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研究生:汪世崇
研究生(外文):Shih-Chung Wang
論文名稱:鴨蛋殼中抗氧化與抗腫瘤物質之萃取與分析
論文名稱(外文):The analysis of the antioxidant and anti-tumor complex in the extract of duck eggshell
指導教授:林佳靜林佳靜引用關係
指導教授(外文):Chai-Ching Lin
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:動物科技學系碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:107
中文關鍵詞:蛋殼抗氧化抗腫瘤
外文關鍵詞:eggshellantioxidantanti-tumor
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台灣養鴨產業發達,其中鴨蛋產品主要以加工方式呈現,因而產生大量的蛋殼廢棄物,目前國內廢棄鴨蛋殼多以焚化方式處理,而雞蛋殼的產量較大,多半以壓碎製成堆肥使用。本研究期望於廢棄蛋殼中開發出具有高經濟價值的保健成分,以提高畜產加工業的經濟效益。
本論文目標有二:由青蛋殼中萃取出具有抗氧化活性的成分物質,已知青蛋殼中含有膽綠素,而膽綠素已被證實具有抗氧化、抗突變與抗發炎等生理活性,本試驗預期由靑蛋殼中開發出具有活性的膽綠素或其他有效抗氧化成分。再者,本研究對民間流傳的青殼蛋具有抗腫瘤之說感到興趣,期望能由青蛋殼萃取物中開發出具抗癌新藥之物質。
在本試驗過程中,已建立一有效的萃取流程,不僅可分離出含有抗氧化活性的蛋殼抽提液,而且於加工後具有抗肝臟腫瘤的功能。進行靑蛋殼中有效成分之抽提,並以光電比色計吸光測試與 HPLC 分析,證實其中含有膽綠素成分。繼之,將蛋殼抽提液進行三種抗氧化能力測試:清除 DPPH 能力、SOD-like 活性與抑制脂質過氧化物能力等。結果顯示:青蛋殼抽提液具有明顯抑制脂質過氧化物的能力,而白蛋殼抽提液則否,推論為膽綠素成分所致;在 SOD-like 活性測試中,靑蛋殼與白蛋殼抽提液兩者皆具有活性,故推測 SOD-like 的活性是由蛋殼抽提液中非膽綠素的其他抗氧化成份所影響。至於清除 DPPH 的能力,則青、白蛋殼抽提液兩者皆無。
再者,進一步將蛋殼抽提液進行加工處理,發現活化的蛋殼萃取物對於肝臟腫瘤細胞株 HepG2 與 huh 7 均具顯著性的抑制效果,其中又以 huh 7之感受性最強。然而,對於鼠胚的初代纖維母細胞 (MEF) 與人類幹細胞株 (HBM01) 的抑制作用則顯著較低 (p < 0.001)。當更深入探討抗腫瘤的作用機制時,發現活化的蛋殼萃取物會導致肝臟腫瘤細胞 huh 7 的粒線體活性降低,並且提升細胞內Caspase-9 與 Caspase-3 的活性,進而導致 huh 7 細胞進行自體凋亡 (apoptosis)。總結上述:本論文為首次發表蛋殼萃取物中具有抗氧化與抗腫瘤之研究,預期將來可由蛋殼萃取物中開發出具有高經濟價值的保健成分或抗癌新藥。
The duck industry is vigorously developed in Taiwan. However, the by-product of eggshell is wasted and may cause the environmental pollution. In order to re-utilize the waste to raise the economical value, we tried to extract the high valuable component from the eggshell. Based on the traditional Chinese medicine, the blue egg shell has been believed to be an important medicine-inducer. Some reports indicated that the pigment of blue egg was biliverdin. Biliverdin has been proven to be an important antioxidant, anti-mutagen and immunoregulator in physiology. We considered that there should be some antioxidants existed in blue eggshell, especially biliverdin.
There were two major objectives in the present study, which were to extract the antioxidative activities of biliverdin and other substrates from blue eggshell, and to assay the anti-tumor ability of the extract. In this study, we obtain the crude extract of eggshell. The biliverdin component in the crude extract was detected by spectrophotometer and by HPLC. Also, the crude extract of blue eggshell has been proven to have the antioxidative ability to prevent the lipid peroxideation, but that of white eggshell didn’t. Moreover, the crude extract was concentrated by vacuum and then heated to be the active extract. The active extract after heating process has been found to strongly inhibit the proliferation and induce the apoptosis of liver tumor cell lines, huh 7 and Hpe G2, especially huh 7. However, the cytotoxicity was much lower to the human bone marrow cell (HBM 01) and the primary culture of mouse embryonic fibroblasts. Also, we proved that the heated eggshell extract could cause huh 7 apoptosis by triggering the pathway of Caspase-9 and Caspase-3 caused by the depolarization of mitochondria.
It is the first time to find the anti-tumor complex existed in eggshell in this study. It may be valuable to develop a new anti-tumor drug from the heated extract of eggshell in the future.
中文摘要 04
英文摘要 06
圖次 07
附圖次 09
附表次 10
壹、前言 11
貳、文獻檢討 13
一、蛋殼生產與再利用 13
(一)、台灣養鴨產業與青殼鴨蛋的生產 13
(二)、蛋殼之形成 15
(三)、蛋殼色素之形成 18
(四)、蛋殼顏色對經濟性狀之影響 21
(五)、蛋殼成分的再利用 24
二、膽綠素 27
(一)、膽汁色素的生理代謝 27
(二)、膽綠素的生理活性 32
參、材料與方法 44
一、試驗大綱 44
二、蛋殼有效成分抽提與萃取 46
(一)、樣品挑選 46
(二)、蛋殼前處理 46
(三)、蛋殼酸解 47
(四)、蛋殼酸解液之液相抽提 47
(五)、真空減壓濃縮 48
(六)、樣品測試 48
三、靑蛋殼抽提液之保健成分分析 49
(一)、蛋殼抽提液之可見光吸光值分析 49
(二)、蛋殼抽提液之高壓液相層析儀分析 49
四、靑蛋殼抽提液之抗氧化能力測定 50
(一)、清除 DPPH 能力之測定 50
(二)、清除超氧化陰離子能力測定 51
(三)、抗脂質氧化能力測定 52
五、活化蛋殼萃取物抗腫瘤能力之測定 53
(一)、細胞培養 54
(二)、細胞染色與型態觀測 55
(三)、細胞存活率測試 56
(四)、細胞自體凋亡測試 57
六、統計分析 61
肆、結果 62
一、靑蛋殼膽綠素萃取與分析 62
(一)、建立膽綠素測定指標 62
(二)、建立最佳之青蛋殼酸解方法 62
(三)、建立最佳之青蛋殼抽提方法 63
(四)、靑蛋殼抽提液之HPLC圖譜分析 64
二、抗氧化能力測定 65
(一)、清除DPPH能力之測定 65
(二)、清除超氧陰離子能力之測定 65
(三)、清除脂質過氧化物能力之測定 65
三、抗腫瘤能力之測定 67
(一)、活化蛋殼萃取物抑制腫瘤細胞能力之研究 69
(二)、活化蛋殼萃取物影響細胞自體凋亡之測定 70
伍、討論 72
陸、結論 80
柒、參考文獻 81
捌、附錄 104
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