µL was injected per run. Altogether, 12.0 mg of the crude ethyl acetate extract obtained from 4L culture medium were purified using the step gradient described in table 2-5. Peaks were collected at 238 nm. Seven fractions were obtained from ethyl acetate extract by semi-preparative HPLC and tested for antimicrobial activity. Since only fraction AF 6 exhibited activity against different bacteria (Gram-positive and Gram-negative) and the yeast Candida maltosa , this fraction was used for structure elucidation (see scheme 2-5).
a Step gradient used for purification of the EtOAc extract by semi- preparative HPLC 0.50 12.50 18.50 22.50 24.50 26.50 95 75 40 0.0 0.0 80.0 5 25 60 100 100 5
Solvent A: H 2O and solvent B: CH 3CN; Flow rate: 3.0 mL/min; HPLC column: Synergi polar- RP80A/250×10mm, 4 micron
Grow th m edium
250ml ethyl acetate/ 3times/ shaking/ 24h at room temparature
Ethyl acetate ext.
- Semi- preparative HPLC Synergi Polar RP - CH 3CN/ H 2O gradient
First AF I AF II AF III AF IV AF V AF VI
Structure elucidation
Extraction, fractionation, and isolation of the secondary metabolites of Anabaena sp.
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Chapter II Materials and methods
a a a a a a Nostoc The methanol extract obtained from extraction of lyophilized biomass from large scale culture of this strain was subjected to silica gel column chromatography, followed by RP C 18 column chromatography, and reversed-phase HPLC (see scheme 2-6). 760 mg of MeOH extract obtained from 3.75 g lyophilized biomass were fractionated using silica gel column. 60 g silica gel (0.040-063 mm) were suspended and saturated in first solvent n-hexane/EtOAc (90:10) for 1 hour at room temperature. This silica gel solution was then poured carefully into the column (60 x 2.5 cm, h x i.d.). The column was equilibrated with first solvent for 1 hour. The extract was previously suspended in a small amount of methanol and carefully applied on the surface of the column protected by a thin layer of sea sand. The column was eluted with 450 mL of each mobile phase with increasing polarity starting with n- hexane/EtOAc (90:10), and followed by n-hexane/EtOAc (40:60), n-hexane/EtOAc
(20:80), MeOH, and MeOH/H 2O (95:5). Each eluting solvent was collected in a different flask. After that 5 fractions (NF I to NF V) were obtained and tested for antibacterial activity. Because fraction NF IV which eluted with MeOH exhibited the strongest antibacterial activity, fraction NF IV was further separated.
360 mg of fraction NF IV was separated by RP C 18 column chromatography.
25 g reverse-phase C 18 silica gel was swollen in 50ml of initial mobile phase
MeOH/H 2O (9:1) for 3 hours at room temperature. The slurry was poured into the column (35 x 2.5 cm, h x i.d.). Column was then rinsed with initial mobile phase for
30 minutes. Amount of 360 mg fraction NF IV was dissolved in a minimum of initial mobile phase and applied on the surface of column. The elution was carried out first with 300 mL MeOH/H 2O (90:10), 300 mL MeOH, and finally with 300 mL DCM. The outflow of the column was collected in different flasks at flow rate 0.4 mL/min.
After that 3 major fractions (NF IV-1 to NF IV-3) were obtained and tested for the antibacterial activity . Because fraction NF IV-1 which eluted with MeOH/H 2O (90:10) exhibited the strongest antibacterial activity, this fraction was further separated.
The purification of fraction NF IV-1 was carried out using the semi- preparative HPLC column Synergi POLAR-RP 80A (250×10mm, 4 micron ) with a flow rate of 3.0 mL/min and detection at 210, 220, 238, 246, 254 and 366 nm. A concentration of 1 mg/50 µL was injected per run. Altogether, 150.0 mg of NF IV-1 were purified using the step gradient described in table 2-6 and the purification was 50
Chapter II Materials and methods monitored by diode array detector (DAD) at wavelength 238 nm. After that, four fractions were collected , among of them, fraction NsF2 exhibited significant antibacterial activity. Thus, fraction NsF2 was used for structure elucidation.
a Step gradient used for purification of fraction NF IV-1 by semi-preparative HPLC 0.50 8.50 12.50 16.50 18.50 60 15 0 0 60 40 85 100 100 40
Solvent A: H 2O+0.05%TFA and solvent B: MeOH; Flow rate: 3.0 mL/min; HPLC column: Synergi polar-RP80A/250×10mm, 4 micron
Lyophilized biomass 3 x n-Hexane Residue n-Hexane ext. 3 x MeOH
MeOH ext. Residue 3 x H 2O
H2O ext.
Silica gel column
n-hexane/EtoAc/MeOH/H 2O gradient
NF I NF II NF III NF IV NF V
Silica gel 60 RP 18 column -MeOH/H 2O=90:10 -MeOH -DCM
NF IV-1 NF IV-2 NF IV-3
- Semi-preparative HPLC Synergi Polar RP
- MeOH/(H 2O+0.05%TFA) gradient
NsF 1 NsF 2 NsF 3 NsF 4
Structure elucidation
Extraction, fractionation, and isolation of the secondary metabolites of Nostoc sp.
a a a a a a Lyngbya majuscula The active MeOH extract obtained from lyophilized biomass was subjected initially to repeated silica gel column chromatography, and finally separated by preparative TLC (see scheme 2-7). The methanol extract (620 mg) obtained from 30 g lyophilized biomass was fractionated on silica gel column. For preparation of the column 150 g of silica gel 60 (0.040-063 mm) were suspended and saturated in first solvent n-hexane/EtOAc
51
Chapter II Materials and methods
(60:40) for one hour at room temperature. This silica gel solution was then poured into the glass column (50 x 4.0 cm, h x i.d.) until the gel bed reached a height of 40 cm. After that, the silica gel column was flushed only with first solvent n- hexane/EtOAc (60:40) for 30 minutes for stabilizing. The methanol extract was dissolved in 1mL MeOH and applied softly on the surface of silica gel bed protected by a thin layer (1 cm) of sea sand, and elution was initiated with 500 mL n- hexane/EtOAc (60:40), followed by 500 mL n-hexane/EtOAc (40:60), 400 mL EtOAc, 400 mL EtOAc/MeOH (60:40), 400 mL EtOAc/ MeOH (40:60), and 400 mL MeOH. The outflow of the column was collected in sub-fractions of 3-7 mL by fraction collector at flow rate 0.3 mL/min. Fifteen major fractions (F 1 to F 15 ) were collected according to the bands detected on TLC developed by using n- hexane/EtOAc/MeOH/CH 3COOH (75:25:5:3), then detected under UV light at 254 and 366 nm or by spraying with anisaldehyde/sulfuric acid reagent and heating. Based on the results of TLC analysis, 7 fractions were chosen for testing cytotoxic activity .
The fraction F 8 eluting with 100% EtOAc exhibited the strong activity and only one main spot in TLC. Thus, further separation of this fraction was necessary.
A portion of the fraction F8 (90 mg) was further separated on silica gel column. Amount of 120 g silica gel 60 (0.040-063 mm) was saturated in the first mobile phase n-hexane/EtOAc (40:60) and poured into the column (50 cm x 4.0 cm, h x i.d.). The column was rinsed with the first mobile phase for 30 minutes. After that, 90 mg of fraction F8 was dissolved in 2 mL of the first mobile phase and loaded on the surface of the silica gel protected by a thin layer (1 cm) of sea sand. 450 mL n- hexane/EtOAc (40:60), 450 mL n-hexane/EtOAc (10:90), and 400 mL MeOH were respectively used to elute substances of fraction F8 from the column. The outflow of the column was collected in sub-fractions of 4 mL automatically by fraction collector at flow rate of 0.4 mL/min. Six major fractions (F8-1 to F8-6) were obtained according the TLC pattern developed in n-hexane/EtOAc/MeOH/CH 3COOH (75:25:5:3), then detected under UV light at 254 and 366 nm or by spraying with anisaldehyde/sulfuric acid reagent and heating. All fractions (F 8-1 to F 8-6) were tested for cytotoxic activity . Because fraction F 8-3 which eluted with 10% n-hexane/EtOAc exhibited a strong cytotoxic activity, this fraction was further purified.
A portion of the fraction F8-3 (30.2 mg) was applied onto a preparative TLC plate (Merck, Silica gel F 254 , thickness 0.25 mm). The chromatogram was developed with mobile phase n-hexane/EtOAc/MeOH/CH 3COOH (75:25:5:3) and five spots 52
Chapter II Materials and methods
with Rf 0.28, 0.22, 0.18, 0.12, and 0.045 were scratched out from plate and resolved in EtOAc by shaking for 3 x 15 minutes. The solutions were centrifuged, filtered and evaporated under reduced pressure. After that, five fractions (F 8-3-1 to F 8-3-5) were obtained. These five fractions (F 8-3-1 to F 8-3-5) were examined by analytical TLC and
HPLC. HPLC analysis showed that fraction F 8-3-2 showed only one peak at R f 0.22, therefore structure elucidation of this fraction was done.
Lyophilized biomass
3 x n-Hexane Residue n-Hexane ext. 3 x MeOH
MeOH ext. Residue 3 x H 2O
Silica gel column H2O ext. 7 solvent system (n-hexane/EtOAc/MeOH gradient)
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 Silica gel column -n-hexane/EtOAc=4:6 -n-hexane/EtOAc=1:9 -MeOH
F8-1 F8-2 F8-3 F8-4 PTLC
n-hexane/EtOAc/MeOH/CH 3COOH 75 : 25 : 5 : 3
F8-3-1 F8-3- 2 F8-3-3 F8-3-4 F8-3-5
Structure elucidation
Extraction, fractionation, and isolation of the secondary metabolites of Lyngbya majuscula (method 1)
The active MeOH extract which obtained from lyophilized biomass of this strain was subjected to silica gel column chromatography followed by reversed-phase HPLC (see scheme 2-8). 240 mg of the methanol extract obtained from 15 g lyophilized biomass were separated on silica gel column. For preparation of the column an amount of 45 g silica gel 60 (0.040-063 mm) was mixed and saturated in the first solvent system for 30 minutes at room temperature. This silica gel solution was then poured into the column (50 x 2.0 cm, h x i.d) and the column was equilibrated by rinsing with first
53
Chapter II Materials and methods solvent system for 30 minutes. 240 mg of the methanol extract were dissolved in 1 mL initial mobile phase and applied carefully on the surface of silica gel bed protected by a thin layer of sea sand. Elution started initially with 450 mL n- hexane/EtOAc (75:25), followed by 250 mL n-hexane/EtOAc (50:50), 250 mL n- hexane/EtOAc (25:75), 150 mL EtOAc, 150mL EtOAc/MeOH (90:10), 150 mL EtOAc/MeOH (25:75), 150 mL EtOAc/MeOH (50:50), 150 mL EtOAc/MeOH (25:75), and finally 150 mL MeOH. The outflow of the column was collected in sub- fractions of 3-7 mL at flow rate 0.3 mL/min by fraction collector except the outflow of the column eluting with 25% EtOAc/MeOH to 100% MeOH, this was collected in different flashes. The sub-fractions were analyzed and combined to main fractions by
TLC with n-hexane/EtOAc/MeOH/CH 3COOH (75:25:5:3) as mobile phase, then detected under UV light at 254 and 366 nm and by spraying with anisaldehyde/sulfuric acid reagent and heating. After that, twenty two major fractions
(F 1 to F 22 ) were obtained. Based on the results of TLC analysis , three fractions F9, F 10 , and F 17 were chosen for testing cytotoxic activity. The fraction F 10 eluting with 25% n-hexane/EtOAc exhibited strong cytotoxic activity, thus, this fraction was further separated.
The purification of fraction F10 was carried out using a semi-preparative HPLC column Synergi POLAR-RP 80A (250×10mm, 4 micron ) with a flow rate of 3.0 mL/min and detection at 210, 220, 238, 254 and 366 nm. A concentration of 500
µg/50 µL was injected per run. Altogether, 14.0 mg F10 were purified using the step gradient described in table 2-7. Five fractions were collected at 210 nm and tested for cytotoxic activity. The fractions F10-3 and F10-5 exhibited significant cytotoxic activity and were therefore used for structural elucidation.
a Step gradient used in purification of fraction F 10 by semi-preparative HPLC 0.50 3.50 14.50 24.50 34.50 39.50 49.50 52.5 80 50 35 25 15 0.0 0.0 80 20 50 65 75 85 100 100 20
Solvent A: H 2O and solvent B: CH 3CN; Flow rate: 3.0 mL/min; HPLC column: Synergi Polar-RP 80A/250×10mm, 4 micron
54
Chapter II Materials and methods
Lyophilized biomass
3 x n-Hexane Residue n-Hexane ext. 3 x EtOAc
EtOAc ext. Residue 3 x MeOH
MeOH ext.
Silica gel column n-Hexane/EtOAc/MeOH gradient
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 F21 F22
-Semi-preparative HPLC Synergi Polar RP
-CH 3CN/H 2O gradient
F10-1 F10-2 F10-3 F10-4 F10-5
Structure elucidation
Extraction, fractionation, and isolation of the secondary metabolites of Lyngbya majuscula (method2)
a a a a Nuclear magnetic resonance spectroscopic and high resolution-mass spectrometric data were recorded in the Helmholtz Centre for Infection Research, Braunschweig and Dr. Victor Wray and Dr. Rolf Jansen both the Helmholtz Centre for Infection Research, Braunschweig provided expert help with the structure elucidation of the secondary metabolites. a a Westiellopsis a Lyngbya majuscula Mass spectrometry: HR-ES-IMS (positive ion mode) were recorded on a high resolution Bruker MaXis mass spectrometer. NMR spectroscopy: 1H, 13 C NMR spectra were recorded at 300 K on a Bruker AVANCE DMX 600 NMR spectrometer locked to the deuterium resonance of the solvent, CDCl 3. Chemical shifts are reference to the residual proton signal of the solvent.
55
Chapter II Materials and methods
a a Calothrix javanica a Scytonema ocellatum
Mass spectrometry: HR-ESI-MS (positive ion mode) were recorded on a Thermo Science LTQ Orbitrap mass spectrometer. NMR spectroscopy: 1D ( 1H) and 2D (COSY, TOCSY, NOESY, HMQC and HMBC) NMR spectra were recorded at 300 K on a Bruker AVANCE DMX 600 NMR spectrometer locked to the deuterium resonance of the solvent, trifluoroethanol- d2/H 2O (1:1). Chemical shifts are reference to the residual proton signal of the solvent (1H: 3.95 and 13 C: 60.85 ppm).
a a Anabaena Mass spectrometry: HR-ESI-MS (positive ion mode) were recorded on a Thermo Science LTQ Orbitrap mass spectrometer. NMR spectroscopy: 1D ( 1H, 13 C and DEPT-135) and 2D (COSY, HMQC and HMBC) NMR spectra were recorded at 300 K on a Bruker AVANCE DMX 600 NMR spectrometer locked to the deuterium resonance of the solvent, trifluoroethanol- d2/H 2O (1:1). Chemical shifts are reference to the residual proton signal of the solvent ( 1H: 3.95 and 13 C: 60.85 ppm).
a a Nostoc Mass spectrometry: ESI-MS were recorded on a Micromass Q-Tof-2 mass spectrometer.
a a a a a Westiellopsis According to data obtained from screening, the ethyl acetate extract from growth medium of this cyanobacterial strain showed a strong antibacterial activity against Gram-positive bacteria B. subtilis , S. aureus and Gram-negative bacteria E. coli , P. aeruginosa as well as yeast Candida maltosa SBUG 700 (IZ of 13.5 mm). This extract was therefore highly appropriate for investigation of antimicrobial compounds. Bioautographic assay was used to locate the active principles of ethyl acetate extract from growth medium of Westiellopsis sp.VN on TLC chromatogram (see 56
Chapter II Materials and methods
2.7.1.2.). The extract was loaded on silica gel TLC plate and developed with three mobile phases: SS1 (n-hexane/ EtOAc/MeOH= 75:25:5)
SS2 (EtOAc/MeOH/H 2O= 100:13.5: 1) SS3 (Toluen/EtOAc/MeOH= 92:5:3)
These TLC chromatograms of ethyl acetate extract always show a very broad active zone so that the antibacterial activity of this extract might be due to the presence of more than one compound. The amount of 6.6 mg ethyl acetate extract was dissolved in 200 mL MeOH, followed by shaking for 30 minutes. Afterwards, the methanol phase was collected by pipetting. The residue which was not dissolved in methanol was dried at room temperature and stored at -20 0C. Methanol phase was dried in vacuum to give the methanol fraction. Since this methanol fraction showed strong antibacterial activity against S. aureus , the active MeOH fraction was analyzed by HPLC with DAD detection and a Synergi-Polar RP (80A) column with a solvent gradient from 5%-100% acetonitrile/water in 30 minutes. Due to the insufficient separation of the components by HPLC, GC-MS was used for identifying the components.
a a a a Identification of fatty acids in the n-hexane extract from Lyngbya majuscula biomass and the MeOH fraction obtained from ethyl acetate extract of Westiellopsis sp .VN medium was done by gas chromatography-mass spectrometry with the help of Dr. Martina Wurster, Department of Phamaceutical Biology, Institute of Pharmacy, Ernst-Moritz-Arndt-University of Greifswald, Germany. The n-hexane extract (1 mg) was hydrolyzed previously. For the hydrolysis 3-4 drops of n-hexane extract were treated with 3 KOH tablets and 5mL of ethanol in a 250 mL flask. The mixture was heated to boiling for 10 min at reflux in a heating mantle. After cooling, the mixture was diluted with 10 mL aqua dest. After this, the pH of the chilled hydrolysate was adjusted with hydrochloric acid up to 2.0 and the fatty acids were extracted 3 times with n-hexane (3x15 mL). The sample was reduced in a rotary evaporator and then the rest was dried with a pinch of sodium sulfate, the supernatant was transferred into an HPLC vial and dried over night at room
57
Chapter II Materials and methods temperature to get the residue. MeOH fraction (1 mg) was hydrolyzed previously as described for n-hexane extract. The derivatization (Liebeke et al., 2010) was performed by adding 40 µL of methoxyamine (MeOX) [20mg ml -1 pyridine] to 1mg dried extract and heating for 3 min in microwave at 240 Watts (W). After this first heating phase, 80 µL of MSTFA (N-methyl-N-trimethylsilyltrifluoroacetamide) was added and heated for further 3 minutes at 240 Watts in the microwave. The solution were then subjected to GC/MS analysis under following conditions
Parameters for the Gas chromatograph Carrier Gas: Helium Carrier Flow: 1.0 mL/min Injection Port Parameters: split, 25:1 Injection Port Temperature: 230 0C Purge flow to split vent: 20 mL/min after 2 min Column: DB-5MS (30 m×0.25 mm×0.25 µm) Column Oven Temperature: 70 0C initial (1 min), Increasing to 76 0C by1.5 0C/min Then to 330 0C by 5 0C/min, keep in 10 min Total: 65.8 min Solvent Delay 6.2 min
Injection Volume: 2.0 µL Injection Liner: Single taper liner MS Transfer Line Temperature: 250 0C Parameter for the mass spectrometer Mode: Electron Ionization (70 eV) Tune: Auto tune with PFTBA (Masses 69, 219, 502) Dwell time: 30 ms Scan-Modus: 35-573 m/z, Scanrate 2.74 scans/sec
The detected compounds were identified by processing of the raw GC-MS data with ChemStation G1701CA software and comparing with the NIST (National
58
Chapter II Materials and methods
Institute of Standards and Technology, Gaithersburg, USA) mass spectral database 2.0 d, and from retention times and mass spectra of standard compounds of the library. Relative amounts of detected compounds were calculated based on the peak areas of the total ion chromatograms (TIC).
a Westiellopsis sp To determine the effects of nitrogen deficiency on biomass production and bioactive compound accumulation of Westiellopsis sp.VN the BG-11 (Rippka, 1979) medium without NaNO 3 was tested in large-scale culture. The standard BG11 medium was used as control. The large-scale culture was performed in a 45 liter-glass fermentor containg 35 L of liquid medium (see 2.5.3) at 28°C under continuous illumination using cool-white fluorescent tubes of 8µmol/m 2. The pH-value of the large-scale culture was adjusted to 7.4 using CO 2 supplementation. 1500 mL of inoculum cultivated for 20 days in Fehrnbach flasks was added to each fermentor After 7 weeks, the cultures were harvested by centrifugation at 6500 rpm in a refrigerated continuous-flow centrifuge. The biomasses were lyophilized (Lyophylizer Alpha 1-4), weighted, and stored at -20 0C. 3 L of every cyanobacterial cultured medium was concentrated to 300 mL by rotary evaporation in vacuum at 40 0C and stored at -20 0C. To investigate the effects of nitrogen deficiency on production of active compounds, lyophilized biomass was extracted with n-hexane, ethyl acetate, and methanol (see 2.6.1) and growth medium was extracted with ethyl acetate (see 2.6.2). The prepared extracts were tested for antibiotic activity against Staphylococcus aureus ATCC 6538 (see 2.7.1.1)
a a The effects of cultivation time on the growth and production of active compounds of Westiellopsis sp.VN strain were investigated in batch cultures. The batch culture experimentw were carried out in 300 mL Erlenmeyer flasks containing
150 mL of liquid medium, BG-11 [Ripka, 1979] medium without NaNO 3 under continuous illumination provided by cool-white fluorescent tubes of 8 µmol/m 2 at room temperature (20 ±2 0C). 20 mL of inoculum cultivated in Fehrbach flasks for 20
59
Chapter II Materials and methods days at the temperature and light conditions described above (according to 12.6 mg lyophilized biomass) was added into each Erlenmeyer flask. The flasks were incubated at the temperature and the light conditions described above and their places were changed randomly everyday. Growth was monitored by measuring the dry weight of biomass after 2, 3, 4, 5, 6, 7, and 8 weeks. At each sampling the contents of three Erlenmeyer flasks were pooled in order to get enough cell material for the analysis. The cells were harvested by centrifugation (3500 rpm/ 10 min/ 10 0C), lyophilized (Lyophylizer Alpha 1-4) and weighted for determining growth curve. To investigate the influence of cultivation time on production of active compounds, the dried cells were extracted by methanol to get methanol extracts that were tested for antibiotic activity against Staphylococcus aureus ATCC 6538 (see 2.7.1.1)
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Chapter III Results
a a a a a a a 12 cyanobacterial strains (see 2.1.1) were cultured in batch cultures (see 2.5.2). After harvesting, both biomass and culture media were used for extraction (see 2.6). The dry weight of extracts from biomass and culture media of the 12 cyanobacterial strains are shown in table 3-1.
a Dry weight of extracts from biomass (1g) and culture media (1L) of 12 cyanobacterial strains
Strain Strain n- Hexane extr. MeOH extr. H2O extr. EtOAc extr. number (mg) (mg) (mg) (mg) Anabaena sp. TVN40 2.80 72.87 101.33 3.80 Nostoc spongiaforme TVN7 3.13 222.26 122.34 7.93 Nostoc coeruleum TVN14 2.60 79.47 112.00 2.53 Nostoc sp. TVN9 4.60 203.73 146.67 8.75 Calothrix elenkinii TVN202 5.80 279.20 152.00 7.20 Calothrix machica TVN201 5.13 144.27 75.33 6.68 var. crassa Calothrix javanica TVN1 5.47 168.15 116.15 5.67 Calothrix sp. TVN20 5.54 150.91 120.42 8.42 Oscillatoria sp. TVN16 8.27 180.00 105.33 3.66 Scytonema ocellatum TVN10 10.55 183.54 102.70 4.20 Scytonema millei TVN12 6.42 152.15 118.9 1.60 Westiellopsis sp.VN TVN22 4.3 188.7 90.1 3.03 extr. = extract
In the majority of cases methanol extracts showed the highest yields while the n-hexane extracts displayed the lowest yields. All the crude extracts were subsequently used for antibacterial screening.
a 48 extracts of biomasses and culture media of 12 different cyanobacterial strains were tested against two Gram-positive and two Gram negative bacteria in agar plate diffusion test for antibacterial activity (see 2.7.1.1). 23 n-hexane, methanol, and ethyl acetate extracts exhibited activity against the Gram positive bacterium Bacillus subtilis ATCC 6051 . The ethyl acetate extract from cultivation medium of Westiellopsis sp.VN (see fig.3-1) exhibited maximum inhibition zone of 25 mm against this bacterium.
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Chapter III Results
Antibacterial activity of cyanobacterial extracts against the Gram positive bacterium Bacillus subtilis ATCC 6501 (n=2; extract concentration 2 mg/6 mm paper disc, agar plate diffusion assay, inhibition zone including the diameter of paper disc).
22 n-hexane, methanol, and ethyl acetate extracts showed activity against Staphylococcus aureus ATCC 6538 (see fig.3-2)
Antibacterial activity of cyanobacterial extracts against the Gram positive bacterium Staphylococcus aureus ATCC 6538 (n=2; extract concentration 2 mg/6 mm paper disc, agar plate diffusion assay, inhibition zone including the diameter of paper disc).
According to the diameter of inhibition zones (mm), the effect of the extracts obtained from the same cyanobacterial strain showed no significant differences in
62
Chapter III Results activity against Staphylococcus aureus and Bacillus subtilis. The methanol extract obtained from biomass of Westiellopsis sp.VN showed the highest activity with inhibition zone of 19 mm and the methanol extracts obtained from biomass of Calothrix elenkinii, Scytonema mileii, Scytonema ocellatum, Calothrix javanica, and Nostoc sp. exhibited moderate activity against Gram-positive bacteria. In addition, the ethyl acetate extracts obtained from medium of the strains Westiellopsis sp.VN and Anabaena sp. showed strong activity. 11 of 48 extracts showed activity against the Gram-negative bacterium Escherichia coli. Of these active extracts, the ethyl acetate extract obtained from cultivation medium of Westiellopsis sp.VN showed the highest activity with inhibition zone of 25 mm, followed by the ethyl acetate extract obtained from cultivation medium of Anabaena sp. with inhibition zone of 21mm. Interestingly, almost all extracts which inhibited the growth of Escherichia coli were prepared from cultivation medium (see fig.3-3). In case of Westiellopsis sp.VN the extracts of biomass and cultivation medium exhibited strong activity.
Antibacterial activity of cyanobacterial extracts against the Gram negative bacterium Escherichia coli ATCC 11229 (n=2; extract concentration 2 mg/6 mm paper disc, agar plate diffusion assay, inhibition zone including the diameter of paper disc).
Activity against the gram negative bacterium Pseudomonas aeruginosa was seldom found. 3 out of 48 extracts inhibited the growth of this bacterium but these three extracts did not result in complete inhibition (see fig.3-4)
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Chapter III Results
Antibacterial activity of cyanobacterial extracts against the Gram negative bacterium Pseudomonas aeruginosa ATCC 22853 (n=2; extract concentration 2 mg/6 mm paper disc, agar plate diffusion assay, inhibition zone including the diameter of paper disc).
In all, a total of 48 lipophilic and hydrophilic extracts obtained from 12 samples of cultured soil cyanobacteria have been screened for their antibacterial activities. Of 48 extracts, 23 (47.92%; 3 n-hexane, 11 MeOH, and 9 EtOAc extracts) showed activity against the Gram-positive bacterium B. subtilis , 22 (45.83%; 3 n- hexane, 11 MeOH, and 8 EtOAc extracts) exhibited activity against the Gram-positive bacterium S. aureus , 11 (22.92%; 1 n-hexane, 4 MeOH, and 6 EtOAc extracts) inhibited the growth of the Gram-negative bacterium E. coli . Three MeOH extracts showed effects on the growth of the Gram-negative bacterium P. aeruginosa but these effects did not result in complete inhibition, and none of the water extracts was active against the test bacteria. Of these active extracts, the methanol extract of Westiellopsis sp.VN showed the highest activity against the Gram-positive bacteria and the ethyl acetate extracts obtained from Westiellopsis sp.VN and Anabaena sp showed the highest activity against Gram-negative bacterium E. coli. Interestingly, 12 of 12 investigated cyanobacteria are able to inhibit the growth of at least one of the test organisms used Based on the results of the antibacterial screening, Westiellopsis sp.VN, Anabaena sp., Calothix javaniva, Scytonema ocellatum and Nostoc sp . strains were chosen for chemical investigation with emphasis on the isolation and structure elucidation of antibacterial active secondary metabolites.
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Chapter III Results
a a a a Westiellopsis a a a a a a Due to the highest antibacterial activity, the methanol extract resulting from extraction of dry biomass was first chosen for chemical investigation with an emphasis on the isolation and structure elucidation of active metabolites. This led to the isolation and identification of the 6 following compounds ambiguine D isonitrile, ambiguine B isonitrile, dechloro-ambiguine B isonitrile, fischerellin A, hydroxy- eicosatetraenoic acid and methoxy-nonadecadienoic acid.
a a a a a a a The methanol extract is usually a complex mixture of organic compounds from non-polarity to polarity. Thus, the methanol extract of Westiellopsis sp. VN was fractionated to remove a large portion of the unwanted material in a fairly low- resolution step. 260 mg of methanol extract were subjected to silica gel column eluting with a stepwise gradient of DCM/EtOAc/MeOH (see 2.8.1) to give 8 fractions evaluated for antibacterial activity against S. aureus using agar diffusion method (see table 3-2).
a Fractionation of methanol extract from Westiellopsis sp. VN biomass by silica gel chromatography and antibacterial activity of fractions to S. aureus
a a a
FI DCM 58.6 22.54 500 21
FII DCM/EtOAc (95:5) 7.7 2.96 500 15
FIII DCM/EtOAc (90:10) 5.2 2.0 500 10 2 FIV DCM/EtOAc (50:50) 40.3 15.5 500 0
FV EtOAc 2.4 0.92 500 0
FVI EtOAc/MeOH (75:25) 4.7 1.81 500 0
FVII EtOAc/MeOH (50:50) 102.2 39.31 500 0
FVIII MeOH 34.9 13.42 500 0 Total 256.0 98.46 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
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Chapter III Results
Three contiguous fractions (FI, FII, and F III ) showed strong antibacterial activity, and the highest activity was detected in fraction FI eluting with 100% DCM with inhibition zone of 21 mm. Moderate activities were found in fractions FII and FIII with inhibition zones of 15 mm and 10 mm, respectively. Because of strong antibacterial activity of these three fractions, they were combined to the mixture which was further purified.
a a a a Sephadex LH-20 column chromatography involves separation based on molecular size of compounds being analyzed. Thus, it was used for separating components of the combined fractions FI, FII, and F III because these three active fractions were eluted with solvent systems not so different in polarity and therefore may contain compounds with the same polar nature. The combined fractions (68.5 mg) were subjected to a sephadex LH-20 column eluted with 90% MeOH in H 2O followed by 100% MeOH, and finally with
50% aceton in H 2O (see 2.8.1) to yield 4 fractions. The antibacterial activity of the fractions against Staphylococcus aureus was tested in agar diffusion assay (see table 3-3).
a Fractionation of combined fractions F I, F II , and F III from Westiellopsis sp. VN biomass by LH-20 chromatography and antibacterial activity of fractions to S. aureus
a a a a
WF 1 1-30 H2O/MeOH (10:90) 51.8 75.62 500 21
WF 2 31-40 H2O/MeOH (10:90) 1.8 2.63 500 15
WF 3 41-70 MeOH 8.6 12.55 500 10 2 WF 4 70-90 H2O/ Aceton (50:50) 5.2 7.59 500 0 Total 67.4 98.39 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
Fraction WF 1 which eluted with 90%MeOH in H 2O showed the highest antibacterial activity with inhibition zone of 21 mm and was further purified.
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Chapter III Results
a a a
Final purification of fraction WF 1 (50.0 mg) was achieved by semi-preparative RP HPLC to give nine fractions. All fractions were evaluated for antibacterial activity against S. aureus (see fig-5 and table 3-4).
W F 1 - 6 WF 1- 9 W F 1 - 5 W F 1 - 8
W F 1 - 3
WF 1- 7 WF 1- 2
WF 1- 4
WF 1- 1
Semi-preparative RP HPLC chromatogram of WF 1 (mobile phase table 2-2) 500 µg/50 µL/injection, detection at 238 nm, column Synergi POLAR-RP 80A (250×10 mm, 4 micron), flow rate 3 mL/min.
a Fractionation of WF 1 from Westiellopsis sp. VN biomass by semi- preparative reversed-phase HPLC and antibacterial activity of fractions to S. aureus
a a
WF 1-1 14.74 2.3 4.6 200 17.0
WF 1-2 23.50 0.6 1.2 200 24.0
WF 1-3 24.19 1.4 2.8 200 28.0
WF 1-4 25.18 0.4 0.8 200 14.0
WF 1-5 30.04 1.7 3.4 200 10.0
WF 1-6 30.85 2.6 5.2 200 8.0
WF 1-7 33.99 0.5 1.0 200 9.0
WF 1-8 41.03 2.8 5.6 200 10.0 2 WF 1-9 41.53-48.00 5.7 11.4 200 0.0 Total 18 36 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
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Chapter III Results
Based on the results of agar diffusion assay and yield of these nine fractions, the four fractions WF 1-3, WF 1-5, WF 1-6, and WF 1-8 were used for structure elucidation.
a a Westiellopsis
a a
Fraction WF 1-3 was found to be the pure compound, ambiguine D isonitrile (see fig.3-6).
Cl 19 CH3 13 12 21 14 20 17 OH 22 23 H3C 15 NC 11 16 10 26 H3C O 18 4 H 3 9 25 5 2 24 28 1 6 CH3 8 N CH3 7 H 27
Ambiguine D isonitrile, molecular formula C 26 H29 ClN 2O3 The high resolution ESI mass spectrum in positive mode of fraction WF 1-3 indicated the presence of one compound in this fraction with the protonated molecular ion peak [M+H] + at m/z 453.1947 (see fig.3-7) corresponding to a molecular formula of C26 H29 ClN 2O3 (calculated 453.1939 for C26 H30 ClN 2O3 ). In addition, 1H NMR chemical shifts of compound1 are identical to those reported for ambiguine D isonitrile isolated by Smitka et al., 1992 (see table 3-5, appendix 1a,b). Thus, the identification of compound 1 was confirmed.
[M+ H] +
[M+ H] +
UV and ESI-MS of fraction WF 1-3 (compound 1)
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Chapter III Results
a 1H NMR data of compound 1 compared with literature data of ambiguine D isonitrile 5 7.12 dd 7.12 dd 6 7.31 m 7.31 m 7 7.32 m 7.32 m 13 4.09 ax 4.09 ax, dd 14 2.47 ax 2.47 ax, q 14 2.17 eq 2.17 eq, ddd 15 1.64 br ddd 17 1.39 s 1.39 s 18 1.35 s 1.35 s 19 1.68 s 1.68 s 20 5.96 dd 5.96 dd 21 5.53 E, d 5.53 E, d 21 5.37 Z, d 5.37 Z, d 25 3.11 d 3.11 d 26 3.55 d 3.55 d 27 1.72 s 1.72 s 28 1,73 s 1.73 s 3-OH 2.91 s 10-OH 4.26 4.26 d *Smitka et al ., 1992
a a
The positive ion ESI mass spectra of fraction WF 1-5 indicated a mixture of three compounds compatible with three peaks. These compounds revealed molecular ion peaks as shown in figure 3-8.
peak1
peak2
peak3
UV and ESI-MS of compounds of fraction WF 1- 5 69
Chapter III Results
The high resolution ESI mass spectrum in positive mode of peak1 showed the presence of one compound with the protonated molecular ion peak [M+H] + at m/z
423.2194 (see fig.3-9) corresponding to a molecular formula of C26 H31 ClN 2O
(calculated 423.2198 for C26 H32 ClN 2O) and this compound was also found in fraction
WF 1-6 . Thus, the presence of this compound in this fraction was indicated.
peak1
[ M+ H] +
MS data of peak 1 of fraction WF 1-5
Peak 2 was identified as the compound ambiguine C isonitrile (compound 2) (see fig.3-10)
19 13 12 CH3 14 17 21 OH 20 H3C 15 23 11 16 10 NC H3C 22 18 4 H 3 25 26 9 5 2 24 28 6 1 CH 8 N CH 3 7 3 H 27
Ambiguine C isonitrile, molecula formula C 26 H32 N2O
The identification of ambiguine C isonitrile (dechloro-ambiguine B isonitrile) with the molecular weight of 388 Dalton compatible with the molecular formula
C26 H32 N2O was deduced from the high resolution ESI mass spectrum in positive mode which displayed a molecular ion peak [M+H] + at m/z 389.2586 (calculated
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Chapter III Results
+ 389.2586 for C26 H33 N2O) and the [M+K] peak at m/z 427.2115 as shown in figure 3- 11
pe a k 2
[M+ K] +
[M+ H] +
MS data of peak 2 of fraction WF 1-5 (compound 2)
Peak 3 was identified as the compound fischerellin A (compound 3) (see figure 3-12)
18 16 19 24 14 17 O 13 26 15 6 20 7 21 N 4 11 3 5 12 1 2 8 N 9 10 22 25 O H 23
Fischerellin A, molecular formula C 26 H36 N2O2
The identification of fischerellin A (compound 3) was established by direct comparison of our spectroscopic data including ESIMS in positive mode, and 1H NMR with those reported in the literature (Hagmann and Jüttner, 1996). First, compound 3 was assigned a molecular formula of C 26 H36 N2O2 by HRESIMS (in positive mode) data which displayed a prominent [M+H] + peak at m/z 409.2846
71
Chapter III Results
+ (calculated 409.2850 for C 26 H37 N2O2) and another [M+K] peak at m/z 447.2402 (see fig 3.13). Next the identification of compound 3 was carried out by searching in DNP using elemental composition indicating there were 5 hit compounds including only fischerellin A isolated from cyanobacteria. Finally direct comparison of 1H NMR data of compound 3 with those of reported in the literature was undertaken (see table 3-6, appendix 2a, b)
peak3
[ M+ H] +
[ M+ K] +
MS data of peak 3 of fraction WF 1-5 (compound 3)
a 1H NMR data of compound 3 compared with literature data* of fischerellin A
6 CH 3.5-3.6 3.56 8 CH 3.8-3.9 3.85 3.83 12 CH 6.2-6.3 6.24 13 CH 5.45-5.5 5.49
26 CH 3 2.9-3.0 2.92 2.93 *Hagmann and Jüttner, 1996 72
Chapter III Results
a a
WF 1-6 was found to be the pure compound, ambiguine B isonitrile (see fig. 3- 14)
Cl 19 12 CH3 14 13 17 21 15 OH 20 H3C 23 11 16 10 NC H3C 22 18 4 H 3 25 26 9 5 2 24 28 6 1 CH 8 N CH 3 7 3 H 27
Ambiguine B isonitrile, molecular formula C 26 H31 ClN 2O
The high resolution ESI mass spectrum in positive mode of fraction WF 1-6 indicated the presence of one compound in this fraction with the protonated molecular ion peak [M+H] + at m/z 423.2195 (see fig.3-15) corresponding to a molecular formula of C26 H31 ClN 2O (calculated 423.2198 for C26 H32 ClN 2O). In addition, the 1H NMR signals of compound 4 were fully consistent with literature values for the known metabolite ambiguine B isonitrile reported earlier (Smitka et al., 1992) (table 3-7 and appendix 3a, b)
[ M+H] +
UV and ESI-MS of fraction WF 1-6 (compound 4)
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Chapter III Results
a Comparison of 1H NMR data of compound 4 with reported data* 1 8.15 br s 8.18 br s 5 7.06 dd 7.07 dd 6 7.15 m 7.17 m 7 7.13 m 7.15 m 11 - 4.68 eq, s 13 - 4.41 ax, dd 14 2.53 ax, q 2.53 ax, q 14 2.30 eq, ddd 2.30 eq, ddd 15 2.41 ddd 2.41 ddd 17 1.23 s 1.23 s 18 1.54 s 1.54 s 19 1.53 s 1.53 s 20 6.04 dd 6.04 dd 21 5.31 E, d 5.31 E, d 21 5.26 Z, d 5.26 Z, d 25 6.33 dd 6.33 dd 26 5.28 E, d 5.28 E, d 5.36 Z, d 5.36 Z, d 27 1.62 s 1.62 s 28 1.64 s 1.64 s 10-OH - 1.67 s * Smitka et al., 1992
a a
The high resolution ESI mass spectrum in positive mode of fraction WF 1-8 displayed a mixture of three peaks (see fig.3-16). Peak 1 with m/z 301.1413 is typical for softener. Its presence was also shown in the 1H NMR spectrum (appendix 4a, b).
peak1
peak2
peak3
ESI-MS of compounds of fraction WF 1- 8
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Chapter III Results
The high resolution ESI mass spectrum in positive mode of peak 2 (see fig.3- 17) showed a protonated molecular ion peak at m/z 335.2555 [M+H] + and other two + + peaks at m/z 351.2498 [M+O] and m/z 367.2242 [M+O 2] , leading to the molecular weight of 334 Dalton. Searching in DNP using molecular weight and 1H NMR spectrum of this fraction which showed typical signals for unsaturated fatty acids (appendix 4a, b) led to the methoxy-eicosatetraenoic acid (compound 5) as the most probable compound with molecular formula C 21 H34 O3 (calculated 335.2581 for
C21 H35 O3).
[M+ O] +
[ M+ H] +
ESI-MS of peak 2 of fraction WF 1- 8 (compound 5) The high resolution ESI mass spectrum in positive mode of peak 3 (see fig.3-18) delivered signals at m/z 311.2596 [M+H] + and 333.2398 [M+Na] + as shown in figure 3.18, leading to the molecular weight of 310 Dalton. Searching in DNP using molecular weight and 1H NMR spectrum of this fraction which showed typical signals for unsaturated fatty acids (shown in appendix 4a,b) led to the hydroxy- nonadecadienoic acid-derivative (compound 6) as the most probable compound with molecular formula C19 H34 O3 (calculated 311.2581 for C19 H35 O3).
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Chapter III Results
[ M+ N a] +
[ M+ H] +
ESI-MS of peak 3 of fraction WF 1- 8 (compound 6) a a a a a a a Westiellopsis
In antimicrobial screening the ethyl acetate extract from cultivation medium of this cyanobacterial strain exhibited a strong antibacterial activity against Gram- positive bacteria B. subtilis , S. aureus and Gram-negative bacteria E. coli , P. aeruginosa as well as yeast Candida maltosa SBUG 700 (IZ of 13.5 mm). This extract was therefore highly appropriate for investigation of antimicrobial compounds. Bioautographic TLC assay was successful used to locate the active principles of ethyl acetate extract from cultivation medium of Westiellopsis sp.VN on TLC chromatograms with three mobile phases SS1, SS2, and SS3, separately. The TLC chromatograms of ethyl acetate extract always show a very broad active zone (see fig.3-19), so that the antibacterial activity of the extract might be due to the presence of more than one compound.
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Chapter III Results
SS1 SS2 SS3
Bioautographic assay of EtOAc extract from culture medium of Westiellopsis sp.VN against S.aureu ; SS1 (n-hexane/EtOAc/MeOH= 75:25:5), SS2 (EtOAc/MeOH/H2O= 100:13.5: 1), SS3 (Toluen/EtOAc/MeOH= 92:5:3)
The ethyl acetate extract of cultivation medium was further separated by shaking with methanol.The methanol fraction obtained from ethyl acetate extract (see 2.10) showed strong antibacterial activity against S. aureus with inhibition zone of 28 mm (C=2.0 mg/disc) in agar diffusion assay. Thus, this active fraction was analyzed by HPLC with a solvent gradient from 5%-100% acetonitrile/water (see fig.3-20).
Analytical HPLC of MeOH fraction of EtOAc extract of cultivation medium of Westiellopsis sp. VN with mobile phase 5%-100% acetonitrile/water for 30minutes, Synergi POLAR- RP 80A column (250×4.6mm, 4 micron), flow rate 1mL/min, detection at 210 nm
Due to the insufficient separation of the components by HPLC, GC-MS was used for identifying the volatile components in this methanol fraction. This fraction was solved in n-hexane and methanol respectively and analyzed after hydrolysis and derivatization (see table 3-8 and 3-9, appendix 5 and 6).
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Chapter III Results
a Compounds of MeOH fraction analyzed as methyl ester in n-hexane after hydrolysis /derivatization
a a a a a a a 21.172 L-Proline, 5-oxo-1- 230, 156, 73, 45 7.330 91.9 (trimethylsilyl)-, trimethylsilyl
ester (C 11 H 23 NO 3Si 2) 32.465 Palmitic acid (C16:0) 313, 269,201, 117, 73, 4.169 89.0 43 35.531 Stearic acids (C18: 0) 339, 222, 117, 73, 55 22.748 84.8 35.532 11-cis- Octadecanoic acid (C18:1) 399, 264, 117, 73, 55, 1.124 81.6 41
51.384 ß-Sitosterol (C 29 H50 O) 486, 396, 357, 255, 1.210 77.5 215, 161, 129 ret. time = retention time; m/z = the mass of an ion divided by the electrical charge of the ion
a Compounds of MeOH fraction analyzed as methyl ester in MeOH after hydrolysis/derivatization
a a a a a a a
13.796 Carbamic acid (CH 3NO 2) 278, 205, 147, 73, 453 0.159 84.1 21.165 L-Proline, 5-oxo-1- 230, 156, 73, 45 2.049 91.1 (trimethylsilyl)-, trimethylsilyl
ester (C 11 H23 NO 3Si 2)
24.373 Lauric acid (C 12 H24 O2) 257, 201, 117, 73, 55 1.143 86.2
24.881 Naphthalene (C 15 H18 ) 183, 83, 39 0.429 85.5 28. 609 Myristic acid (C14:0) 458, 368, 392, 247, 1.298 88.1 129, 73 32.055 Palmitic acid, isopropyl ester 298, 256, 213, 157, 0.734 71.1 (C16:0) 129, 102, 60, 43 32.465 Palmitic acid (C16:0) 313, 269,201, 117, 73, 2.456 88.3 43 36.015 Octadecanoic acid (C18:0) 341, 201, 117, 73, 43 1.345 90.9 ret. time = retention time; m/z = the mass of an ion divided by the electrical charge of the ion
GC-MS analysis of MeOH fraction obtained from ethyl acetate extract of the cultivation medium, revealed the presence of different saturated fatty acids and the
78
Chapter III Results unsaturated 11-cis- octadenoic acid. Furthermore naphthalene, carbamic acid and 5- oxoproline were found. However, the GC-MS technique is most suitable for thermostable and volatile substances, but not for non-volatile ones and chromatogram obtained from analytical HPLC of this methanol fraction revealed the presence of other compounds. Thus, further separation of the mixture of other substances in this methanol fraction is necessary to verify whether only compounds identified so far (see table 3.8 and 3.9) were responsible for very strong antimicrobial activity of this methanol fraction. a Westiellopsis Cultivation of Westiellopsis sp. VN in BG11 without nitrate and in normal
BG11 medium containing 1.5 g/L NaNO 3 (see fig. 3-21) showed that the amounts of dry biomass and the yields of methanol extracts did not differ significantly at different nitrogen concentrations. The yields of the lipophilic n-hexane and EtOAc extracts in the nitrogen-free medium were higher than those in nitrogen-containing medium Fermenters for cultivation of Westiellopsis sp.VN containing standard BG11 medium and BG-11 medium without NaNO 3, illuminated continuously with 8µmol/m2 at 28°C, pH 7.4, CO 2 supplementation
Based on diameter of inhibition zone (IZ), the concentration of antibacterial compounds in methanol extracts of biomass and ethyl acetate extracts of growth
79
Chapter III Results medium did not change markedly while antibacterial compound concentration of n- hexane and ethyl acetate extracts of biomass changed significantly (see table 3-10, fig. 3-22).
a Dry biomass and antibacterial activity against S.aureus of extracts in BG-11 medium
a a
Lyophilized biomass 14.750g 14.884 g Yield of methanol extract from biomass 1153.6 mg 1192.3 mg Yield of EtOAc extract from biomass 157.7 mg 91.8 mg Yield of EtOAc extract from medium 98.7 mg 70.0 mg Yield of n-hexane extract from biomass 132.1 mg 51.1 mg IZ of n- hexane extract from biomass 18.0 mm 9.0 mm IZ of EtOAc extract from biomass 18.0 mm 12.5 mm IZ of MeOH extract from biomass 17.0 mm 19.0 mm IZ of EtOAc extract from medium 29.0 mm 27.0 mm IZ {Diameter of I nhibition zone including diameter of paper disc (6 mm)}
Biomass Growth medium
BG11 0 BG11 BG11 BG11 hex. 0 hex.
EtOAc. EtOAc
MeOH MeOH EtOAc. EtOAc.
Agar diffusion test of extracts prepared from biomass and cultivation medium of Westiellopsis sp.VN grown in BG-11 media S.aureus, C=2mg/disc
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Chapter III Results
a a a a a a The biomass yield and dry weight respectively increased continuously to the end of the 7-to 8- week growth period. Extracts prepared from biomass harvested after 4 weeks of cultivation showed an increased diameter of inhibition zone in agar diffusion assay against S. aureus . Synthesis of antibiotic substances seems to be constant over the next 2 weeks and increases slightly during the last two weeks of cultivation time (see fig.3-23a and 3-23b).
50 25 45 40 20 35 30 15 25 20 10 15 10 5 5
Dry weight (mg/ 100ml0 culture) 0 0 1 2 3 4 5 6 7 8 9 Diameter of Inhibition zone (mm) I ncubation time (weeks)
Dry weight Diameter of Inhibition zone
a The effect of incubation time on dry weight and antibiotic production of Westiellopsis sp.VN
Agar diffusion test of MeOH extracts of Westiellopsis sp.VN biomass Methanol extracts prepared from biomass cultivated for 2, 3, 4, 5, 6, 7, and 8 weeks, respectively. Test organisms S.aureus, C=2mg/disc
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Chapter III Results
a a Calothrix javanica The lyophilized biomass obtained from batch culture (see 2.5.2) was extracted with n-hexane, methanol, and water, respectively to give three extracts. The methanol extract exhibited the strongest activity against B. subtilic and S.aureus and was therefore selected for chemical investigation. The investigation of this active methanol extract led to isolation of a new cyclic peptide named daklakapeptin according to the locality of the strain. a a a a a a a
210 mg of MeOH extract was chromatographed on a reverse-phase C 18 column eluting with 5 solvent systems starting with MeOH/EtOH/H 2O (45:45:10), followed by MeOH/H 2O (9:1), MeOH, and washing with MeOH/C 3H6O (1:1) and
CH 2Cl 2, respectively to yield 10 fractions (CJF I to CJF X) (see 2.8.2). All obtained fractions were evaluated for their antibacterial activities against S. aureus in agar diffusion assay. The result of this assay revealed that fraction CJF II eluting with
MeOH/EtOH/H 2O (45:45:10) possesses the strongest activity (see table 3-11), therefore it was further purified.
a Fractionation of methanol extract from Calothrix javanica biomass by RP C 18 chromatography and antibacterial activity of fractions to S. aureus a a a a 2 CJF I 1-8 MeOH/EtOH/H 2O (45: 45:10) 33.8 16.10 500 0
CJF II 9-24 MeOH/EtOH/H 2O (45: 45:10) 89.9 42.81 500 9.0
CJF III 25-30 MeOH/EtOH/H 2O (45: 45:10) 1.8 0.86 500 7.0
CJF IV 31-34 MeOH/EtOH/H 2O (45: 45:10) 1.2 0.57 500 0
CJF V 35-40 MeOH/EtOH/H 2O (45: 45:10) 1.4 0.67 500 0
CJF VI 41-50 MeOH/EtOH/H 2O (45: 45:10) 8.4 4.00 500 7.0
CJF VII 51-100 MeOH/ H 2O (90:10) 17.8 8.48 500 7.5
CJF VIII 101-150 MeOH 14.5 6.90 500 0
CJF IX 151-200 MeOH/CH 3COCH 3 (50:50) 14.2 6.76 500 0
CJF X 201-250 DCM 22.8 10.86 500 0 Total 205.8 98.01 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
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Chapter III Results
a a a a a
First, the active fraction CJF II was analyzed by HPLC with a solvent gradient of acetonitrile/water (table 3-12, figure 3-24).
a Step gradient used in HPLC analysis of CJF II by analytical HPLC 0.50 25.5 26.50 30.50 32.00 95 5 0 0.0 95 5 95 100 100 5 Solvent A: H 2O and solvent B: CH 3CN; Flow rate: 1.0 mL/min; HPLC column: Synergi polar- RP80A/250×4.6mm, 4 micron
Analytical HPLC chromatogram of fraction CJF II with mobile phase of acetonitrile/water (table 3-12) and 2mg/mL /injection, Synergi POLAR-RP 80A column (250×4.6mm, 4 micron), flow rate 1mL/min, detection at 220, 246 nm, respectively.
Chromatogram obtained from analytical HPLC revealed that purification of fraction CJF II by reversed-phase HPLC was possible. To optimize separation process, the mobile phase was changed and the mobile phase of MeOH/ water acidified with 0.05% TFA was tested. With this mobile phase a better separation was achieved (see table 2-3) used for a successful separation of fraction CJF II by semi-preparative HPLC. Altogether, 86 mg of fraction CJF II were purified. 7 peaks CJF II-1 to CJF II-7 were collected and controlled at the wave length of 220 nm to afford 7 seven fractions (see table 3-13).
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Chapter III Results
a Separation of CJF II from Calothrix javanica biomass by semi- preparative reversed-phase HPLC and activity of the fractions to S. aureus
a a
CJF II -1 3.66 1.3 1.51 100 6.5 2 CJF II -2 4.54 43.4 50.47 200 0
CJF II -3 11.38 0.7 0.81 100 0
CJF II -4 12.93 3.8 4.42 200 12.5
CJF II -5 21.28 3.0 3.49 100 7.0
CJF II -6 26.63 4.4 5.12 100 15.0
CJF II -7 30.44 1.6 1.86 100 0 Total 58.2 67.68 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
Seven fractions were analyzed by analytical HPLC using a step gradient of MeOH/ water + 0.05% TFA as mobile phase described in table 3-14 . In analytical
HPLC fraction CJF II-4 was the most pure one (see fig.3-25) and therefore used for structural elucidation.
a Step gradient used in HPLC analysis of seven fractions by analytical HPLC 0.5 16.5 24.5 30.5 31.0 50 15 0 0 50 50 85 100 100 50 Solvent A: H 2O+0.05%TFA and solvent B: MeOH; Flow rate: 1.0 mL/min; HPLC column: Synergi polar-RP80A/250×4.6mm, 4 micron
Analytical HPLC chromatogram of fraction CJF II-4 (mobile phase see table 3-14), 2mg/mL /injection, detection at 220 nm, column Synergi POLAR-RP 80A (250×4.6 mm, 4 micron), flow rate 1mL/min.
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Chapter III Results
a a
Fraction CJF II-4 was found to be a new cyclic peptide named daklakapeptin.
Daklakapeptin (compound 7) The structure of the peptide was elucidated using a combination of high- resolution ESI MS and 2D NMR techniques. Initially spin systems in the homonuclear 2D 1H TOCSY spectrum were identified starting from the backbone amide protons in the region 8.2 to 7.7 ppm. All protons of ten residues were found of which five could be specifically identified from their characteristic chemical shifts, namely Tyr, Gln, Leu, Ile and Thr (see table 3-15, systems 3, 4, 5, 7 and 10). The shifts of four others (1, 2, 6 and 9) indicated the presence of unusual spin systems. Careful integration of the 1D spectrum at 7.8 ppm indicated the α- and β-protons of a Thr residue (system 8) overlap at 4.21 ppm. The presence of Tyr and Gln (3 and 4) was confirmed from the identification of their aromatic and terminal CONH 2 group, respectively, in the region 7.5 to 6.5 ppm. Two further spin systems lacking amide protons were then identified from correlations in the Hα region of the spectrum, 5.0 to 4.0 ppm. These, from the characteristic shifts, corresponded to Ile and Pro residues (see table 3-15, systems 11 and 12). Overlap in the amide region of the spectrum that caused some ambiguities in system identification was clarified by comparison with the spin system patterns in the Hα region and the high-field region of the spectrum. Comparison of these data with the cross peaks in the 2D COSY spectrum confirmed the intra-residue assignments and established the nature of the unusual spin systems (see table 3-15). Sequence specific assignments were determined from the cross-peaks in the 2D 1H NOESY spectrum based on short observable distances between HN, Hα and
85
Chapter III Results
Hß of amino acid i and HN of amino acid i+1. The data, table 3-16, afforded two possible combinations for the major sequence fragment of 10-2-12-3-8-9-7-1-6-4/5 or 2-10-12-3-8-9-7-1-6-4/5 together with a minor fragment 4-11. This same spectrum indicated that the substituted Ile residue (system 11) was in fact an N-methyl substituted unit. As in previous work heteronuclear 2D correlations were then recorded with the intention of providing further sequence information through the observation of correlations in the HMBC spectrum of HN with the amide carbonyl carbons. Unfortunately most of carbonyl carbons were restricted to a narrow band in the 13 C spectrum that prevented an unambiguous assignment. Thus only the sequence 9-7 was confirmed. However the combination of the HMQC and HMBC data did allow a complete assignment of the 13 C shifts of the side-chains of the individual units (see table 3-15) that confirmed the nature of these residues. The structure of the molecule was finally established using high-resolution ESI mass spectrometric data. The protonated molecular ion in the HR-ESI-MS of + [M+H] at 1432.8289 corresponded with a molecular composition of C 68 H114 O20 N13 which agreed with the molecular formula C 68 H113 O20 N13 and mass calculated independently from the structure of the individual residues deduced from the NMR data (see table 3-17) and indicated the molecule was a cyclic peptide. A detailed analysis was then made of the fragmentation pattern in the high-resolution ESI MS. This indicated the cyclic peptide initially underwent cleavage at the amide bond directly prior to the proline residue. Subsequent fragmentation of this linear molecular ion then afforded an unambiguous sequence that is independent, but compatible with one of the possible sequences deduced from the NMR data, (b) in the footnote of table 3-16. The comparison of the sequence from the fragmentation in the HR-ESI-MS and the NMR data is shown in table 3-18 and the corresponding complete structure of the molecule is shown in the figure 3.26.
a NMR data of CJF II-4 1 NH 8.14 CO Cα 82.0 4.57 Cβ 79.7 3.74 Cγ 32.8 1.75 Cδ 20.3, 19.9 1.09, 0.92 2-Complex NH 8.12 CO 174.3 Cα 64.8 4.65 Cβ 73.1 4.27 Cγ 64.8 3.60 86
Chapter III Results
3-Tyr NH 8.04 CO Cα 57.3 4.64 Cβ 38.2 3.17, 2.91 Arom C1:130.3, C2,6: 132.2, H2/6: 7.13, H3/5: 6.86 C3,5:117.6, C4: 157.0 4-Gln NH 8.03 CO Cα 51.6 4.85 Cβ 28.3 2.08, 1.93 Cγ 32.8 2.32 CONH 2 179.5 7.28, 6.59 5-Leu NH 8.03 CO Cα 55.1 4.37 Cβ 42.2 1.65 Cγ 26.3 1.60 Cδ 23.7, 21.9 0.95, 0.91 6-Complex NH 7.99 CO Cα 53.3 4.60 Cβ 35.3 2.18, 1.95 Cγ 60.0 3.70, 3.59 7-Ile NH 7.85 CO Cα 61.4 4.26 Cβ 38.3 1.93 Cγ 27.0, 16.5 1.57, 1.25, 0.98 Me Cδ 11.6 0.91 8-Thr NH 7.79 CO Cα 62.8 4.21 Cβ 69.1 4.21 Cγ 20.3 1.09 9§ CO -1 175.6 CH 2-2 42.1 2.56 CH(NH-)-3 49.9 4.18, 7.78 NH CH 2-4 35.1 1.57 CH 2-5 27.6 1.38 CH-6 33.2 1.31 (CH 3)2-7 24.0, 14.7 1.31, 0.88 10-Thr NH 7.77 CO Cα 59.0 4.66 Cβ 68.8 4.28 Cγ 20.8 1.24 11-IleNMe N-CH 3 32.9 3.19 CO Cα 64.6 4.67 Cβ 34.2 2.11 Cγ 26.7, 16.5 1.40, 1.05; 0.97 Me Cδ 11.2 0.90 12-Pro CO 175.5 Cα 63.4 4.39 Cβ 30.9 2.13, 1.70 Cγ 26.5 2.00, 1.96 Cδ 50.1 3.82, 3.68 Footnote § 9 is (CH 3)2.CH.CH 2.CH 2.CH(NH-).CH 2 CO- 87
Chapter III Results
a Sequence information deduced from the NOE’s found in the 2D NOESY spectrum of CJF II-4
12 (Pro)-3 12α-8NH? 3-8 3-8 3ß-8NH(x2) 3H2/6 – 8NH 8-9 9(CH 2)-7 9(CHß)-7NH 7-1 7-1 7ß-1NH, 7γ-1NH 7α-6NH? 1-6 6-4or 5 4-11Nme
2-10 or 10-2 2α and 10α overlap 2α /10α-12α(x2), 2α/10α-12ß(x2) (or reverse) 4γ-CONH2γ Summary: Possible sequence: (a) 2-10-12-3-8-9-7-1-6-4/5 or (b) 10-2-12-3-8-9-7-1-6-4/5, 4-11
a Calculation of the molecular mass from the structure of the residues deduced from the NMR data.
a a a 1 C6 H 11 NO 2 129 (CH 3)2 CHCH(OH)CH(NH)CO 2 C4 H 7 NO 3 117 HOCH 2CHOHCH(NH)CO 3-Tyr C9 H 9 NO 2 163.2 4-Gln C5 H 8 N 2 O 2 128.1 5-Leu C6 H 11 NO 113.2 6 C4 H 7 NO 2 101 HOCH 2CH 2CH(NH)CO 7-Ile C6 H 11 NO 113.2 8-Thr C4 H 7 NO 2 71.1 9 C8 H 15 NO 141 (CH 3)2.CH.CH 2.CH 2.CH(NH-).CH 2CO- 10-Thr C4 H 7 NO 2 101.1 11-IleNMe C7 H 13 NO 127.2 12-Pro C5 H 7 NO 97.1
Total C68 H113 N13 O20 1431
88
Chapter III Results
a Comparison of the sequence from the high-resolution ESI-MS data with that from the NMR data. Sequential loss of 261.1234 101.0477 141.1154 113.0841 129.0788 101.0479 113.0840 128.0586 127.0988 50.5237 x2 58.5212 x2 exact fragment C14 H17 O3N masses in the HR- 2 ESI-MS [M+H] + Molecular formula C5H7NO + C4H7NO 2 C8H15 NO C6H11 NO C6H11 NO 2 C4H7NO 2 C6H11 NO C5H8N2O2 C7H13 NO C4H7NO 2 C4H7NO 3 of fragments C9H9NO 2 sequentially lost in the HR-ESI-MS Sequence from the Pro - Tyr Thr (Leu+2CH 2) Leu Leu+O Thr Leu Gln Ile-NMe Thr Thr+O NMR analysis a b c Footnote: The structures of the complex residues are as follows: a) (CH 3)2CH.CH 2CH 2CH(NH-)CH 2CO-, b) (CH 3)2CHCH(OH)CH(NH-)CO-, c) HOCH 2CHOHCH(NH-)CO-.
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Chapter III Results
a a Scytonema ocellatum The investigation of the active methanol extract obtained from lyophilized biomass from batch culture of this strain led to isolation of a cyclic peptide which was the same as that isolated from Calothrix javanica .
a a a a a a a a In the first purification step, 230 mg of the methanol extract were fractionated on RP C 18 column with three solvent systems (see 2.8.3) to get 8 fractions (SOF I to SOF VIII ) which were evaluated for antibacterial activity against S. aureus using agar diffusion method (see table 3-19).
a Fractionation of methanol extract from Scytonema ocellatum biomass by RP-18 chromatography and antibacterial activity of fractions to S. aureus
a a a a 2 SOF I 1-2 MeOH/EtOH/H 2O 66.6 28.96 1.0 0 (45:45:10)
SOF II 3-10 MeOH/EtOH/H 2O 76.3 32.74 1.0 10.0 (45:45:10)
SOF III 11-12 MeOH/EtOH/H 2O 1.7 0.74 1.0 0 (45:45:10)
SOF IV 13-20 MeOH/EtOH/H 2O 9.5 4.13 1.0 0 (45:45:10)
SOF V 21-23 MeOH/EtOH/H 2O 8.4 3.65 1.0 0 (45:45:10)
SOF VI 24-40 MeOH/EtOH/H 2O 19.3 8.39 1.0 0 (45:45:10)
SOF VII 41-80 MeOH 43.3 18.83 1.0 0
SOF VIII 81-120 MeOH/CH 3COCH 3 2.5 1.09 1.0 0 (10:10) Total 226.6 98.53 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
Because only fraction SOF II showed strong antibacterial activity, this fraction was further separated.
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Chapter III Results
a a a a
To remove more unwanted substances still remaining in the active fraction
SOF II, silica gel column chromatography was employed.
74.3 mg of fraction SOF II were subjected to silica gel column eluting with a step gradient of n-hexane/EtOAc/MeOH (see 2.8.3) to yield 10 fractions. All these fractions were tested for their antibacterial activity against S. aureus in agar diffusion assay (see table 3-20)
a Separation of SOF II from Scytonema ocellatum biomass by silical gel chromatography and antibacterial activity of the fraction to S. aureus
a a a a 2 SOF II-1 1-4 n-hexane/EtOAc 5.9 7.94 500 0 (25:75)
SOF II-2 5-20 n-hexane/EtOAc 1.1 1.48 500 0 (25:75)
SOF II-3 21-36 EtOAc 1.1 1.48 500 0
SOF II-4 37-43 EtOAc/MeOH (50:50) 1.1 1.48 500 0
SOF II-5 44-45 EtOAc/MeOH (50:50) 17.8 23.96 500 9.0
SOF II-6 46-51 EtOAc/MeOH (50:50) 11.7 15.75 500 8.0
SOF II-7 52-63 MeOH 5.8 7.81 500 0
SOF II-8 64-67 MeOH 10.9 14.68 500 0
SOF II-9 68-70 MeOH 3.8 5.11 500 0
SOF II-10 71-72 MeOH 2.4 3.23 500 0 Total 61.6 82.92 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
The fractions SOF II-5 and SOF II-6 eluting with 50% EtOAc/MeOH exhibited approximately the same antibacterial activity. Thus, these two fractions were pooled and further purified.
a a a
Final purification of the pooled fractions SOF II-5 and SOF II-6 (26.0 mg) was achieved by reversed-phase HPLC (see 2.8.3) to yield three fractions. All these fractions were evaluated for antibacterial activity against S. aureus (see fig.3-27 and
91
Chapter III Results table 3-21).
FSO 2
FSO 3
FSO 1
Semi-preparative RP HPLC chromatogram of the pooled fractions SOF II-5 and SOF II-6 with mobile phase (table 2-4) and 500µg/50 µL /injection, detection at 220 nm, column Synergi POLAR-RP 80A (250×10mm, 4 micron), flow rate 3mL/min.
a Separation of SOF II-5 and SOF II-6 from Scytonema ocellatum biomass by semi-preparative reversed-phase HPLC and antibacterial activity of the fractions to S. aureus
a a 2 FSO 1 3.08 1.0 3.85 200 0
FSO 2 4.29 13.4 51.54 200 0
FSO 3 14.32 2.4 9.23 200 12.0 Total 16.8 64.62 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
The active fraction FSO 3 was analyzed by analytical HPLC (see fig.3-28) with a step gradient as described in table 3- 22 and then used for structure elucidation.
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Chapter III Results
a Step gradient used in HPLC analysis of FSO 3 by analytical HPLC 0.50 18.50 21.50 24.50 25.00 95 5 0 0.0 95 5 95 100 100 5 Solvent A: H 2O and solvent B: CH 3CN; Flow rate: 1.0 mL/min; HPLC column: Synergi polar- RP80A/250×4.6mm, 4 micron
Analytical HPLC chromatogram of fraction FSO 3 (mobile phase see table 3-22), 2mg/mL /injection, detection at 220 nm, column Synergi POLAR-RP 80A (250×4.6 mm, 4 micron), flow rate 1mL/min.
a a
Fraction FSO 3 was found to be the same new cyclic peptide also found in the biomass of Calothrix javanica (see 3.3.3).
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Chapter III Results
a a Anabaena The investigation of the ethyl acetate extract resulting from the cultivation medium of this strain led to isolation of fluourensadiol. Because the crude extracts obtained from Anabaena sp. strain revealed a strong and wide range of antimicrobial activity in our antibacterial screening (see 3.1.2), this strain was chosen for bioassay-guided isolation of active compounds. In order to pursuit sufficient material for isolation and structure elucidation of active metabolites, this strain was cultured in large scale as described in 2.5.3. The biomass and cultivation medium were separated by centrifugation and filtration (see 2.5.3). The lyophilized biomass was then extracted with n-hexane, methanol, and water, respectively as described in 2.6.1 to yield three extracts (n-hexane, methanol, and water extracts) and the microscopically cell-free cultivation medium was extracted with ethyl acetate as described in 2.6.2 to afford ethyl acetate extract. All these extracts were tested for antimicrobial activity against Gram-positive and Gram- negative bacteria and yeast Candida maltosa . It is interesting that among of all extracts obtained from large scale culture, all extracts prepared from biomass exhibited no activity against all test microorganisms while only the ethyl acetate extract from the microscopically cell-free cultivation medium exhibited very strong activity against all test microorganisms (see table 3-23). Thus, this active ethyl acetate extract was chosen for further bioassay-guided isolation.
a Antibacterial activity of extracts from Anabaena sp. cultivated in large scale Diameter of inhibition zone (mm) 1 B.subtilic S.aureus E.coli C. maltosa Ethyl acetate ext. from growth medium 15.0 16.0 24.0 16.0 Methanol ext. from biomass 02 0 0 0 n-hexane ext. from biomass 0 0 0 0 Water ext. from biomass 0 0 0 0 Ext. =extract; 1Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect.
a a a a a Based on the result of the HPLC analysis, separation of the active crude ethyl acetate extract by reversed-phase HPLC was done. Altogether, 12 mg of ethyl
94
Chapter III Results acetate extract obtained from culture medium was purified with a step gradient of
CH 3CN/H 2O as mobile phase described in table 2-5. The collection of the separated peaks was controlled at 238 nm and 254 nm. Seven peaks at retention times of 12.75, 15.76, 17.71, 18.33, 19.32, 20.77, and 22.77 min were collected respectively to yield 7 fractions (see fig.3-29). All these fractions were tested for antimicrobial activity (table 3-24, fig.3-30)
AF 3
AF 5
AF 6
AF 1
AF 2
AF 4
firstfirst
Semi-preparative RP HPLC chromatogram of the crude EtOAc extract from culture medium (mobile phase see table 2-5), 1mg/50 µL/injection, detection at 238 nm, column Synergi POLAR-RP 80A (250×10 mm, 4 micron), flow rate 3 mL/min.
a Separation of EtOAc extract from Anabaena sp.culture medium by semi- preparative reversed-phase HPLC and antibacterial activity of the fractions to E. coli a a first 12.75 0.3 2.50 200 02
AF 1 15.76 0.6 5.00 200 0
AF 2 17.71 0.2 1.67 200 0
AF 3 18.33 0.4 3.33 200 0
AF 4 19.32 0.3 2.50 200 0
AF 5 20.77 0.2 1.67 200 0
AF 6 22.77 1.3 10.83 200 20.0 Total 3.3 27.50 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
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Chapter III Results
Agar diffusion test of 7 fractions obtained from ethyl acetate extract of culture medium of Anabaena sp. Test organism E.coli with c=0.2mg/disc
Because only fraction AF 6 exhibited antimicrobial activity, this fraction was used for structure elucidation.
a a
Fraction AF 6 was found to be the the pure substance flourensadiol (see fig.3- 31)
Flourensadiol, molecular formula C 15 H26 O2
The HR-ESI-MS showed a sodiated molecular ion at m/z 261.1825 that was compatible with the molecular formula C 15 H26 O2. The 2D COSY spectrum allowed the unambiguous identification of two fragments in the molecule, namely
CH 3.CH.CH 2.CH 2 and CH 2.CH.CH.CH, together with signals of an isolated CH 2OH 13 and two singlet CH 3 groups. The C and DEPT-135 NMR spectra indicate the presence of 15 carbon atoms consisting of 3 methyl, 5 methylene, 5 methine and 2 quaternary carbons. A HMQC spectrum allowed correlation of these with the system 96
Chapter III Results identified in the COSY spectra and identified a further complex methylene system and methine system (see table 3-25). Two and three through-bond correlations in the long-range 2D HMBC spectrum identified two systems incorporating the singlet methyl groups and the isolated CH 2OH group as CH 3.C*(CH 2OH) and CH 3.C*(OH) where C* are quaternary carbons (see table 3-25). At this stage the Chapman and Hall data bank was searched using the molecular formula and the characteristic CH 3.C*(CH 2OH) fragment. Thirty one compounds were found of which only one, Flourensadiol (Murakami et al., 2001 and Kingston et al., 1975) was compatible with the other fragments found in the molecule. Further detailed inspection of the COSY and HMBC spectra confirmed that the structure deduced from the NMR data were indeed compatible with the structure of fluorensadiol originally determined by X-ray crystallography (Pettersen et al., 1975). Characteristic NOE’s observed in the 2D ROESY spectrum (see table 3-25) confirmed the relative stereochemistry was identical with the structure reported previously. We report the complete NMR data for the first time in the table 3-25.
a NMR data of AF 6 (Flourensadiol) in trifluoroethanol-d2/H 2O (1:1)
a 1 77.4 s 2 A 1.77 m 38.4 t B 1.64 m C1, C8 3 A 1.78 m 20.0 t C1 B 1.47 ddd 14.3,11.8, 11.8 C2, C4, C5/C6, C1 4 0.83 ddd 11.8, 9.4, 5.8 29.8 d C5/C6, C13 5 25.0 s 6 0.38 dd 9.3, 9.3 25.1 d C4, C5, C3, C11, H4, H7, C13, C14 H9B, H10B, H12, H13 7 1.87 m 40.8 d C1 8 1.87 m 59.1 d 9 A 1.70 m 26.4 t B 1.59 m C8 10 A 1.82 m 29.6 t B 1.30 m C9 11 2.00 m 39.8 d --- 12 0.96 d 6.8 16.8 q 13 1.11 s 24.2 q C5, C4, C6, C14 14 A 3.77 d 11.6 65.3 t C5/C6, C4, C13 H7, H3B, B 3.71 d 11.6 C5/C6, C4, C13 H13 15 1.17 s 32.1 q C1, C2, C8 H2A, H2B,H8, H9A 97
Chapter III Results
a a Nostoc The lyophilized biomass obtained from large scale culture (see 2.5.3) of Nostoc sp. was extracted with n-hexane, methanol, and water, respectively to give three extracts. Of three extracts, methanol extract exhibited the strongest activity against Gram-positive bacteria ( B. subtilic and S.aureus) and Gram-negative bacteria (E.coli) and was therefore chosen for bioassay-guided isolation of antibacterial metabolites.
a a a a a a a 760 mg of MeOH extract was first fractionated on silica gel column eluting with a step gradient of n-hexane/EtOAc/MeOH/H 2O (see 2.8.5) to yield 5 fractions which were evaluated for their antibacterial activities against S. aureus using agar diffusion assay (see table 3- 26).
a Fractionation of methanol extract from Nostoc sp. biomass by silical gel chromatography and antibacterial activity of fractions to S. aureus
a a a 2 NF I n-hexane/EtOAc (90:10) 20.0 2.63 1.0 0.0
NF II n-hexane/EtOAc (40:60) 93.5 12.30 1.0 6.5
NF III n-hexane/EtOAc (20:80) 36.1 4.75 1.0 0.0
NF IV MeOH 363.6 47.84 1.0 10.0
NF V MeOH/H 2O (95:5) 26.3 3.46 1.0 0.0 Total 539.5 70.98 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
Because fraction NF IV showed the strongest activity, this fraction was further separated. a a a a a a
360 mg of the active fraction NF IV was subjected to C 18 column eluting with three solvent systems (see 2.8.5) to afford three fractions which were evaluated for
98
Chapter III Results their antibacterial activities against S. aureus using agar diffusion assay (see table 3- 27).
a Fractionation of NF IV from Nostoc sp. biomass by RP C 18 chromatography and antibacterial activity of fractions to S. aureus
a a a
NF IV-1 MeOH/H 2O (90:10) 151.6 42.11 0.5 11.0 2 NF IV-2 MeOH 96.3 26.75 0.5 0.0
NF IV-3 DCM 42.8 11.89 0.5 0.0 Total 290.7 80.75 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
Only fraction NF IV-1 exhibited antibacterial activity and was further purified.
a a a a a a Final purification of the active fraction NF IV-1 (150 mg) was achieved by repeated reversed-phase HPLC to afford 4 fractions. All obtained fractions were evaluated for antibacterial activity against S. aureus using agar diffusion assay (see fig.3-32, table 3-28)
NsF 1
NsF 3 NsF 4
Semi-preparative RP HPLC chromatogram of fraction NF IV-1 with mobile phase of MeOH/ water + 0.05% TFA (see table 2.6) and 1mg/50 µL/injection, detection at 238 nm, column Synergi POLAR-RP 80A (250×10 mm, 4 micron), flow rate 3 mL/min.
99
Chapter III Results
a Fractionation of NF IV-1 from Nostoc sp. biomass by semi-preparative RP HPLC and antibacterial activity of fractions to S. aureus a a 2 NsF 1 4.73 25.4 16.93 500 0.0
NsF 2 5.48 3.6 2.40 500 10.0
NsF 3 6.26 3.5 2.33 500 0.0
NsF 4 7.71 1.7 1.13 500 0.0 Total 1. Diameter of inhibition zone (mm) includes Ø disc (6mm), 2. no effect
Only fraction NsF 2 showed antibacterial activity, this fraction was therefore used for structure elucidation.
a a
The low resolution ESI-MS of fraction NsF 2 which exhibited antibacterial activity against Staphylococcus aureus with diameter of inhibition zone of 10.0mm in concentration of 500mg/dics showed signal at m/z 426 [M+H] +. The NMR and MS characterization of compound in this fraction NsF 2 is in progress.
a a Lyngbya majuscula
Chemical investigation of this strain using two methods resulted in the isolation of the 3 cytotoxic compounds anhydrodebrommoaplysiatoxin, debromoaplysiatoxin, and anhydroaplysiatoxin. Method1: The methanol extract resulting from lyophilized biomass of L.majuscula prepared as described in 2.8.6.1 was separated by repeated silica gel chromatography followed by preparative TLC to afford 17-debromo-3,4-didehydro-3- deoxy-aplysiatoxin {(anhydrodebrommoaplysiatoxin) , 1.8 mg} with a yield of 0.12% based on the mass of the crude extract. Method 2: The methanol extract from lyophilized biomass of L.majuscula prepared as described in 2.8.6.2 was separated by silica gel chromatography followed by reversed-phase HPLC to afford debromoaplysiatoxin, anhydroaplysiatoxin (3.8 mg and 2.5mg ) with a yield of 0.12% and 0.21% , respectively, based on the mass of crude extract.
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Chapter III Results
a a a a a a The lyophilized biomass of this strain was extracted with n-hexane, methanol, and water, respectively to give three extracts. Because the methanol extract exhibited considerable cytotoxic activity, it has been chosen for isolation of the cytotoxic substances using bioassay-guided fractionation.
a a a a a a a In the first purification step, silica gel column chromatography of the methanol extract (620 mg) using a stepwise gradient of n-hexane/EtOAc/ MeOH (see
2.8.6.1) yielded 15 major fractions (F 1 to F 15 ). Based on the results of TLC analysis, 7 fractions of these 15 fractions were tested for their cytotoxic activity (see table 3-29 and fig.3-33)
a Fractionation of methanol extract from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions against cell line 5637
a a
a
F1 1-20 n-hexane/EtOAc (60:40) 1.86 0.3 n.e
F2 21-35 n-hexane/EtOAc (60:40) 8.06 1.3 4.2
F3 36-60 n-hexane/EtOAc (60:40) 24.18 3.9 10.0
F4 61-70 n-hexane/EtOAc (60:40) 2.79 0.45 0.1
F5 71-154 n-hexane/EtOAc (40:60) 40.61 6.55 6.5
F6 155-216 n-hexane/EtOAc (40:60) 60.45 9.75 n.e
F7 217-240 100% EtOAc 19.22 3.1 23.3
F8 241-308 100% EtOAc 119.6 19.29 8.4
F9 309-423 EtOAc/ MeOH (60:40) 17.98 2.9 2.1
F10 424 EtOAc/MeOH (40:60) 0.31 0.05 n.e
F11 425-431 EtOAc/MeOH (40:60) 0.93 0.15 n.e
F12 432-434 EtOAc/MeOH (40:60) 33.17 5.35 n.e
F13 435-437 EtOAc/MeOH (40:60) 17.05 2.75 n.e
F14 438-556 EtOAc/MeOH (40:60) 171.12 27.60 n.e
F15 557-640 100% MeOH 99.51 16.05 n.e Total 616.84 99.49 n.e. not estimated
101
Chapter III Results
Thin layer chromatogram of 15 fractions obtained from silica gel column eluted with a stepwise gradient of n-hexane/EtOAc/MeOH (method1). {Mobile phase: n-hexane/EtOAc/MeOH/CH 3COOH (75+25+5+3); Detection: Vis after spraying with Anisaldehyde/sulfuric acid reagent}
Because fraction F 8 eluting with 100% EtOAc exhibited a strong cytotoxic activity against 5637 cells (bladder cancer cell line) and only one main spot in TLC was detected, it was selected for further separation.
a a a a a a
A portion of fraction F8 (90 mg) was further purified by silica gel column chromatography with a stepwise gradient from 60% n-hexane to 90%EtOAc to 100% MeOH as mobile phase ( see 2.8.6.1) to give six main fractions which were tested for their cytotoxic activity (see table 3-30).
a Separation of fraction F 8 from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions to 5637 cell line
a a a
F8-1 1-112 n-hexane/EtOAc (40:60) 9.6 10.67 0.16
F8-2 113-148 n-hexane/EtOAc (10:90) 118 13.11 0. 029
F8-3 149-208 n-hexane/EtOAc (10:90) 31.21 34.68
F8-4 209-214 n-hexane/EtOAc (10:90) 2.9 3.22
F8-5 215-225 n-hexane/EtOAc (10:90) 8.4 9.33
F8-6 226-325 100%MeOH 20.09 22.32 Total 84.00 93.33
Of these six fractions, fraction F8-3 exhibited the strong cytotoxic activity and the highest yield and therefore it was selected for further purification.
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Chapter III Results
a a a a
30.2 mg of fraction F8-3 was further purified by preparative TLC. The chromatogram was developed with n-hexane/EtOAc/MeOH/CH 3COOH (75:25:5:3), five spots were scratched out to yield five fractions (see table 3-31).
a Separation of fraction F8-3 from L.majuscula biomass by preparative TLC
a a
F8-3-1 0.28 1.2 3.97
F8-3-2 0.22 3.1 10.26
F8-3-3 0.18 1.7 5.62
F8-3-4 0.12 2.0 6.62
F8-3-5 0.045 0.1 0.33 Total 8.1 26.8
All five fractions (F 8-3-1 to F 8-3-5) were examined by analytical TLC and
HPLC. The result of HPLC analysis showed that fraction F 8-3-2 (3.1 mg) was pure enough for structure elucidation by ESI-MS and NMR.
a a
Fraction F 8-3-2 was found to be the pure compound, 17-debromo-3,4- didehydro-3-deoxy-aplysiatoxin (anhydrodebromoaplysiatoxin), a known metabolite (see fig.3-34)
25 26 5 CH3 H3C 4 6 CH 7 3 3 24 32 2 O 8 O CH3O 1 9 17 10 12 14 16 O O O 11 13 15 18 27 30 19 H3C CH3 CH3 21 31 29 O 23 22 28 20 OH OH
17-debromo-3, 4didehydro-3-deoxy –aplysiatoxin, molecular formula C 32 H 46 O9
The high resolution ESIMS in positive mode of compound 9 showed ions at m/z 543.2947 [M+H-MeOH] +, m/z 575.3209 [M+H] +, m/z 597.3027 [M+Na] +, and m/z 613.6773 [M+K] + (see fig.3-35) and the molecular weight was deduced to be 574
103
Chapter III Results
Dalton compatible with the molecular formula C32 H 46 O9 (calculated 575.3215 for C32
H47 O9).
[ M + H - M e O H ] +
[ M+ K] +
[M+ H] + [ M + N a ] +
ESI-MS spectrum of fraction F 8-3-2 (compound 9)
In addition, the loss of methanol in the mass spectrum requires the presence of a methoxy group in the structure and in comparison with the literature data (Nagai et al., 1998) and based on 1H NMR spectrum (see table 3-32 and appendix 10a,b) structure of compound 9 was finally confirmed.
a Comparison of 1H NMR data of compound 9 with reported data *
a a 2 3.05 (d) 3.05 (br. d, 13.4 ) 3.32 (d) 3.32 (br. d, 12.1) 8 1.73 (dd) 1.73(ax, dd, 3.6 and 14.8) 2.22 (dd) 2.22 (eq, dd, 2.7 and 14.8) 9 4.84 (q) 4.84 (q, 2.9, 2.9 and 3.0) 11 3.77 (dd) 3.77 (dd, 1.8 and 10.7) 15 3.99 (t) 3.99 (t, 6.4 and 6.7) 17 6.82 (d) 6.82 (br. d, 7.6) 18 7.15 (t) 7.15 (t, 7.8 and 7.8) 19 6.72 (ddd) 6.72 (ddd, 1.0, 2.5 and 8.0) 21 6.86 (t) 6.86 (t, 1.7 and 2.3) 22 0.83 (3H, d) 0.83 (3H, d, 6.0) 23 0.82 (3H, d) 0.82 (3H, d, 6.0) 24 0.95 (3H,s) 0.95 (3H, s) 25 0.82 (3H,s) 0.82 (3H, s) 26 1.59 (3H,s ) 1.59 (3H, s) 28 2.76(dd) 2.76 (dd, 3.8 and 17.9)
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Chapter III Results
2.72 (dd) 2.72 (dd, 10.6 and 17.7) 29 5.30 (dt) 5.30 (dt, 3.8, 3.8 and 10.4) 30 3.84 (m) 3.84 (m) 31 1.11 (3H, d) 1.11 (3H, d, 6.5) 32 3.17 (3H,s) 3.17 (3H,s) (CH3O-) * Nagai. et al., 1998 1. Spectra determined in CDCl 3 2. Spectra determined in acetone-d6; data reported in ppm
a a a a a a a a a a a a a
To remove a portion of unwanted components in the methanol extract the dried residue of lyophilized biomass was extracted with methanol solvent after initial extraction with n-hexane and ethyl acetate solvent, respectively. A portion of the methanol extract (240 mg) was applied onto a silica gel column and eluted with a stepwise gradient starting with 75% n-hexane to 100%
EtOAc to 100% MeOH (see 2.8.6.2) to yield twenty major fractions (F 1 to F 22 ). Based on the results of the TLC analysis, three fractions (F9, F10, and F 17 ) were chosen for testing cytotoxic activity against 5637 cell line (see fig3-36 and table 3-33).
Thin layer chromatogram of 22 fractions obtained from silica gel column eluted with a stepwise gradient of n-hexane/EtOAc/ MeOH (method 2); [Mobile phase: n-hexane/EtOAc/MeOH/CH 3COOH (75:25:5:3); Detection: Vis after spraying Anisaldehyde/sulfuric acid reagent]
105
Chapter III Results
a Fractionation of methanol extract from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions against cell line 5637
a a
a
F1 1-3 n-hexane/EtOAc (75:25) 4.4 1.83 n.e
F2 4-8 n-hexane/EtOAc (75:25) 11.0 4.58 n.e
F3 9-16 n-hexane/EtOAc (75:25) 4.8 2.0 n.e
F4 17-27 n-hexane/EtOAc (75:25) 17.6 7.3 n.e
F5 28-48 n-hexane/EtOAc (75:25) 25.7 10.7 n.e
F6 49-80 n-hexane/EtOAc (75:25) 16.9 7.04 n.e
F7 81-104 n-hexane/EtOAc (50:50) 6.3 2.63 n.e
F8 105-107 n-hexane/EtOAc (50:50) 5.3 2.21 n.e
F9 108 n-hexane/EtOAc (50:50) 1.8 0.75 5.5
F10 109-120 n-hexane/EtOAc (50:50) 15.3 6.38 6.3
F11 121-138 n-hexane/EtOAc (25:75) 4.4 1.83 n.e
F12 139-154 n-hexane/EtOAc (25:75) 1.9 0.79 n.e
F13 155-159 n-hexane/EtOAc (25:75) 5.5 2.9 n.e
F14 160 100% EtOAc 11.7 4.88 n.e
F15 161-173 EtOAc/MeOH (90:10) 12.3 5.13 n.e
F16 174-180 EtOAc/MeOH (90:10) 0.1 0.04 n.e
F17 181 EtOAc/MeOH (90:10) 0.7 0.29 0.95
F18 182-195 EtOAc/MeOH (90:10) 0.9 0.38 n.e
F19 196 EtOAc/MeOH (25:75) 4.0 1.67 n.e
F20 197 EtOAc/MeOH (50:50) 16.7 6.96 n.e
F21 198 EtOAc/MeOH (25:75) 15.8 6.58 n.e
F22 199 100% MeOH 38.9 16.20 n.e Total 222 93.07 n.e. not estimated.
According to the results of cytotoxicity test, the fraction F 10 eluting with 50% n-hexane/EtOAc which exhibited strong cytotoxic activity and did not show so many spots in TLC was analyzed by HPLC to isolate pure compounds. HPLC analysis of fraction F 10 revealed that further separation by semi preparative HPLC was possible.
106
Chapter III Results
a a a a a
The active fraction F 10 was analyzed by HPLC with DAD detection. At 210 nm the separation of fraction F 10 into five substances was successful using a Synergi- RP (80A) column and a solvent gradient from 20%-100% acetonitrile/water. Based on the results of analytical HPLC, the mobile phase (acetonitrile/water) for semi preparative HPLC was established (see table 2.7). To optimize separation process, different sample amounts (200, 400, 500, 1000, 1500, 2000 µg/50 µL injection volume) for application on the column and different flow rates (2, 3, 3.5, 4, 5 mL/min) were tested. For a successful separation a sample amount of 500 µg/50 µL injection volume and a flow rate of 3 mL/min were used.
Altogether, 14.0 mg of fraction F 10 were purified using the step gradient described in 2.8.1.2 and the collection of the separated peaks was controlled at the wave length of 210 nm. Five peaks F10-1, F 10-2, F 10-3, F 10-4, and F 10-5 were collected to give 5 fractions. All these fractions were tested for their cytotoxic activity against bladder cancer cell line 5637 (see fig.3-37 and table 3-34)
F1 0 - 3
F1 0 - 4
F1 0 - 2 F1 0 - 5
F1 0 - 1
Semi-preparative RP HPLC chromatogram of F 10 (mobile phase see table 2.7) 500 µg/50 µL injection volume, detection at 210nm, column Synergi POLAR-RP 80A (250×10 mm, 4 micron), flow rate 3mL/min
107
Chapter III Results
a Separation of fraction F 10 from Lyngbya majuscula biomass by semi- preparative RP HPLC and cytotoxic activity of fractions against cell line 5637
a
F10-1 16.33 1.0 7.14 550
F10-2 19.23 1.1 7.86 380
F10-3 19.69 5.3 37.86 86
F10-4 23.50 1.9 13.57 No activity
F10-5 36.08 1.5 10.71 40 Total 10.8 77.14
Because fractions F10-3 and F 10-5 exhibited strong activity (IC 50 = 86ng/mL and 40ng/mL, respectively), the structure of including compounds was elucidated by ESI –MS and NMR.
a a a
a a
Fraction F10-3 was recognized as the pure compound debromoaplysiatoxin (see fig.3-38)
25 26 5 CH3 H3C 4 6 CH 7 3 3 24 32 2 O 8 O CH3O 1 OH 9 17 10 12 14 16 O O O 11 13 15 18 27 30 19 H3C CH3 CH3 21 31 29 23 22 28 O 20 OH OH
Debromoaplysiatoxin, molecular formula C 32 H48 O10
The identification of debromoaplysiatoxin (compound 10) was established by direct comparison of our spectroscopic data, including 1H NMR, 13 C NMR, and HRMS in negative mode with those reported in the literature. 1H NMR, 13 C NMR data of compound 10 were consistent with literature values for the known metabolite debromoaplysiatoxin (Nagai et al., 1997) (see table 3-35 and appendix 11 a, b)
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Chapter III Results
a Comparison of 13 C NMR and 1H NMR data of compound 10 with literature values of *Debromoaplysiatoxin
a a a a ( 1 169.19 169.2 (0) - - 2 46.91 46.9 (2) 2.52 (β, d) 2.52 (β, d, 12.7) 2.76 (α,d) 2.76 (α, d, 12.5) 3 98.81 98.8 (0) - - 4 35.68 35.7 (1) 1.86 ( m) 1.86 (m) 5 41.16 41.1 (2) 1.62 (ax, t) 1.62 (ax, t, 13.1 and 13.1) 1.05 (eq, dd) 1.05 (eq, dd, 3.6 and 13.4) 6 38.98 39.0 (0) - - 7 100.85 100.8 (0) - - 8 33.66 33.6 (2) 1.71( ax, dd) 1.71 (ax, dd, 3.6 and 14.8) 2.68 (eq, dd) 2.68 (eq, dd, 3.0 and 14.7) 9 73.21 73.2 (1) 5.23 (m) - 10 35.42 35.4 (1) 1.71 (m) 1.71 ( m) 11 69.88 69.8 (1) 3.93 (dd) 3.93 (dd, 2.1and 10.9) 12 34.24 34.2 (1) 1.52 (m) 1.52 (m) 13 31.24 31.2 (2) 1.31 (m) 1.31 (m) 1.39 (m) 1.39 (m) 14 36.12 36.1 (2) 1.64 (m) 1.63 (m) 1.97 (m) 1.97 (m) 15 85.83 85.8 (1) 4.0 (t) 4.0 (t, 6.5 and 6.5) 16 145.93 145.9 (0) - - 17 119.34 119.3 (1) 6.84 (dt) 6.84 (dt, 1,1 and 7.9) 18 129.79 129.8 (1) 7.13 (t) 7.13 (t,7.8 and 7.8) 19 114.98 115.0 (1) 6.71 (ddd) 6.71 (dt,1,1 and 7.9) 20 158.34 158.3 (0) - - 21 114.67 114.6 (1) 6.92 (m) 6.92 (t,1 and 1) 22 13.52 13.6 (3) 0.79 (d) 0.79 (d,6.8) 23 13.04 13.0 (3) 0.71 (d) 0.71 (d,6.9) 24 26.79 26.8 (3) 0.83 (s) 0.83 (s) 25 23.61 23.6 (3) 0.8 (s) 0.8 (s) 26 16.48 16.5 (3) 0.86 (d) 0.86 (d,6.8) 27 170.36 170.4 (0) - - 28 34.69 34.6 (2) 2.92 (α, dd) 2.92 (α, dd, 11.1 and 2.88 (β, m) 18.2) 2.87 (β, dd, 2.8 and 18.1) 29 74.30 74.3 (1) 2.23 (m) 5.23 (m) 30 67.08 67.0 (1) 4.03 (m) 4.03 (m) 31 17.72 17.7 (3) 1.12 (d) 1.12 (d, 6.4) 32 56.60 56.6 (3) - 3.17 (s) * Nagai et al., 1997 1. Spectra determined in CDCl 3; data reported in ppm 2. Spectra determined in acetone-d6; data reported in ppm. Carbon types determined by a DEPT experiment and reported as 0 (quaternary), 1(CH), 2(CH 2), or 3(CH 3) 3. Spectra determined in CDCL 3, 600 MHz; data reported in ppm 4. Spectra determined in acetone-d6, 500 MHz; data reported in ppm
In addition, the HRMS in negative mode of compound 10 showed a molecular ion peak at m/z [M-H+HCl] - 627.2942 (see fig.3-39) and the molecular
109
Chapter III Results weight was deduced to be 592 Dalton compatible with the molecular formula
C32 H48 O10.
[ M - H + H C l ] -
UV and ESI-MS spectrum of compound 10
a a
Fraction F10-5 was identified as the pure compound 3,4-didehydro-3-deoxy- aplysiatoxin (figure 3-40)
25 26 5 CH3 H3C 4 6 CH3 3 7 24 32 2 O 8 O CH3O Br 1 9 17 10 12 14 16 O O O 11 13 15 18 27 30 19 H3C CH3 CH3 21 31 29 23 22 28 O 20 OH OH
3,4-didehydro-3-deoxy-aplysiatoxin, molecular formula C 32 H45 BrO 9
The high resolution ESIMS in positive mode of compound 11 showed two peaks as a molecular ion peak at m/z 653.2329 [M] + and an isotope ion peak at m/z 655. 2311 [M+2] + which were very similar in height (see fig. 3-41) indicating that compound 11 contains a bromine atom and the molecular weight was deduced to be
653 Dalton compatible with the molecular formula C32 H 45 BrO9 (calculated 653.2320 for C32 H 46 BrO9).
110
Chapter III Results
[M+ 2] +
[ M ] + [M+ 2] +
UV and ESI-MS spectrum of compound 11
1H NMR data of compound 11 were fully consistent with literature values for the known metabolite 3,4-didehydro-3-deoxy- aplysiatoxin (Moore et al., 1984) (see table 3-36 and appendix 12a,b).
a Comparison of 1H NMR data of compound 11 with reported data *
a a 2 3.070 (dm) 3.070 (br dm) 3.342 (d) 3.342 (d) 5 2.185 (dm) 2.185 (br dm) 11 3.805 (dd) 3.805 (dd) 15 4.447 (dd) 4.447 (dd) 18 7.357 (dd) 7.357 (dd) 19 6.715 (d) 6.715 (d) 21 7.008 (d) 7.008 (d) 22 0.845 (d) 0.845 (d) 23 0.856 (d) 0.856 (d) 24 0.956 (s) 0.956 (s) 25 0.829 (s) 0.829 (s) 28 2.739 (dd) 2.739 (dd)
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Chapter III Results
2.781 (dd) 2.781 (dd) 29 5.357 (dt) 5.357 (dt) 30 3.854 (qd) 3.854 (qd) 4.07 (d) 4.07 (d)
OCH 3 3.210 (s) 3.210 (s) *Moore et al., 1984 1. Spectra determined in CDCl 3; data reported in ppm 2. Spectra determined in acetone-d6; data reported in ppm
a a a a
The mixture of compounds in n-hexane extract of L. majuscula was separated by gas chromatography and the summary of fatty acids in this extract was shown in table 3-37 and appendix 13.
a Fatty acids analyzed as methyl ester in n-hexane extract a a a a a a a 28.498 Myristic acid (C14:0) 285, 201, 117, 73, 43 0.925 71.5 29.888 Pentadecanoic acid (C15:0) 299, 201, 117, 73, 40 0.663 74.8 32.257 Palmitic acid (C16:0) 313, 201, 117, 73, 47 3.64 88.0 35.072 Linoleic acid (C18:2) 337,262,73 0.391 73.3 35.531 Stearic acid (C18:0) 339, 222, 117, 73, 55 22.748 84.8 38.788 Arachidic acid (C20:0) 367, 292, 185,117, 73 0.606 75.4 48.997 Cholesterol 458, 368, 392, 247, 1.062 83.1 129, 73 ret. time = retention time; m/z = the mass of an ion divided by the electrical charge of the ion
It was shown, that in n-hexane extract of Lyngbya majuscula the main fatty acids were stearic acid and palmitic acid. Beside this, cholesterol, the unsaturated linoleic acid and other fatty acids were also found.
112
Chapter IV Discussion
a a a a a a a a a a a
Antibiotics were considered to be "miracle drugs" when they first became available half a century ago, but their popularity rapidly led to overuse (Saleem et al., 2010). Over the last decade, it has become clear that antimicrobial drugs are losing their effectiveness due to the evolution of pathogen resistance, as resistance to antibiotics in bacterial population increased dramatically with time and usage of antimicrobial drugs. There is therefore a continuing need to explore, search for and develop newer and more effective broad spectrum antimicrobial agents. Against the growing problem of antibiotic-resistant bacteria, alternative antimicrobial drugs sources which are nontoxic/ less toxic to human and without side effects for human and for environment must be found. Natural products are both fundamental sources of new chemical diverse and integral components of today’pharmaceutical compendium (Saleem et al., 2010) and natural antimicrobials will undoubtedly have an important role in protecting against infection. This new direction in research has been the subject of many studies on antimicrobial effects of various organisms including cyanobacteria. The medicinal qualities of cyanobacteria were first appreciated as early as 1500 BC, when Nostoc species were used to treat gout, fistula and several forms of cancer. Yet, not much attention was paid till the turn of the century when during 1990, workers at University of Hawaii, Oregon State had begun to screen extracts of cyanobacteria, mostly strains of Microcystis and Anabaena spp. for various biological activities. It was reported that nearly 4000 strains of freshwater and marine cyanobacteria were screened inferring that cyanobacteria are a rich source of potentially useful natural products (6% having anticancer, antiproliferative activity). Later, several screening processes were initiated with important targets such as antibacterial, antifungal, anti-AIDS, anticancer and other activities. Screening of cyanobacteria for antibiotics has opened a new horizon for discovering new drugs. Numerous screening programs have revealed the potential of cyanobacteria in the production of novel antimicrobial compounds (Schlegel et al., 1999; Mian et al., 2003; Soltani et al., 2005; Pawar &Puranik, 2008). Various strains of cyanobacteria are known to produce intracellular and extracellular metabolites with
113
Chapter IV Discussion antibacterial activity (Falch et al., 1995; Jaki et al ., 1999a, 2000a,b; Harvey, 2000; Mundt et al. , 2001; Volk &Furkert, 2006; Asthana et al., 2006; Rao et al., 2007; Kaushik &Chauhan, 2008). Many studies have assessed the antibacterial activity of some cyanobacteria and their extracts ( Kreitlow et al., 1999; Schlegel et al., 1999; Mian et al ., 2003; Ghasemi et al., 2003; Mundt et al., 2003; Ghasemi et al ., 2004; Soltani et al., 2005; Volk &Furkert, 2006; Asthana et al., 2006; Taton et al ., 2006; Abedin &Taha, 2008; Martins et al., 2008; Abdel-Raouf & Ibraheem, 2008; Patil et al., 2009). There are numerous reports on biological active compounds isolated from cyanobacteria living in both freshwater and marine environments (e.g. Østensvik et al., 1998; Harada, 2004; Fassanito et al., 2005; Tan, 2007). But little work has done to screen cyanobacteria isolated from rice, cotton and coffee fields with regard to their production of antibacterial substances. In one study, the culture media of cyanobacteria belonging to Nostocaceae, Microchaetaceae and Scytonematacaea isolated from the Argentinian paddy-fields, were found to be active against S. aureus (De Caire et al ., 1993). Investigations of another group showed that cyanobacteria from paddy fields of northern Thailand produce bioactive substances with antibiotic activity against B. subtilis (Chetsumon et al ., 1993). Also, Soltani et al., 2005 isolated 76 cyanobacterial strains from Iranian paddy fields. 22.4% of them (17 cyanobacteria belonging to Stigonemataceae, Nostocaceae, Oscillatoriaceae, and Chrococcaceae) exhibited antimicrobial effects and growth of B. subtilis PTCC 1204, Staphylococcus epidermidis PTCC 1114, E. coli PTCC 1047, and Salmonella typhi PTCC 1108 was inhibited by 12, 14, 8, and 2 strains of cyanobacteria, respectively. Only few reports publish data about screening of Vietnamese cyanobacteria with regard to their production of bioactive compounds (Bui et al ., 2007), and none relate to antibacterial screening and new biological active compounds of terrestrial cyanobacteria from South Vietnam. Thus, screening of 12 cyanobacterial strains isolated from rice, cotton and coffee fields of Dak Lak province located in South Vietnam for their antibacterial activities is among the first studies done for assessment of antibacterial activity of Vietnamese terrestrial cyanobacteria with the objective of finding new antibacterial compounds which could serve as a source of new lead structures for development of antibacterial drugs or chemical leads useful in facilitating the development of new therapeutic or commercial agents in the future.
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Chapter IV Discussion
12 cyanobacterial strains belonging to 6 genera isolated from rice, cotton and coffee fields 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.
a a a The BG-11 medium was selected as the primary cultivation medium as it was designed for culturing cyanobacteria (Watanabe, 2005) and has been used for culture 12 selected cyanobacterial strains successfully. The temperature used for cyanobacterial cultures in this work was room temperature (20±2 0C), this temperature is consistent with a general rule that the optimum temperature for cyanobacteria is 15-20 0C (Castenholz, 1988; Andersen, 2005). The light source used for cyanobacterial culture in this work were cool fluorescence lamps because the spectral range of light absorbed by cyanobacteria requires the use of fluorescent light (i.e. cool-white, warm-white, daylight), other light sources have a great proportion of the output in the far red and near-infrared regions which are not available to oxygenic phototrophs. The growth period of the samples lasted 4-6 weeks to ensure that the majority of cultures were in stationary phase and therefore most of likely producing secondary metabolites (Lincoln et al ., 1996). The cultures were harvested by centrifugation followed by filtration with filter paper to separate cells (biomass) and growth media. The harvested biomasses were freeze dried and extracted. In order to separate unknown chemical compounds from the totality of cyanobacterial metabolites, procedures modified from methods of plant natural products isolation (Cannell, 1998) were used. The dried biomasses of cyanobacteria were successively extracted with solvents of increasing polarity: n- hexane, methanol, and water. By modifying the extraction method for extracellular metabolites from bacteria (Cannell, 1998), the harvested growth media were extracted with ethyl acetate by liquid- liquid extraction to get ethyl acetate extracts. All extracts were tested in vitro for their antibacterial activity.
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a a a Our data revealed that of 48 extracts, 23 (47.92%; 3 n-hexane, 11 MeOH, and 9 EtOAc extracts) showed activity against the Gram-positive bacterium B.subtilis , 22 (45.83%; 3 n-hexane, 11 MeOH, and 8 EtOAc extracts) exhibited activity against the Gram-positive bacterium S. aureus , 11 (22.92%; 1 n-hexane, 4 MeOH, and 6 EtOAc extracts) inhibited the growth of the Gram-negative bacterium E. coli , 3 (MeOH extracts) showed effects on the growth of the Gram-negative bacterium P. aeruginosa, but these effects did not result in complete inhibition, and none of the water extracts was active against test bacteria. These results indicate that the extracts contained different antibacterial substances and reflected the variety of secondary metabolites and are also in agreement with results in the literature. For example, Jaki et al., 1999b found that of 86 extracts of cyanobacteria, 14 (16.3%; 9 DCM/MeOH
2:1 and 5 MeOH/H 2O 7:3 extracts) showed activity against Gram-positive bacteria. 5 extracts (5.8%; 3 DCM/MeOH 2:1 and 2 MeOH/H 2O 7:3 extracts) exhibited activity against Gram-negative bacteria. Similarly, Falch et al. 1995 reported that the petroleum ether, dichloromethane, ethyl acetate, methanol and 80% aqueous methanol or water extracts of cyanobacteria showed different antibacterial effects in bioautographic assays with B. subtilis and E. coli. Soltani et al., 2005 reported that the petroleum ether, methanol, and aqueous, extracts of cyanobacteria showed antimicrobial activity. The results of this study showed that the growth of Gram-positive bacteria was more inhibited in comparison with Gram-negative bacteria, this was in agreement with the results of previous studies (e.g. Jaki et al., 1999b; Mundt et al., 2001; Mian et al., 2003; Ghasemi et al ., 2003; Soltani et al., 2005; Taton et al ., 2006, Asthana et al., 2006, Volk &Furkert, 2006; Martins et al., 2008). This finding may be related to the fact that cyanobacteria possess features familiar to Gram-negative bacteria (Dahms et al ., 2006) and the most Gram-negative bacteria are resistant to toxic agents in the environment due to the barrier of lipopolysaccharides of their outer membrane (Dixon et al ., 2004; Martins et al ., 2008). The difference in toxicity against Gram- positive and Gram-negative bacteria can also indicate that the mechanism of toxicity is different in the two types of cells caused by different permeability to the cyanobacterial compounds (Martins et al., 2008). In addition, in ecological consideration, cyanobacteria possess morphology of Gram-negative bacteria; the fact that the bioactive cyanobacterial metabolites are effective more strongly against
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Chapter IV Discussion
Gram-positive bacteria approves the idea that the antibiotic substances are secondary metabolites produced for defense purpose in cyanobacterial life (Piccardi et al ., 2000; Bhadury &Wright, 2004). In this study, all investigated cyanobacteria (12/12) showed an antibacterial activity to at least one of the test organisms while in the literature17 of 76 investigated cyanobacterial strains (22.4%) exhibited antimicrobial effects (Soltani et al., 2005). Thus our results provide evidence to further support the use of cyanobacteria in drug discovery efforts for antibiotic lead compounds and it points out the necessity of exploring cyanobacteria of local habitats as potentially excellent sources of these compounds. As mentioned in 1.2, most of bioactive compounds isolated from cyanobacteria belong to the groups of peptides (e.g. cyclic depsipeptides, cyclic peptides, and lipopeptides), fatty acids, polyketides, alkaloids, amides, terpenoids, lactones, pyrroles, and others. From our screening, because different activities were observed in extracts obtained with different organic solvents we can suggest that compounds with different polarities are involved. Especially, antibacterial activities were found mostly in methanol and ethyl acetate extracts, therefore substances of low polarity to middle polarity such as fatty acid, polyketides, peptides, alkaloids, and terpernoids were expected. Of the twelve investigated cyanobacteria, all crude extracts of Westiellopsis sp.VN and the crude ethyl acetate extract of Anabaena sp. showed very strong antibacterial activity against Gram-positive and Gram-negative bacteria, as well as the crude methanol extracts obtained from Calothrix javanica, Scytonema ocellatum, Nostoc sp. showed strong antibacterial activity (see 3.1.2). Therefore these strains were selected for chemical investigation with an emphasis on the isolation and structure elucidation of antimicrobial secondary metabolites.
a a a a Westiellopsis
Westiellopsis
The strain Westiellopsis sp.VN isolated from rice field soil of Dak Lak province in Southern Vietnam was described on morphological characteristics and
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Chapter IV Discussion identified based on its partial 16S rRNA gene sequence and molecular-phylogenetic tree constructed based on 16S rRNA gene sequences of this cyanobacterial strain and 11 other cyanobacterial strains belong to genera Westiellopsis and Fischerella in GenBank (Ho et al., 2005a; Ho, 2007). It is interesting to note that in Vietnam, species of genus Westiellopsis have not been described yet; therefore Westiellopsis sp. VN strain was described for the first time by Ho et al., 2005a. Up to now, there is no literature available on chemical investigations of Vietnamese Westiellopsis genus and the results of screening demonstrated that all the crude extracts of Westiellopsis sp. VN exhibited very strong antibacterial activity against Gram-positive and Gram-negative bacteria (see 3.1.2), as well as yeast Candida maltosa SBUG 700 {(IZ of 16.5mm including diameter of disc (6mm) with C=2mg/disc in agar diffusion assay}. Thus, the Westiellopsis sp.VN strain was first selected for the detailed investigation with an emphasis on the isolation and structure elucidation of antimicrobial secondary metabolites.
a a a Westiellopsis a
Branched filamentous cyanobacteria belonging to the order Stigonematales are known to produce antibacterial, antifungal, antialgal and cytotoxic compounds, with diverse chemical structures. To date, examination of the genus Fischerella , Hapalosiphon , and Westiella resulted in the isolation of isonitrile-containing indole alkaloids such as hapalindoles (Moore et al., 1984a; Huber et al., 1998), ambiguines isonitriles (Smitka et al ., 1992; Park et al ., 1992; Huber et al ., 1998; Raveh &Carmeli, 2007, Mo et al ., 2009), fischerindoles (Park et al., 1992), and welwitindolinones (Stratmann et al., 1994). However, only few biologically active compounds were identified from the genus Westiellopsis , e.g. westiellamide, a bistratamide-related cyclic peptide from terrestrial cynobacterium Westiellopsis prolifica Janet exhibited cytotoxic activity against KB and LoVo cell lines at 2 µg/mL, but not solid tumor selectivity in the Corbett assay (Prinsep et al., 1992). This compound appears to be identical with a bistratamide-type compound isolated from the ascidian Lissoclinum bistratum, suggesting that cyanophyte symbionts in Lissoclinum bistratum are responsible for synthesis of the bistratamides (Patterson et al ., 1992). The chemical investigation of the methanol extract prepared from dry biomass of Westiellopsis sp.VN using bioassay-guided isolation led to the isolation
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Chapter IV Discussion and/or identification of the six intracellular compounds ambiguine D isonitrile, ambiguine B isonitrile, dechloro-ambiguine B isonitrile, and fischerellin A as well as hydroxy-eicosatetraenoic acid and methoxy-nonadecadienoic acid. Identification of these active compounds was established by direct comparison of our spectroscopic data, including 1H NMR and HR-ESI-MS with those reported in the literature. Ambiguine D isonitrile was first isolated from Westiellopsis prolifica EN-3-1 and identified conclusively by 1H NMR and TLC analysis. This compound displayed moderate antifungal activity to Candida albicans , Trichophyton mentagrophytes, and Aspergillus fumigatus with MIC values of 1.25, >80.0, and > 80.0 µg/mL, respectively (Smitka et al., 1992). Ambiguine D isonitrile was also isolated from Fischerella sp. (Raveh &Carmeli, 2007) and only in trace amounts detected by LC- MS in Fischerella ambigua UTEX 1903 (Mo et al., 2009). In our experiments, ambiguine D isonitrile was isolated from Westiellopsis sp.VN and this compound exhibited antibacterial activity against S. aureus with diameter of inhibition zone of 28.0mm in concentration of 200mg/dics (see table 3-4). Ambiguine B isonitrile was isolated from the terrestrial cyanobacterium Fischerella ambigua UTEX 1903. This compound showed moderate antifungal activity to Candida albicans , Trichophyton mentagrophytes, and Aspergillus fumigatus with MIC values of 1.25, >80.0, 20.0 µg/mL, respectively (Smitka et al., 1992) and moderate antimicrobial activity to Bacillus anthracis, Staphylococcus aureus, Mycobacterium smegmatis, Candida albicans with MIC values of 3.7, 10.9,
27.8, 1.7 µM, respectively, as well as cytotoxic activity to Vero cells with IC 50 value of 58.6 µM (Mo et al., 2009). Ambiguine B isonitrile was also isolated from terrestrial cyanobacterium Hapalosiphon delicatulus (Huber et al., 1998) and Fischerella sp. (Raveh & Carmeli, 2007). In this work, ambiguine B isonitrile was isolated from Westiellopsis sp.VN and showed antibacterial activity against S. aureus with diameter of inhibition zone of 8.0mm in concentration of 200mg/dics (see table 3-4). Ambiguine C isonitrile was isolated from the terrestrial cyanobacterium Fischerella ambigua UTEX 1903. This compound showed moderate antifungal activity to Candida albicans , Trichophyton mentagrophytes, and Aspergillus fumigatus with MIC values of 2.5, >80.0, >80.0 µg/mL, respectively (Smitka et al., 1992) and moderate antibacterial activity to Mycobacterium tuberculosis, Bacillus anthracis, Staphylococcus aureus, Mycobacterium smegmatis, Candida albicans with MIC values of 7.0, 16.1, 7.4, 59.6, <1 µM, respectively, as well as cytotoxic activity 119
Chapter IV Discussion
to Vero cells with IC 50 value of 78.3 µM (Mo et al., 2009). In our case, ambiguine C isonitrile was observed in a mixture with other compounds of fraction WF 1-5 which showed antibacterial activity against S .aureus with diameter of inhibition zone of 10.0mm in concentration of 200mg/dics (see table 3-4). Fischerellin A, a photosystem II-inhibiting allelochemical with antifungal and herbicidal activity (Hagmann & Jüttner, 1996) was isolated from Fischerella muscicola UTEX 1829. In our work this compound was observed also as a mixture in fraction WF 1-5 which showed antibacterial activity against S .aureus with IZ of 10.0 mm (see table 3-4). Two derivatives of fatty acids, methoxy-eicosatetraenoic acid and hydroxy- nonadecadienoic acid have been identified in fraction WF 1-8. They were found as a mixture in fraction WF 1-8 which showed antibacterial activity against S.aureus with diameter of inhibition zone of 10.0mm in concentration of 200mg/dics (see table 3-4). However, in contrast to clear structure elucidation of the 4 compounds ambiguine B, C, D isonitriles and fischerellin A, the chemical structure of the two derivatives of methoxy-eicosatetraenoic and hydroxy-nonadecadienoic acid has not clearly elucidated. In summary, bioassay–guided fractionation of the methanol extract obtained from biomass of our Westiellopsis sp. VN led to the isolation of ambiguine B,D isonitriles and the identification of ambiguine C isonitrile and fischerellin A. Furthermore, two derivatives of fatty acids methoxy-eicosatetraenoic acid and hydroxy-nonadecadienoic acid have been identified. These compounds have not been described as constituents of Westiellopsis sp. VN previously. Interestingly, up to the present study, 15 ambiguine A-O isonitriles have been found in Westiellopsis prolifica EN-3-1, Hapalosiphon delicatulus , Fischerella sp, Fischerella ambigua UTEX 1903 but not yet in Fischerella muscicola UTEX 1829, while fischerellin A only has been isolated from Fischerella muscicola UTEX 1829 and other strain of genus Fischerella so far. In our Westiellopsis sp VN ambiguine B, C, D isonitriles and fischerellin A were detected. Moore et al., 1987 proposed a common biogenesis of the hapalindoles, ambiguines, fischerindoles, and welwitindolinones and this proposal has supported by Strantman et al., 1994; Raveh & Carmeli, 2007; Van Wagoner et al., 2007; Gademann & Portman, 2008; Mo et al., 2009. Several polycyclic tryptophan/isoprenoid compounds have been reported from genera Fischerella, 120
Chapter IV Discussion
Hapalosiphon, Westiella , and Westiellopsis, order Stigonematales. The rich diversity in their cyclic structures highlights the flexibility isoprenoid chemistry in forming carbon-carbon bonds. Some of schemes presenting possible biogenetic relationships among some of the dozens of reported compounds of the family Stigonemataceae were published by Stratmann et al., 1994, Raveh & Carmeli, 2007; Van Wagoner et al., 2007; Gademann & Portman, 2008. It has been suggested in literature that co- occurrence of nonchlorinated and chlorinated ambiguine isonitriles in cyanobacteria of the order Stigonematales may be due to some imperfection in the biosynthesis and the resulting arrays of compounds have been proposed to provide an ecological advantage (Raveh & Carmeli, 2007). In our study, the identification of the nonchlorinated ambiguine C isonitrile together with the chlorinated ambiguine B, D isonitriles found in Westiellopsis sp.VN, was therefore not surprising. The Fischerellins (see fig. 4-1), previously isolated only from genus Fischerella , are enediyne metabolites that showed activity against photosynthetic microorganisms including cyanobacteria (Gross et al., 1991; Hagmann &Jüttner, 1996; Papke et al ., 1997).
Fischerellins from cyanobacteria
The configuration of the fischerellin enediyne moiety (ene/yne/yne) is topologically distinct from that seen in the more famous anticancer enediynes from actinomycetes. An interesting difference between fischerellin A and B is the presence of methyl branching on the side chain in the former, and absence of methylation but extension of the side chain by one carbon in the latter. No biosynthetic studies have been undertaken for the fischerellins until now (Van Wagoner et al., 2007). It is interesting to note that occurrence of fischerellin A has not been described for genus Westiellopsis, but the close relationship between the genera Fischerella and Westiellopsis could allow a gene transfer between the organisms. Otherwise it was not excluded, that genetic information for biosynthesis of
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Chapter IV Discussion fischerellins was repressed in Westiellopsis strains and can be activated by different endogenic or exogenic factors.
a a a a Westiellopsis a
Bioactive metabolites of cyanobacteria which have been isolated so far have been mostly accumulated in the cyanobacterial biomass. Moreover, cyanobacteria excrete various organic compounds into their environment and, until now, a couple of biologically active compounds were also identified among these extracellular metabolites, e.g. some antibacterial diterpenoids in Nostoc commune (Jaki et al., 1999a, 2000a), antifungal peptides in Tolypotrix byssoidea (Jaki et al ., 2001), and the antialgal indol alkaloid norhamane (Volk &Furkert, 2006). In our investigations of ethyl acetate extract of culture medium of Westiellopsis sp.VN we found a strong antibacterial activity of this extract. Based on this we have developed isolation procedure for the active compounds, excreted by Westiellopsis sp.VN strain into its environment. Ethyl acetate extract was extracted with different solvents and only methanol fraction of this extract showed antibacterial activity. Microorganisms typically produce an extracellular product in low concentration (<3%) (Gailliot, 1998) resulting in small amount of methanol fraction. Separation of the components of this fraction by HPLC was not successful, though different solvent gradients and conditions have been used. Therefore GC-MS was used for identifying the volatile components. Results of GC-MS revealed that this fraction contains different saturated fatty acids with 14, 16, and 18 carbons, the unsaturated 11-cis-octadecanoic acid, naphthalene, carbamic acid and 5-oxoproline. In many studies, it has been shown that fatty acids and volatile components are responsible for a broad spectrum of antibacterial activity and other activities (Mundt et al., 2003; Ozdemir et al ., 2004). Thus, the presence of identified compounds in this methanol fraction is not surprising, however to verify whether only these compounds are the cause for very strong antimicrobial activity of this methanol fraction, further separation of this methanol fraction is necessary.
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Chapter IV Discussion
a a Westiellopsis a
Clearly, cyanobacteria produce a large number of biologically active metabolites with potential pharmaceuticals but in general there are still some serious obstacles to the development of drugs from cyanobacteria. The most obvious are their slower growth rate in culture and lower biomass yield as well as the smaller quantities of secondary metabolites produced in comparison with bacteria and fungi. Additionally, a number of studies (Carmichael, 1986; Borowitzka, 1995) have verified that the desired bioactive compounds may decline, alteration or be lost entirely during culture, though reasons for this phenomenon are so far inexplicable. Even when seemingly stable strains of drug –producing cyanobacteria are obtained using conventional cloning techniques, drug production sometimes disappears on repeated subculturing, especially if culture conditions are modified, such as anatoxin- a toxicity in Anabaena flos-aquae NRC-44-1 disappeared when the medium was changed from ASM-1 to the nitrate- richer BG 11; similarly, repeated subculturing of Anabaena flos-aquae S-23-g, a strain that produces the neurotoxin anatoxin-d, with ASM-1 followed by BG11 resulted in loss of neurotoxicity and expression of hepatotoxicity similar to that observed in Microcystis (Carmichael, 1986). Thus, it is of great importance to optimize the cultivation conditions and production of metabolites in cyanobacteria. Temperature during incubation period, pH of the culture medium, phosphate concentration, length of incubation period, salinity, medium constituents, light intensity are important factors influencing biomass and bioactive secondary metabolite production (e.g. Repka et al., 2001; Griffiths & Saker, 2003; Ame et al ., 2003; Hirata et al., 2003; Noaman et al ., 2004; Abu et al., 2007; Abedin & Taha, 2008; Imai et al., 2009). Carbon and nitrogen sources fed to the antimicrobial agent producing cyanobacteria may exhibit significant roles in orienting the secondary metabolic pathways (Sailer et al., 1993; Griffiths & Saker, 2003). Medium studies and optimization methods are commonplace with respect to increasing the yield or activity of a given product. Until now, there have been some papers concerned with the optimization of growth rate and bioactive secondary metabolite production, especially antibiotic production by cyanobacteria (Moore et al., 1988; Bloor & England, 1991; Chetsumon et al., 1993; Noaman et al., 2004; Panda et al ., 2006; Silva & Silva, 2007). Such examples are:
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Chapter IV Discussion
- The seeking out the optimum with respect to antibiotic production by Nostoc muscorum was undertaken by Bloor &England, 1991 and the results of their experiments showed that an increased level of nitrate and a decreased level of iron, compared with the starting basal medium, promoted antibiotic activity. Furthermore it was seen that growth was limited at lower iron concentrations (3 µM and 0 µM) and it is likely that available iron in the growth medium represses biosynthesis of enzymes that are necessary for antibiotic production. It is not yet clear whether iron can inhibit antibiotic production after the organism has started production. - Optimization of antibiotic production by the cyanobacterium Scytonema sp.TISTR 8208 immobilized on polyurethane foam was studied by Chetsumon et al., 1993. This study showed that the Scytonema sp.TISTR 8208 produced an antibiotic with a broad spectrum in the post-exponential growth phase. Modification of the -1 composition of the BGA medium by adding 1.5 g L NaNO 3, increasing the -1 Fe 2(SO 4)3.x6H 2O concentration to 0.025 g L , not adding NaCl, using an initial pH of 7.0, incubating at 35 0C, and at the light intensity of 90µmol photon m -2s-1 enabled a 28-fold increase of antibiotic production. Optimization of antibiotic production by this strain in the continuous culture was undertaken and it was shown that three times more antibiotic was produced in the continuous culture than in batch culture at the 16th day (Chetsumon et al. , 1995). - Examining the effect of culture conditions on growth and accumulation of three scytophycin compounds produced by Scytonema ocellatum undertaken by Patterson & Bolis, 1993 showed that the optimal temperature for production was 25 0C and continuous illumination at an intensity of at least 25 µmol photons m -2 s -1 was required for maximum yield. Growth and metabolites production were optimal in the pH range of 8.0 to 8.5. - According to Noaman et al., 2004, an antimicrobial agent is produced by the cyanobacterium Synechococcus leopoliensis which was found to be active against the Gram-positive S. aureus. The effects of temperature, pH, incubation period, some media and different nitrogen and carbon sources on both growth and antimicrobial activity were investigated. Temperature of 35 0C and pH 8 were optimum for growth and antimicrobial agent production. Maximum of growth and antimicrobial activity were estimated after 14 and 15 days of incubation, in BG-11medium. No antimicrobial activity could be detected by the use of G medium, moderate activity was recorded with Chu 10 medium, while high activity was reported in BG-11 124
Chapter IV Discussion medium. Leucine was the best nitrogen source for antimicrobial activity, while maximum antimicrobial activity was reached by using the carbon sources, citrate and acetate. Very high antimicrobial activity was detected by using the carbon source galactose in combination with the nitrogen source alanine or by using arabinose with methionine. - Soltani et al., 2007 showed the effect of salinity (NaCl-free, 0.5 and 1%) on growth and antimicrobial activity of the cyanobacterium Fischerella sp. FS18. Aqueous, petroleum ether and methanol extracts of the strain were examined for activity against 4 bacteria and 2 fungi. The results indicated that the growth rate was enhanced in NaCl-free medium but the antibacterial activity was higher in medium with 1% NaCl. It could be explained not only by the role of NaCl in the life cycle of bacteria but also by decrease in the growth rate of cyanobacteria. - In studies of Abedin &Taha, 2008, Spirulina platensis was evaluated for bioactivity against Aspergilus flavus, Fusarium moniliforme, Candida albicans, Bacillus subtilis, and Pseudomonas aeruginosa by operating the statistical design of Plackett-Burman for the degree of significance of 8 different trials by using 7 independent variables. The results obtained from Plackett-Burman design revealed that highest main effect and t-value were detected with NaCl in case of A. flavus while they were detected with MgSO 4 and micronutrients (a) in case of F. moniliforme , with
FeSO 4 and micronutrienst (a) with C. albicans . On the other hand, the results revealed that highest main effects and t-value were detected with micronutrients (b) to B. subtilis while they were detected with NaCl and K 2SO 4 in case of P. aeruginosa. - According to Silva & Silva, 2007, the influence of the mineral nutrients on the growth of Tolypothrix tenuis was studied and the optimization of mineral nutrients in culture medium of T. tenius allowed a 73% increase in the final biomass level. - Optimization studies of biomass production and protein biosynthesis by Spirulina sp. undertaken by Abu et al., 2007 showed that biomass and protein produced were significantly (P=0.05) higher under the optimized conditions (pH 9.0; 0 2- temperature 30 C, 25 mg/L NO 3 ; so on). According to Hirata et al., 2003, in a laboratory culture of Noctoc spongiaeforme , the production of Nostocine A was enhanced at higher temperature (30 0C) and more intense light (30 W/m 2) than during cultivation under basal conditions at 25 0C and 10 W/m 2.
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Chapter IV Discussion
- Imai et al ., 2009 reported that in the laboratory experiments where Microcystic aeruginosa and Microcystic wesenbergii were cultured under various temperatures, growth rates of M. aeruginosa were significantly higher than those of M. wesenbergii at high temperatures (30 and 35 0C) but growth rates of these two species were similar at lower temperature (20 and 25 0C). - The production of phycocyanin and interactions between sodium nitrate, calcium chloride, trace metal mix and citric acid were investigated and modeled by Singh et al., 2009. These results showed that under optimized conditions Phormidium ceylanicum produced a 2.3-fold increased concentration of phycocyanin in comparison to common used BG-11 medium in 32 days. In general, optimization of growth media and hence biomass yield will have profound effects on cell metabolism and subsequently the quantities and types of secondary compounds synthesized. Secondary metabolite production, considered non- essential to a cell′s initical survival may not relate directly to high biomass yield; synthesis of these compounds is often triggered under conditions not conducive to high growth rates, such as nutrient deprivation (Olaizolá, 2003). Therefore, optimization of culture conditions should seek to establish a balance between secondary metabolism and growth rate. As for any microorganisms, the bioprocess intensification of cyanobacteria is different for every species. This work is an experiment to optimize the conditions for enhancing the growth rate and the production of antimicrobial agents by the cyanobacterium Westiellopsis sp. VN. This strain was selected for culture optimization experiments because all extracts of Westiellopsis sp.VN possessed the highest strength and widest range of antimicrobial activity, however, the nutrient factors controlling the production of these antibiotics by this strain have not been studied so far. First step was to scale up cultivation from the 500 mL volume in Fehrnbach flasks to a 35L volume in a fermentor with aeration using air and CO 2 to adjust the pH to 8.5. In contrast to the cyanobacteria Anabaena sp., Nostoc sp., Calothrix elenkinii, and Scytonema millei which strains did not produce antimicrobial active metabolites or lower concentrations of these compounds in large scale culture (see appendix 7), the strain Westiellopsis sp. VN exhibited good growth and production of antimicrobial active compounds. Because nitrogen sources fed to the antimicrobial agent producing cyanobacteria may exhibit significant roles in orienting the secondary metabolic 126
Chapter IV Discussion pathways (Sailer et al., 1993; Griffiths & Saker, 2003), we have investigated effect of nitrogen deficiency on biomass production and antibacterial compound accumulation of this strain. Westiellopsis sp.VN was cultured with BG-11 medium without NaNO 3 for 7 weeks under the following culture conditions: temperature 28 0C, pH 7.4, and continuous illumination using cool-white fluorescent tubes with an intensity of 8µmol/m 2 in a 45 liter-glass fermenter. Cultivation of the strain with the BG-11 standard medium under the culture conditions above-mentioned was used as control. For evaluation the effects of nitrogen deficiency on yield of lyophilized biomass were measured. Furthermore the yield of n-hexane, EtOAc, MeOH extracts of biomass and EtOAc extract of growth medium was estimated and their activity was tested against S. aureus . Our studies have shown that the dry biomass weights did not significantly differ between BG-11 medium without NaNO 3 and BG-11 standard medium, indicating that NaNO 3 does not play a role in biomass production of Westiellopsis sp.VN. In the literature is described for the Nodularia strain GR8b in batch culture that the highest nitrate concentrations resulted in reduced dry weight (Repka et al., 2001). Lehtimäki et al., 1994 showed that in laboratory batch cultures, the biomass of Nodularia spumigena and Aphanizomenon flos-aquae decreased with unnaturally high inorganic nitrogen concentrations. According to Piorreck et al. 1983, in batch culture, increasing N level led to an increase of the biomass. This study supports a conclusion of Soltani et al., 2007 that the good or poor growth of cyanobacteria not only must be due to their efficiency to metabolize nitrogen but is actually the sum of the entire physiology and genetics of these organisms.
Concerning the effect of NaNO 3 deficiency on synthesis of antimicrobial substances by this strain in the large scale, cultivation with BG-11 medium without
NaNO 3 seems to increase the production of antimicrobial substances in comparison to BG-11 standard medium. The diameter of inhibition zone of n-hexane and ethyl acetate extracts from biomass and culture medium respectively cultured in BG-11 without NaNO 3 (18.0 mm, 18.0mm, respectively) were bigger than those cultured in BG-11 standard medium (9.0 mm, 12.5 mm, respectively). Similar results have been published by Rapala et al., 1997. They have investigated two Anabaena sp. strains, 90 and 202A 1 and have shown that the highest levels of microcystins were determined in a nitrogen-free growth medium. Growth in N-free medium showed that the cells of Anabaena and Aphanizomenon strains contained more toxin than growth in N-rich 127
Chapter IV Discussion medium (Rapala et al., 1993). Otherwise decreased toxin production upon removal or reduction of nitrate from medium was reported for Microcystis aeruginosa (Utkilen and Giolme, 1995) and for production of nodularin by Nodularia spumigena and Aphanizomenon flos-aquae at high inorganic nitrogen concentrations (42.000 mg/L) (Lehtimäki et al., 1997). High toxin production in Oscillatoria agardhii strains also correlated with high nitrogen concentrations (test range, 0.42 to 84 mg of N per liter) (Sivonen, 1990). Several species of Microcystis and Oscillatoria synthesize elevated quantities of toxic secondary metabolites under high nitrogen conditions (Borowitzka, 1999). Thus, different cyanobacteria seem to differ in their responses to external nitrogen concentrations. In summary, this study showed that limitation of nitrogen appeared to be suitable stimulants for accumulation of antimicrobial active substances and nitrogen concentration had no significant effect on the growth of Westiellopsis sp.VN in the large scale cultivation. Our results are in general agreement with the results of Chetsumon et al., 1995 who found that in batch culture immobilized cells of Scytonema sp. TISTR 8208, secreted the antibiotic after the complete depletion of nitrate in the medium, but different from results reported by other authors, e.g, Boor & England, 1991, who reported that increasing nitrate from its base level of 8mM to 26.4mM allowed an increase in antibiotic production of Nostoc muscorum .
a a a a a a a Westiellopsis a Effect of incubation time on biomass production and antimicrobial compound accumulation of Westiellopsis sp.VN in batch cultivation with BG 11 medium without 0 NaNO 3 at temperature 20±2 C, pH 7.4, and continuous illumination provided by cool- white fluorescent tube of 8µmol/m 2 was investigated. Growth rate of Westiellopsis sp.VN was monitoring by measure dry biomass weight after 2, 3, 4, 5, 6, 7, and 8 weeks as well as the production of antimicrobial compounds was evaluated by measuring the diameter of inhibition zone of methanol extracts obtained from dry biomass in agar diffusion assays using S. aureus . The results showed that the amount of biomass increased during cultivation time and the biomass yield and diameter of inhibition zone increased towards the end of the 7-to 8- week growth period but the inhibition zone of the extract after 8 weeks cultivation is the most clear. From these
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Chapter IV Discussion results we conclude that antimicrobial substances were released over the whole cultivation time by Westiellopsis sp.VN strain. Antimicrobial activity increased fast at the beginning of the exponential phase (log phase) and continued to increase gradually over the whole incubation time during log phase to stationary phase. Antimicrobial activity was the highest after cultivation time of 8- weeks, during the late stationary phase of the culture. To date, effect of cultivation age on bioactive compound accumulation of cyanobacteria was investigated by several groups. Codd et al ., 1989 reported that the cyclic peptide hepatotoxins produced by Microcystis aeruginosa are retained within the cells during the lag and growth phases, with significant amounts being released only after the culture becomes senescent and cells begin to lyse. Lehtimäki et al., 1994 described that in batch cultures, nodularin concentrations in growth media increased with incubation time indicating release of intracellular nodularin when cells lysed. In contrast to this Westhuizen & Eloff, 1983 who studied effect of culture age on toxicity of the blue-green alga Microcystis aeguginosa showed that toxicity increased gradually during the exponential growth phase to a maximum (LD 50 = 18 mg kg -1) at the beginning of the stationary phase and then decreased. Borowitzka, 1995 postulated that synthesis of cyanobacterial metabolites generally occurs during the late exponential and early stationary phase of organism growth. From our own results and results of other authors above-mentioned, we conclude that the production of the active compounds already starts in the exponential growth phase, but the synthesis enhances clearly during the stationary phase. For isolation of active compounds in higher yields harvest of the biomass seems to be meaningful in the stationary phase of the culture. Influence of physicochemical factors on synthesis of secondary, bioactive compounds of Westiellopsis sp.VN requires further investigation. The role of other factors such as pH, temperature, illumination intensity, different N, P, and C sources, and micronutrients on antimicrobial production should be determined. Furthermore the influence of competitors in the culture for example heterotrophic bacteria or other cyanobacteria or microalgae, normally living together with this cyanobacterium in its natural environment should also be investigated. Future studies are also needed to determine the genetic or biochemical factors regulating synthesis of antimicrobial substances by cyanobacteria.
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Chapter IV Discussion
a a Calothrix javanica a Scytonema ocellatum Calothrix javanica In screening 12 different cyanobacterial strains isolated from soil samples in Dak Lak province of Southern Vietnam for antibacterial activity, we have found that the extracts of the cyanobacterium Calothrix javanica showed activity against Gram- positive (B.subtilic and S.aureus) and Gram-negative (E.coli) bacteria. This strain was therefore selected for bioassay-guided isolation of the active substances. Morphological identification of the Calothrix javanica isolated from a sample of rice-field soil collected from EaRoc, Ea Sup, Dak Lak, Vietnam was made in accordance with traditional phycological system and by using RAPD-PCR technique (Ho & Vo, 2004; Ho et al., 2006; Ho, 2007). Scytonema ocellatum The extracts obtained from this strain exhibited also strong antimicrobial activity against Gram-positive bacteria ( B. subtilic and S.aureus ) in our screening and this strain was also selected for further chemical investigation. Morphological identification of the cyanobacterium Scytonema ocellatum isolated from a sample of rice-field soil collected from EaRoc, Ea Sup, Dak Lak, Vietnam was made in accordance with traditional phycological system (Ho & Vo, 2004; Vo et al., 2006; Ho, 2007).
Calothrix javanica a Scytonema ocellatum
According to Houghton & Raman, 1998, extracts from cyanobacteria, leaves and other green parts of plants made with ethanol, methanol, chloroform and solvents of similar polarity will contain large amounts of chlorophyll. For removal of chlorophyll, the reverse-phase column chromatography was used for alcoholic extracts. This method exploits the non-polar nature of chlorophyll so that it is retained on a reverse-phase adsorbent while more polar components pass through. With aforementioned reasons reverse-phase C 18 column chromatography was employed for first purification of the active methanol extract from biomass of Calothrix javanica . Bioassay-guided fractionation of the active methanol extract prepared from the lyophilized biomass of the Calothrix javanica strain led to the isolation of a new
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Chapter IV Discussion cyclic peptide (3.8mg, 1.89% yield, based on the mass of the crude methanol extract) exhibiting strong antibiotic activity. The structure of this new compound was elucidated by exhaustive 1D ( 1H) and 2D (COSY, TOCSY, NOESY, HMQC, HMBC) NMR spectroscopy in combination with HR-ESI-MS. The new cyclic peptide was named as daklakapeptin. Daklakapeptin was found to have totally 12 residues including 6 proteinogenic amino acids (Pro, Tyr, Ile, Leu, Gln, Thr), 4 complexes (X,Y,T,Z) and the methyl derivative of Ile. The exact sequence of daklakapeptin is shown in figure 4-2
IleNMe Thr Z Pro Tyr Thr Sequence Gln Leu T Y Ile X of daklakapeptin
Up to now, there have been only a few scattered reports of the chemistry and biological activity of the Calothrix genus in the literature. The first mentioned calophycin, a cyclic decapeptide containing a novel (2R, 3R, 4S)-3-amino-2-hydroxy- 4-methylpalmitic acid unit (Hamp), is a potent broad-spectrum fungicide from Calothrix fusca EU-10-1 with the MIC against Candida albicans of 1.25µg/ml (Moon et al., 1992). Then, calothrixins A and B, pentacyclic indolophenanthridines, isolated from two Calothrix strains, inhibited the growth in vitro of a chloroquine resistant strain of Plasmodium falciparum , the most virulent strain of plasmodia to humans, with IC 50 values of 58 nM and 180 nM, respectively. These calothrixins also exhibited potent activity against the human cervical cancer cell line, HeLa, with IC 50 values of 40, 350 nM, respectively (Rickards et al., 1999). Calothrixin A also inhibited RNA synthesis and DNA replication (Doan et al., 2000; 2001). Abarzua et al., 1999 reported that crude extract of Calothrix brevissima showed very low antialgal effects against the diatom Nitzschia pusilla . Recently, Berry et al., 2008 reported that five isolates of Calothrix ( Calothrix 13-3, Calothrix 30-1-13, Calothrix 3-26, Calothrix 3-27, Calothrix 67-1) from the Florida Everglades and South Florida contained Calothrixin A. According to Becher et al., 2009, a screening for cyanobacterial cholinesterase inhibitors had shown that low inhibitory effects on BChE (butyrylcholinesterase) activity was observed for crude extract of Calothrix anomala and Calothrix 7507 caused increased activities of BChE compared to the 100% control.
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Chapter IV Discussion
In present study our new peptide showed antibacterial activity against S .aureus with the diameter of inhibition zone of 12.5mm in concentration of 200mg/dics (see table 3-13). Chemical investigation of the active methanol extract resulting from lyophilized biomass of Scytonema ocellatum led to the isolation of a pure active compound (2.4mg, 1.21% yield, based on the mass of the crude methanol extract) which exhibited strong antimicrobial activity with the diameter of inhibition zone of 12 mm in concentration of 200mg/dics (see table 3-20). Interestingly, the pure active compound from Scytonema ocellatum strain displayed protonated molecular ion peak at m/z 1432 [M+H] + in ESIMS positive mode, the same that was found for the new peptide isolated from the biomass of Calothrix javanica (see appendix 14c). Moreover, the 1H NMR forms of both compounds was almost the same (see appendix 14 a,b). In order to verify that the two compounds isolated from Calothrix javanica and Scytonema ocellatum strains were identical, the crude methanol extracts prepared from Calothrix javanica and Scytonema ocellatum strains were analyzed by HPLC with the same conditions. The results of analytical HPLC revealed that both crude methanol extracts from Calothrix javanica and Scytonema ocellatum displayed the same peak with t R 15.35 min (see figure 4-3, 4-4).
Analytical HPLC chromatogram of methanol extract of Calothrix javanica with mobile phase of Acetonitrile/ H 2O (from 5% to 100% Acetonitrile in 32min) and 2 mg/mL/injection, detection at 246nm, column Synergi POLAR-RP 80A (250×4.6mm, 4 micron), flow rate 1mL/min. 132
Chapter IV Discussion
Analytical HPLC chromatogram of methanol extract of Scytonema ocellatum with mobile phase of Acetonitrile/ H 2O (from 5% to 100% Acetonitrile in 32min) and 2 mg/mL/injection, detection at 246nm, column Synergi POLAR-RP 80A (250×4.6mm, 4 micron), flow rate 1mL/min.
Thus, it was confirmed that these two compounds isolated by us from Calothrix javanica and Scytonema ocellatum strains were identical and the same peptide was synthesized by these two different species. This is interesting but not surprising because in the literature there are several reports on strains of different genera within the same family or even on strains of different families can synthesize identical compounds (Sivonen & Börner, 2008). In our case, Calothrix javanica belongs to genus Calothrix and Scytonema ocellatum strain belongs to genus Scytonema, and both of these genera belong to the order Nostocales (Komárek & Anagnostidis, 1989; 16S rRNA tree showing the close relationships of both genera shown in fig.4-5).
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Chapter IV Discussion
The phylogenetic relationships of cyanobacteria inferred from 16S rRNA nucleotide sequence (Tomitani et al., 2006)
To date, several strains of the genus Scytonema have been proven to be producers of bioactive secondary metabolites. Of the genus Scytonema the freshwater cyanobacterium, Scytonema hofmanni , is known to be highly toxic toward other cyanobacteria and green algae (Mason et al., 1982). It has been suggested that Scytonema hofmanni produces allelopathic substances like the chlorine-containing γ- lactone, cyanobacterin, allowing this slow-growing organism to compete with more prolific organisms (Pignatello et al., 1983). Besides this, several depsipeptides with biological activities, especially protease inhibition, have been reported from this species (Matern et al. , 2001; 2003). Additionally, nostodione A previously isolated from Nostoc commune was also isolated from this strain and nostodione A is known to be proteasome inhibitor with an IC 50 value of 50 µM (Hee et al., 2008). Scytophycins macrolides that inhibit the proliferation of a wide variety of mammalian cells were isolated from the genera Cylindrospermum, Scytonema , and Tolypothrix (Patterson et al ., 1994); Scytophycins from Scytonema pseudohofmanni displayed cytotoxic and antimycotic activity (Ishibashi et al., 1986). Tantazoles and mirabazoles
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Chapter IV Discussion are unusual alkaloids from the terrestrial cyanobacterium Scytonema mirable which show murine and human solid tumour selective cytotoxicity (Carmeli et al., 1990a; 1991, 1993); Scytonemin A, a cyclic peptide, the major metabolite of a Scytonema sp. strain, possesses potent calcium antagonistic properties on atria at 5µg/mL (diltiazem was active at 2.5µg/mL) and on portal vein of rat at 20 µg/mL (diltiazem showed activity at 0.5 µg/mL). Also scytonemin A is weakly active against a wide spectrum of bacteria and fungi, and is mildly cytotoxic (Helms et al., 1988). A group of known and new diacylated sulfoglycolipids were isolated from a strain of Scytonema sp. (Reshef et al., 1997). Up to now, there have been several reports of the chemistry and biological activity of the Scytonema ocellatum strain in the literature. Tolytoxin (6-hydroxy-7- O-methylscytophycin B) and two scytophycins (19-O- demethylscytophycin C, and 6- hydroxy-7-O-methylscytophycin E) were isolated from this species. These pure compounds showed MICs of 1-5 ng/ML and 10-50 ng/mL against KB and LoVo, respectively (Carmeli et al., 1990b). Tolyxin also is a potent antifungal antibiotic, exhibiting MIC s in the range of 0.25 to 8 nM (Patterson & Carmeli, 1992) and functions as a phytoalexin, a defense agent produced in response to fungal attack (Patterson & Bolis, 1997). In the present work, new cyclic peptide shows antibacterial activity against S. aureus . Both marine and freshwater cyanobacteria have the ability to produce a large number of peptide metabolites. These cyanopeptide metabolites have enormous structure diversity ranging from linear to cyclic and multicyclic with simple peptides, depsipeptides, and lipopeptides structures encompassing a size range of 300-2000 Da. Many peptides contain nonproteinogenic residues, like D-amino acids, β-amino acids, hydroxylated and N-methylated amino acids, in addition to proteinogenic amino acids, which add further to their structure diversity (Van Wagoner et al., 2007). Several biosynthesis pathways of cyanobacterial peptides have yet to be studied (Welker & von Döhren, 2006; Van Wagoner et al., 2007, Sivonen et al., 2007; Gerwick et al ., 2008; Jones et al., 2010). These cyanopeptide metabolites possess a varietyof bioactivity, e.g. antifungal activity (Neuhof et al ., 2005), antimalaria activity (Linington et al., 2007; McPhail et al., 2007), antiviral activity (Zainuddin et al ., 2007), protease inhibition activity (Baumann et al., 2007, Taori et al., 2008), cytotoxic activity (Linington et al ., 2008, Gutiérrez et al., 2008; Tripathi et al., 2009),
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Chapter IV Discussion antiprotozoal activity (Simmons et al ., 2008), and antimicrobial activity (Zainuddin et al ., 2009). The cyclic peptides are of particular interest because of their generally high bioactivity and structural diversity, and the fact that they are phylogenetically very old (Rantala et al., 2004). The synthesis of cyclic peptides is nonribosomal and controlled by cassettes of enzymes encoded by gene clusters (Meissner et al., 1996). The gene clusters are subjected to natural recombination {imperfect repeats, gene loss and horizontal gene transfer} (Mikalsen et al., 2003), which can explain the large variation in cyclic peptide sequence. It has been postulated that the gene clusters coding the nonribosomal peptide synthesis pathways can be engineered to produce peptides with desired activity such as antibiotic, immunosuppressant or anti-cancer activity (Neilan et al ., 1999). Amino and carboxy termini are themselves linked together with a peptide bond, forming a circular chain. A number of cyclic peptides have been discovered in nature and they can range from a few amino acids in length to hundreds. Cyclic peptides tend to be extremely resistant to digestion, allowing them to survive intact in the human digestive tract. Several cyclic peptides from cyanobacteria exhibit antimicrobial activity. For example, kawaguchipeptins A and B are two cyclic undecapeptides with antibacterial activity isolated from Microcystis aeruginosa . They inhibit the growth of the Gram-positive bacterium Staphylococcus aureus at a concentration of 1 µg/mL (Ishida et al., 1997). In our work, new cyclic peptide showed antibacterial activity against S. aurerus with diameter of inhibition zone of 12.5 mm in concentration of 200mg/dics. Further tests for activity to other bacteria and cytotoxic activity are in progress.
a a Anabaena Anabaena
In our antibacterial screening, the crude extracts obtained from Anabaena sp. strain cultivation medium revealed a strong and wide range of antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as yeast Candida maltosa. Biologically active metabolites isolated from cyanobacteria so far have been mostly accumulated in the cyanobacterial biomass and so far only a couple of bioactive compounds have been isolated from the cultivation medium e.g., some antibacterial
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Chapter IV Discussion diterpenoids from Nostoc commune (Jaki et al., 1999a; 2000a), antifungal peptides from Tolypotrix byssoidea (Jaki et al., 2001), or the antialgal, antibacterial and antifungal indole alkaloid norharmane from Nodularia harveyana as well as the 4,4′- dihydroxybiphenyl from Nostoc insulare (Volk, 2005; Volk & Furkert, 2006). Thus, this strain was very interesting for bioassay guided isolation of the components in medium responsible for the antibacterial activity. Morphological identification of the cynobacterium Anabaena sp. strain isolated from a sample of industrial cultivating soil (cotton) collected Dak Lak province of Vietnam was made in accordance with traditional phycological system (Ho et al., 2005; Ho, 2007).
Anabaena
Bioassay-guided isolation of the ethyl acetate extract from the microscopically cell-free cultivation medium resulted in identification of the aromadendrane sesquiterpene, flourensadiol, (1.3mg, and 10.83% of the dry extract weight) which was previously isolated from the common western shrub Flourensia cernus collected near Big Bend, Texas, USA (Kingston et al., 1975, Pettersen et al., 1975). This plant is a deciduous shrub endemic to the Chihuahuan Desert of the southwestern United States and northern Mexico. The structure of flourensadiol was established using an extensive array of 1D (1H and 13 C, and DEPT-135) and 2D (HMQC, COSY, and HMBC) NMR and HR- ESI-MS experiments as described in detail in 3.7. To the best our knowledge, this is the first report on occurrence of an antibacterial sesquiterpenoid compound in a cyanobacterium and the antibacterial activity of flourensadiol was also reported for the first time. Kingston et al., 1975 and Pettersen et al., 1975 determined the structure of the compound by X-ray analysis of the crystallized compound and published MS, IR, and proton NMR data. In our work, the structure of flourensadiol was established using an extensive array of 1D ( 1H , 13 C, DEPT-135) and 2D (HMQC, COSY, HMBC) NMR and HR-ESI-MS experiments, and the complete NMR data of flourensadiol are reported for the first time. Occurrence of terpenoids in cyanobacteria is uncommon (Prinsep et al ., 1996; Jaki et al., 1999a, 2000a,b). Some authors published the occurrence of diterpenoids with antibacterial (Jaki et al., 1999a, 2000b; Gutiérrez et al., 2008), cytotoxic and
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Chapter IV Discussion molluscicidal activities (Jaki et al ., 2000a), as well as anti-inflammatory activity (Prinsep et al., 1996). Moreover, further examples are the triterpenoid bacteriophanes (Simonin et al., 1992) isolated from several species of cyanobacteria. The antifungal hapalindoles, hapalindolinones, and ambiguines as well as the welwitindolinones are metabolites of mixed biosynthetic origin containing isoprene units and have been found in several species of the family Stigonemataceae (Prinsep et al., 1996; Jaki et al., 1999a). Up to now, there is no information in literature about pharmacological effects of flourensadiol except a report of Kingston et al., 1975 which indicated that the plant Flourensia cernua from which flourensadiol was isolated, possess toxicity to sheep and goat. The toxicity was found in the petrol-insoluble portion which contained flourensadiol together with other components. In the report of Estell et al., 1994 it was shown that several of the terpenes identified in tarbush which was collected in a heavily infested tarbush area on the Jornada Experimental Range exhibited antimicrobial activity in ruminants. It is of interest to note that in our study flourensadiol exhibited antibacterial activity against E.coli with diameter of inhibition zone of 20.0mm in concentration of 200mg/dics but due to the very small amount (1.3 mg) of flourensadiol isolated by our work it was not performed in bioassays for further activities, but this strain should be investigated for further biological activities. The natural function of cyanobacterial metabolites so far is unknown but exometabolites in all likelihood are associated with the interaction between competing microorganisms of the same habitat (Volk & Furkert, 2006), particularly in resource- limited environments for example in case of the isolated fluorensiadiol. The common western shrub Flourensia cernus was collected near Big Bend, Texas, USA and the Anabaena sp. was isolated from soil of cotton fields of Dak Lak province, both habitats with poor nutrients and high temperature. It could be most likely that these sesquiterpenes area involved in defense reactions against cohabitants and improve chances of survival in their environment.
a a Nostoc Nostoc
The extracts obtained from this strain exhibited strong antimicrobial activity against Gram-positive bacteria ( B. subtilic and S.aureus ) and Gram-negative bacteria
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Chapter IV Discussion
(E.coli) in our screening for cyanobacterial antibacterial activity. Thus, this strain also was selected for further chemical investigated. Morphological identification of the cynobacterium Nostoc sp. isolated from a sample of rice-field soil collected from Dak Lak province, Vietnam was made in accordance with traditional phycological system (Ho, 2007).
Nostoc
The low resolution ESI-MS of fraction NsF 2 which exhibited antibacterial activity against Staphylococcus aureus with diameter of inhibition zone of 10.0mm in concentration of 500mg/dics showed signal at m/z 426 [M+H] +. The NMR and MS characterization of compound in this fraction NsF 2 is in progress.
a a a Lyngbya majuscula a a a L. majuscula a
Marine cyanobacteria would probably ranked along side the actinomycetes and myxobacteria as a prolific producer of unique natural products. Over the past 30 years, the research for bioactive secondary metabolites (natural products) from marine organisms has yielded a wealth of new molecules (estimated at approximately 17.000) with many fundamentally new chemotypes and extraordinary potential for biomedical research and applications (Blunt et al . 2008). Marine cyanobacteria belong to the most fruitful sources of marine natural products, with nearly 700 compounds described (Tan, 2007; Jones et al., 2009). Majority of the papers is dominated by cyanobacterial collections from reef systems in Hawaii, the Caribbean, Madagascar, and Papua New Guinea. However, a few is known on the chemistry of marine cyanobacteria from other parts of the world, such as South East Asia where biodiversity is high (Tan, 2006). At present, a few is known on biological activity and chemistry of marine cyanobacteria from Vietnam. Here is the opportunity to search for novel cyanobacterial biomolecules from these marine areas which have not been studied intensively so far. The filamentous marine cyanobacterium Lyngbya majuscula (Gomont) is of particular importance, as approximately 35% of all cyanobacterial bioactive
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Chapter IV Discussion compounds identified so far have been isolated from the genus Lyngbya, with 76% of these coming from Lyngbya majuscula (Jones et al. 2009) . The compounds isolated from Lyngbya majuscula exhibit a variety of biological activities including antimicrobial, antiproliferative, immunosuppressant activities. Thus, it is accepted that the marine cyanobacterium Lyngbya majuscula is an exceptional source of novel potential pharmaceuticals. Although a lot of studies have been carried out and are still going on in the research for novel bioactive compounds from Lyngbya majuscula , there is little report on bioactive compounds isolated from this marine cyanobacterium growing near the coasts of Vietnam. In our studies, chemical investigation of a Lyngbya majuscula strain collected in Vietnam at Hon Khoi locality in Khanh Hoa province was undertaken. The n-hexane, methanol, and water extracts prepared from lyophilized biomass of this strain were screened for biological activities including antibacterial, antifungal and cytotoxic activity. Interestingly, the methanol extract showed strong cytotoxic activity against 5678 human cell line but weak antimicrobial activity against Gram-positive bacteria and a yeast ( B. subtilis, S. aureus , and C. maltosa with an inhibition zone of 8, 7, and 9 mm, respectively), and no antibacterial activity against Gram-negative bacteria ( E. coli and P. aeruginosa ); therefore this strain was investigated for compounds responsible for cytotoxic activity.
a a a Lyngbya majuscula a
The chemical investigation with an emphasis on the isolation and structure elucidation of cytotoxic compounds of the methanol extract resulting from this strain led to the isolation and identification of the 3 cytotoxic compounds anhydrodebrommoaplysiatoxin (compound 9), debromoaplysiatoxin (compound 10), and anhydroaplysiatoxin (compound 11). Identification of these cytotoxic compounds was established by direct comparison of our spectroscopic data, including 1D ( 1H, 13 C) NMR and HR-ESI-MS with those reported in the literature as described in 3.7.1 and 3.7.2. Debrommoaplysiatoxin (compound 10) was first isolated from the digestive gland of the sea hare Stylocheilus longicauda (Kato & Scheuer, 1974, 1975) and then from the marine blue-green alga Lyngbya majuscula (Moore et al., 1984b), but also
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Chapter IV Discussion from a mixture of two blue-green algae Schizothrix calcicola and Oscillatoria nigroviridis (Moore et al., 1984). Compound 10 has been showed to be an activator of protein kinase C and to be a potent tumor promoter (Fujiki et al ., 1981, 1984; Nagai et al ., 1997) and displayed some antineoplastic activity (Mynderse et al., 1977). In our studies, debrommoaplysiatoxin (compound 10) exhibited cytotoxic activity against bladder cancer cell line 5637 with IC 50 of 86ng/ml. Anhydrodebromoaplysiatoxin (compound 9) and anhydroaplysiatoxin (compound 11) were first obtained as artifact during the purification of debromoaplysiatoxin and aplysiatoxin from the sea hare Stylocheilus longicauda (Kato & Scheuer, 1976). However, Moore et al., 1984 and Nagai et al., 1998 found compound 9 as a natural product of L. majuscula collected in Marshall Islands and the Hawaii Red Alga Gracilaria coronopifolia , respectively . Compound 9 exhibited antineoplastic activity (Mynderse et al. , 1977) and had been reported as potent tumor promoter (Fujiki et al., 1982; Nagai et al., 1998). In our study, compound 9 and 11 have been identified in the biomass of L. majuscula , the anhydroaplysiatoxin exhibited cytotoxic activity against bladder cancer cell line 5637 with IC 50 of40 ng/ml but anhydrodebrommoaplysiatoxin was not yet tested for cytotoxic activity. Notably, Chlipala et al., 2010 reported that nhatrangins A and B, anhydrodebrommoaplysiatoxin, and anhydroaplysiatoxin have been isolated from a Vietnamese collection of Lyngbya majuscula . Nhatrangins A and B are described as two polyketide metabolites in biosynthesis of the aplysiatoxins by this cyanobacterium. The carbon skeleton of these molecules appears to be related to the carbon skeleton of the aplysiatoxins and may give an insight into the biosynthesis of these metabolites. In addition, it is of interest to note that the biomass of the marine cyanobacterium L.majuscula used by Chlipala et al., 2010 and used in this work were collected in Nhatrang of Khanh Hoa province, Vietnam, but our sample was collected in August while the Chlipala sample was collected in May. This may be the reason for differences in our results comparing with the results of Chlipala et al., 2010, since differences in environmental conditions in the dry season and the rainy season might change the production of biologically active metabolites.
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Chapter IV Discussion
In our chemical investigation, chemically different and interesting compounds have been isolated from cyanobacteria. These results promise that further chemical and biological investigations may provide interesting findings. In further studies, following points should be included: - The new cyclic peptide daklakapeptin isolated from Calothrix javanica and Scytonema ocellatum as well as flourensadiol isolated from Anabaena sp. should be isolated in sufficient amounts for further biological investigations with a view to future therapeutic applications. - The influence of the composition of the cultivation media on the growth rate and chemical production of cyanobacteria have to be optimized to improve the synthesis of interesting substances. - For compounds isolated from the cultivation medium such as flourensadiol, it would be interesting to study the release mechanisms of these compounds into the media to optimize the culture conditions and the release rate. - Because cyanobacteria often produce substances with unusual structure with bioactive diversity, it is of great important to study the biosynthetic pathways of these cyanobacteria. - The hazardous effects, ecological role, and physiological functions of the cyanobacterial secondary metabolites up to now are poor understood. Thus, these aspects especially the ecological role of secondary metabolites should be investigated. - A comparison of the chemical and morphological characteristics of cyanobacterial strains which have been cultivated for long time under laboratory conditions with re-collected organisms from nature should be undertaken in all likelihood to reveal the environment influences on these organisms.
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Summary
a
Cyanobacteria are a diverse and ancient group of photosynthetic prokaryotic organisms that can inhabit a wide range of environments including extreme conditions such as hot springs, desert soils and the Antarctic. They are abundant producers of natural products well recognized for their bioactivity and utility in drug discovery and biotechnology applications. Novel intracellular and extracellular compounds from various cultured and field cyanobacteria with diverse biological activities and a wide range of chemical classes have considerable potential for development of pharmaceuticals and other biomedical applications. However, cyanobacteria are still viewed as unexplored source of potential drugs. Especially the collections of cyanobacterial strains from South East Asia where biodiversity is high are still largely unexplored. Thus, we investigated twelve soil cyanobacterial strains isolated from soil samples collected from rice, cotton, and coffee fields in Dak Lak province of Vietnam and one marine strain, Lyngbya majuscula collected from Khanh Hoa province of Vietnam for the search for new compounds with antimicrobial and cytotoxic activities.
From the 12 soil cyanobacterial strains, 48 extracts prepared with n-hexane, methanol, and water for biomasses and ethyl acetate for growth media were screened for antibacterial activity against Gram-positive bacteria ( Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 6538) and Gram-negative bacteria ( Escherichia coli ATCC 11229 , Pseudomonas aeruginosa ATCC 27853). Of 48 extracts, 47.92% and 45.83% showed activity against Bacillus subtilis and Staphylococcus aureus , respectively, while 22.92% and 6.25% exhibited activity against Escherichia coli and Pseudomonas aeruginosa , respectively. All investigated cyanobacteria (12/12) showed antibacterial activity to at least one of the test organisms applied. Among the active extracts, extracts obtained from 5 cyanobacterial strains, Westiellopsis sp. VN, Calothrix javanica , Scytonema ocellatum , Anabaena sp. and Nostoc sp. showed the highest strength and range of antibacterial activity and therefore were selected for chemical investigation with an emphasis on the isolation and structure elucidation of antimicrobial compounds.
Bioassay-guided fractionation of the methanol extract prepared from biomass of Westiellopsis . by silica gel chromatography, followed by sephadex LH-20 chromatography and reversed-phase HPLC led to isolation and identification of 6 compounds as ambiguine D isonitrile, ambiguine B isonitrile, dechloro-ambiguine B isonitrile,
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fischerellin A, hydroxy-eicosatetraenoic acid and methoxy-nonadecadienoic acid. Identification of these active compounds was established by direct comparison of our spectroscopic data, including 1H NMR and HR-ESI-MS with those reported in the literature. All these compounds showed biological activity. The identification of fatty acids and other volatile components by GS-MS in the active MeOH fraction obtained from EtOAc extract of growth medium was done before commencing further fractionation processes.
Culture optimization of Westiellopsis sp.VN showed that NaNO 3 deficiency increased accumulation of antimicrobial compounds. Biosynthesis of antimicrobial compounds increased over cultivation time resulting in increased diameter of inhibition zone of the methanol extract towards the end of the 7-to 8- week growth period, but the most clear inhibition zone of this extract was detected after cultivation time of 8 weeks.
Bioassay-guided fractionation of the methanol extract prepared from biomass of either Calothrix javanica by C 18 chromatography followed by reversed-phase HPLC or
Scytonema ocellatum by C 18 chromatography followed by silica gel chromatography and reversed-phase HPLC led to isolation and structure elucidation of new cyclic peptide named daklakapeptin. Structure of daklakapeptin was elucidated by exhaustive 1D ( 1H) and 2D (COSY, TOCSY, NOESY, HMQC, HMBC) NMR spectroscopy in combination with HR- ESI-MS. Daklakapeptin was found to have totally 12 residues including 6 proteinogenic amino acids (Pro, Tyr, Ile, Leu, Gln, Thr), 4 complexes (X,Y,T,Z) and the methyl derivative of Ile. The exact sequence of daklakapeptin is shown in following figure
IleNMe Thr Z Pro Tyr Thr
Gln Leu T Y Ile X with X: (CH 3)2CHCH 2CH 2CH(NH-)CH 2CO-, Y:(CH 3)2CHCH(OH)CH(NH-)CO-,
T: HOCH 2CH 2CH(NH-)CO-, Z: HOCH 2CHOHCH(NH-)CO- This new cyclic peptide exhibited antibacterial activity against Staphylococcus aureus with diameter of inhibition zone of 12.5 mm in concentration of 200 mg/disc. Further test for activity to other bacteria and for cytotoxic activity are in progress.
Using reversed-phase HPLC to separate compounds in the crude ethyl acetate extract obtained from culture medium of Anabaena led to isolation and structure elucidation of flourensadiol. The structure of flourensadiol was established using an extensive array of 1D (1H, 13 C, DEPT-135) and 2D (HMQC, COSY, HMBC) NMR and HR-ESI-MS experiments.
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Flourensadiol was isolated previously from the common western shrub Flourensia cernua. However, only MS, IR, and proton NMR data but no reports on biological activity were available. In this study, we report the complete NMR data of flourensadiol for the first time. Flourensadiol was found to be very strong antibacterial active against Escherichia coli with diameter of inhibition zone of 20.0 mm in concentration of 200 mg/disc. Further test for activity to other bacteria and cytotoxic activity are in progress.
Bioassay-guided fractionation of the methanol extract from biomass of Nostoc by silica gel chromatography followed by C 18 chromatography and reversed phase HPLC led to isolation of the active fraction NsF 2 which exhibited antibacterial activity against Staphylococcus aureus with diameter of inhibition zone of 10.0 mm in concentration of 500 + mg/disc. The low resolution ESI-MS of fraction NsF 2 showed signal at m/z 426 [M+H] . The
NMR and MS characterization of compounds in fraction NsF 2 is in progress.
Bioassay-guided fractionation of the methanol extract prepared from biomass of marine cyanobacterium Lyngbya majuscula collected from Khanh Hoa province of Vietnam by various chromatographic methods (CC, PTLC, HPLC) afforded 3 cytotoxic compounds anhydrodebromoaplysiatoxin, debromoaplysiatoxin, and anhydroaplysiatoxin. Identification of these cytotoxic compounds was established by direct comparison of our spectroscopic data, including ( 1H, 13 C) NMR and HR-ESI-MS with those reported in the literature. In our study, debromoaplysiatoxin and anhydroaplysiatoxin exhibited cytotoxic activity against bladder cancer cell line 5637 with IC 50 of 86 ng/ml and 40 ng/ml, respectively but anhydrodebromoaplysiatoxin was not yet tested for cytotoxic activity. The identification of fatty acids by GS-MS technique in the n-hexane extract obtained from biomass of this marine cyanobacterium was undertaken before commencing further fractionation processes. The presented results prove that soil cyanobacteria are a promising source to yield chemical and pharmaceutical interesting compounds.
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This PhD thesis work was carried out at Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst-Moritz-Arndt University Greifswald, Germany with help from many individuals and organizations. First and foremost, I would like to express my deepest gratitude and thanks to my supervisor, PD. Dr. Sabine Mundt who gave me the great opportunity to be involved in cyanobacterial natural product research in her group with for generous support, continuous encouragement, direct expert guidance and admirable advice throughout my work. She will always have a special place in my memories. I wish to express my special gratitude to Prof. Dr. Ulrike Lindequist for giving me an opportunity to study in her Institute, for guiding, supporting and providing working facilities during my work, and for partial financial supports in finishing my study. I am deeply indebted to Dr. Victor Wray and Dr. Rolf Jansen, Helmholt Center for Infection Research Braunschweig for structure elucidation of the isolated natural products. Also I would like to express my sincere thanks to Dr. Victor Wray for giving me a chance to be in his laboratory and for his guidance, fruitful discussion, constructive advises, and valuable moral support in finishing this thesis. I would like to express my great thanks to Dr. Dang Diem Hong in Institute of Biotechnology, Vietnam Academy of Science and Technology, Ha Noi, Vietnam; Dr. Ho Sy Hanh in Pedagogical College, Dak Lak, Vietnam; M.Sc. Pham Huu Tri in Institute of Oceanography, Vietnam Academy of Science and Technology, Nha Trang, Vietnam for providing cyanobacterial strains. I would like to express my sincere thanks to Dr. Kristian Wende in our institute for cytotoxic tests and Dr. Martina Wurster in our institute for identification of the volatile components and fatty acids of some my samples together with their kind help and useful suggestions during my work. I would like to express my great thanks to Mrs. H. Bathrow not only for her help, advise, encouragement during culture of cyanobacteria, but also for moral support, nice atmosphere, and familiar situation during my stay in Greifswald. My special thanks come to PD. Dr. Michael Lalk in our institute for his help in recording HRMS and NMR spectra of some compounds and for supporting me as
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well as spending time on my questions. The kind help of Mr. Dipl. Phys. K.H. Lichtnow in the aspect of computer is also appreciated. I would like to express my gratitude to Prof. Dr. Le Tran Binh, Dr. Jörn Kasbohm, Dr. Le Thi Lai (Join Graduate Education Program) and Dr. Nguyen Van Ngu (Ministry of Education and Training of Vietnam) for their kindness, generous considerations, precious advices, unwavering support, and continuous encouragement and for giving me a chance to study in Greifswald. My special thanks to Prof. Dr. Ramzi A.A. Mothana in Faculty of Pharmacy, Sana’a-University, Yemen; Prof. Dr. Sahar Hussein in National Research Center, Egypt; Dr. Wajid Rehman in Department of Chemistry, Hazara University, Pakistan; Dr. Gerold Lukowski in Institute of Marine Biotechnology, Greifswald, Germany for their useful discussions and scientific advices to broaden my knowledge. I wish to thanks all members of Pharmaceutical Biology Department, Institute of Pharmacy, Greifswald, former and present, for the friendly and comfortable working atmosphere. Especially, I would like to thanks Mrs. R.Ball, Mrs. Matthias, Mrs. Fenske, PD. Dr. B. Haertel, M. Preisitsch, E. Puhlmann, W. Poggendorf, B.T. Huong, M.Harm, S. Blackert, K. Tarman, M. Shushni, A. Hassan, K. Eiden, Z. Alresly, C. Bäcker for technical assistance, exchanging experiments and nice discussion related to work and life in Greifswald. Their friendship really helped me to warm up my time of stay in Greifswald and help me to understand more about the people and the culture of Germany. I wish to express my thanks to all members of board of direction of Hong Duc University and all my colleagues of Natural Sciences faculty and in Department of Plant, Hong Duc university, my friends and my students in Vietnam and all over the world for their supporting, sharing and caring. My deep thanks to all members at the Department of Algal Biotechnology, Institute of Biotechnology, VAST for their kind help and sharing a nice atmosphere, and fruitful discussions during my time in this department, especially I would like my express deepest gratitude to Dr. Dang Diem Hong the leader of this department not only for providing soil cyanobacterial strains but also for providing me an opportunity to work in her laboratory and her guidance, valuable support, fruitful discussions, and constructive advices, in algae culturing techniques and molecular biology of cyanobacteria.
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Prof. Dr. Vo Hanh in Vinh University and Dr. Duong Thi Thuy in Institute of Environmental Technology, VAST, Ha Noi are thanked for their kind help and fruitful discussion on identification and culturing of cyanobacteria. Also Dr. Nguyen Huu Dai, the leader of Department of Marine Botany, Institute of Oceanography, VAST, Nha Trang, Vietnam is thanked for his kind help, support, and fruitful discussion during my stay in Nha Trang for collecting the marine cyanobacterium Lyngbya majuscula sample. Many thanks go to all my friends in Greifswald for their sharing in working and life during my stay here, especially I would like to thanks Nguyen Hien, K.R. Baswani for their valuable discussion in chemistry and my deep thanks also to Nghiem Quynh Huong for her precious help in dissertation formatting. Special thanks go to the laboratory of the Baltic-Analytics GmbH for performing GC-MS experiments of some my samples. I would like to acknowledge the Ministry of Education and Training (MOET), Vietnam and German Academic Exchange Service (DAAD) for providing me with the doctoral fellowship. Also my special thanks go to Institute of Marine Biotechnology (IMAb), Greifswald and Dr. G.Roth from Foreign Students Office, AAA, for providing me a partial financial support in finishing my study. Last, but not least, I would like to express my deepest gratitude and thanks to my mother, my great family, and my husband for their eternal support, continuous encouragement during my work. Especially to my lovely son, Hoang An, who bring me happiness, a lot of joys, hopes, and promotion to fulfill this work.
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a a a Name: Le Thi Anh Tuyet Gender: Female Date of birth: 19 May 1973 Place of birth: Thanh Hoa, Vietnam Nationality: Vietnamese Marital status: Married a a a a a Ph.D student at the Institute of Pharmacy, Ernst-Moritz- Arndt University of Greifswald, Germany Research internship in Algal Culturing Techniques at Algal Biotechnology Department, Institute of Biotechnology, Ha Noi,Vietnamese Academy of Science and Technology Training course on Methods for Natural Products Isolation at Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University, Düsseldorf, Germany Post-graduate training course in Molecular Biology at Environmental Health Research Institute (IUF), Heinrich- Heine-University Düsseldorf gGmbH, Düsseldorf, Germany German language course in Carl Duisburg Centren Dortmund, Germany supported by DAAD Master of Science (M.Sc.) degree in Biology at Hanoi University for Teacher’s Training, Vietnam National University-Hanoi, Vietnam (now is Hanoi National University of Education). The thesis experiments were carried at Animal Gene Technology Department, Institute
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of Biotechnology (IBT), Ha Noi, Vietnamese Academy of Science and Technology (VAST) Bachelor of Science (B.Sc.) degree in Biology at Vinh University, Vietnam Worked as a lecturer at Natural Sciences Faculty, Hong Duc University, Thanh Hoa, Vietnam Worked as a teacher at High School in Thanh Hoa, Vietnam Worked as a teacher at High School in Thanh Hoa, Vietnam
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1. , Victor Wray, Rolf Jansen, Manfred Nimtz, Ho Sy Hanh, Dang Diem Hong, Sabine Mundt. Daklakapeptin, a new cyclic peptide with antibacterial activity from the soil cyanobacteria Calothrix javanica and Scytonema ocellatum from Vietnam (close to submission) 2. , Victor Wray, Manfred Nimtz, Ho Sy Hanh, Dang Diem Hong, Sabine Mundt. An extracellular sesquiterpenoid with antibacterial activity from the soil cyanobacterium Anabaena sp. from Vietnam (close to submission) 3. , Rolf Jansen, Victor Wray, Martina Wurster, Ho Sy Hanh, Dang Diem Hong, Sabine Mundt. Antibiotic constituents from the Vietnamese soil cyanobacterium Westiellopsis sp .VN (close to submission) 4. Wajid Rehman, Amin Badshah, Salimullah Khan and (2009) Synthesis, characterization, antimicrobial and antitumor screening of some diorganotin(IV) complexes of 2-[(9 H-Purin-6-ylimino)]-phenol. European Journal of Medicinal Chemistry 44(10), 3981-3985. 5. Nguyen Van Cuong, (1997) Initial results of the study on generating gene transferred fish. Vietnam Journal of Genetics and Applications 2:10- 16.
1. and Sabine Mundt. Screening of soil cyanobactria from Vietnam for antibacterial activity. In "13 th International symposium on phototrophic prokaryotes", August 9-14, 2009, Montreal, Quebec, Canada.
173
1 a H NMR of fraction WF 1-3 of Westiellopsis sp. VN 174
1 H NMR of fraction WF 1-3 of Westiellopsis sp. VN
175
1 a H NMR of fraction WF 1-5 of Westiellopsis sp. VN
176
1 H NMR of fraction WF 1-5 of Westiellopsis sp. VN 177
1 a H NMR of fraction WF 1-6 of Westiellopsis sp. VN
178
1 H NMR of fraction WF 1-6 of Westiellopsis sp. VN 179
1 a H NMR of fraction WF 1-8 of Westiellopsis sp. VN
180
1 H NMR of fraction WF 1-8 of Westiellopsis sp. VN 181
Abundance 4600000 TIC: 010714.D 4400000 4200000 4000000 3800000 3600000 3400000 3200000 3000000 2800000 2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Time-->
GC-MS spectrum for the fatty acids of fraction MeOH of Westiellopsis sp. VN in n-hexane hydrolysis /derivatization
182
Abundance 5800000 TIC: 010715.D 5600000 5400000 5200000 5000000 4800000 4600000 4400000 4200000 4000000 3800000 3600000 3400000 3200000 3000000 2800000 2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Time-->
GC-MS spectrum for the fatty acids of fraction MeOH of Westiellopsis sp. VN in MeOH hydrolysis /derivatization
183
a a a a a a B.s. a S.a. b E.e. c P.a. d C.m. e a n-hexane ext. 0.0 0.0 0.0 0.0 n.t. Batch MeOH ext. 14,0 9.0 0.0 0.0 11.0 Anabaena EtOAc ext. 18.0 16.0 21.0 0.0 n.t. sp. n-hexane ext. 0.0 0.0 0.0 n.t. 0.0 Large scale MeOH ext. 0.0 0.0 0.0 n.t. 0.0 EtOAc ext. 15.0 16.0 24.0 n.t. 16.0 n-hexane ext. 0.0 0.0 0.0 0.0 n.t. Batch MeOH ext. 11.0 13.0 8.0 0.0 n.t. Nostoc sp. EtOAc ext. 8.0 0.0 0.0 0.0 n.t. n-hexane ext. 8.0 7.0 0.0 0.0 n.t. Large scale MeOH ext. 10.0 15.0 0.0 0.0 n.t. EtOAc ext. n.t. n.t. n.t. n.t. n.t. n-hexane ext. 0.0 0.0 0.0 0.0 n.t. Batch MeOH ext. 17.0 19.0 12.0 12.0 n.t. Calothrix EtOAc ext. 7.0 6.5 6.5 0.0 n.t. elenkinii n-hexane ext. 15.5 8.0 7.5 0.0 n.t. Large scale MeOH ext. 8.0 8.0 7.0 0.0 n.t. EtOAc ext. n.t. n.t. n.t. n.t. n.t. n-hexane ext. 0.0 0.0 0.0 0.0 n.t. Batch MeOH ext. 17.0 18.0 8.0 0.0 n.t. Scytonema EtOAc ext. 14.0 12.0 0.0 0.0 n.t. millei n-hexane ext. 12.0 0.0 7.0 9.0 n.t. Large scale MeOH ext. 0.0 7.0 7.0 0.0 n.t. EtOAc ext. n.t. n.t. n.t. n.t. n.t. IZ=Inhibition zone; Ext.=extract; n-hexane ext. and MeOH ext. from dry biomass; EtOAc ext. from culture medium; n.t.= not test. 1Diameter of inhibition zone (mm) includes Ø disc (6mm) aBacillus subtilic; b Staphylococcus aureus; cEscherichia coli; dPseudomonas aeruginos; eCandida maltosa
Diameter of inhibition zone of extracts from three cyanobacterial strains in large scale cultivation in comparison with batch cultivation against test organisms in vitro
184
1 a H NMR of fraction CJF II-4 of Calothrix javanica
185
1 H NMR of fraction CJF II-4 of Calothrix javanica
186
1 a H NMR of fraction AF 6 of Anabaena sp.
187
1 H NMR of fraction AF 6 of Anabaena sp.
188
1 a H NMR of fraction F8-3-2 of Lyngbya majuscula
189
1 H NMR of fraction F8-3-2 of Lyngbya majuscula
190
1 a H NMR of fraction F10-3 of Lyngbya majuscula
191
13 C NMR of fraction F10-3 of Lyngbya majuscula
192
1 a H NMR of fraction F10-5 of Lyngbya majuscula
193
1 H NMR of fraction F10-5 of Lyngbya majuscula
194
Abundance TIC: M210411.D 2900000 2800000 2700000 2600000 C1 8 2500000 2400000 2300000 2200000 2100000 2000000 1900000 1800000 1700000 1600000 1500000 1400000 1300000 1200000 1100000 1000000 900000 800000 700000 C1 6 600000 500000 400000 300000 200000 C1 4 100000 C2 0 0 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Time-->
GC-MS spectrum for the fatty acids of n-hexane extract of Lyngbya majuscula
195
1 a H NMR data of fraction CJF II-4 of Calothrix javanica compared with fraction FSO 3 of Scytonema ocellatum
196
1 H NMR data of fraction CJF II-4 of Calothrix javanica compared with fraction FSO 3 of Scytonema ocellatum
197
MS of methanol extract of Calothrix javanica compared with MS of methanol extract of Scytonema ocellatum
198