Chemical and biological investigations of Vietnamese cyanobacteria [Elektronische Ressource] / vorgeelgt von Le Thi Anh Tuyet

De
Ernst Moritz Arndt-Universität Greifswald Mathematisch-Naturwissenschaftliche Fakultät Chemical and Biological Investigations of Vietnamese Cyanobacteria Inauguraldissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Le Thi Anh Tuyet geboren am 19.05.1973 in Thanh Hoa, Vietnam Greifswald, Juli 2010 Dekan: Prof. Dr. Klaus Fesser……………………………………………… 1. Gutachter: Prof. Dr. Johannes F. Imhoff 2. Gutachter: PD. Dr. Sabine Mundt Tag der Promotion:……16.09.2010…………………………………………………………… Hiermit erkläre ich, daß diese Arbeit bisher von mir weder an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald noch einer anderen wissenschaftlichen Einrichtung zum Zwecke der Promotion eingereicht wurde. Ferner erkläre ich, daß ich diese Arbeit selbständig verfaßt und keine anderen als die darin angegebenen Hilfsmittel benutzt habe.
Publié le : vendredi 1 janvier 2010
Lecture(s) : 186
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Source : D-NB.INFO/1009144715/34
Nombre de pages : 212
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Ernst Moritz Arndt-Universität Greifswald
Mathematisch-Naturwissenschaftliche Fakultät




Chemical and Biological
Investigations of Vietnamese
Cyanobacteria

Inauguraldissertation
zur
Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)
an der Mathematisch-Naturwissenschaftlichen Fakultät
der
Ernst-Moritz-Arndt-Universität Greifswald

vorgelegt von Le Thi Anh Tuyet
geboren am 19.05.1973
in Thanh Hoa, Vietnam







Greifswald, Juli 2010
























Dekan: Prof. Dr. Klaus Fesser………………………………………………

1. Gutachter:
Prof. Dr. Johannes F. Imhoff

2. Gutachter:
PD. Dr. Sabine Mundt

Tag der
Promotion:……16.09.2010……………………………………………………………






Hiermit erkläre ich, daß diese Arbeit bisher von mir weder an der
Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität
Greifswald noch einer anderen wissenschaftlichen Einrichtung zum Zwecke der
Promotion eingereicht wurde.

Ferner erkläre ich, daß ich diese Arbeit selbständig verfaßt und keine anderen
als die darin angegebenen Hilfsmittel benutzt habe.




Greifswald, den 19 Juli 2007 Unterschrift




Le Thi Anh Tuyet

a


.....................................................................................................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


...........................................................................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

..............................................................................................................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 WF (compound 1) ....................................... 68 1-3
3.2.1.4.2 Structure elucidation of fraction WF ............................................................. 69 1-5
3.2.1.4.3 Structure elucidation of fraction WF (compound 4)........................................ 73 1-6
3.2.1.4.4 Identification of compounds in fraction WF .................................................. 74 1-8
3.2.2 Chemical investigation of the active ethyl acetate extract resulting from cultivation medium of
Westiellopsis sp. VN .............................................................................................................. 76
3.2.3 Culture optimization of Westiellopsis sp.VN .................................................................. 79
3.2.3.1 Nitrogen deficiency............................................................................................... 79
3.2.3.2 The effect of incubation time on biomass production and antibacterial production........ 81
3.3 Chemical investigation of Calothrix javanica............................................................ 82
3.3.1 Fractionation of methanol extract from biomass by RPC chromatography........................ 82 18
3.3.2 Purification of fraction CJF by semi-preparative reversed-phase HPLC .......................... 83 II

3.3.3 Structure elucidation of fraction CJF (compound 7)....................................................... 85 II-4
3.4 Chemical investigation of Scytonema ocellatum...................................................... 90
3.4.1 Fractionation of methanol extract obtained from biomass by RP C column chromatography18
............................................................................................................................................. 90
3.4.2 Separation of fraction SOF using silica gel column ....................................................... 91 II
3.4.3 Purification of the pooled fractions using reversed-phase RP HPLC................................. 91
3.4.4 Structure elucidation of fraction FSO ........................................................................... 93 3
3.5 Chemical investigation of Anabaena sp. .................................................................... 94
3.5.1 Purification of EtOAc extract using semi-preparative reversed-phase HPLC ..................... 94
3.5.2 Structure elucidation of fraction AF (compound 8) ........................................................ 96 6
3. 6 Chemical investigation of Nostoc sp. ........................................................................ 98
3.6.1 Fractionation of methanol extract obtained from biomass using silica gel column.............. 98
3.6.2 Separation of the acitve fraction NF using RP C column chromatography.................... 98 IV 18
3.6.3 Purification of the active fraction NF using semi-preparative reversed-phase HPLC........ 99 IV-1
3.6.4 Structure elucidation of fraction NsF .......................................................................... 100 2
3.7 Chemical investigation of Lyngbya majuscula ........................................................ 100
3.7.1 Isolation of cytotoxic compounds of methanol extract obtained from biomass according to
method 1 ............................................................................................................................. 101
3.7.1.1 Fractionation of methanol extract by silica gel column chromatography .................. 101
3.7.1.2 Separation of fraction F by silica gel column chromatography............................... 102 8
3.7.1.3 Purification of fraction F by preparative TLC ..................................................... 103 8-3
3.7.1.4 Structure elucidation of fraction F (compound 9).............................................. 103 8-3-2
3.7.2 Isolation of cytotoxic compounds of methanol extract obtained from biomass according to
method 2 ............................................................................................................................. 105
3.7.2.1 Separation of methanol extract by silica gel column chromatography ...................... 105
3.7.2.2 Purification of fraction F using semi-preparative reversed-phase HPLC ................ 107 10
3.7.2.3 Structure elucidation of fractions F and F ..................................................... 108 10-3 10-5
3.7.2.3.1 Structure elucidation of fraction F (compound 10)...................................... 108 10-3
3.7.2.3.2 Structure elucidation of fraction F (compound 11)........................................ 110 10-5
3.7.3 Fatty acid analysis...................................................................................................... 112
!......................................................................................................113
4.1 Screening of crude extracts for antibacterial activity ............................................. 113
4.1.1 Selection of antibiotic screening and cyanobacterial strains ........................................... 113
4.1.2 Cultivation and extraction........................................................................................... 115
4.1.3 Antibacterial activity.................................................................................................... 116
4.2 Chemical investigation and culture optimization of Westiellopsis sp. VN............ 117
4.2.1 Selection of Westiellopsis sp.VN................................................................................. 117
4.2.2 Active intracellular metabolites of Westiellopsis sp.VN strain.......................................... 118
4.2.3 Chemical composition of volatile extracellular compounds of Westiellopsis sp.VN strain .. 122
4.2.4 Cultivation optimization of Westiellopsis sp.VN strain.................................................... 123
4.2.5 Effect of incubation time on biomass production and antimicrobial compound accumulation
of Westiellopsis sp.VN strain ................................................................................................ 128
4.3 Chemical investigation of Calothrix javanica and Scytonema ocellatum ............... 130
4.3.1 Selection of Calothrix javanica ................................................................................... 130
4.3.2 Selection of Scytonema ocellatum ................................................................................. 130
4.3.3 New cyclic peptide of Calothrix javanica and Scytonema ocellatum............................... 130
4.4 Chemical investigation of Anabaena sp. .................................................................... 136
4.4.1 Selection of Anabaena sp............................................................................................ 136
4.4.2 Active compound of Anabaena sp. .............................................................................. 137

4.5 Chemical investigation of Nostoc sp. ......................................................................... 138
4.5.1 Selection of Nostoc sp. ................................................................................................. 138
4.5.2 Active compounds of Nostoc sp. ................................................................................... 139
4.6 Chemical investigation of the marine Lyngbya majuscula....................................... 139
4.6.1 Selection of the marine cyanobacterium L. majuscula collected in Vietnam .................... 139
4.6.2 Cytotoxic compounds of the marine cyanobacterium Lyngbya majuscula collected in
Vietnam .............................................................................................................................. 140
4.7 Conclusion ................................................................................................................... 142
"..............................................................................................................143

#.........................................................................................................143

$%&
.......................................................................................168
'
..........................................................................................171

#()


#'
.173
((* ...............................................................................................................174

+#+
#+,- Crytophycin 1 and crytophycin 8................................................................ 11
#+,- Norharmane from cyanobacteria................................................................. 12
#+, - Cryptophycin 1 and 52, potent antitumor agents from cyanobacteria........ 14
#+,!- Prominent anticancer marine cyanobacterial secondary metabolites and
synthetic analogues ........................................................................................................... 15
#+,.- Borophycin from cyanobacteria.................................................................. 17
#+,/- Nostocarboline from Nostoc 78-12A.......................................................... 21
#+,- Map of Vietnam with the locality of 12 cyanobacterial strains in Dak
Lak province ..................................................................................................................... 23
#+,- Morphology of 12 cyanobacterial strains ................................................... 28
#+, - Map of Vietnam with the collection area and collection place of
Lyngbya majuscula in Khanh Hoa province indicated ..................................................... 29
collection place of Lyngbya majuscula in Khanh Hoa province indicated ...................... 29
#+,!- Natural habit of Lyngbya majuscula and microscopic view of filament
of Lyngbya majuscula....................................................................................................... 31
#+,.- Agilent 6890N gas chromatograph and mass selective detector ............... 37
#+,/- Cultivation of cyanobacteria ....................................................................... 39
#+ ,- Antibacterial activity of cyanobacterial extracts against the Gram
positive bacterium Bacillus subtilis ATCC 6501.............................................................. 62
#+ ,- Antibacterial activity of cyanobacterial extracts against the Gram positive
bacterium Staphylococcus aureus ATCC 6538 ................................................................ 62
#+ , - Antibacterial activity of cyanobacterial extracts against the Gram
negative bacterium Escherichia coli ATCC 11229 .......................................................... 63
#+ ,!- Antibacterial activity of cyanobacterial extracts against the Gram
negative bacterium Pseudomonas aeruginosa ATCC 22853 ........................................... 64
#+ ,.- Semi-preparative RP HPLC chromatogram of WF .................................... 67 1
#+ ,/- Ambiguine D isonitrile ............................................................................... 68
#+ ,0- UV and ESI-MS of fraction WF ............................................................. 68 1-3
#+ ,1- UV and ESI-MS of compounds of fraction WF ..................................... 69 1- 5

#+ ,2- MS data of peak 1 of fraction WF ........................................................... 70 1-5
#+ ,3- Ambiguine C isonitrile.............................................................................. 70
#+ ,- MS data of peak 2 of fraction WF ........................................................ 71 1-5
#+ ,- Fischerellin A............................................................................................ 71
#+ , - MS data of peak 3 of fraction WF ........................................................ 72 1-5
#+ ,!- Ambiguine B isonitrile.............................................................................. 73
#+ ,.- UV and ESI-MS of fraction WF ........................................................... 73 1-6
#+ ,/- ESI-MS of compounds of fraction WF ................................................ 74 1- 8
#+ ,0- ESI-MS of peak 2 of fraction WF ........................................................ 75 1- 8

#+ ,1- ESI-MS of peak 3 of fraction WF ......................................................... 76 1- 8
#+ ,2- Bioautographic assay of ethyl acetate extract from cultivation medium
of Westiellopsis sp.VN against S.aureu ............................................................................ 77
#+ ,3- Analytical HPLC of MeOH fraction of EtOAc extract of cultivation
medium of Westiellopsis sp. VN ...................................................................................... 77
#+ ,- Fermenters for cultivation of Westiellopsis sp.VN ................................ 79
#+ ,- Agar diffusion test of extracts prepared from biomass and cultivation
medium of Westiellopsis sp.VN grown in BG-11 media ................................................. 80

#+ , a- The effect of incubation time on dry weight and antibiotic production
of Westiellopsis sp.VN...................................................................................................... 81
#+ ,

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