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研究生:許亞嫺
研究生(外文):Ya-hsien
論文名稱:不同產地、不同品種之蕎麥萃取物抗氧化與保肝活性之探討
論文名稱(外文):Comparison of antioxidative and hepatoprotective activities of various buckwheat extracts
指導教授:蔡惠燕蔡惠燕引用關係
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:應用化學系碩士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:108
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蕎麥在以前是備荒救災的作物,現在已轉變成經濟作物,其經濟價值高,因含有芸香苷(rutin)、槲黃素(quercetin)等類黃酮物質,具有降低心血管疾病、抗氧化、消除自由基等功能,芸香苷和槲黃素的抗氧化能力比維他命C和E高,農作物栽培季節與品種均會影響此類物質之含量,故本研究探討台灣生長之甜蕎、苦蕎及蒙古進口之甜蕎的萃取物,對肝細胞的保肝活性。藉由t-BHP誘導體外培養之肝細胞的氧化傷害,探討蕎麥萃取物對肝細胞的保護能力。結果顯示所有萃取物皆具有保護作用,可減少t-BHP對肝細胞造成的傷害。利用FRAP assay檢測萃取物的總抗氧化能力,萃取物隨著酒精萃取濃度升高,其抗氧化效果也越好。用HPLC定量芸香苷和槲黃素含量,結果顯示,苦蕎的芸香苷含量皆比甜蕎的含量更高,而在不同產地(二林與蒙古)、不同部位(蕎仁與蕎殼),皆可發現芸香苷的存在,且甜蕎殼中,芸香苷含量比槲黃素含量高,苦蕎仁的芸香苷含量和槲黃素含量皆比苦蕎殼的含量高,此結果與多篇文獻結果符合。但這些萃取物中,也有許多物質未被鑑定出來,而這些物質可能降低肝細胞的保護作用,綜合掉其他成分抗t-BHP氧化傷害之能力,另外在一些萃取物中含有過多類黃酮(芸香苷和槲黃素)物質時,也有上述之情況產生。台灣二林生產的甜蕎仁萃取物比進口的蒙古甜蕎仁萃取物在肝細胞實驗中,具有更佳的保護作用,此結果顯示,本地生產之蕎麥有更高之附加價值。

Buckwheat used to be a product for natural disaster relief, but is now an industrial crop with high economic value. Bcckwheat is recognized as a valuable source of flavonoids. Flavonoids such as rutin and quercetin, are anti-oxidants known for reducing cardiovascular diseases and radical scavenging activity. Rutin and quercetin are more antioxidative effect than that of vitamin C and vitamin E. The phytochemical content are varied for variety of breads and cultivation. Therefore, this study compared the liver protection activity of extracts from different breeds, cultivation of buckwheat. By treating hepatocytes with t-BHP that induces the oxidative damage of in-vitro hepatocytes, the protective activity of buckwheat on the hepatocytes was determined. All the extracts demonstrated protective activity and reduced the extent of t-BHP damage on the hepatocytes. Total antioxidative activity of the extracts was measured by FRAP assay. As the concentration of the ethanol of the extracting solvents increased, the extract displayed better antioxidative effect. The rutin and quercetin of the extracts were analyzed by HPLC-UV. The results indicate that the rutin content of tartary buckwheat (Fagopyrum tataricum Gaertn) is higher than that of common buckwheat (Fagopyrium esculentum Moench), while the rutin was found in the groat and hull of buckwheat cultivated in Erlin and Mongolia. The rutin content is higher than the quercetin content in the hull of common buckwheat, and the rutin and quercetin content in the groat of the tartary buckwheat are higher than those of the hull of the tartary buckwheat. These results are consistent with those reported in the literatures. However, there are many substances not been identified in present work. These unidentified substances may cause cytotoxicity and set off the antioxidative effect of other components. Some extracts containing excessive flavonoids such as rutin and quercetin, may case over dose problems. The extracts of groat of common buckwheat cultivated in Taiwan show better protective activity compared to those imported from Mongolia. The results indicate that the locally cultivated buckwheat has higher added value.

摘要 I
Abstract II
目錄 IV
圖目錄 VII
表目錄 XI
第一章、前言 1
第一節、自由基與抗氧化物 1
1-1.自由基 1
1-2.活性氧 1
1-3.抗氧化劑 2
1-4.類黃酮(flavonoids)的簡介與分類 4
第二節、蕎麥 6
2-1.蕎麥的種類 6
2-2.蕎麥的營養成分 7
2-3.蕎麥的植物性化學成分(Phytochemicals) 9
2-4.蕎麥中之芸香苷(rutin)與槲黃素(quercetin)之介紹 12
第三節、體外培養初代肝細胞之介紹 13
第四節、實驗方法原理 15
4-1.MTT assay(methyl thiazolyl tetrazolium assay) 15
4-2.抗氧化能力試驗 15
4-3.高效能液相層析儀(High-performance liquid chromatography,HPLC)之原理 16
4-4.tert-Butyl hydroperoxide(t-BHP)的作用機制及原理 17
第二章、實驗目的 19
第三章、材料與方法 20
第一節、實驗儀器 20
第二節、藥品試劑 21
第三節、實驗步驟 23
3-1.蕎麥萃取 23
3-2.初代大鼠肝細胞之培養 23
3-3.蕎麥萃取物對t-BHP誘發初代大鼠肝細胞損傷之細胞活性分析 24
3-4.總抗氧化能力試驗(FRAPassay) 24
3-5.利用高效能液相層析儀(HPLC)分析蕎麥萃取物中類黃酮之含量 25
3-6.高效能液相層析儀(HPLC)之方法確效 26
3-7.統計分析 27
第四章、結果與討論 28
第一節、蕎麥萃取物對初代大鼠肝細胞抗t-BHP的氧化傷害之保護作用 30
1-1.劑量的探討 30
1-2.比較不同萃取方式的蕎麥萃取物對t-BHP誘導大鼠初代肝細胞氧化傷害之保肝作用 50
第二節、總抗氧化能力試驗(FRAP assay) 75
2-1.待測樣品之干擾程度 75
2-2.總抗氧化力校正曲線之建立 77
2-3.蕎麥萃取物之總抗氧化能力 80
第三節、高壓液相層析(HPLC)之結果 83
3-1.校正曲線的建立 83
3-2.方法確效 85
3-3.偵測極限(LOD)與定量極限(LOQ) 86
3-4.HPLC圖譜之比較 86
3-5.蕎麥萃取物中芸香苷(rutin)、槲黃素(quercetin)的含量 91
3-6.蕎麥萃取物其它未被鑑定出成分之相對含量百分比 94
第四節、抗t-BHP的存活率、總抗氧化能力與經HPLC定量之芸香苷、槲黃素含量之比較 97
第五章、結論 101
第六章、未來與展望 104
第七章、參考文獻 105


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