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Chemical and Biological Investigations of Vietnamese cyanobacteria

  • 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, 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. Bioassay-guided fractionation of the methanol extract prepared from biomass of 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 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. 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. Bioassay-guided fractionation of the methanol extract prepared from biomass of marine cyanobacterium Lyngbya majuscula collected from Khanh Hoa province of Vietnam by various chromatographic methods (CC, PTLC, HPLC) afforded 3 cytotoxic compounds anhydrodebromoaplysiatoxin, debromoaplysiatoxin, and anhydroaplysiatoxin. Identification of these cytotoxic compounds was established by direct comparison of our spectroscopic data, including (1H, 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.
  • Chemische und biologische Untersuchungen Vietnamesischer Cyanobakterien

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Metadaten
Author: Le Thi Anh Tuyet
URN:urn:nbn:de:gbv:9-000848-1
Title Additional (English):Chemical and Biological Investigations of Vietnamese cyanobacteria
Title Additional (German):Chemische und biologische Untersuchungen Vietnamesischer Cyanobakterien
Advisor:PD Dr. Sabine Mundt, Prof. Dr. Johannes F. Imhoff
Document Type:Doctoral Thesis
Language:English
Date of Publication (online):2010/09/21
Granting Institution:Ernst-Moritz-Arndt-Universität, Mathematisch-Naturwissenschaftliche Fakultät (bis 31.05.2018)
Date of final exam:2010/09/16
Release Date:2010/09/21
Tag:Cyanobacteria
GND Keyword:Cyanonacteria
Faculties:Mathematisch-Naturwissenschaftliche Fakult√§t / Institut f√ľr Pharmazie
DDC class:500 Naturwissenschaften und Mathematik / 540 Chemie