頭髮菜porphyra-334離子特徵及萃取之研究

The ionic characteristics and extraction of porphyra-334 from Bangia atropurpurea

莊立凡(Li-Fan Chuang)

指導教授 ： 周宏農

國立臺灣大學/生命科學院/漁業科學研究所/博士(2016年)

https://doi.org/10.6342/NTU201601727

摘要

類蕈孢素胺基酸(mycosporine-like amino acids, MAAs)存在於研究對象的頭髮菜Bangia atropurpurea中，可能為已知生物中含量最高，故選定頭髮菜及其所含最主要的MAA, porphyra-334 (p-334)為研究的對象。以2008年購自莆田存放於冰箱的頭髮菜為材料，經水萃取後，粗萃物通過陽離子交換樹酯AG® 50W-X8以行吸附，再以0.2 % acetic acid沖提脫附後可獲得63 %純度的p-334，最終經C-18高效層析管柱純化。透過1H-NMR及13C-NMR測定p-334在不同酸鹼度下之化學位移變化。其中H2-9, H-11, H-13及C-1, C-2, C-3的化學位移隨著pH的逐漸增加而有顯著的向高場移動的趨勢，從而推斷p-334在1. pD 0.9時，帶兩個正電荷，2. pD 3時，一個正電荷及3. pD大於4時零淨電荷。此推論同時可驗證樹酯脫附p-334之條件。

產業化製程當中以−萃取最為重要。從市售烘乾後的頭髮菜為材料，可由沖水滲漉方式快速萃得70 % (以文獻中所標示25% methanol在45oC經過2小時的萃取，並重複三次萃取所得為生物體內MAAs 100%的含量)以上藻體所含的MAAs。另採自東北角石城海岸的同種海藻，比較藻體萃取前的處理，藉以了解該等物質存在藻體中之狀態以及能夠被萃取出來的最有效率操作。研究中設計了萃取前的處理方式，以模擬藻體在自然生長於高潮線環境下，當受到退潮後的日曬與下雨或漲潮後的復水，以及一般養殖在採收後的清洗與乾燥過程中，對藻體內MAAs溶出的影響。結果發現海水及淡水浸泡都會引起少量MAAs的滲出，但海水浸泡所引起的滲出低於淡水浸泡的損失，可說明在自然漲退潮下，生鮮藻自有保護機制以防止MAAs的滲出，而經過數日陰乾後的藻體，會增加其滲出MAAs量。推測生鮮藻體中的MAAs可能非屬游離態，在淡、海水中藻體的浸泡均不易溶出，但隨著長時間陰乾處理後可能讓MAAs轉化成易滲漉的游離態分子或離子並存在於細胞壁外。本研究為文獻中首次以NMR實驗討論p-334在不同酸鹼值下酸鹼基的離子狀態，及過去文獻中不曾有過的從頭髮菜大量製備p-334的初步探討，期望對未來p-334的產業化萃取與提純提供參考。

關鍵字

頭髮菜 ； porphyra-334 ； 類蕈孢素胺基酸 ； 萃取 ； 滴定 ； 離子狀態

並列摘要

Mycosporine-like amino acids (MAAs) exist in Bangia atropurpurea which probably has the highest content of MAAs, compared with the other studied organisms. Porphyra-334 (p-334) was found the major component of MAAs in B. atropurpurea. Hence, we choose the species as the object material for extraction and purification for p-334. The major material applied for the p-334 isolation was purchased from Putian in 2008 and has been stored in refrigeration since then. Water extract of B. atropurpurea was forced to pass through the cationic AG® 50W-X8 resins, then eluted by 0.2% acetic acid to obtain the p-334 of 63% purity, and futher purified by a C-18 reversed phase high performance chromatography. Tedious 1H-NMR and 13C-NMR measurements of p-334 were performed following a series of pH changes from titration. It was found that a siginificant upfield change of chemical shifts at H2-9, H-11, H-13 and C-1, C-2, C-3 with the increase of pH. We assumed that the structure of p-334 was deprotonated following the increase of pH. (1) bisprotonated at pD 0.9; (2) monoprotonated at pD 3; and (3) zero net charge at a pD over 4. This result also verified the condition of p-334 desorption from resins.

Based on the literatures which indicate a complete MAAs extraction can be got after three subsequent extractions of 25% methanol at 45oC for 2 hours on any organisms. We found that more than 70% of the MAAs in the B. atropurpurea of commercial product could be easily extracted by a simple percolation. In such cases, some of the materials were treated by mimicking the natural conditions or the commercial operations on the specimen after collected from the northeast coast of Shicheng. In order to understand the chemical nature of p-334 in thalli and the optimal extraction methodology that can be applied in the commercial operation, we performed various treatments. We observed the leaching of MAAs of the dried specimen were less in sea water than in fresh water, and less in freshly prepared sample than old storage sample. From these observations, we presumed the algae inhabiting at the high tide zone developed extracellular MAAs as sunscreen to protect the cells during low tide and remained as a bound form, and gradually become dissociated form during the storage.

並列關鍵字

Bangia atropurpurea ； porphyra-334 ； mycosporine-like amino acids ； extraction ； titration ； ionic state

參考文獻

1 Takano, S., Nakanishi, A., Uemura, A. & Hirata, Y. Isolation and structure of a 334 nm UV-absorbance substance, porphyra-334 from the red alga Porphyra Tenera kjellman. Chem. Lett. 26, 419-420 (1979).

2 Chioccara, F., Misuraca, G., Novellino, E. & Prota, G. Occurrence of two new mycosporine-like aminoacids, Mytilins A snd B in the edible mussel, Mytilus galloprovincialis. Tetrahedron Lett. 34, 3181-3182 (1979).

3 Cockell, C. S. & Knowland, J. Ultraviolet radiation screening compounds. Biol. Rev. Camb. Philos. Soc. 74, 311-345 (1999).

4 Carreto, J. I., Carignan, M. O. G. & Montoya, N. A high-resolution reverse- phase liquid chromatography method for the analysis of mycosporine-like amino acids (MAAs) in marine organisms. Mar. biol. 146, 237-252 (2005).

5 Mason, D. S., Schafer, F., Shick, J. M. & Dunlap, W. C. Ultraviolet radiation absorbing mycosporine-like amino acid (MAAs) are acquired from their diet by medaka fish (Oryzias latipes) but not by SKH-1 hairless mice. Comp. Biochem. Physiol. A 120, 587-598 (1998).