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研究生: 曾佳宜
Zheng, Jia-Yi
論文名稱: 木奶果葉萃取物之抗氧化活性及對人類大腸直腸癌細胞 Caco-2 抑制活性之研究
Antioxidant activity and inhibitory effect against caco-2 colorectal carcinoma cells from Baccaurea ramiflora Lour leaf extracts
指導教授: 廖遠東
Liaw, Ean-Tun
學位類別: 碩士
Master
系所名稱: 農學院 - 食品科學系所
Department of Food Science
論文出版年: 2022
畢業學年度: 110
語文別: 中文
論文頁數: 108
中文關鍵詞: 木奶果葉抗氧化活性大腸直腸癌細胞細胞凋亡
外文關鍵詞: Baccaurea ramiflora Lour leaves, Antioxidant activity, Colorectal carcinoma cells, Cell apoptosis
DOI URL: http://doi.org/10.6346/NPUST202200367
相關次數: 點閱:30下載:0
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  • 木奶果(Baccaurea ramiflora)為大戟科之藥用植物,於中國少數民族
    傣族的醫藥學中可作為消炎止痛藥。經研究顯示,木奶果葉片甲醇萃
    取物具有抗氧化、降血糖及抗發炎等功效。由於甲醇具有較強之毒性,
    且會使人體受到傷害,因此本研究將木奶果葉片以 95℃熱水及不同
    濃度乙醇 (25%、50%、75%、95%) 進行萃取後,分析生物活性成分、
    抗氧化活性及對人類大腸直腸癌細胞 Caco-2 之抑制活性。結果顯示,
    總酚以 75%乙醇萃取物含量最高達 354.6±0.764 mg/g;總類黃酮以
    95%乙醇萃取含量為最高達 60.3±1.967 mg/g。經 HPLC-UV/Vis 分析,
    熱水含有較多酚類化合物,分別為綠原酸(3.86±0.07 mg/g)、兒茶素
    (0.65±0.04 mg/g)、咖啡酸(1.63±0.01 mg/g)及槲皮素(5.95±0.02 mg/g)。
    於抗氧化試驗中,DPPH、ABTS 及還原能力以 75%乙醇萃取最佳,
    其 IC50 值分別為 15.732±0.309 g/mL 、168.236±10.97 g/mL 與
    209.89±16.49 g/mL。於細胞存活試驗,75%乙醇萃取物於 800 g/mL
    濃度下作用 72 小時後,抑制癌細胞存活率達 80 %以上,並透過流式
    細胞儀分析細胞週期及細胞凋亡,結果顯示 75%乙醇萃取物隨著濃度
    及時間的增加,增加 SubG1 期之細胞比例及總凋亡百分比。利用西
    方墨點法進一步分析細胞凋亡途徑中凋亡蛋白含量,75%乙醇萃取物
    透過增加 Bax 蛋白及降低 pro-caspase3 蛋白使細胞以內部粒線體途徑
    走向凋亡。綜上所述,木奶果葉片含有較高活性物質及抗氧化能力,
    且於大腸直腸癌細胞存活試驗,75%乙醇萃取物有較佳之細胞毒殺效
    果,並以濃度及時間依賴性方式增加 SubG1 期及總凋亡百分比,因
    此木奶果葉片有助於未來作為保健食品之開發。

    Baccaurea ramiflora is a medicinal plant of the Euphorbiaceae family,
    which is used as an anti-inflammatory and analgesic in the medicine of the
    Chinese minority Dai ethnic minority. Previous studies indicate that
    Baccaurea ramiflora Lour leaves extracted with methanol showed
    antioxidant, hypoglycemic, and anti-inflammatory effects. Since methanol
    is highly toxic and can cause damage to humans, in this study, Baccaurea
    ramiflora Lour leaves were extracted with hot water at 95°C and different
    concentrations with ethanol (25%, 50%, 75%, 95%) to analyze the
    bioactive components, antioxidant activity and inhibitory activity against
    human colorectal cancer cells Caco-2.
    The results showed that 75% ethanol extract demonstrated the highest
    total phenol content with 354.6±0.764 mg/g, while 95% ethanol extract
    exhibited the greatest flavonoid content with 60.3±1.967 mg/g. Following
    HPLC-UV/Vis analysis, The hot water contained more phenolic
    compounds, chlorogenic acid (3.86±0.07 mg/g), catechin (0.65±0.04
    mg/g), caffeic acid (1.63±0.01 mg/g) and quercetin (5.95±0.02 mg/g). For
    the antioxidant assay, DPPH, ABTS, and reducing power showed the
    strongest IC50 values of 15.732±0.309 g/mL, 168.236±10.97 g/mL, and
    209.89±16.49 g/mL for 75% ethanol extract. In cell experiments, 75%
    ethanol extract was concentrated at 800 g/mL for 72 hours, inhibiting the
    cancer cell viability up to 80%. The cell cycle and apoptosis were analyzed
    by flow cytometry, and the results showed that 75% ethanol extract
    increased the subG1 phase and total apoptosis percentages with the
    increase in concentration and time. Using western blots to further analyze
    apoptotic proteins in the apoptotic pathway, 75% ethanol extracts caused
    apoptosis by increasing Bax protein and decreasing pro-caspase3 protein
    in the internal pathway. Conclusively, Baccaurea ramiflora Lour leaves
    could be useful for the future development of healthy food.

    摘要 I
    第一章 前言 1
    第二章 文獻回顧 3
    2.1自由基 3
    2.1.1自由基的種類 3
    2.2抗氧化劑 7
    2.2.1內源性抗氧化劑 7
    2.2.2外源性抗氧化劑 8
    2.2.3合成抗氧化劑 14
    2.3癌症概況及生成 15
    2.3.1大腸直腸癌介紹 19
    2.4細胞週期簡介 22
    2.5 細胞死亡 24
    2.5.1 細胞凋亡(Apoptosis) 24
    2.5.2 細胞壞死( Necrosis) 25
    2.6木奶果 28
    第三章 材料與方法 32
    3.1實驗架構與設計 32
    3.2實驗材料 33
    3.2.1 樣品來源 33
    3.2.2人類大腸直腸癌細胞株 33
    3.2.3化學實驗藥品 33
    3.2.4 細胞實驗藥品 34
    3.2.5儀器設備 35
    3.2.6細胞實驗設備 35
    3.3實驗方法 37
    3.3.1木奶果萃取物製備 37
    3.3.2生物活性成分分析 37
    3.3.3抗氧化活性評估 39
    3.3.4細胞培養 41
    3.3.5大腸直腸癌細胞試驗 44
    3.3.6統計分析 49
    第四章 結果與討論 52
    4.1木奶果葉片生物活性成分分析 52
    4.1.1萃取率 52
    4.1.2總酚含量測定(Total phenols content, TPC) 54
    4.1.3總類黃酮含量測定(Total flavonoid content , TFC) 54
    4.1.4 高效液相層析儀(HPLC-UV/Vis) 56
    4.2抗氧化活性 61
    4.2.1 DPPH自由基清除能力(DPPH˙+ scavenging ability assay) 61
    4.2.2 ABTS自由基清除能力(ABTS˙+ scavenging activity) 61
    4.2.3還原能力(Reducing Power) 61
    4.3相關性分析 67
    4.4細胞實驗 69
    4.4.1細胞存活率試驗 69
    4.4.2細胞型態觀察 72
    4.4.3細胞週期 74
    4.4.4細胞凋亡 79
    4.4.5凋亡蛋白表現量 84
    五、結論 86
    六、參考文獻 87

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