細基龍鬚菜萃取產物之免疫與抗癌活性分析

Immunological and antitumor activity assays for the marine red alga Gracilaria tenuistipitata.

任雅罄(Ya-Ching Jen)

指導教授 ： 黃穰

國立臺灣大學/理學院/海洋研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10323

摘要

台灣細基龍鬚菜為菊花心種，而本論文實驗針對細基龍鬚菜進行多醣萃取與PBS蛋白質液萃取、分離與純化。並將所得之萃取液進行細胞培養實驗，藉以瞭解龍鬚菜萃取物質在活化免疫細胞上扮演的角色與其是否具有抗腫瘤活性。結果顯示龍鬚菜以多種方式萃取，均能獲得促進免疫細胞增殖與抑制乳癌、肝癌及胰臟癌細胞的活性物質。龍鬚菜經由熱水萃取與異丙醇沉澱之多醣體均能促進巨噬細胞增殖。異丙醇沉澱之多醣體效果較佳，在濃度glucose 15.6μg/ml時，提高巨噬細胞增殖達到最大48.7%。龍鬚菜以熱水萃取，加熱到60℃ 180分鐘所得之萃取液對乳癌、肝癌、胰臟癌細胞均有顯著抑制效果，癌細胞存活率降為46%~59%。龍鬚菜以PBS萃取，並經過硫酸銨沉澱、離子交換層析與膠體過濾純化，可純化出龍鬚菜免疫活性蛋白(GTP)。GTP濃度為0.05~0.1μg/ml時，對巨噬細胞有顯著的刺激增殖效果，可提高達79~83%。GTP濃度為25.6μg/ml時，對乳癌、肝癌、胰臟癌細胞的抑制效果最好，細胞存活率分別下降為19.1%、23.7%及25.9%。證實GTP為有效抑制癌細胞之活性物質。SDS-PAGE電泳分析結果顯示此純化蛋白質為雙聚體蛋白，於20kDa與35kDa各有一蛋白帶；蛋白質帶有藻紅素，外觀呈現粉紅螢光，於498 nm、546 nm及565 nm具有吸收高峰，為藻紅蛋白。血球凝集測試顯示此蛋白對人類A、B、O及AB四種血型之紅血球皆無凝集活性。GTP對酸鹼值變化較不具有耐受性，僅能在弱酸~弱鹼環境下維持高免疫活性。GTP在80℃下加熱30分鐘，免疫活性下降至62%，表示GTP不能耐受較高溫度，在高溫下容易失去活性。本論文實驗結果證實細基龍鬚菜具有免疫活性及抗腫瘤蛋白質與多醣類，值得進一步研究以期待未來可應用在保健及醫藥上。

關鍵字

細基龍鬚菜 ； 海藻

並列摘要

The marine red alga Gracilaria tenuistipitata (Gracilariaceae) was assayed in vitro for proliferation stimulation in murine macrophage and antitumor activity against MDAMB-231, MIA Paca-2 and Hep G2. The bioactive substances were extracted in phosphate buffer saline and hot water, followed by purification and biochemical characterization. It appeared that both saline and hot-water extracts could significantly stimulate macrophage proliferation and, on the contrary, inhibit tumor cell growth. However, the isopropanol -precipitated polysaccharide had greater stimulation activity than hot-water extract, with maximal activity, i.e. proliferation increased by 48.7%, at concentration of 15.6 μg/ml. The extract at 60℃ for 3 hr inhibited markedly the growth of tumor cells used, i.e. cells survivability decreased by 46~59% as compared with the control. A novel protein assigned as GTP was obtained from the reported alga after being extracted in saline, then precipitated by saturated ammonium sulfate, and finally isolated by ion-exchange and gel filtration column chromatography. The isolated protein was a dimmer in structure, having two subunits of 20 and 35 kDa as explained on SDS-PAGE and gel filtration and absorbance at 498,546 and 565 nm. As shown in the results, macrophage proliferation increased by 79~83% and, on the contrary, tumor cell survivability reduced by 74.1~80.9% in the presence of GTP at concentration as low as 0.05~0.1 μg/ml. However, it did not agglutinate human A, B, O and AB erythrocytes. Additionally, GTP was not tolerable to high temperature, as well as acidic and alkaline solutions; when the stimulation activity reduced greatly while subjected to 80 ℃ and or pH<6.0 or >8.0. The above findings suggested that Gracilaria tenuistipitata is a promising source of bioactive polysaccharides and proteins in use for health care and medicine.

並列關鍵字

Gracilaria tenuistipitata ； alga

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