Chemical and Biological Investigations of Vietnamese ... · Lyngbya majuscula in Khanh Hoa province...

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……………………………………………………………

Erklärung

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

Table of Contents

1 INTRODUCTION.....................................................................................................1

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

2 MATERIALS AND METHODS ...........................................................................23

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

3 RESULTS ..............................................................................................................61

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 WF1-3 (compound 1) ....................................... 68 3.2.1.4.2 Structure elucidation of fraction WF1-5............................................................. 69 3.2.1.4.3 Structure elucidation of fraction WF1-6 (compound 4)........................................ 73 3.2.1.4.4 Identification of compounds in fraction WF1-8 .................................................. 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 RPC18 chromatography ........................ 82 3.3.2 Purification of fraction CJFII by semi-preparative reversed-phase HPLC .......................... 83

3.3.3 Structure elucidation of fraction CJFII-4 (compound 7)....................................................... 85

3.4 Chemical investigation of Scytonema ocellatum...................................................... 90 3.4.1 Fractionation of methanol extract obtained from biomass by RP C18 column chromatography............................................................................................................................................. 90 3.4.2 Separation of fraction SOFII 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 FSO3 ........................................................................... 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 AF6 (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 NFIV using RP C18 column chromatography .................... 98 3.6.3 Purification of the active fraction NFIV-1 using semi-preparative reversed-phase HPLC ........ 99 3.6.4 Structure elucidation of fraction NsF2 .......................................................................... 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 F8 by silica gel column chromatography ............................... 102 3.7.1.3 Purification of fraction F8-3 by preparative TLC ..................................................... 103 3.7.1.4 Structure elucidation of fraction F8-3-2 (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 F10 using semi-preparative reversed-phase HPLC ................ 107 3.7.2.3 Structure elucidation of fractions F10-3 and F10-5 ..................................................... 108

3.7.2.3.1 Structure elucidation of fraction F10-3 (compound 10)...................................... 108 3.7.2.3.2 Structure elucidation of fraction F10-5 (compound 11) ........................................ 110

3.7.3 Fatty acid analysis...................................................................................................... 112

4 DISCUSSION ......................................................................................................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

SUMMARY ..............................................................................................................143

REFERENCES.........................................................................................................143

ACKNOWLEDGMENTS .......................................................................................168

CURRICULUM VITAE..........................................................................................171

LIST OF PUBLICATIONS AND OTHER SCIENTIFIC ACHIEVEMENTS .173

APPENDIX ...............................................................................................................174

List of Figures Figure 1-1: Crytophycin 1 and crytophycin 8................................................................ 11

Figure 1-2: Norharmane from cyanobacteria................................................................. 12

Figure 1-3: Cryptophycin 1 and 52, potent antitumor agents from cyanobacteria ........ 14

Figure 1-4: Prominent anticancer marine cyanobacterial secondary metabolites and synthetic analogues ........................................................................................................... 15

Figure 1-5: Borophycin from cyanobacteria.................................................................. 17

Figure 1-6: Nostocarboline from Nostoc 78-12A.......................................................... 21

Figure 2-1: Map of Vietnam with the locality of 12 cyanobacterial strains in Dak Lak province ..................................................................................................................... 23

Figure 2-2: Morphology of 12 cyanobacterial strains ................................................... 28

Figure 2-3: 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

Figure 2-4: Natural habit of Lyngbya majuscula and microscopic view of filament of Lyngbya majuscula ....................................................................................................... 31

Figure 2-5: Agilent 6890N gas chromatograph and mass selective detector ............... 37

Figure 2-6: Cultivation of cyanobacteria ....................................................................... 39

Figure 3-1: Antibacterial activity of cyanobacterial extracts against the Gram positive bacterium Bacillus subtilis ATCC 6501.............................................................. 62

Figure 3-2: Antibacterial activity of cyanobacterial extracts against the Gram positive bacterium Staphylococcus aureus ATCC 6538 ................................................................ 62

Figure 3-3: Antibacterial activity of cyanobacterial extracts against the Gram negative bacterium Escherichia coli ATCC 11229 .......................................................... 63

Figure 3-4: Antibacterial activity of cyanobacterial extracts against the Gram negative bacterium Pseudomonas aeruginosa ATCC 22853 ........................................... 64

Figure 3-5: Semi-preparative RP HPLC chromatogram of WF1 .................................... 67

Figure 3-6: Ambiguine D isonitrile ............................................................................... 68

Figure 3-7: UV and ESI-MS of fraction WF1-3 ............................................................. 68

Figure 3-8: UV and ESI-MS of compounds of fraction WF1- 5..................................... 69

Figure 3-9: MS data of peak 1 of fraction WF1-5 ........................................................... 70

Figure 3-10: Ambiguine C isonitrile.............................................................................. 70

Figure 3-11: MS data of peak 2 of fraction WF1-5 ........................................................ 71

Figure 3-12: Fischerellin A............................................................................................ 71

Figure 3-13: MS data of peak 3 of fraction WF1-5 ........................................................ 72

Figure 3-14: Ambiguine B isonitrile.............................................................................. 73

Figure 3-15: UV and ESI-MS of fraction WF1-6 ........................................................... 73

Figure 3-16: ESI-MS of compounds of fraction WF1- 8 ................................................ 74

Figure 3-17: ESI-MS of peak 2 of fraction WF1- 8 ........................................................ 75

Figure 3-18: ESI-MS of peak 3 of fraction WF1- 8......................................................... 76

Figure 3-19: Bioautographic assay of ethyl acetate extract from cultivation medium of Westiellopsis sp.VN against S.aureu ............................................................................ 77

Figure 3-20: Analytical HPLC of MeOH fraction of EtOAc extract of cultivation medium of Westiellopsis sp. VN ...................................................................................... 77

Figure 3-21: Fermenters for cultivation of Westiellopsis sp.VN ................................ 79

Figure 3-22: Agar diffusion test of extracts prepared from biomass and cultivation medium of Westiellopsis sp.VN grown in BG-11 media ................................................. 80

Figure 3-23a: The effect of incubation time on dry weight and antibiotic production of Westiellopsis sp.VN...................................................................................................... 81

Figure 3-23b: Agar diffusion test of MeOH extracts of Westiellopsis sp.VN biomass ............................................................................................................................. 81

Figure 3-24: Analytical HPLC chromatogram of fraction CJFII .................................. 83

Figure 3-25: Analytical HPLC chromatogram of fraction CJFII-4 .................................. 84

Figure 3-26: Daklakapeptin .......................................................................................... 85

Figure 3-27: Semi-preparative RP HPLC chromatogram of the pooled fractions SOFII-5 and SOFII-6 ........................................................................................................... 92

Figure 3-28: Analytical HPLC chromatogram of fraction FSO3 ................................... 93

Figure 3-29: Semi-preparative RP HPLC chromatogram of the crude EtOAc extract from culture medium ........................................................................................................ 95

Figure 3-30: Agar diffusion test of 7 fractions obtained from ethyl acetate extract of culture medium of Anabaena sp. ..................................................................................... 96

Figure 3-31: Fluourensadiol........................................................................................... 96

Figure 3-32: Semi-preparative RP HPLC chromatogram of fraction NFIV-1 ................ 99

Figure 3-33: Thin layer chromatogram of 15 fractions obtained from silica gel column ............................................................................................................................ 102

Figure 3-34: 17-debromo-3, 4didehydro-3-deoxy –aplysiatoxin ................................ 103

Figure 3-35: ESI-MS spectrum of fraction F8-3-2 ........................................................ 104

Figure 3-36: Thin layer chromatogram of 22 fractions obtained from silica gel column ............................................................................................................................ 105

Figure 3-37: Semi-preparative RP HPLC chromatogram of F10 .................................. 107

Figure 3-38: Debromoaplysiatoxin............................................................................. 108

Fgure 3-39: UV and ESI-MS spectrum of compound 10 ............................................ 110

Figure 3-40: 3,4-didehydro-3-deoxy-aplysiatoxin....................................................... 110

Figure 3-41: UV and ESI-MS spectrum of compound 11........................................... 111

Figure 4-1: Fischerellins from cyanobacteria .............................................................. 121 Figure 4-2: Sequence of daklakapeptin ....................................................................... 131 Figure 4-3: Analytical HPLC chromatogram of methanol extract of Calothrix javanica .......................................................................................................................... 132

Figure 4-4: Analytical HPLC chromatogram of methanol extract of Scytonema ocellatum ........................................................................................................................ 133

Figure 4-5: The phylogenetic relationships of cyanobacteria inferred from 16S rRNA nucleotide sequence ........................................................................................................ 134

List of Tables Table 1-1: The orders of cyanobacteria according to the botanical classification and their correspondence to the subsections of the bacteriological code .................................. 4

Table 2-1: Cyanobacterial strains .................................................................................. 24

Table 2-2: Step gradient used in purification of fraction WF1 by semi-preparative HPLC ................................................................................................................................ 44

Table 2-3: Step gradient used in purification of fraction CJFII by semi-preparative HPLC ................................................................................................................................ 46

Table 2-4: Step gradient used in purification the pooled fractions (SOFII-5,SOFII-6) .... 48

Table 2-5: Step gradient used for purification of the EtOAc extract by semi-preparative HPLC ............................................................................................................. 49

Table 2-6: Step gradient used for purification of fraction NFIV-1 by semi-preparative HPLC ................................................................................................................................ 51

Table 2-7: Step gradient used in purification of fraction F10 by semi-preparative HPLC ................................................................................................................................ 54

Table 3-1: Dry weight of extracts from biomass (1g) and culture media (1L) of 12 cyanobacterial strains........................................................................................................ 61

Table 3-2: Fractionation of methanol extract from Westiellopsis sp. VN biomass by silica gel chromatography and antibacterial activity of fractions to S. aureus ................. 65

Table 3-3: Fractionation of combined fractions FI, FII, and FIII from Westiellopsis sp. VN biomass by LH-20 chromatography and antibacterial activity of fractions to S. aureus................................................................................................................................ 66

Table 3-4: Fractionation of WF1 from Westiellopsis sp. VN biomass by semi-preparative reversed-phase HPLC and antibacterial activity of fractions to S. aureus .... 67

Table 3-5: 1H NMR data of compound 1 compared with literature data of ambiguine

D isonitrile ........................................................................................................................ 69

Table 3- 6: 1H NMR data of compound 3 compared with literature data* of

fischerellin A..................................................................................................................... 72

Table 3-7: Comparison of 1H NMR data of compound 4 with reported data*.............. 74

Table 3-8: Compounds of MeOH fraction analyzed as methyl ester in n-hexane after hydrolysis /derivatization.................................................................................................. 78

Table 3-9: Compounds of MeOH fraction analyzed as methyl ester in MeOH after hydrolysis/derivatization................................................................................................... 78

Table 3-10: Dry biomass and antibacterial activity against S.aureus of extracts in BG-11 medium.................................................................................................................. 80

Table 3-11: Fractionation of methanol extract from Calothrix javanica biomass by RP-18 chromatography and antibacterial activity of fractions to S. aureus ..................... 82

Table 3-12: Step gradient used in HPLC analysis of CJFII by analytical HPLC ........... 83

Table 3-13: Separation of CJFII from Calothrix javanica biomass by semi-preparative reversed-phase HPLC and activity of the fractions to S. aureus ................... 84

Table 3-14: Step gradient used in HPLC analysis of seven fractions by analytical HPLC ................................................................................................................................ 84

Table 3-15: NMR data of CJFII-4..................................................................................... 86

Table 3-16: Sequence information deduced from the NOE’s found in the 2D NOESY spectrum of CJFII-4 ............................................................................................................ 88

Table 3-17: Calculation of the molecular mass from the structure of the residues deduced from the NMR data............................................................................................. 88

Table 3-18: Comparison of the sequence from the high-resolution ESI-MS data with that from the NMR data. ................................................................................................... 89

Table 3-19: Fractionation of methanol extract from Scytonema ocellatum biomass by RP-18 chromatography and antibacterial activity of fractions to S. aureus ................ 90

Table 3-20: Separation of SOFII from Scytonema ocellatum biomass by silical gel chromatography and antibacterial activity of the fraction to S. aureus ............................ 91

Table 3-21: Separation of SOFII-5 and SOFII-6 from Scytonema ocellatum biomass by semi-preparative reversed-phase HPLC and antibacterial activity of the fractions to S. aureus................................................................................................................................ 92

Table 3-22: Step gradient used in HPLC analysis of FSO3 by analytical HPLC........... 93

Table 3-23: Antibacterial activity of extracts from Anabaena sp. cultivated in large scale................................................................................................................................... 94

Table 3-24: 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

Table 3-25: NMR data of AF6 (Fluourensadiol) ........................................................... 97

Table 3-26: Fractionation of methanol extract from Nostoc sp. biomass by silical gel chromatography and antibacterial activity of fractions to S. aureus ................................ 98

Table 3-27: Fractionation of NFIV from Nostoc sp. biomass by RP C18 chromatography and antibacterial activity of fractions to S. aureus ................................ 99

Table 3-28: Fractionation of NFIV-1 from Nostoc sp. biomass by semi-preparative RP HPLC and antibacterial activity of fractions to S. aureus .............................................. 100

Table 3-29: Fractionation of methanol extract from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions against cell line 5637..... 101

Table 3-30: Separation of fraction F8 from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions to 5637 cell line ............................ 102

Table 3-31: Separation of fraction F8-3 from Lyngbya majuscula biomass by preparative TLC.............................................................................................................. 103

Table 3-32: Comparison of 1H NMR data of compound 9 with reported data *.......... 104

Table 3-33: Fractionation of methanol extract from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions against cell line 5637..... 106

Table 3-34 Separation of fraction F10 from Lyngbya majuscula biomass by semi-preparative RP HPLC and cytotoxic activity of fractions against cell line 5637 ........... 108

Table 3-35: Comparison of 13C NMR and 1H NMR data of compound 10 with literature values of *Debromoaplysiatoxin..................................................................... 109

Table 3-36: Comparison of 1H NMR data of compound 11 with reported data *........ 111

Table 3-37: Fatty acids analyzed as methyl ester in n-hexane extract.......................... 112

List of Schemes Scheme 2-1: Scheme of extraction................................................................................. 40

Scheme 2- 2: Extraction, fractionation, and isolation of the secondary metabolites of Westiellopsis sp.VN.......................................................................................................... 45

Scheme 2-3: Extraction, fractionation, and isolation of the secondary metabolites of Calothrix javanica ............................................................................................................ 46

Scheme 2-4: Extraction, fractionation, and isolation of the secondary metabolites of Scytonema ocellatum ........................................................................................................ 48

Scheme 2-5: Extraction, fractionation, and isolation of the secondary metabolites of Anabaena sp...................................................................................................................... 49

Scheme 2-6: Extraction, fractionation, and isolation of the secondary metabolites of Nostoc sp........................................................................................................................... 51

Scheme 2-7: Extraction, fractionation, and isolation of the secondary metabolites of Lyngbya majuscula (method 1)......................................................................................... 53

Scheme 2-8: Extraction, fractionation, and isolation of the secondary metabolites of Lyngbya majuscula (method2).......................................................................................... 55

Abbreviations

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

IC50 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

1

1 Introduction

1.1 Cyanobacteria

1.1.1 Cyanobacterial physiology and morphology

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 CO2 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)


2

1.1.2 Ecology of cyanobacteria

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).


3

1.1.3 Classification of cyanobacteria

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 &


4

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).

Table 1-1: The orders of cyanobacteria according to the botanical classification and their correspondence to the subsections of the bacteriological code

Botanical

classification

Bacteriological

classification5

Main morphological features

and occurrence in the

environment

Example

Order

Chroococcales1

Subsection I Unicellular cyanobacteria that

reproduce by binary cell division

in one, two or three plane; or

budding, either single cells or in

colonies held together by

Synechocystis

Microcystis


5

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

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.

Pleurocapsa

Order

Oscillatoriales2 Subsection III Organisms form trichomes of

vegetative cells, mostly

uniseriate, without differentiated

cells such as heterocytes and

akinetes (resting cells, spores).

The trichomes show not true

branches but in some genera false

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.

Oscillatoria

Spirulina

Lyngbya

Planktothrix

Limnothrix


6

Order

Nostocales3 Subsection IV Binary division in one plane

giving rise to 1-seriate trichomes,

though sometimes with "false"

branches; one or more cells per

trichome differentiate into

heterocysts, at least when

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.

Anabaena

Nodularia

Nostoc

Scytonema

Calothrix

Order

Stigonematales4 Subsection V Binary division periodically or

commonly in more than one

plane, giving rise to multiseriate

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.

Fischerella

Hapalosiphon

Westiellopsis

1. Komárek & Anagnostidis, 1999; 2. Komárek & Anagnostidis, 2005; 3. Komárek & Anagnostidis,

1989; 4. Anagnostidis & Komárek, 1990; 5. Rippka et al., 1979, Castenholz, 2001

1.2 Cyanobacteria–a new and rich source of novel bioactive

compounds with pharmaceutical potential

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


7

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.,


8

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).


9

1.2.1 Antimicrobials

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.,


10

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


11

efficiency and lower toxicity than cryptophycin 1 in vivo (Moore et al., 1996; Liang et

al., 2005).

Figure 1-1: 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).


12

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).

Figure 1-2: Norharmane from cyanobacteria

1.2.2 Cytotoxic and antitumoural activities

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


13

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 MICs< 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


14

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).

Figure 1-3: Cryptophycin 1 and 52, potent antitumor agents from cyanobacteria


15

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 (1), dolastatin 10(2), and dolastatin 15 (3) served as lead structures for the

development of synthetic analogues. e.g. compound 4, TZT-1027 (5), ILX-651 (6),

and LU-103793 (7), usually with improved pharmacokinetic properties (Wipf et al.,

2004; Mita et al., 2006; Wantanabe et al., 2006; Tan, 2007; Butler, 2008).

Figure 1-4: 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


16

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 (4), 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) (5) are still

being evaluated (http://www.clinicaltrials.gov/ct2/results?term=soblidotin; Patel et

al., 2006; Gerwick et al., 2008; Butler, 2008).

Dolastatin 15 (3) 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 (6) and LU103793

(7) (Simmons et al., 2005; Ray et al., 2007). ILX-651(tasidotin or synthadotin) (6) is

an orally active third generation synthetic dolastatin 15 (3) 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


17

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).

Figure 1-5: 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 IC50 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).


18

1.2.3 Antiviral activity

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;


19

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).

1.2.4 Toxins and other pharmacologically active compounds

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


20

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 IC50 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


21

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.

Figure 1-6: 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).


22

1.3 Aim of the work

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.

Chapter II Materials and methods

23

2 Materials and methods

2.1 Biological materials

2.1.1 Soil cyanobacteria

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).

Figure 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,


24

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).

Table 2-1: Cyanobacterial strains

Scientific

name

Strain

number

Morphological feature Classification

Oscillatoria sp.

TVN16

Trichomes long small without any

branching, not constricted at the cross-

walls, sheath absent; cells discoid, no

heterocysts in filament.

Oscillatoriaceae

Oscillatoriales

Anabaena sp.

TVN40

Trichomes constricted at the cross-

walls, look like string of beads; cells

barrel-shaped, mostly longer than

broad; no heterocysts in filament; no

branches.

Nostocaceae

Nostocales

Nostoc

spongiaeforme

Agardh ex

Born. et Flah.

TVN7

Filaments flexuous, loosely entangled;

sheath thin; cells cylindrical or oblong;

heterocysts subspherical; spores in long

chains.

Nostoc

coeruleum

Lyngbye ex

Born. et Flah.

TVN14

Filaments densely entangled, flexuous,

sheath mostly indistinct; trichomes 5.1-

6.8 µm broad; cells short, barrel-shaped;

heterocysts subspherical; spores not

known.

Nostoc sp.

TVN9

Filaments single, flexuous, entangled;

cells nearly spherical, no heterocysts in

filament, no branches.

Nostocaceae

Nostocales

Calothrix

javanica de

Filaments single, pale blue-green to

olive green, lamellated sheath,


25

Wilde

TVN1

gradually attenuated to a pointed apex;

trichomes 5.1-6 µm broad; heterocysts

basal, 4-5.1 µm broad, 4.8-6 µm in

length; spores single or two together,

about 8.5-9 µm broad, 6-10 µm long

Calothrix elenkinii

Kossinsk.

TVN202

Filaments 80-250 µm, united in tuff,

swollen at the base, 6-9 µm broad;

sheath close to the trichome, thin, not

lamellated, open at the ends; trichomes

at the base 5.1-6.8 µm broad, apical hair

not formed; cells quadratic or somewhat

shorter; heterocysts single, basal, 4.1-

6.8 µm broad

Calothrix sp. TVN20 Filaments single, unbranched;

heterocysts basal.

Calothrix

marchica var.

crassa Rao,

C.B.

TVN201 Filaments in groups, irregularly bent

and closely entangled, 8.5-13.6 µm

broad; sheath thin, firm; trichomes 8.5-

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.

Rivulariaceae

Nostocales

Scytonema

millei Bornet

ex Born. et

Flah.

TVN12 Filaments interwoven, false branches

erect; sheath firm brownish; heterocysts

discoid

Scytonema

ocellatum

Lyngbye ex.

Born. et Flah.

TVN10 Filaments up to 3 mm long, 10.2-16.3

µm; false branched; sheath firm, often

lamellated; trichomes 6.8-8.5 µm broad;

cells shorter than broad or quadratic;

heterocysts subquadratic, size 6.8-6.8

µm

Scytonemataceae

Nostocales


26

Westiellopsis

sp.VN

TVN22 Filaments with true branching

filaments of two kinds, primary

filaments and secondary filaments.

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

Stigonemataceae

Stigonematales

Oscillatoria sp. (x 100) Anabaena sp. (x 100)


27

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)


28

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

Figure 2-2: Morphology of 12 cyanobacterial strains


29

2.1.2 Marine cyanobacteria

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

Figure 2-3: 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)

1A

1B


30

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


31

b c

Figure 2-4: a and b Natural habit (took photograph during low tide) of Lyngbya majuscula c Microscopic view of filament (x 40) of Lyngbya majuscula

2.1.3 Bacteria, yeast, and cancer cell lines as test organisms

• 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)

2.2 Chemicals

2.2.1 Cultivation of cyanobacteria

All media were prepared and autoclaved at 1210C 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.

BG-11 medium (Rippka et al., 1979)

Ingredient Stock solution (g/100 mL)

Nutrient solution (mL/L)

NaNO3 15.0 10 K2HPO4. x 3 H2O 0.4 10 MgSO4. x 7 H2O 0.75 10 CaCl2 x 2 H2O 0.36 10 Citric acid 0.06 10 Ferric ammonium citrate 0.06 10 EDTA (disodium magnesium salt) 0.01 10 Na2CO3 0.2 10 *[Trace metal mix A5+Co] 1 Distilled water 919


32

*[Trace metal mix A5+Co]

Ingredient Stock solution (g/L)

Nutrient solution (mL/L)

H3BO3 2.86 MnCl2 x 4H2O 1.81 ZnSO4. x 7H2O 0.222 CuSO4. x 5H2O 0.079 Na2MoO4. x 2H2O 0.390 Co (NO 3)2. x 6H2O 0.0494 Distilled water 1.0 L

1

Modified BG-110 medium (BG-11 without nitrate)

2.2.2 Cultivation of bacteria and yeast as test organisms

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 1210C),

pH: 7.5±0.2 at 250C

2.2.3 General laboratory chemicals

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

2.2.4 Chemical reagents

• Anisaldehyd- sulphuric acid (AS) reagent (Merck, Darmstadt, Germany):


33

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).

• Iodonitrotetrazolium chlorid (INT) (Sigma, Steinheim, Germany): 5 mg of

the INT is dissolved in 1 mL of ethanol 50%.

2.2.5 Fatty acid analysis

Ethanol Merck, Darmstadt, Germany

KOH VWR, Darmstadt, Germany

HCl Bayer, Leverkusen, Germany

Na2SO4 VWR, Darmstadt, Germany

Methoxyamine (MeOX) Sigma- Aldrich, Munich, Germany

N-methyl-N-

trimethylsilyltrifluoroacetamide (MSTFA)

CS Chromatography Service,

Langerwehe, Germany)

2.3 Solvents

Acetone Carl Roth GmbH & Co.KG, Karlsruhe, Germany

Acetic acid Carl Roth GmbH & Co.KG, Karlsruhe, Germany

Acetonitrile (HPLC gradient

grade)

Prolabo VWR International company, made in EC

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

grade)

Prolabo VWR International company, made in EC

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.


34

2.4 Equipment in generally company, town, country

2.4.1 Cultivation of cyanobacteria

- 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

pH 170)

Wissenschaftlich – Technische Werkstätten GmbH,

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-

25 µm pore size

Schleicher & Schüll MicroscienceGmbH, Dassel,

Germany

Deep Freezer GTL 2811WS Bauknecht, Berlin Germany

Autoclave Varioklav, H+P Labortechnik, Germany

Sterilizer Memmert, Schwabach, Germany

2.4.2 Extraction

Laboratory sea sand Merck, Darmstadt, Germany

Porcelain mortar and pestle

250 mL and 500 mL

Erlenmeyer flasks

Merck, Darmstadt, Germany

System of rotatory vacuum

evaporator: pump B-178,

vacuum controller B-721, Rota

vapor R-114, water bath B-480

Büchi Labortechnik AG, Flawil, CH, Büchi & Co,

Berlin, Germany

Stirrer, Heidolph MR 3000 Schwabach, Germany

Shaker Kika Labortecknik Staufen, Janke & Kunkel

GmbH & Co.KG, Germany

Banderlin Sonorex RK103H Bandelin electronic, Berlin, Germany


35

Ultrasonic cleaner

Centrifuge (Rotanta 460R) Hettich Zentrifugen, Tuttlingen; Germany

100 mL centrifuge tubes, round

bottom

Carl Roth GmbH & Co.KG, Karlsruhe, Germany

Lyophylizer Alpha 1-4 Christ Gefriertrocknungsanlagen, Osterode,

Germany

Filter paper Ø 185 mm with 12-

25 µm pore size

Schleicher & Schüll MicroscienceGmbH, Dassel,

Germany

Banderlin Sonorex RK103H

Ultrasonic cleaner

Banderlin electronic, Berlin, Germany

2.4.3 Isolation of secondary metabolites

2.4.3.1 Thin layer chromatography (TLC)

TLC sheets: Silica gel 60, F254,

20x20 cm


TLC tank Desaga GmbH, Heidelberg, Germany

Capillaries Hirschmann Laborgeräte GmbH & Co. KG, Eberstadt,

Germany

UV lamp Desaga SARSTEDT-

GRUPPE HP-UVIS

Desaga GmbH, Heidelberg, Germany

UVPMultiDoc-It, Digital

Imaging System

Cambridge, UK

Thermoplate Desaga

SARSTEDT-GRUPPE

Desaga GmbH, Heidelberg, Germany

2.4.3.2 Preparative TLC

Preparative TLC plate: PLC Silica gel F254, 2mm, 20 x 20 cm (Merck,

Darmstadt, Germany)

2.4.3.3 Open column chromatography

Glass column: 60 x 1.2 cm,

50 x 4.0 cm; 50 x 2.0 cm;

Schott Duran, Mainz, Germany


36

35 x 2.5 cm; 35 x 1.2 cm (h x i.d.)

50 mL, 100 mL, 250 mL, 500

mL, and 1000 mL Erlenmeyer

flasks


Test tubes (10 mL) Schott Duran, Mainz, Germany

Fraction Collector Model 2110 Bio-Rad Laboratories, Richmond, USA

Silica gel 60 ( 0.015-0.040 mm;

0.040-0.063 mm)

Merck, Darmstadt,Germany

Sephadex LH-20 Amersham Biosciences AB, Uppsala, Sweden

Silica gel 60RP-18 ( 0.040-0.063

mm)

Merck, Darmstadt,Germany

Pasteur pipettes, 150 mm, 230 mm VWR GmbH, Darmstadt, Germany

2.4.3.4 HPLC

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)

2.4.4 Agar plate diffusion test

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


37

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

2.4.5 Bioautographic TLC assay

Thin layer chromatography sheets

silica gel 60 F254, 20x20 cm

Merck KgaA, Darmstadt, Germany

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

2.4.6 Fatty acid analyses

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)


38

2. 5 Cultivation of cyanobacteria

2.5.1 The stock culture

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 ±20C,

under continuous illumination of 8µmol/m2 by cool fluorescence lamps. The stock

cultures were maintained for 20-30 days.

2.5.2 The batch culture

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 BG11 medium in 1.5 liter Fehrnbach flasks. These

samples were cultivated at 20 ±20C, 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/

100C) 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 400C and extracted immediately with EtOAc solvent.

2.5.3 The large scale culture

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 CO2 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


39

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

continuous-flow centrifuge and lyophilized, then stored at -200C.

a

b c

Figure 2-6: Cultivation of cyanobactria: a. Stock culture;

b. Batch culture; c. Large scale culture

2.6 Extraction

2.6.1 Extraction of intracellular compounds

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


40

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 400C

to get dry extracts. The water was reduced by rotary evaporation at 400C and removed

completely by lyophilization (lyophylizer Alpha 1-4). The dried extracts were

weighed and stored at -200C until use.

2.6.2 Extraction of extracellular compounds

For media extraction, 3L of cyanobacterial culture medium were

concentrated to 250-300 mL by rotary evaporation in vacuum at 400C. 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 -200C

until use.

Cyanobacte r ia

5g freeze-dried biomass Media

n- hexane ext ract

2 50m l n-hexane/ 3 t im es/ st ir ring

Residue

Methanol ext ract

2 50m l methanol/ 3 t im es/ st ir ring

Residue

W ater ext ract

2 5 0m l w ate r/ 3 t im es/ st ir ring

Residue

Ethyl acet ate ext ract

2 50m l ethyl aceta te/

3 t im es/ shaking

Bioassay

Suspension

- Crushed w ith n-hexane and see sand in mortar

-Ult rasonicated for 7min

Scheme 2-1: Scheme of extraction


41

2.7 Bioassays

2.7.1 Assays for antimicrobial activity

2.7.1.1 Agar diffusion assay

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 370C 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 370C 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


42

nutrient agar should be below 400C. 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.

2.7.1.2 Bioautographic TLC assay

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, Rf 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 Rf values of the control chromatogram so

that the active compounds were located on TLC.


43

2.7. 2 Assays for cytotoxic activity

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).

2.8 Fractionation and isolation of the secondary metabolites of 6

cyanobacterial strains

2.8.1 Fractionation and isolation of the secondary metabolites of Westiellopsis

sp.VN

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 (FI to FVIII)

were obtained, and solvents were removed in vacuo using a rotary evaporator. All

fractions were tested for their antibacterial activity. The fractions FI, FII, FIII eluting

with 100% DCM, 95% DCM/EtOAc, and 90% DCM/EtOAc were pooled since they

all exhibited approximately the same antibacterial activity.


44

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 H2O/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 H2O/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 (WF1 to WF4)

were collected and tested for antibacterial activity. Because fraction WF1 which eluted

with 10% H2O/MeOH exhibited the highest antibacterial activity, fraction WF1 was

further purified.

The fraction WF1 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 (WF1-1-WF1-9) were collected under

UV of 238nm and then tested for the activity against S .aureus. Fractions WF1-3, WF1-

5, WF1-6, and WF1-8 exhibited significant antibacterial activity were therefore used for

structural elucidation.

Table 2-2: Step gradient used in purification of fraction WF1 by semi-preparative HPLC

Time (min) 0.50 3.50 40.50 42.50 50.50 52.50

Solvent A (%) 80 50 15 0.0 0.0 80.0

Solvent B (%) 20 50 85 100 100 20

Solvent A: H2O and solvent B: CH3CN; Flow rate: 3.0 mL/min; HPLC column: Synergi polar-

RP80A/250×10mm, 4 micron


45

3 x MeOH

WF1-1 WF1-2 WF1-3 WF1-4 WF1-5 WF1-6 WF1-7 WF1-8 WF1-9

WF1 WF2 WF3 WF4

LH-20 column

H2O/MeOH (1:9), MeOH, H2O/Aceton (1:1)

3 x n-Hexane

3 x EtOAc

Lyophilized biomass

n-Hexane ext.Residue

EtOAc ext. Residue

MeOH ext.

FI FII FIII FIV FV FVI FVII FVIII

Silica gel column

DCM/ EtOAc/ MeOH gradient

-Semi-preparative HPLC Synergi-Polar RP-CH3CH/H2O gradient

Structure elucidation

Scheme 2- 2: Extraction, fractionation, and isolation of the secondary metabolites of Westiellopsis

sp.VN

2.8.2 Fractionation and isolation of the secondary metabolites of Calothrix

javanica


biomass from batch culture of this strain was subjected to RP C18 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 C18 column chromatography. For preparation of the

column 15 g reverse-phase C18 silica gel was swollen in 50mL MeOH/EtOH/H2O

(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/H2O (45: 45:10), followed by 200 mL MeOH/H2O (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 (CJFI to


46

CJFX) were received and tested for the antibacterial activity. The fraction CJFII eluted

with MeOH/EtOH/H2O (45:45:10) was further purified because this fraction showed

antibacterial activity.

The purification of fraction CJFII 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 CJFII 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 CJFII-4 was the most pure, this

fraction was therefore used for structural elucidation.

Table 2-3: Step gradient used in purification of fraction CJFII by semi-preparative HPLC

Time (min) 0.50 16.50 24.50 30.50 31.50

Solvent A (%) 30 15 0 0 30

Solvent B (%) 70 85 100 100 70

Solvent A: H2O+0.05%TFA and solvent B: MeOH; Flow rate: 3.0 mL/min; HPLC column: Synergi

polar-RP80A/250×10 mm, 4 micron

3 x n-Hexane

Lyophilized biomass

n-Hexane ext.3 x MeOH

Residue

MeOH ext.

Silica gel 60 RP18 column

- MeOH/ EtOH/H2O= 45:45:10

- MeOH/ H2O= 90:10

- MeOH

- MeOH/ Acetone= 1:1

- DCM

- Semi-preparative HPLC Synergi Polar RP- MeOH/(H2O+0.05%TFA) gradient

CJFI CJFII CJFIII CJFIV CJFV CJFVI CJFVII CJFVIII CJFIX CJFX

CJFII-1 CJFII-2 CJFII-3 CJFII-4 CJFII-5 CJFII-6 CJFII-7


H2O ext.

Residue3 x H2 O

Scheme 2-3: Extraction, fractionation, and isolation of the secondary metabolites of Calothrix javanica


47

2.8.3. Fractionation and isolation of the secondary metabolites of Scytonema

ocellatum


biomass from batch culture of this strain was first fractionated using RP C18 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 C18 column chromatography. An amount of 15 g reverse-phase C18

silica gel was swollen in 50mL MeOH/H2O (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/H2O (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 (SOFI to SOFVIII) were obtained and tested for the

antibacterial activity. Because only fraction SOFII which eluted with

MeOH/EtOH/H2O (45:45:10) exhibited strong antibacterial activity, this fraction was

further separated.

74.3 mg of fraction SOFII 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/H2O (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


48

with the same spots on TLC were combined to main fraction. After TLC analyzing

ten major fractions (SOFII-1 to SOFII-10) were collected and tested for the antibacterial

activity. The fractions SOFII-5 and SOFII-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


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 FSO3 exhibited antibacterial activity, this

fraction was used for structural elucidation.

Table 2-4: Step gradient used in purification the pooled fractions (SOFII-5, SOFII-6) by semi-preparative HPLC

Time (min) 0.50 18.50 21.50 24.50 25.50

Solvent A (%) 95 5 0 0 95

Solvent B (%) 5 95 100 100 5

Solvent A: H2O and solvent B: CH3CN; Flow rate: 3.0 mL/min; HPLC column: Synergi Polar-


Lyophilized biomass

Residue

Silica gel 60 RP18 column

- MeOH/ EtOH/H2O= 45:45:10

- MeOH

- MeOH/Acetone= 1:1

SOFI SOFII SOFIII SOFIV SOFV SOFVI SOFVII SOFVIII

Silica gel column

- n-hexane/EtOAc =25:75

- EtOAc

- EtOAc/MeOH =50:50

- MeOH

SOFII-1 SOFII-2 SOFII-3 SOFII-4 SOFII-5 SOFII-6 SOFII-7 SOFII-8 SOFII-9 SOFII-10

- Semi-preparative HPLC Synergi Polar RP

- CH3CN/H2O gradient

FSO1 FSO2 FSO3


Residue

3 x n-Hexane

n-Hexane ext.

3 x H2OMeOH ext.

3 x MeOH

H2O ext.

Scheme 2-4: Extraction, fractionation, and isolation of the secondary metabolites of Scytonema ocellatum


49

2.8.4 Fractionation and isolation of the secondary metabolites of Anabaena sp.

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 AF6 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).

Table 2-5: Step gradient used for purification of the EtOAc extract by semi-preparative HPLC

Time (min) 0.50 12.50 18.50 22.50 24.50 26.50

Solvent A (%) 95 75 40 0.0 0.0 80.0

Solvent B (%) 5 25 60 100 100 5



Grow th medium

- Semi- preparative HPLC Synergi Polar RP- CH3CN/ H2O gradient

Structure elucidat ion

250ml ethyl acetate/ 3times/ shaking/ 24h

at room temparature

Ethyl acetate ext .

First AFI AFII AFIII AFIV AFV AFVI

Scheme 2-5: Extraction, fractionation, and isolation of the secondary metabolites of Anabaena sp.


50

2.8.5 Fractionation and isolation of the secondary metabolites of Nostoc sp.

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 C18 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/H2O (95:5). Each eluting solvent was collected in a

different flask. After that 5 fractions (NFI to NFV) were obtained and tested for

antibacterial activity. Because fraction NFIV which eluted with MeOH exhibited the

strongest antibacterial activity, fraction NFIV was further separated.

360 mg of fraction NFIV was separated by RP C18 column chromatography.

25 g reverse-phase C18 silica gel was swollen in 50ml of initial mobile phase

MeOH/H2O (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 NFIV 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/H2O (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 (NFIV-1 to NFIV-3) were obtained and tested for the

antibacterial activity. Because fraction NFIV-1 which eluted with MeOH/H2O (90:10)

exhibited the strongest antibacterial activity, this fraction was further separated.

The purification of fraction NFIV-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 NFIV-1

were purified using the step gradient described in table 2-6 and the purification was


51

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.

Table 2-6: Step gradient used for purification of fraction NFIV-1 by semi-preparative HPLC

Time (min) 0.50 8.50 12.50 16.50 18.50

Solvent A (%) 60 15 0 0 60

Solvent B (%) 40 85 100 100 40


polar-RP80A/250×10mm, 4 micron

Lyophilized biomass

Residue

NFI NFII NFIII NFIV NFV

NFIV-1 NFIV-2 NFIV-3

- Semi-preparative HPLC Synergi Polar RP

- MeOH/(H2O+0.05%TFA) gradient

NsF1 NsF2 NsF3 NsF4


Residue

3 x n-Hexane

n-Hexane ext.

3 x H2OMeOH ext.

3 x MeOH

H2O ext.

Silica gel column

n-hexane/EtoAc/MeOH/H2O

gradient

Silica gel 60 RP18 column-MeOH/H2O=90:10

-MeOH

-DCM

Scheme 2-6: Extraction, fractionation, and isolation of the secondary metabolites of Nostoc sp.

2.8.6 Fractionation and isolation of the secondary metabolites of Lyngbya

majuscula

2.8.6.1 Method 1

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


52

(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 (F1 to F15) were

collected according to the bands detected on TLC developed by using n-

hexane/EtOAc/MeOH/CH3COOH (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 F8 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/CH3COOH

(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 (F8-1 to F8-6) were tested

for cytotoxic activity. Because fraction F8-3 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 F254, thickness 0.25 mm). The chromatogram was developed

with mobile phase n-hexane/EtOAc/MeOH/CH3COOH (75:25:5:3) and five spots


53

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 (F8-3-1 to F8-3-5) were

obtained. These five fractions (F8-3-1 to F8-3-5) were examined by analytical TLC and

HPLC. HPLC analysis showed that fraction F8-3-2 showed only one peak at Rf 0.22,

therefore structure elucidation of this fraction was done.

MeOH ext.

3 x n-Hexane

Lyophilized biomass

n-Hexane ext.3 x MeOH

Residue

H2O ext.

Residue

3 x H2O

Silica gel column7 solvent system

(n-hexane/EtOAc/MeOH gradient)

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15

F8-1 F8-2 F8-3 F8-4

PTLC

n-hexane/EtOAc/MeOH/CH3COOH

75 : 25 : 5 : 3

F8-3-1 F8-3- 2 F8-3-3 F8-3-4 F8-3-5


Silica gel column-n-hexane/EtOAc=4:6

-n-hexane/EtOAc=1:9-MeOH

Scheme 2-7: Extraction, fractionation, and isolation of the secondary metabolites of Lyngbya majuscula (method 1)

2.8.6.2 Method 2

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


54

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/CH3COOH (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

(F1 to F22) were obtained. Based on the results of TLC analysis, three fractions F9, F10,

and F17 were chosen for testing cytotoxic activity. The fraction F10 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


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.

Table 2-7: Step gradient used in purification of fraction F10 by semi-preparative HPLC

Time (min) 0.50 3.50 14.50 24.50 34.50 39.50 49.50 52.5

Solvent A (%) 80 50 35 25 15 0.0 0.0 80

Solvent B (%) 20 50 65 75 85 100 100 20

Solvent A: H2O and solvent B: CH3CN; Flow rate: 3.0 mL/min; HPLC column: Synergi Polar-RP

80A/250×10mm, 4 micron


55

3 x n-Hexane

3 x EtOAc

Lyophilized biomass

n-Hexane ext.

Residue

EtOAc ext.

3 x MeOH

Residue

MeOH ext.

Silica gel columnn-Hexane/EtOAc/MeOH gradient

F10-1 F10-2 F10-3 F10-4 F10-5

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

-CH3CN/H2O gradient

Scheme 2-8: Extraction, fractionation, and isolation of the secondary metabolites of Lyngbya majuscula (method2)

2.9 Structure elucidation of the isolated secondary metabolites

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.

2.9.1 Structure elucidation of compounds isolated from Westiellopsis sp.VN and

Lyngbya majuscula

Mass spectrometry: HR-ES-IMS (positive ion mode) were recorded on a high

resolution Bruker MaXis mass spectrometer.

NMR spectroscopy: 1H, 13C NMR spectra were recorded at 300 K on a

Bruker AVANCE DMX 600 NMR spectrometer locked to the deuterium resonance of

the solvent, CDCl3. Chemical shifts are reference to the residual proton signal of the

solvent.


56

2.9.2 Structure elucidation of compounds isolated from Calothrix javanica and

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/H2O (1:1). Chemical shifts are reference to the residual proton signal of the solvent

(1H: 3.95 and 13C: 60.85 ppm).

2.9.3 Structure elucidation of compounds isolated from Anabaena sp.

Mass spectrometry: HR-ESI-MS (positive ion mode) were recorded on a

Thermo Science LTQ Orbitrap mass spectrometer.

NMR spectroscopy: 1D (1H, 13C 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/H2O (1:1). Chemical shifts are reference to the residual proton signal of the

solvent (1H: 3.95 and 13C: 60.85 ppm).

2.9.4 Structure elucidation of compounds isolated from Nostoc sp.

Mass spectrometry: ESI-MS were recorded on a Micromass Q-Tof-2 mass

spectrometer.

2.10 Investigation of the active ethyl acetate extract of Westiellopsis

sp. VN growth medium

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


57

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/H2O= 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 -200C. 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.

2.11 Gas chromatography-mass spectrometry

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


58

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: 2300C

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: 700C initial (1 min),

Increasing to 760C by1.5 0C/min

Then to 3300C by 50C/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: 2500C

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


59

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).

2.12 Culture optimization of Westiellopsis sp.VN

2.12.1 The effects of nitrogen deficiency

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 NaNO3 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/m2. The pH-value of the

large-scale culture was adjusted to 7.4 using CO2 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 -200C. 3 L of every cyanobacterial cultured

medium was concentrated to 300 mL by rotary evaporation in vacuum at 400C and

stored at -200C.

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)

2.12.2 The effects of cultivation time (culture age)

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 NaNO3 under

continuous illumination provided by cool-white fluorescent tubes of 8 µmol/m2 at

room temperature (20 ±20C). 20 mL of inoculum cultivated in Fehrbach flasks for 20


60

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/ 100C),

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)

Chapter III Results

61

3 Results

3.1 Screening of antibacterial activity

3.1. 1 Extract preparation

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.

Table 3-1: Dry weight of extracts from biomass (1g) and culture media (1L) of 12 cyanobacterial strains

Strain Strain number

n- Hexane extr. (mg)

MeOH extr. (mg)

H2O extr. (mg)

EtOAc extr. (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

var. crassa TVN201 5.13 144.27 75.33 6.68

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.

3.1.2 Screening of crude extracts

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.

Chapter III Results

62

Figure 3-1: 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)

Figure 3-2: Antibacterial activity of cyanobacterial extracts against the Gram positive bacterium Staphylococcus aureus ATCC 6538


According to the diameter of inhibition zones (mm), the effect of the extracts

obtained from the same cyanobacterial strain showed no significant differences in

Chapter III Results

63

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.

Figure 3-3: Antibacterial activity of cyanobacterial extracts against the Gram negative bacterium Escherichia coli ATCC 11229


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)

Chapter III Results

64

Figure 3-4: Antibacterial activity of cyanobacterial extracts against the Gram negative bacterium Pseudomonas aeruginosa ATCC 22853


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.

Chapter III Results

65

3.2 Chemical investigation and culture optimization of Westiellopsis

sp. VN

3.2.1 Chemical investigation of methanol extract obtained from biomass

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.

3.2.1.1 Fractionation of methanol extract by silica gel column chromatography

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).

Table 3-2: Fractionation of methanol extract from Westiellopsis sp. VN biomass by silica gel chromatography and antibacterial activity of fractions to S. aureus

Yield

Fractions

Mobile phase (mg) %

recovered

Dose

(µg/disc)

Diameter of inhibition

zone (mm)1

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

FIV DCM/EtOAc (50:50) 40.3 15.5 500 02

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

Chapter III Results

66

Three contiguous fractions (FI, FII, and FIII) 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.

3.2.1.2 Seperation of the combined fractions (FI, FII, and FIII) by sephadex LH-20

column

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 FIII 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 H2O followed by 100% MeOH, and finally with

50% aceton in H2O (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).

Table 3-3: Fractionation of combined fractions FI, FII, and FIII from Westiellopsis sp. VN biomass by LH-20 chromatography and antibacterial activity of fractions to S. aureus

Yield

Fractions

No of sub

Fractions

pooled

Mobile phase (mg) %

recovered

Dose

(µg/disc)

Diameter of

inhibition

zone (mm)1

WF1 1-30 H2O/MeOH (10:90) 51.8 75.62 500 21

WF2 31-40 H2O/MeOH (10:90) 1.8 2.63 500 15

WF3 41-70 MeOH 8.6 12.55 500 10

WF4 70-90 H2O/ Aceton (50:50) 5.2 7.59 500 02

Total 67.4 98.39


Fraction WF1 which eluted with 90%MeOH in H2O showed the highest

antibacterial activity with inhibition zone of 21 mm and was further purified.

Chapter III Results

67

3.2.1.3 Purification of fraction WF1 using reversed-phase HPLC

Final purification of fraction WF1 (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).

WF1- 1

WF1- 2

WF1 -3

WF1- 4

WF1 - 5

WF1 - 6

WF1- 7

WF1 -8

WF1- 9

Figure 3-5: Semi-preparative RP HPLC chromatogram of WF1 (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.

Table 3-4: Fractionation of WF1 from Westiellopsis sp. VN biomass by semi-preparative reversed-phase HPLC and antibacterial activity of fractions to S. aureus

Yield Fractions Retention

time (mg) % recovered

Dose

(µg/disc)


zone (mm)1

WF1-1 14.74 2.3 4.6 200 17.0

WF1-2 23.50 0.6 1.2 200 24.0

WF1-3 24.19 1.4 2.8 200 28.0

WF1-4 25.18 0.4 0.8 200 14.0

WF1-5 30.04 1.7 3.4 200 10.0

WF1-6 30.85 2.6 5.2 200 8.0

WF1-7 33.99 0.5 1.0 200 9.0

WF1-8 41.03 2.8 5.6 200 10.0

WF1-9 41.53-48.00 5.7 11.4 200 0.02

Total 18 36


Chapter III Results

68

Based on the results of agar diffusion assay and yield of these nine fractions,

the four fractions WF1-3, WF1-5, WF1-6, and WF1-8 were used for structure elucidation.

3.2.1.4 Structure elucidation of isolated compounds of Westiellopsis sp.VN

3.2.1.4.1 Structure elucidation of fraction WF1-3 (compound 1)

Fraction WF1-3 was found to be the pure compound, ambiguine D isonitrile

(see fig.3-6).

NH

CH3

H3C

H3C

CH3CH3

H

OH NC

16

2

34

5

7

8

9

1011

1213

14

15

16

17

18

19

2021

22 23

24

25

26

27

28

Cl

O

Figure 3-6: Ambiguine D isonitrile, molecular formula C26H29ClN2O3

The high resolution ESI mass spectrum in positive mode of fraction WF1-3

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 C26H29ClN2O3 (calculated 453.1939 for C26H30ClN2O3 ).

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 ] +

Figure 3-7: UV and ESI-MS of fraction WF1-3 (compound 1)

Chapter III Results

69

Table 3-5: 1H NMR data of compound 1 compared with literature data of ambiguine

D isonitrile

Position

Compound 1( in CDCl3)

δH

*Ambiguine D isonitrile in CDCl3

δH

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

3.2.1.4.2 Structure elucidation of fraction WF1-5

The positive ion ESI mass spectra of fraction WF1-5 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

Figure 3-8: UV and ESI-MS of compounds of fraction WF1- 5

Chapter III Results

70

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 C26H31ClN2O

(calculated 423.2198 for C26H32ClN2O) and this compound was also found in fraction

WF1-6 . Thus, the presence of this compound in this fraction was indicated.

peak1

[ M+ H] +

Figure 3-9: MS data of peak 1 of fraction WF1-5

Peak 2 was identified as the compound ambiguine C isonitrile (compound 2)

(see fig.3-10)

NH

CH3

H3C

H3C

CH3

CH3

H

OH

NC

16

2

34

5

7

8

9

1011

1213

14

15

16

17

18

19

20

21

22

23

24

25 26

27

28

Figure 3-10: Ambiguine C isonitrile, molecula formula C26H32N2O

The identification of ambiguine C isonitrile (dechloro-ambiguine B isonitrile)

with the molecular weight of 388 Dalton compatible with the molecular formula

C26H32N2O 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

Chapter III Results

71

389.2586 for C26H33N2O) and the [M+K]+ peak at m/z 427.2115 as shown in figure 3-

11

[ M + K] +

pe a k 2

[ M + H ] +

Figure 3-11: MS data of peak 2 of fraction WF1-5 (compound 2)

Peak 3 was identified as the compound fischerellin A (compound 3) (see figure

3-12).

N

N

O

O H

1 23

45

67

89

10

1112

1314

15

16

17

18

19

2021

22

23

24

25

26

Figure 3-12: Fischerellin A, molecular formula C26H36N2O2

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 C26H36N2O2 by HRESIMS (in

positive mode) data which displayed a prominent [M+H]+ peak at m/z 409.2846

Chapter III Results

72

(calculated 409.2850 for C26H37N2O2) 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] +

Figure 3-13: MS data of peak 3 of fraction WF1-5 (compound 3)

Table 3- 6: 1H NMR data of compound 3 compared with literature data* of

fischerellin A

Position Multiplicity Proton shift of

compound 3 in CDCl3

*Proton shift of fischerellin

A in CDCl3

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 CH3 2.9-3.0 2.92

2.93

*Hagmann and Jüttner, 1996

Chapter III Results

73

3.2.1.4.3 Structure elucidation of fraction WF1-6 (compound 4)

WF1-6 was found to be the pure compound, ambiguine B isonitrile (see fig. 3-

14)

NH

ClCH3

H3C

H3C

CH3

CH3

H

OH

NC

16

2

34

5

7

8

9

1011

121314

15

16

17

18

19

20

21

22

23

24

25 26

27

28

Figure 3-14: Ambiguine B isonitrile, molecular formula C26H31ClN2O


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 C26H31ClN2O (calculated 423.2198 for C26H32ClN2O).

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]+

Figure 3-15: UV and ESI-MS of fraction WF1-6 (compound 4)

Chapter III Results

74

Table 3-7: Comparison of 1H NMR data of compound 4 with reported data*

Position

Compound 4 ( in CDCl3)

δH

*Ambiguine B isonitrile in CDCl3

δH

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

5.36 Z, d 5.28 E, 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

3.2.1.4.4 Identification of compounds in fraction WF1-8


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

Figure 3-16: ESI-MS of compounds of fraction WF1- 8

Chapter III Results

75

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+O2]+, 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 C21H34O3 (calculated 335.2581 for

C21H35O3).

[ M + O] +

[ M+ H] +

Figure 3-17: ESI-MS of peak 2 of fraction WF1- 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 C19H34O3 (calculated 311.2581 for C19 H35O3).

Chapter III Results

76

[ M+ Na] +

[ M+ H] +

Figure 3-18: ESI-MS of peak 3 of fraction WF1- 8 (compound 6)

3.2.2 Chemical investigation of the active ethyl acetate extract resulting from

cultivation medium of Westiellopsis sp. VN

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.

Chapter III Results

77

SS1 SS2 SS3

Figure 3-19: 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).

Figure 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).

Chapter III Results

78

Table 3-8: Compounds of MeOH fraction analyzed as methyl ester in n-hexane after hydrolysis /derivatization

ret.

time (min)

trivial name main mass spectral

fragment (m/z)

percent

(%)

match

(%)

21.172 L-Proline, 5-oxo-1-

(trimethylsilyl)-, trimethylsilyl

ester (C11 H23NO3Si2)

230, 156, 73, 45 7.330 91.9

32.465 Palmitic acid (C16:0) 313, 269,201, 117, 73,

43

4.169 89.0

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,

41

1.124 81.6

51.384 ß-Sitosterol (C29H50O) 486, 396, 357, 255,

215, 161, 129

1.210 77.5

ret. time = retention time; m/z = the mass of an ion divided by the electrical charge of the ion

Table 3-9: Compounds of MeOH fraction analyzed as methyl ester in MeOH after hydrolysis/derivatization

ret.

time [min]


fragment (m/z)

percent

(%)

match

(%)

13.796 Carbamic acid (CH3NO2) 278, 205, 147, 73, 453 0.159 84.1

21.165 L-Proline, 5-oxo-1-

(trimethylsilyl)-, trimethylsilyl

ester (C11H23NO3Si2)

230, 156, 73, 45 2.049 91.1

24.373 Lauric acid (C12H24O2) 257, 201, 117, 73, 55 1.143 86.2

24.881 Naphthalene (C15H18) 183, 83, 39 0.429 85.5

28. 609 Myristic acid (C14:0) 458, 368, 392, 247,

129, 73

1.298 88.1

32.055 Palmitic acid, isopropyl ester

(C16:0)

298, 256, 213, 157,

129, 102, 60, 43

0.734 71.1

32.465 Palmitic acid (C16:0) 313, 269,201, 117, 73,

43

2.456 88.3

36.015 Octadecanoic acid (C18:0) 341, 201, 117, 73, 43 1.345 90.9


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

Chapter III Results

79

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.

3.2.3 Culture optimization of Westiellopsis sp.VN

3.2.3.1 Nitrogen deficiency

Cultivation of Westiellopsis sp. VN in BG11 without nitrate and in normal

BG11 medium containing 1.5 g/L NaNO3 (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.

Figure 3-21: Fermenters for cultivation of Westiellopsis sp.VN containing standard BG11 medium and BG-11 medium without NaNO3, illuminated continuously with 8µmol/m2 at 28°C, pH 7.4, CO2

supplementation

Based on diameter of inhibition zone (IZ), the concentration of antibacterial

compounds in methanol extracts of biomass and ethyl acetate extracts of growth

Chapter III Results

80

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).

Table 3-10: Dry biomass and antibacterial activity against S.aureus of extracts in BG-11 medium

BG11 without NaNO3

(BG-110)

BG11 with NaNO3

(BG-11)

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)}

hex .

EtOAc.

MeOH

hex .

EtOAc

MeOH

BG110 BG11

BG110 BG11

EtOAc. EtOAc.

Biomass Grow th m edium

Figure 3-22: 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

Chapter III Results

81

3.2.3.2 The effect of incubation time on biomass production and antibacterial

production

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).

0

5

10

15

20

25

30

35

40

45

50

0 1 2 3 4 5 6 7 8 9

I ncuba t ion t im e ( weeks)

Dry w

eight (mg/100ml culture)

0

5

10

15

20

25

Dry weight

Diameter of Inhibition zone

Figure 3-23a: The effect of incubation time on dry weight and antibiotic production of Westiellopsis sp.VN

Figure 3-23b: 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

Dia

mete

r of

Inhib

itio

n z

one (m

m)

Chapter III Results

82

3.3 Chemical investigation of 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.

3.3.1 Fractionation of methanol extract from biomass by RPC18 chromatography

210 mg of MeOH extract was chromatographed on a reverse-phase C18

column eluting with 5 solvent systems starting with MeOH/EtOH/H2O (45:45:10),

followed by MeOH/H2O (9:1), MeOH, and washing with MeOH/C3H6O (1:1) and

CH2Cl2, respectively to yield 10 fractions (CJFI to CJFX) (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 CJFII eluting with

MeOH/EtOH/H2O (45:45:10) possesses the strongest activity (see table 3-11),

therefore it was further purified.

Table 3-11: Fractionation of methanol extract from Calothrix javanica biomass by RP C18 chromatography and antibacterial activity of fractions to S. aureus

Yield

Fractions

No of

sub

fractions

pooled

Mobile phase (mg) %

recovered

Dose

(µg/disc)

Diameter

of IZ

(mm)1

CJFI 1-8 MeOH/EtOH/H2O (45: 45:10) 33.8 16.10 500 02

CJFII 9-24 MeOH/EtOH/H2O (45: 45:10) 89.9 42.81 500 9.0

CJFIII 25-30 MeOH/EtOH/H2O (45: 45:10) 1.8 0.86 500 7.0

CJFIV 31-34 MeOH/EtOH/H2O (45: 45:10) 1.2 0.57 500 0

CJFV 35-40 MeOH/EtOH/H2O (45: 45:10) 1.4 0.67 500 0

CJFVI 41-50 MeOH/EtOH/H2O (45: 45:10) 8.4 4.00 500 7.0

CJFVII 51-100 MeOH/ H2O (90:10) 17.8 8.48 500 7.5

CJFVIII 101-150 MeOH 14.5 6.90 500 0

CJFIX 151-200 MeOH/CH3COCH3 (50:50) 14.2 6.76 500 0

CJFX 201-250 DCM 22.8 10.86 500 0

Total 205.8 98.01


Chapter III Results

83

3.3.2 Purification of fraction CJFII by semi-preparative reversed-phase HPLC

First, the active fraction CJFII was analyzed by HPLC with a solvent gradient

of acetonitrile/water (table 3-12, figure 3-24).

Table 3-12: Step gradient used in HPLC analysis of CJFII by analytical HPLC

Time (min) 0.50 25.5 26.50 30.50 32.00 Solvent A (%) 95 5 0 0.0 95 Solvent B (%) 5 95 100 100 5


RP80A/250×4.6mm, 4 micron

Figure 3-24: Analytical HPLC chromatogram of fraction CJFII 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 CJFII 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 CJFII by semi-preparative HPLC. Altogether, 86 mg of fraction CJFII were

purified. 7 peaks CJFII-1 to CJFII-7 were collected and controlled at the wave length of

220 nm to afford 7 seven fractions (see table 3-13).

Chapter III Results

84

Table 3-13: Separation of CJFII from Calothrix javanica biomass by semi-preparative reversed-phase HPLC and activity of the fractions to S. aureus



Dose

(µg/disc)


zone (mm)1

CJFII -1 3.66 1.3 1.51 100 6.5

CJFII -2 4.54 43.4 50.47 200 02

CJFII -3 11.38 0.7 0.81 100 0

CJFII -4 12.93 3.8 4.42 200 12.5

CJFII -5 21.28 3.0 3.49 100 7.0

CJFII -6 26.63 4.4 5.12 100 15.0

CJFII -7 30.44 1.6 1.86 100 0

Total 58.2 67.68


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 CJFII-4 was the most pure one (see fig.3-25) and therefore used for

structural elucidation.

Table 3-14: Step gradient used in HPLC analysis of seven fractions by analytical HPLC

Time (min) 0.5 16.5 24.5 30.5 31.0 Solvent A (%) 50 15 0 0 50 Solvent B (%) 50 85 100 100 50


polar-RP80A/250×4.6mm, 4 micron

Figure 3-25: Analytical HPLC chromatogram of fraction CJFII-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.

Chapter III Results

85

3.3.3 Structure elucidation of fraction CJFII-4 (compound 7)

Fraction CJFII-4 was found to be a new cyclic peptide named daklakapeptin.

Figure 3-26: 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 CONH2 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

Chapter III Results

86

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 13C 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 13C 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 C68H114O20N13

which agreed with the molecular formula C68H113O20N13 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.

Table 3-15: NMR data of CJFII-4 Residue C/H No. δC δH

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

Chapter III Results

87

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,

C3,5:117.6, C4: 157.0 H2/6: 7.13, H3/5: 6.86

4-Gln NH 8.03 CO Cα 51.6 4.85 Cβ 28.3 2.08, 1.93 Cγ 32.8 2.32 CONH2 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 CH2-2 42.1 2.56 CH(NH-)-3 49.9 4.18, 7.78 NH CH2-4 35.1 1.57 CH2-5 27.6 1.38 CH-6 33.2 1.31 (CH3)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-CH3 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 (CH3)2.CH.CH2.CH2.CH(NH-).CH2 CO-

Chapter III Results

88

Table 3-16: Sequence information deduced from the NOE’s found in the 2D NOESY spectrum of CJFII-4

α-N (i, i+1) N-N (i, i +1) Others α-N(i,i+2)

12 (Pro)-3 12α-8NH? 3-8 3-8 3ß-8NH(x2)

3H2/6 – 8NH

8-9 9(CH2)-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

Table 3-17: Calculation of the molecular mass from the structure of the residues deduced from the NMR data.

Residue Molecular Formula Mass

1 (CH3)2 CHCH(OH)CH(NH)CO

C6 H11 NO2 129

2 HOCH2CHOHCH(NH)CO

C4 H7 NO3 117

3-Tyr C9 H9 NO2 163.2 4-Gln C5 H8 N2 O2 128.1 5-Leu C6 H11 NO 113.2 6

HOCH2CH2CH(NH)CO C4 H7 NO2 101

7-Ile C6 H11 NO 113.2 8-Thr C4 H7 NO2 71.1 9

(CH3)2.CH.CH2.CH2.CH(NH-).CH2CO- C8 H15 NO 141

10-Thr C4 H7 NO2 101.1 11-IleNMe C7 H13 NO 127.2 12-Pro C5 H7 NO 97.1

Total C68H113N13O20 1431

Chapter III Results

89

Table 3-18: Comparison of the sequence from the high-resolution ESI-MS data with that from the NMR data.

Sequential loss of exact fragment masses in the HR-ESI-MS

261.1234 C14H17O3N

2 [M+H]+

101.0477 141.1154 113.0841 129.0788 101.0479 113.0840 128.0586 127.0988 50.5237 x2 58.5212 x2

Molecular formula of fragments sequentially lost in the HR-ESI-MS

C5H7NO + C9H9NO2

C4H7NO2 C8H15NO C6H11NO C6H11NO2 C4H7NO2 C6H11NO C5H8N2O2 C7H13NO C4H7NO2 C4H7NO3

Sequence from the NMR analysis

Pro - Tyr Thr (Leu+2CH2) a

Leu Leu+O b

Thr Leu Gln Ile-NMe Thr Thr+O c

Footnote: The structures of the complex residues are as follows: a) (CH3)2CH.CH2CH2CH(NH-)CH2CO-, b) (CH3)2CHCH(OH)CH(NH-)CO-, c) HOCH2CHOHCH(NH-)CO-.

Chapter III Results

90

3.4 Chemical investigation of 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.

3.4.1 Fractionation of methanol extract obtained from biomass by RP C18

column chromatography

In the first purification step, 230 mg of the methanol extract were

fractionated on RP C18 column with three solvent systems (see 2.8.3) to get 8

fractions (SOFI to SOFVIII) which were evaluated for antibacterial activity against S.

aureus using agar diffusion method (see table 3-19).

Table 3-19: Fractionation of methanol extract from Scytonema ocellatum biomass by RP-18 chromatography and antibacterial activity of fractions to S. aureus

Yield

Fractions

No of sub

fractions

pooled

Mobile phase (mg) %

recovered

Dose

(mg/disc)

Diameter

of IZ

(mm)1

SOFI 1-2 MeOH/EtOH/H2O

(45:45:10)

66.6 28.96 1.0 02

SOFII 3-10 MeOH/EtOH/H2O

(45:45:10)

76.3 32.74 1.0 10.0

SOFIII 11-12 MeOH/EtOH/H2O

(45:45:10)

1.7 0.74 1.0 0

SOFIV 13-20 MeOH/EtOH/H2O

(45:45:10)

9.5 4.13 1.0 0

SOFV 21-23 MeOH/EtOH/H2O

(45:45:10)

8.4 3.65 1.0 0

SOFVI 24-40 MeOH/EtOH/H2O

(45:45:10)

19.3 8.39 1.0 0

SOFVII 41-80 MeOH 43.3 18.83 1.0 0

SOFVIII 81-120 MeOH/CH3COCH3

(10:10)

2.5 1.09 1.0 0

Total 226.6 98.53


Because only fraction SOFII showed strong antibacterial activity, this fraction

was further separated.

Chapter III Results

91

3.4.2 Separation of fraction SOFII using silica gel column

To remove more unwanted substances still remaining in the active fraction

SOFII, silica gel column chromatography was employed.

74.3 mg of fraction SOFII 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)

Table 3-20: Separation of SOFII from Scytonema ocellatum biomass by silical gel chromatography and antibacterial activity of the fraction to S. aureus

Yield

Fractions

No of sub

fractions

pooled

Mobile phase (mg) %

recovered

Dose

(µg/disc)

Diameter

of IZ

(mm)1

SOFII-1 1-4 n-hexane/EtOAc

(25:75)

5.9 7.94 500 02

SOFII-2 5-20 n-hexane/EtOAc

(25:75)

1.1 1.48 500 0

SOFII-3 21-36 EtOAc 1.1 1.48 500 0

SOFII-4 37-43 EtOAc/MeOH (50:50) 1.1 1.48 500 0

SOFII-5 44-45 EtOAc/MeOH (50:50) 17.8 23.96 500 9.0

SOFII-6 46-51 EtOAc/MeOH (50:50) 11.7 15.75 500 8.0

SOFII-7 52-63 MeOH 5.8 7.81 500 0

SOFII-8 64-67 MeOH 10.9 14.68 500 0

SOFII-9 68-70 MeOH 3.8 5.11 500 0

SOFII-10 71-72 MeOH 2.4 3.23 500 0

Total 61.6 82.92


The fractions SOFII-5 and SOFII-6 eluting with 50% EtOAc/MeOH exhibited

approximately the same antibacterial activity. Thus, these two fractions were pooled

and further purified.

3.4.3 Purification of the pooled fractions using reversed-phase RP HPLC

Final purification of the pooled fractions SOFII-5 and SOFII-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

Chapter III Results

92

table 3-21).

FSO1

FSO3

FSO2

Figure 3-27: Semi-preparative RP HPLC chromatogram of the pooled fractions SOFII-5 and SOFII-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.

Table 3-21: Separation of SOFII-5 and SOFII-6 from Scytonema ocellatum biomass by semi-preparative reversed-phase HPLC and antibacterial activity of the fractions to S. aureus



Dose (µg/disc) Diameter of inhibition

zone (mm)1

FSO1 3.08 1.0 3.85 200 02

FSO2 4.29 13.4 51.54 200 0

FSO3 14.32 2.4 9.23 200 12.0

Total 16.8 64.62


The active fraction FSO3 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.

Chapter III Results

93

Table 3-22: Step gradient used in HPLC analysis of FSO3 by analytical HPLC

Time (min) 0.50 18.50 21.50 24.50 25.00 Solvent A (%) 95 5 0 0.0 95 Solvent B (%) 5 95 100 100 5


RP80A/250×4.6mm, 4 micron

Figure 3-28: Analytical HPLC chromatogram of fraction FSO3 (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.

3.4.4 Structure elucidation of fraction FSO3

Fraction FSO3 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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94

3.5 Chemical investigation of Anabaena sp.

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.

Table 3-23: 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.

3.5.1 Purification of EtOAc extract using semi-preparative reversed-phase

HPLC

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

Chapter III Results

95

acetate extract obtained from culture medium was purified with a step gradient of

CH3CN/H2O 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)

f irst

AF1

AF3

AF5

AF6

AF2

AF4

f irst

Figure 3-29: 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.

Table 3-24: Separation of EtOAc extract from Anabaena sp.culture medium by semi-preparative reversed-phase HPLC and antibacterial activity of the fractions to E. coli




zone (mm)1

first 12.75 0.3 2.50 200 02

AF1 15.76 0.6 5.00 200 0

AF2 17.71 0.2 1.67 200 0

AF3 18.33 0.4 3.33 200 0

AF4 19.32 0.3 2.50 200 0

AF5 20.77 0.2 1.67 200 0

AF6 22.77 1.3 10.83 200 20.0

Total 3.3 27.50


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96

Figure 3-30: 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 AF6 exhibited antimicrobial activity, this fraction was

used for structure elucidation.

3.5.2 Structure elucidation of fraction AF6 (compound 8)

Fraction AF6 was found to be the the pure substance flourensadiol (see fig.3-

31)

Figure 3-31: Flourensadiol, molecular formula C15H26O2

The HR-ESI-MS showed a sodiated molecular ion at m/z 261.1825 that was

compatible with the molecular formula C15H26O2. The 2D COSY spectrum allowed

the unambiguous identification of two fragments in the molecule, namely

CH3.CH.CH2.CH2 and CH2.CH.CH.CH, together with signals of an isolated CH2OH

and two singlet CH3 groups. The 13C 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

Chapter III Results

97

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 CH2OH group as CH3.C*(CH2OH) and CH3.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 CH3.C*(CH2OH) 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.

Table 3-25: NMR data of AF6 (Flourensadiol) in trifluoroethanol-d2/H2O (1:1)

Carbon

No.

1H (ppm)

Multiplicity

J (Hz) 13C (ppm)

Multiplicity

HMBC (H—>C) ROESY

1 77.4 s 2 A 1.77 m

B 1.64 m 38.4 t

C1, C8

3 A 1.78 m B 1.47 ddd

14.3,11.8, 11.8

20.0 t C1 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,

C13, C14 H4, H7,

H9B, H10B, H12, H13

7 1.87 m 40.8 d C1 8 1.87 m 59.1 d 9 A 1.70 m

B 1.59 m 26.4 t

C8

10 A 1.82 m B 1.30 m

29.6 t 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

B 3.71 d 11.6 11.6

65.3 t C5/C6, C4, C13 C5/C6, C4, C13

H7, H3B, H13

15 1.17 s 32.1 q C1, C2, C8 H2A, H2B,H8, H9A

Chapter III Results

98

3. 6 Chemical investigation of Nostoc sp.

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.

3.6.1 Fractionation of methanol extract obtained from biomass using silica gel

column

760 mg of MeOH extract was first fractionated on silica gel column eluting

with a step gradient of n-hexane/EtOAc/MeOH/H2O (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).

Table 3-26: Fractionation of methanol extract from Nostoc sp. biomass by silical gel chromatography and antibacterial activity of fractions to S. aureus

Yield

Fractions

Mobile phase (mg) % recovered

Dose

(mg/disc)

Diameter of IZ

(mm)1

NFI n-hexane/EtOAc (90:10) 20.0 2.63 1.0 0.02

NFII n-hexane/EtOAc (40:60) 93.5 12.30 1.0 6.5

NFIII n-hexane/EtOAc (20:80) 36.1 4.75 1.0 0.0

NFIV MeOH 363.6 47.84 1.0 10.0

NFV MeOH/H2O (95:5) 26.3 3.46 1.0 0.0

Total 539.5 70.98


Because fraction NFIV showed the strongest activity, this fraction was

further separated.

3.6.2 Separation of the acitve fraction NFIV using RP C18 column

chromatography

360 mg of the active fraction NFIV was subjected to C18 column eluting with

three solvent systems (see 2.8.5) to afford three fractions which were evaluated for

Chapter III Results

99

their antibacterial activities against S. aureus using agar diffusion assay (see table 3-

27).

Table 3-27: Fractionation of NFIV from Nostoc sp. biomass by RP C18

chromatography and antibacterial activity of fractions to S. aureus

Yield

Fractions

Mobile phase (mg) % recovered

Dose

(mg/disc)

Diameter of IZ

(mm)1

NFIV-1 MeOH/H2O (90:10) 151.6 42.11 0.5 11.0

NFIV-2 MeOH 96.3 26.75 0.5 0.02

NFIV-3 DCM 42.8 11.89 0.5 0.0

Total 290.7 80.75


Only fraction NFIV-1 exhibited antibacterial activity and was further purified.

3.6.3 Purification of the active fraction NFIV-1 using semi-preparative reversed-

phase HPLC

Final purification of the active fraction NFIV-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)

NsF1

NsF2

NsF3 NsF4

Figure 3-32: Semi-preparative RP HPLC chromatogram of fraction NFIV-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.

Chapter III Results

100

Table 3-28: Fractionation of NF IV-1 from Nostoc sp. biomass by semi-preparative RP HPLC and antibacterial activity of fractions to S. aureus




zone (mm)1

NsF1 4.73 25.4 16.93 500 0.02

NsF2 5.48 3.6 2.40 500 10.0

NsF3 6.26 3.5 2.33 500 0.0

NsF4 7.71 1.7 1.13 500 0.0

Total


Only fraction NsF2 showed antibacterial activity, this fraction was therefore

used for structure elucidation.

3.6.4 Structure elucidation of fraction NsF2

The low resolution ESI-MS of fraction NsF2 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 NsF2 is in progress.

3.7 Chemical investigation of 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.

Chapter III Results

101

3.7.1 Isolation of cytotoxic compounds of methanol extract obtained from

biomass according to method 1

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.

3.7.1.1 Fractionation of methanol extract by silica gel column chromatography

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 (F1 to F15). 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)

Table 3-29: Fractionation of methanol extract from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions against cell line 5637

Yield Fracti

ons

No of sub-

fractions pooled

Mobile phase

mg % recovered

IC50

µg/L

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


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

Chapter III Results

102

Figure 3-33: 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/CH3COOH (75+25+5+3); Detection: Vis after spraying with Anisaldehyde/sulfuric acid reagent}

Because fraction F8 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.

3.7.1.2 Separation of fraction F8 by silica gel column chromatography

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).

Table 3-30: Separation of fraction F8 from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions to 5637 cell line

Yield

Fractions

No of sub-

fractions

pooled

Mobile phase mg % recovered

IC50

µg/L

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 < 0.05

F8-4 209-214 n-hexane/EtOAc (10:90) 2.9 3.22 < 0.05

F8-5 215-225 n-hexane/EtOAc (10:90) 8.4 9.33 < 0.05

F8-6 226-325 100%MeOH 20.09 22.32 < 0.05

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.

Chapter III Results

103

3.7.1.3 Purification of fraction F8-3 by preparative TLC

30.2 mg of fraction F8-3 was further purified by preparative TLC. The

chromatogram was developed with n-hexane/EtOAc/MeOH/CH3COOH (75:25:5:3),

five spots were scratched out to yield five fractions (see table 3-31).

Table 3-31: Separation of fraction F8-3 from L.majuscula biomass by preparative TLC

Yield

Fractions

Retention factor mg % recovered

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 (F8-3-1 to F8-3-5) were examined by analytical TLC and

HPLC. The result of HPLC analysis showed that fraction F8-3-2 (3.1 mg) was pure

enough for structure elucidation by ESI-MS and NMR.

3.7.1.4 Structure elucidation of fraction F8-3-2 (compound 9)

Fraction F8-3-2 was found to be the pure compound, 17-debromo-3,4-

didehydro-3-deoxy-aplysiatoxin (anhydrodebromoaplysiatoxin), a known metabolite

(see fig.3-34)

O

O O

H3C

H3C

O

OH

CH3O

CH3

O

CH3

OH

CH3

CH3

O

1

2

3

45

6

7

8

910

1112

1314

1516

17

18

19

2021

2223

2526

24

27

2829

30

31

32

Figure 3-34: 17-debromo-3, 4didehydro-3-deoxy –aplysiatoxin, molecular formula C32 H46O9

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

Chapter III Results

104

Dalton compatible with the molecular formula C32 H46O9 (calculated 575.3215 for C32

H47O9).

[ M + H -M eOH ] +

[ M + H ] + [ M+ N a ] +

[ M+ K] +

Figure 3-35: ESI-MS spectrum of fraction F8-3-2 (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.

Table 3-32: Comparison of 1H NMR data of compound 9 with reported data *

Position

Compound 9 *Anhydrodebromoaplysiatoxin

1δH

2δH (J in Hz)

2 3.05 (d) 3.32 (d)

3.05 (br. d, 13.4 ) 3.32 (br. d, 12.1)

8 1.73 (dd) 2.22 (dd)

1.73(ax, dd, 3.6 and 14.8) 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)

Chapter III Results

105

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

(CH3O-) 3.17 (3H,s) 3.17 (3H,s)

* Nagai.et al.,1998 1. Spectra determined in CDCl3 2. Spectra determined in acetone-d6; data reported in ppm

3.7.2 Isolation of cytotoxic compounds of methanol extract obtained from

biomass according to method 2

3.7.2.1 Separation of methanol extract by silica gel column chromatography

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 (F1 to F22). Based

on the results of the TLC analysis, three fractions (F9, F10, and F17) were chosen for

testing cytotoxic activity against 5637 cell line (see fig3-36 and table 3-33).

Figure 3-36: 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/CH3COOH (75:25:5:3); Detection: Vis after spraying Anisaldehyde/sulfuric acid reagent]

Chapter III Results

106

Table 3-33: Fractionation of methanol extract from Lyngbya majuscula biomass by silica gel chromatography and cytotoxic activity of fractions against cell line 5637

Yield Fractions No of sub

fractions pooled

Mobile phase

mg % recovered

IC50

µg/ml









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




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 F10 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 F10 revealed that further separation by semi preparative HPLC was possible.

Chapter III Results

107

3.7.2.2 Purification of fraction F10 using semi-preparative reversed-phase

HPLC

The active fraction F10 was analyzed by HPLC with DAD detection. At 210

nm the separation of fraction F10 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 F10 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, F10-2, F10-3, F10-4, and F10-5 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)

F10 -3

F1 0 -4

F1 0 -5

F10 -2

F1 0 - 1

Figure 3-37: Semi-preparative RP HPLC chromatogram of F10 (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

Chapter III Results

108

Table 3-34 Separation of fraction F10 from Lyngbya majuscula biomass by semi-preparative RP HPLC and cytotoxic activity of fractions against cell line 5637

Yield

Fractions

Retention time mg % recovered

IC50 (ng/ml)

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 F10-5 exhibited strong activity (IC50= 86ng/mL

and 40ng/mL, respectively), the structure of including compounds was elucidated by

ESI –MS and NMR.

3.7.2.3 Structure elucidation of fractions F10-3 and F10-5

3.7.2.3.1 Structure elucidation of fraction F10-3 (compound 10)

Fraction F10-3 was recognized as the pure compound debromoaplysiatoxin (see

fig.3-38)

O

O O

H3C

H3C

O

OH

CH3O

CH3

O

CH3

OH

CH3

CH3

OH

O

1

2

3

45

6

7

8

910

1112

1314

1516

17

18

19

2021

2223

2526

24

27

2829

30

31

32

Figure 3-38: Debromoaplysiatoxin, molecular formula C32H48O10

The identification of debromoaplysiatoxin (compound 10) was established by

direct comparison of our spectroscopic data, including 1H NMR, 13C NMR, and

HRMS in negative mode with those reported in the literature. 1H NMR, 13C 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)

Chapter III Results

109

Table 3-35: Comparison of 13C NMR and 1H NMR data of compound 10 with literature values of *Debromoaplysiatoxin

Position

Compound

10

*Debromo-

aplysiatoxin

Compound

10

*Debromo-

aplysiatoxin 1

δC 2δC

3δH

4δH (J in Hz)

1 169.19 169.2 (0) - - 2 46.91 46.9 (2) 2.52 (β, d)

2.76 (α,d) 2.52 (β, d, 12.7) 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)

1.05 (eq, dd) 1.62 (ax, t, 13.1 and 13.1)

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)

2.68 (eq, dd) 1.71 (ax, dd, 3.6 and 14.8) 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)

1.39 (m) 1.31 (m) 1.39 (m)

14 36.12 36.1 (2) 1.64 (m) 1.97 (m)

1.63 (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)

2.88 (β, m) 2.92 (α, dd, 11.1 and

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 CDCl3; 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(CH2), or 3(CH3) 3. Spectra determined in CDCL3, 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

Chapter III Results

110

weight was deduced to be 592 Dalton compatible with the molecular formula

C32H48O10.

[ M - H + H C l ] -

Fgure 3-39: UV and ESI-MS spectrum of compound 10

3.7.2.3.2 Structure elucidation of fraction F10-5 (compound 11)

Fraction F10-5 was identified as the pure compound 3,4-didehydro-3-deoxy-

aplysiatoxin (figure 3-40)

O

O O

H3C

H3C

O

OH

CH3O

CH3

O

CH3

OH

CH3

CH3

O

1

2

3

45

6

7

8

910

1112

1314

1516

17

18

19

2021

2223

2526

24

27

2829

30

31

32

Br

Figure 3-40: 3,4-didehydro-3-deoxy-aplysiatoxin, molecular formula C32H45BrO9

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 H45 BrO9 (calculated 653.2320

for C32 H46BrO9).

Chapter III Results

111

[ M ] + [ M + 2 ] +

[ M + 2 ] +

Figure 3-41: 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).

Table 3-36: Comparison of 1H NMR data of compound 11 with reported data *

Position

Compound 11 *3,4-didehydro-3-deoxy-

aplysiatoxin

1δH

2δH

2 3.070 (dm)

3.342 (d)

3.070 (br dm)

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)

Chapter III Results

112

2.781 (dd) 2.781 (dd)

29 5.357 (dt) 5.357 (dt)

30 3.854 (qd)

4.07 (d)

3.854 (qd)

4.07 (d)

OCH3 3.210 (s) 3.210 (s)

*Moore et al., 1984 1. Spectra determined in CDCl3; data reported in ppm 2. Spectra determined in acetone-d6; data reported in ppm

3.7.3 Fatty acid analysis

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.

Table 3-37: Fatty acids analyzed as methyl ester in n-hexane extract

ret.

time [min]


fragment (m/z)

percent

(%)

match

(%)

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,

129, 73

1.062 83.1


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.

Chapter IV Discussion

113

4 Discussion

4.1 Screening of crude extracts for antibacterial activity

4.1.1 Selection of antibiotic screening and cyanobacterial strains

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


114

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.


115

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.

4.1.2 Cultivation and extraction

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±20C), this temperature is consistent with a general rule that the

optimum temperature for cyanobacteria is 15-200C (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.


116

4.1.3 Antibacterial activity

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/H2O 7:3 extracts) showed activity against Gram-positive bacteria. 5

extracts (5.8%; 3 DCM/MeOH 2:1 and 2 MeOH/H2O 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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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.

4.2 Chemical investigation and culture optimization of Westiellopsis

sp. VN

4.2.1 Selection of Westiellopsis sp.VN

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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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.

4.2.2 Active intracellular metabolites of Westiellopsis sp.VN strain

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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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 IC50 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


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to Vero cells with IC50 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 WF1-5 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 WF1-5 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 WF1-8. They were found as a

mixture in fraction WF1-8 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,


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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).

Figure 4-1: 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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fischerellins was repressed in Westiellopsis strains and can be activated by different

endogenic or exogenic factors.

4.2.3 Chemical composition of volatile extracellular compounds of Westiellopsis

sp.VN strain

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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4.2.4 Cultivation optimization of Westiellopsis sp.VN strain

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:


124

- 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

composition of the BGA medium by adding 1.5 g L-1 NaNO3, increasing the

Fe2(SO4)3.x6H2O concentration to 0.025 g L-1, not adding NaCl, using an initial pH of

7.0, incubating at 350C, 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 250C

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 350C 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


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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 MgSO4 and micronutrients (a) in case of F. moniliforme, with

FeSO4 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 K2SO4 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;

temperature 300C, 25 mg/L NO3 2-; 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

(300C) and more intense light (30 W/m2) than during cultivation under basal

conditions at 250C and 10 W/m2.


126

- 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 350C) but growth rates of these two

species were similar at lower temperature (20 and 250C).

- 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 CO2 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


127

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 NaNO3

for 7 weeks under the following culture conditions: temperature 280C, pH 7.4, and

continuous illumination using cool-white fluorescent tubes with an intensity of

8µmol/m2 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 NaNO3 and BG-11 standard medium,

indicating that NaNO3 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 NaNO3 deficiency on synthesis of antimicrobial

substances by this strain in the large scale, cultivation with BG-11 medium without

NaNO3 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 NaNO3 (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 202A1 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


128

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.

4.2.5 Effect of incubation time on biomass production and antimicrobial

compound accumulation of Westiellopsis sp.VN strain

Effect of incubation time on biomass production and antimicrobial compound

accumulation of Westiellopsis sp.VN in batch cultivation with BG 11 medium without

NaNO3 at temperature 20±20C, pH 7.4, and continuous illumination provided by cool-

white fluorescent tube of 8µmol/m2 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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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 (LD50 = 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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4.3 Chemical investigation of Calothrix javanica and Scytonema

ocellatum

4.3.1 Selection of 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).

4.3.2 Selection of 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).

4.3.3 New cyclic peptide of Calothrix javanica and 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 C18 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


131

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

Figure 4-2: Sequence

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 IC50 values of 58 nM and 180 nM, respectively. These calothrixins also

exhibited potent activity against the human cervical cancer cell line, HeLa, with IC50

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.

Thr Z Pro Tyr Thr

XIleYTLeuGln

IleNMe


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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 tR 15.35 min (see

figure 4-3, 4-4).

Figure 4-3: Analytical HPLC chromatogram of methanol extract of Calothrix javanica with mobile phase of Acetonitrile/ H2O (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.


133

Figure 4-4: Analytical HPLC chromatogram of methanol extract of Scytonema ocellatum with mobile phase of Acetonitrile/ H2O (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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Figure 4-5: 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 IC50 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


135

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 MICs 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),


136

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.

4.4 Chemical investigation of Anabaena sp.

4.4.1 Selection of Anabaena sp.

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


137

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).

4.4.2 Active compound of Anabaena sp.

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 13C, 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 , 13C,

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


138

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.

4.5 Chemical investigation of Nostoc sp.

4.5.1 Selection of Nostoc sp.

The extracts obtained from this strain exhibited strong antimicrobial activity

against Gram-positive bacteria (B. subtilic and S.aureus) and Gram-negative bacteria


139

(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).

4.5.2 Active compounds of Nostoc sp.

The low resolution ESI-MS of fraction NsF2 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 NsF2 is in progress.

4.6 Chemical investigation of the marine Lyngbya majuscula

4.6.1 Selection of the marine cyanobacterium L. majuscula collected in Vietnam

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


140

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.

4.6.2 Cytotoxic compounds of the marine cyanobacterium Lyngbya majuscula

collected in Vietnam

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, 13C) 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


141

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 IC50 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 IC50 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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4.7 Conclusion

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.

Summary

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Summary

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 sp. VN 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,

Summary

144

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 NaNO3 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.


either Calothrix javanica by C18 chromatography followed by reversed-phase HPLC or

Scytonema ocellatum by C18 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

Thr Z Pro Tyr Thr

XIleYTLeuGln

IleNMe

with X: (CH3)2CHCH2CH2CH(NH-)CH2CO-, Y:(CH3)2CHCH(OH)CH(NH-)CO-,

T: HOCH2CH2CH(NH-)CO-, Z: HOCH2CHOHCH(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 sp. led to isolation and structure elucidation of

flourensadiol. The structure of flourensadiol was established using an extensive array of 1D

(1H, 13C, DEPT-135) and 2D (HMQC, COSY, HMBC) NMR and HR-ESI-MS experiments.

Summary

145

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 sp. by

silica gel chromatography followed by C18 chromatography and reversed phase HPLC led to

isolation of the active fraction NsF2 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 NsF2 showed signal at m/z 426 [M+H]+. The

NMR and MS characterization of compounds in fraction NsF2 is in progress.


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, 13C) 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 IC50 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.

146

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356. Wilmotte, A., and Golubic, S. (1991). Morphological and genetic criteria in the taxonomy of Cyanobacteria/ Cyanobacteria. Algo. Stud. 64, 1-24.

357. Wilmotte, A., and Herdman, M. (2001). Phylogenetic relationships among the cyanobacteria based on 16S RRNA sequences. In: Boone, D.R. and Castenholz R.W. (Eds.) Bergey's Manual of Systematic Bacteriology. Second edition, Springer, New York. 487-493.

358. Wilmotte, A., Stam, W. and Demoulin, V. (1997). Taxonomic study of marine oscillatoriacean strains (Cyanophyceae, Cyanobacteria) with narrow trichomes. III. DNA-DNA hybridization studies and taxonomic conclusions. Algol Stud. 87, 11-28.

359. Woese, C. R. (1987). Bacterial evolution. Microbiol Rev. 51, 221–271.

360. Woese, C. R., Sogin, M., Stahl, D., Lewis, B.J., and Bonen, L. (1976). A comparison of the 16S ribosomal RNAs from mesophilic and thermophilic bacilli: Some modifications in the Sanger method for RNA sequencing. J. Mol. Evol. 7, 197- 213.

361. Xiong, S., Fan, J., and Kitazato, K., (2010). The antiviral protein cyanovirin-N: the current state of its production and applications. Appl Microbiol Biotechnol. 86, 805-812.

362. Zang, L. H., Longley, R., and Koehn, F.E. (1997). Antiproliferative and immunosuppressive properties of microcolin A, a marine-derived lipopeptide. Life Sci. 60, 751-762

168

Acknowledgments

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

169

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.

170

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.

171

Curriculum Vitae

Personal Data

Name: Le Thi Anh Tuyet

Gender: Female

Date of birth: 19 May 1973

Place of birth: Thanh Hoa, Vietnam

Nationality: Vietnamese

Marital status: Married

Educational Background

January 2008 to present Ph.D student at the Institute of Pharmacy, Ernst-Moritz-

Arndt University of Greifswald, Germany

2007 Research internship in Algal Culturing Techniques at

Algal Biotechnology Department, Institute of

Biotechnology, Ha Noi,Vietnamese Academy of Science

and Technology

Oct. 2006-Dec. 2006 Training course on Methods for Natural Products

Isolation at Institute of Pharmaceutical Biology and

Biotechnology, Heinrich-Heine-University, Düsseldorf,

Germany

Oct. 2005-Sept. 2006 Post-graduate training course in Molecular Biology at

Environmental Health Research Institute (IUF), Heinrich-

Heine-University Düsseldorf gGmbH, Düsseldorf,

Germany

Aug. 2005-Sept. 2005 German language course in Carl Duisburg Centren

Dortmund, Germany supported by DAAD

1995-1997 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

172

of Biotechnology (IBT), Ha Noi, Vietnamese Academy of

Science and Technology (VAST)

1990-1994 Bachelor of Science (B.Sc.) degree in Biology at Vinh

University, Vietnam

Employment Record

Oct. 1998-Aug. 2005 Worked as a lecturer at Natural Sciences Faculty, Hong

Duc University, Thanh Hoa, Vietnam

Jun. 1997-Sept. 1998 Worked as a teacher at High School in Thanh Hoa,

Vietnam

Sept. 1994-April 1995 Worked as a teacher at High School in Thanh Hoa,

Vietnam

173

List of publications and other scientific achievements 1. Le Thi Anh Tuyet, 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. Le Thi Anh Tuyet, 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. Le Thi Anh Tuyet, 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 Le Thi Anh Tuyet (2009)

Synthesis, characterization, antimicrobial and antitumor screening of some

diorganotin(IV) complexes of 2-[(9H-Purin-6-ylimino)]-phenol. European Journal of

Medicinal Chemistry 44(10), 3981-3985.

5. Nguyen Van Cuong, Le Thi Anh Tuyet (1997) Initial results of the study on

generating gene transferred fish. Vietnam Journal of Genetics and Applications 2:10-

16.

Poster

1. Le Thi Anh Tuyet and Sabine Mundt. Screening of soil cyanobactria from

Vietnam for antibacterial activity. In "13th International symposium on phototrophic

prokaryotes", August 9-14, 2009, Montreal, Quebec, Canada.

174

Appendix

Appendix 1a: 1H NMR of fraction WF1-3 of Westiellopsis sp. VN

175

Appendix 1b: 1H NMR of fraction WF1-3 of Westiellopsis sp. VN

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Appendix 5: GC-MS spectrum for the fatty acids of fraction MeOH of Westiellopsis sp. VN in n-hexane hydrolysis /derivatization

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Appendix 6: GC-MS spectrum for the fatty acids of fraction MeOH of Westiellopsis sp. VN in MeOH hydrolysis /derivatization

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Diameter of inhibition zone in mm1

Strain

Cultivation of

cyanobacteri

a

Extract B.s.a S.a.

b E.e.

c

P.a.d

C.m.e

n-hexane ext. 0.0 0.0 0.0 0.0 n.t.

MeOH ext. 14,0 9.0 0.0 0.0 11.0

Batch

EtOAc ext. 18.0 16.0 21.0 0.0 n.t.

n-hexane ext. 0.0 0.0 0.0 n.t. 0.0

MeOH ext. 0.0 0.0 0.0 n.t. 0.0

Anabaena

sp.

Large scale

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.

MeOH ext. 11.0 13.0 8.0 0.0 n.t.

Batch

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.

MeOH ext. 10.0 15.0 0.0 0.0 n.t.

Nostoc sp.

Large scale

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.

MeOH ext. 17.0 19.0 12.0 12.0 n.t.

Batch

EtOAc ext. 7.0 6.5 6.5 0.0 n.t.

n-hexane ext. 15.5 8.0 7.5 0.0 n.t.

MeOH ext. 8.0 8.0 7.0 0.0 n.t.

Calothrix

elenkinii

Large scale


n-hexane ext. 0.0 0.0 0.0 0.0 n.t.

MeOH ext. 17.0 18.0 8.0 0.0 n.t.

Batch

EtOAc ext. 14.0 12.0 0.0 0.0 n.t.

n-hexane ext. 12.0 0.0 7.0 9.0 n.t.

MeOH ext. 0.0 7.0 7.0 0.0 n.t.

Scytonema

millei

Large scale


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

Appendix 7: Diameter of inhibition zone of extracts from three cyanobacterial strains in large scale

cultivation in comparison with batch cultivation against test organisms in vitro

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Appendix 8a: 1H NMR of fraction CJFII-4 of Calothrix javanica

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Appendix 8b: 1H NMR of fraction CJFII-4 of Calothrix javanica

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Appendix 9a: 1H NMR of fraction AF6 of Anabaena sp.

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Appendix 9b: 1H NMR of fraction AF6 of Anabaena sp.

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Appendix 10a: 1H NMR of fraction F8-3-2 of Lyngbya majuscula

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Appendix 10b: 1H NMR of fraction F8-3-2 of Lyngbya majuscula

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Appendix 11a: 1H NMR of fraction F10-3 of Lyngbya majuscula

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Appendix 11b: 13C NMR of fraction F10-3 of Lyngbya majuscula

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Appendix 12a: 1H NMR of fraction F10-5 of Lyngbya majuscula

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Appendix 12b: 1H NMR of fraction F10-5 of Lyngbya majuscula

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C14

C16

C18

C20

Appendix 13: GC-MS spectrum for the fatty acids of n-hexane extract of Lyngbya majuscula

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Appendix 14a: 1H NMR data of fraction CJFII-4 of Calothrix javanica compared with fraction FSO3 of Scytonema ocellatum

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Appendix 14b:

1H NMR data of fraction CJFII-4 of Calothrix javanica compared with fraction FSO3 of Scytonema ocellatum

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Appendix 14c: MS of methanol extract of Calothrix javanica compared with MS of methanol extract of Scytonema ocellatum

Chemical and Biological Investigations of Vietnamese ... · Lyngbya majuscula in Khanh Hoa province...

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