博士 / 國立臺灣大學 / 海洋研究所 / 89 / Thirty-five clones, belonging to twelve species of dinoflagellates were planktons isolated from brackish water ponds or epiphytes isolated from the surface wash-off of Sargassum spp. These isolates were identified as species of Amphidinium carterae, A. klebsii, Gyrodinium instriatum, Coolia monotis, Gambierdiscus toxicus, Ostreopsis lenticularis, Prorocentrum lima, P. mexicanum, P. mininum and some unknown species belonging to Gonyaulax, Gymnodinium and Prorocentrum. Methanolic extracts of the filtered cells were diluted with water to make 60% methanol solution and partitioned with n-hexane and chloroform, successively. Both chloroform and aqueous fractions were screened for their toxicity in mouse (i.p. injection) and brine shrimp (solution). Methanolic extracts of 14 clones and chloroform extraxts of 6 clones showed mouse toxicity under the tested doses. It was summarized that 28.6% of the total tested fractions (20/70 fractions) and 42% of the tested clones (15/35 clones) were toxic. The toxic species were G. toxicus, O. lenticularis, P. lima, A. klebsii and some clones of A. carterae and Prorocentrum sp. Extracts from 19 clones, belonging to 7 species, showed brine shrimp toxicity (LD50<1000 ppm). Ratio of toxic clones was counted for 56% of the total tested (19/34). Among the toxic clones clone #2 and #4 of A. carterae, clone #1 of A. klebsii and clone #1 of G. instriatum were found the most toxic ones (LD50 ~100 ppm). Toxins extracted in different solvents from these algal isolates were further studied for their chemical nature.
Four macrolides were isolated from n-butanol extracts of two toxic Prorocentrum clones, P. lima (PL01) and Prorocentrum sp. (PM08). Among them 4-hydroxyprorocentrolide and 14-O-acetyl 4-hydroxyprorocentrolide were purified from the P. lima (PL01) and recognized as new compounds. In addition, a new compound, spiro-prorocentrimine and a known toxin, prorocentrolide have been isolated from Prorocentrum sp. (PM08). Spiro-prorocentrimine has similar macrocyclic structure as prorocentrolide, but a smaller macrolide moiety. The unique feature of spiro-prorocentrimine is the spiro-linked cyclic imine with the ortho, para-disubstituted 3’-cyclohexene, rather than the hexahydroisoquinoline moiety on 4-hydroxyprorocentrolide, 14-O-acetyl 4-hydroxyprorocentrolide and prorocentrolide. Spiro-prorocentrimine is the first compound of this group of marine toxins with a clear stereochemistry by x-ray crystallographic studies.
In addition to the macrolides, two okadaic acid (OA) derivatives, DTX4b and DTX6, were isolated form butanol extract of P. lima (PL01). They were identified as two new sulfated compounds with 1472 Da and 1486 Da molecular weights, respectively. OA and four OA analogues were also isolated from chloroform extracts of P. lima (strain PL01). Among them, okadaic acid, methyl ester of OA, dinophysistoxin-1 (DTX-1), and two diol esters of OA, OA-D8 and OA-D9d, were identified by NMR and LC-MS(/MS) analysis. One of the compounds, OA-D9d was recognized as a new compound in the literature. It was also noted that a comparable amount of OA and DTX-1 were in the cell-free media of P. lima cultures.
Totally, eleven compounds, including toxins and their congeners of marine Prorocentrum spp. were isolated and identified in this research. Structural elucidation of these compounds was done by intensive NMR spectroscopy that included 1H, 13C-NMR, and COSY, HSQC, HMBC and NOESY 2D techniques. In addition to the x-ray crystallography that was applied to the crystal structure of spiro-prorocentrimine, MS, UV and IR spectrum were also measured to support the structural elucidation.
| Identifer | oai:union.ndltd.org:TW/089NTU00279001 |
| Date | January 2000 |
| Creators | Chung-Kuang Lu, 盧重光 |
| Contributors | Rang Huang, Hong-Nong Chou, 黃穰, 周宏農 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 192 |
Page generated in 0.0093 seconds