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研究生中文姓名:歐倩妤
研究生英文姓名:Ou, Chein-Yu
中文論文名稱:庫達海馬乙醇萃取物之抗氧化性及抗腫瘤性評估
英文論文名稱:Evaluation on Antioxidant and Antitumor of Ethanol Extracts from Seahorse Hippocampus kuda
指導教授姓名:黃登福
口試委員中文姓名:教授︰鄭森雄
教授︰黃耀文
教授︰周薰修
業界委員︰陳石松
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學號:10432060
請選擇論文為:學術型
畢業年度:106
畢業學年度:105
學期:
語文別:中文
論文頁數:66
中文關鍵詞:庫達海馬成分分析抗氧化性抗腫瘤性
英文關鍵字:Hippocampus kudaBiochemical compositionAntioxidant activitiesCell viability
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海馬 (seahorse) 為一種硬骨海洋動物,廣泛應用於中藥材,多以泡製為藥酒之方式供人類食用。本研究以常見海馬品種之一─庫達海馬 (Hippocampus kuda, HK) 凍乾研磨為粉後,進行成分分析,得知粗蛋白 69.16% (以乾重計,DW)、粗脂肪 0.86%、灰分 28.06%、總醣類 1.00 mg/g、總酚 0.31 mg/g及總類黃酮 0.03 mg/g。再以 10 倍量 15% 及 70% 乙醇搖晃萃取 6 hr,萃取液凍乾後分別作為 15HK 及 70HK,另取部分 15HK 經蛋白酶 pronase E 處理,凍乾後作為 15PHK。其中 15HK 之蛋白質、總醣類、總酚及總類黃酮含量,測定結果分別為 29.33、13.60、9.53 mg/g DW 及未檢出 (not detected, ND);15PHK 分別為 12.13、11.75、13.28 mg/g DW 及 ND;70HK 分別為7.97、31.53、11.87 及 2.43 mg/g DW。接著以測定 2,2-diphenyl-1-picrylhydrazyl 自由基清除活性 (DPPH radical scavenging activity)、還原力 (reducing power) 及 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) 自由基清除活性 (ABTS radical scavenging activity) 等測定 15HK、15PHK 及 70HK 抗氧化活性,顯示 15PHK 有較高之抗氧化效果,3 種抗氧化活性之測定結果分別為 43.57%、0.12 Abs. 及 25.56%。接著將 15PHK 透過快速蛋白質層析儀 (fast protein liquid chromatography; FPLC) 搭配 Hiprep DEAE FF 16/10 column 進行純化,得到 5 種 fractions,再分別測定抗氧化性,結果顯示 5 種 fractions 純化後之抗氧化效果較未純化之 15PHK 差。
其次為進行抗腫瘤之細胞實驗,將 3 種抽出物針對人類子宮頸癌細胞 Hela、人類神經母瘤細胞 SH-SY5Y、人類肺癌細胞 A549 及人類黑色素瘤細胞 A375.S2 細胞進行抗腫瘤活性實驗,結果顯示以 70HK 對 A549 細胞具較佳抑制效果,進而測定此細胞之乳酸去氫酶釋放量,結果顯示隨著樣品處理濃度越高則數值隨之增加,表示細胞損傷程度越嚴重。一氧化氮 (nitric oxide, NO) 生成量測定實驗中,隨著樣品處理濃度越高則數值隨之增加,表示細胞透過發炎反應使其產生 NO。活性氧物質 (reactive oxygen species, ROS) 生成量測定,亦有隨樣品處理濃度越高,生成量上升之現象,ROS 含量過多可使細胞快速分裂,對人體造成損傷。於粒線體膜電位 (mitochondrion membrane potential, MMP) 測定中,隨著樣品處理濃度越多,則表現量越低,是由於細胞發生凋亡現象所導致。最後進行細胞凋亡壞死分析,結果可發現細胞發生凋亡現象,且隨樣品處理濃度越高,而逐漸趨向壞死之表現。
綜上所述,海馬 3 種抽出物中,抗氧化效果以 15PHK 為最佳,而 70HK 對人類肺癌細胞 A549 可有效降低細胞活性,其機制推測為透過 NO 及 ROS 生成導致細胞發生凋亡。
Seahorse is a small marine teleost animal. It has been used as traditional medicine for thousands of years in Eastern Asia because it has positive effects on human’s health. In this study, seahorses were collected from Penghu, freezing-dried and grounded into powder. The biochemical compositions of Hippocampus kuda (HK) powder were analyzed as follows: crude protein 69.16% (DW), crude lipid 0.86%, ash 28.06%, total saccharide 1.00 mg/g, total phenolic content 0.31 mg/g, and total flavonoids 0.03 mg/g. Furthermore, seahorses were derived into three parts. Two parts were extracted with 15% and 70% ethanol, and freezing-dried as 15HK and 70HK extracts. One part was extracted by 15% ethanol and further hydrolyzed by pronase E. The hydrolysate was as 15PHK hydrolysate. The protein content was 29.33, 12.13 and 7.97 mg/g DW in terms of 15HK extract, 15PHK hydrolysate and 70HKextract, respectively, and respective total saccharide content was 13.60, 11.75 and 31.53 mg/g, respective total phenolic content was 9.53, 13.28 and 11.87 mg/g, respective total flavonoid content was ND, ND and 2.43 mg/g. Three groups were also tested for their antioxidant activities including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power and 2,2'-azino-bis(3-ethylbenzothiazoline -6 -sul-
phonic acid (ABTS) radical scavenging activity The results showed that 15PHK has the potential ability of antioxidation. The results of antioxidant activities were 43.57%, 0.12 Abs. and 25.56%, respectively. 15PHK was purified with Hiprep DEAE FF 16/10 column by fast protein liquid chromatography (FPLC). Five fractions were abtained and tested their antioxidant activities. The results showed that 5 purified fractions of 15PHK appeared less antioxidant activity.
Antitumor cell experiments were performed to incubate human cervix cancer cell line HeLa, human neuroblastoma cell line SH-SY5Y, human malignant melanoma cell line A375.S2, and human lung adenocarcinoma epithelial cell line A549 with 15HK, 15PHK and 70HK treatment. In MTT assay, A549 cell could be inhibited cell viability obviously by 70HK. A549 cell shrank after 24 hr 70HK incubation by observing. Moreover, lactate dehydrogenase (LDH) was released, nitric oxide (NO) was formed, and reactive oxygen (ROS) was measured corresponding with the more amount of 70HK. In contrast, mitochondrion membrane potential (MMP) was decreased. It showed that A549 cell incubated with 70HK treatment happened to apoptosis, and the more amount of 70HK treated, the more necrosis happened.
摘要 I
Abstract II
目次 III
Figure Contents V
Table Contents VI
壹、研究背景 1
一、 海洋生物之生理活性物質 2
(一) 海洋生物 2
(二) 生理活性物質 2
(三) 生理活性物質分類 3
(四) 海洋生理活性醣類 5
(五) 海洋生理活性胜肽 5
二、 海馬 7
(一) 簡介 7
(二) 海馬種類 7
(三) 營養價值及機制 7
(四) 庫達海馬 8
三、 研究動機及目的 10
貳、研究內容 11
第一章、庫達海馬乙醇萃取物之抗氧化性探討 12
一、 前言 12
二、 材料與方法 13
三、 結果 24
四、 討論 26
五、 結果表格與圖 28
第二章、庫達海馬乙醇萃取物之抗腫瘤性探討 36
一、 前言 36
二、 材料與方法 37
三、 結果 45
四、 討論 47
五、 結果表格與圖 49
參、綜合結論 57
肆、參考文獻 58
附錄一、庫達海馬之圖片 65
謝誌 66
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