| Summary: | 碩士 === 國立臺灣大學 === 漁業科學研究所 === 91 === It has been known that dinoflagellate are rich in various bioactive compounds. Among our cultured dinflagellate clones we paid our attention to the strain of Amphidinium carterae AC09, due to its toxicities to mice and brine shrimp larvae. AC09 was cultured under controlled conditions for consistent results. Ethanol extract of AC09 Cell biomass was partitioned to CHCl3 and BuOH further separation. Directed by cell line cytotoxicity, both extracts went through a series of column chromatographic separations, in an order of LH-20 gel permeation, Si flash column, and C18 reversed-phase high pressure column, and 3 pheophorbides and 2 polyketides were obtained in pure form to show bioactivity.
In a batch of accumulated culture of 1000L of AC09, 3.6x1011 cells were subject for separation and 10 mg pheophorbide a, 25 mg pheophorbide a methyl ester and 6.5 mg pyropheophorbide a were isolated. Structures of these three known pheoporbides were confirmed through intensive high resolution FAB/MS and 1D and 2D NMR spectroscopic studies and a comparison with the published data. The difference of structure between these pheophorbides lies in the existence or not of —COOCH3 at C-7b and C-10. Pyropheophorbide a has only one carboxylic acid at C-7b, and pheophorbide a has two carboxylic acids, with the additional one at C-10 and forms methyl ester, while pheophorbide a methyl ester has two carboxylic acids, and both methylated. Nuclear magnetic resonance spectra of these three peophorbides are alike in most of the signals except the additional carbonyl and oxymethylcarbons. Two polyketides, compound 4 (28 mg) and compound 5 (10 mg) were also isolated from the same batch. Their molecular weight are 1402 and 1024 respectively. The conjugated diene at one end and the two 6-membered cyclic ethers of the long chain molecule of compound 4 indicated it an analogue of amphidinol. Compound 5 was characterized by its 9-membered cyclic ether and an epoxide. Although complete structures of these two polyketides are yet elucidated, their identified partial structures showed they are two new compounds.
All three peophorbides and compound 4 showed cytotoxicity, an ED50 between 1.21 to 5.53 g/ml, against KB and Hela cancer cell lines, but not toxic to HONE-1 and NUGC-3 cell lines at the same level.
It was found that the contents of pyropheoporbide a varied in different species of dinoflagellate growing in the same culture condition. Species of Gymnodinium and Amphidinium contains up to 1g per g dry cell. This is the first report to reveal such a high amount in living cells of normal growth. Coolia monotis contained 0.1g per g dry cell, and species of Prorocentrum and Gyrodinium contained less or nothing. It was also found the unit cell content of pyropheophorbide a increased with the log phase growth of AC09, suggesting the compound was not a degraded chlorophyll a from aged cells.
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