臺灣博碩士論文加值系統

雨生紅球藻(Haematococcus pluvialis)在生長環境受脅迫下能大量合成具有抗氧化能力之蝦紅素，紅球藻會視其生長環境如：營養源、光照、pH、溫度等因素的改變而合成蝦紅素來保護自己免受環境傷害 。 當雨生紅球藻暴露在不利的生長條件或環境壓力下，如營養源(氮或磷)缺乏、鹽度過高或強光照射下，會刺激綠色細胞開始轉變成紅色細胞，進行二級代謝物蝦紅素的累積。諸多因素，都會影響到雨生紅球藻累積蝦紅素的含量 。 在各項研究中指出，蝦紅素具商業開發和極高的營養價值。市售蝦紅素主要分為化學合成及生物萃取二種，生物萃取的主要來源為雨生紅球藻 (Haematococcus pluvialis) 、法夫酵母菌 (Phaffia rhodozyma)及磷蝦油(Krill oil)等。其中以雨生紅球藻所獲得的蝦紅素含量最高。
本研究使用兩階段培養雨生紅球藻，第一階段以添加 0.5%的 S 醱酵液之培養基配合白、紅、藍光培養至生長對數期，第二階段照射紫外光刺激二級代謝物產生。比較不同條件培養下其萃取物的抗氧化差異、進行 HPLC 檢測不同 LED
光培養，進而造成的化合物變化及對正常細胞及腫瘤細胞的存活率測試。
測試結果 5000Lux 培養紅球藻 ， 以 95%乙醇萃取紅球藻萃取物進行 ABTS、DPPH 自由基清除率，依序為藍光培養組別(BA60)>白光培養組別(WA60) >紅光培養組別(RA60)；三種 LED 光培養雨生紅球藻之萃取物進行 HPLC 分析結果蝦紅素異構物面積依序為白光培養組別(WA60)>紅光培養組別(RA60)>藍光培養組別(BA60) 。 白光、藍光、紅光培養雨生紅球藻萃取物濃度為 25µg/mL 對 CHO-K1細胞作用具有使正常細胞增生的作用；以 100µg/mL 對 MDA-MB231 細胞作用24 小時存活率 WA60 為 63% 、RA60 為 58%、BA60 為 62%，而經過48小時後存活率沒有隨時間下降。

Astaxanthin is a carotenoid which is known as “the king of antioxidant”,
Astaxanthin exhibits high antioxidant activity superior to most of the hydrophobic
antioxidants, showing pharmaceutical and nutraceutical value to be developed in
commercial production, mainly in aquatic plants and animals. When the red cyst
exposed to adverse growth conditions or environmental stress, such as the lack of
nutrient sources (nitrogen or phosphorus), salt is too high or strong light irradiation
will stimulate the green cells begin to turn into red cells and begin accumulation of
secondary metabolites-astaxanthin.
Therefore, this research aims to increase astaxanthin and the biomass of the
microalga by using different combination of illumination on with 95% EtOH extracts ,
we compare the result of different concentration level of antioxidant and survival rate
of normal cells and tumor cells.
The test results of ABTS, DPPH show that the radical scavenging rate of extract,
extracted by 95% EtOH which composed of Haematococcus pluvialis cultured under
5000 Lux, obtained the following performance order from highest to lowest: Blue
group (BA60)> White group (WA60)> Red group (RA60), respectively. The HPLC
indicates that the extracts of Haematococcus pluvialis cultured under three kinds of
LED light obtain the following results, showed in the order of the area of astaxanthin
isoforms from greatest to least: White light group (WA60), Red light group (RA60),
Blue light group (BA60), respectively. Cultured under White light, Blue light and Red
light, the Haematococcus pluvialis extracted by 95% EtOH, with a concentration of
25μg/mL, can induce survival rate in CHO-K1.With 100μg/mL on MDA-MB231, the
survival rate after 24 hours for the three groups WA60, BA60, RA60 displayed the following results: 63%, 62%, 58%, respectively. As a result, the survival rate did not decrease over time.
These results demonstrate that Haematococcus pluvialis cultured under red and
blue light with UV-irradiated can not only shorten the time to generate secondary
metabolites, but also increase the antioxidant properties and antitumor activities.

目次................................................................................................................................ 1
圖目錄............................................................................................................................ 3
表目錄............................................................................................................................ 4
中文摘要........................................................................................................................ 5
Abstract .......................................................................................................................... 6
第一章 緒論.................................................................................................................. 8
第二章 文獻回顧........................................................................................................ 10
2.1 雨生紅球藻(Haematococcus pluvialis)簡介........................................................ 10
2.2 雨生紅球藻中的蝦紅素應用................................................................................ 12
2.3 雨生紅球藻之生長影響參數................................................................................ 14
2.4 雨生紅球藻生產二級代謝物之影響參數............................................................ 17
2.5 自由基與氧化壓力之介紹.................................................................................... 21
第三章 研究動機與目的............................................................................................ 23
3.1 研究目的................................................................................................................ 23
3.2 實驗架構............................................................................................................... 24
第四章 實驗材料與方法............................................................................................ 25
4.1 實驗材料............................................................................................................... 25
4.3 實驗材料、儀器設備........................................................................................... 27
4.4 實驗方法............................................................................................................... 28
4.5 測量方式............................................................................................................... 29
4.6 細胞實驗............................................................................................................... 30
4.7 抗氧化能力評估................................................................................................... 33
4.7.1 清除 ABTS 自由基(2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) ...33
4.7.1.1 清除 ABTS 自由基能力原理 ....................................................................... 33
4.7.1.2 ABTS 藥品配置及實驗步驟 ....................................................................... 34
4.7.2 清除 1,1-diphenyl-2-picryl hydrazyl（DPPH） ................................................ 35
4.7.2.1 DPPH 清除原理 ............................................................................................. 35
4.6.2.2 DPPH 藥品配置 ............................................................................................. 35
4.7.2.3 清除 DPPH 實驗步驟 .................................................................................. 36
4.8.1 HPLC 偵測波長及動相條件的選擇 ................................................................. 37
第五章 結果與討論.................................................................................................... 38
5.1 不同培養參數對雨生紅球藻生長情形之探討.................................................... 38
5.1.1 不同光照條件培養雨生紅球藻生長之影響..................................................... 38
5.1.2 不同培養基對雨生紅球藻生長之探討............................................................. 42
5.2 不同培養條件對雨生紅球藻萃取液抗氧化能力之影響................................... 44
5.2.1 不同光照條件對雨生紅球藻萃取液之清除 ABTS 自由基能力測定 ........... 44
5.2.2 不同光照條件培養雨生紅球藻萃取液清除 DPPH 自由基能力測定 ........... 46
5.3 以不同光照條件培養雨生紅球藻之酒萃物對倉鼠卵巢細胞(CHO-K1)及人類
乳癌細胞(MDA-MB-231)生長之影響 ....................................................................... 48
5.3.1 以不同光照條件培養雨生紅球藻之酒萃物對倉鼠卵巢細胞(CHO-K1)生長
之影響.......................................................................................................................... 48
5.3.2 以不同光照條件培養雨生紅球藻之酒萃物對人類乳癌細胞(MDA-MB-231)
生長之影響.................................................................................................................. 51
5.4 經由不同光照條件培養雨生紅球藻所得萃取物進行 HPLC 分析 .................. 53
5.5 結論........................................................................................................................ 57
附錄.............................................................................................................................. 59
第七章 參考文獻........................................................................................................ 61

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