臺灣博碩士論文加值系統

已知渦鞭毛藻富含多類活性物質，針對實驗室已建立之具小白鼠與豐年蝦幼苗生物毒性的底棲性渦鞭毛藻Amphidinium carterae AC09藻株，經由人工控制下之生長環境大量培養，及癌細胞株毒性分析的輔助，從藻細胞的氯仿與正丁醇的萃取液中，利用LH-20膠濾層析管柱、矽膠快速層析管柱與C18逆相高效液相層析管柱等一系列依序的層析分離模式，純化出三個已知的葉綠素降解物pheophorbides及兩個化合物。
從1000L培養液的3.6x1011 AC09細胞中獲得有pheophorbide a (10mg)、pheophorbide a methyl ester (25mg)和pyropheophorbide a (6.5mg)。這些pheophorbides的化學結構鑑定，經由高解析質譜MS和一維及二維核磁共振光譜NMR的詳細解析與文獻中相關光譜資料的比對而確認。這三個pheophorbide在化學分子上的區別主要在於C-7b和C-10上有無 -COOCH3 的取代，而在碳原子數上及氫譜上有其特殊訊號，其他在核磁共振光譜上則非常的近似。pyropheophorbide a僅有一個碳酸基在C-7b上，pheophorbide a除C-7b的碳酸基外，於C-10上另有一甲基酯化的碳酸根，而pheophorbide a methyl ester的兩碳酸根均已甲酯化。另外純化出兩長鏈polyketides，化合物4 (28mg)和5 (10mg)，分子量分別為1402與1024。化合物4長鏈的一端具有的共軛烯和兩六員環醚，與已知的amphidinol類化合物相似，但其他結構部分證實4為新化合物；化合物5則具有一個三員環醚 (epoxide) 和一個九員環醚，是為其特徵，也未曾在文獻上發現相同的化合物，這兩polyketide化合物分子大，類似官能基的氫譜訊號重疊嚴重，以目前的光譜資料尚無法解出完整的結構。
細胞毒性分析顯示三個pheophorbides和化合物4在KB和Hela癌細胞株上都具有顯著細胞毒性，ED50值在1.21-5.53 g/ml之間。在HONE-1與NUGC-3癌細胞株上則不顯著或無毒性。
從培養中的一些渦鞭毛藻株的pyropheophorbide a含量分析中，發現Gymnodinium及Amphidinium屬藻株含較高量pyropheophorbide a，而Coolia monotis 含量較少，Prorocentrum與Gyrodinium則含微量或不存在，此為首次在正常培養中的藻細胞內，發現到有較高含量pyropheophorbide a的存在，每g乾藻含量為0.1~1 g。在AC09藻株培養中也發現到單位細胞中pyropheophorbide a的含量隨著細胞的對數生長而增加，顯示化合物並非一般所認知，由老化細胞中的葉綠素降解而來。

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 1g per g dry cell. This is the first report to reveal such a high amount in living cells of normal growth. Coolia monotis contained 0.1g 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.

摘要…………………………………………………………...ii
Abstract……………………………………………………….iv
壹、前言 1
貳、材料與方法 7
一、藥品及重要儀器 7
二、藻體的來源與培養 8
三、活性物質的萃取及純化 8
四、光譜分析與結構鑑定 11
五、pyropheophorbide a 的分析 12
六、癌細胞毒殺活性試驗 13
參、結果與討論 15
一、化合物結構證明 15
二、細胞毒殺活性結果 71
三、pyropheophorbide a分析結果 73
肆、參考文獻 77
附錄一 84

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