臺灣博碩士論文加值系統

檸檬學名 Citrus limon 為芸香科 (Rutaceae) 柑橘屬 (Citrus) 的常綠 喬木，原產於印度及東南亞。現今東亞之主要產區為中國大陸長江以南， 台灣全島皆有栽種，其中屏東縣為臺灣主要的檸檬產區，約佔全島 80% 的產量。
檸檬是屏東縣的重要農產品，尤其在屏東平原北部的高樹鄉、里港 鄉、九如鄉和鹽埔鄉出產的檸檬以香氣濃郁著名。檸檬不僅是里港鄉重 要的經濟作物，食用檸檬也對當地居民的健康狀況產生正向的影響。
檸檬向來以榨汁食用為主，榨汁後剩下的大量副產品如皮、渣和籽 通常被當廢棄物處理，造成多餘的環境負擔。然而民間醫療已有將檸檬 應用於治療幾種疾病的先例，化學分析亦顯示檸檬副產物確實具有幾種 有益健康的特性，因此原為廢棄物的副產物再利用，應是具潛力的未來 發展方向，例如應用於醫療或保健製劑。故本研究將探討檸檬萃取物的
細胞毒殺活性，同時分析化學成分，以確定其為具藥用特性的化合物。
本研究的實驗步驟略述如下：先將乾燥的檸檬種子使用甲醇浸泡， 浸泡液經過濾、濃縮得濃稠液後加水使之懸浮，依次以正己烷 (n-Hexane) 與水以體積比 1:1 進行三次分配萃取，得正己烷層產物，水層接著與乙酸 乙酯 (ethyl acetate, EA) 以體積比 1:1 進行三次分配萃取，得乙酸乙酯層 產物，最後水層再與正丁醇 (n-BuOH) 進行三次萃取，得正丁醇層產物， 而剩餘的為水層部份。各層萃取物在口腔癌細胞 SAS、OCEM-1 和 NOK 毒殺試驗中，發現粗萃物具活性，且正己烷層活性較佳，其次是乙酸乙 酯層，因此將正己烷層及乙酸乙酯層萃取物利用管柱層析及高效液相層 析儀分離純化，得到三種三萜類化合物，其為 obacunone (T1)、limonin (T2) 及 nomilin (T3)。
細胞毒殺活性方面，檸檬種子之正己烷層萃取物對於口腔上皮癌細 胞及正常口腔上皮細胞皆有明顯的細胞毒殺效果，而 obacunone 具有最佳 的活性。本實驗以體外試驗 (in vitro) 初步測試檸檬萃取物的結論發現其 細胞毒殺效果並無腫瘤專一性。

Citrus lemon (Rutaceae) (Citrus) is mostly cultivated in India, Southeast Asia and near Yangtze River in China. In Taiwan, lemon production is concentrated in the central and south part of the country, with Pingtung accounting for 80% of the production.
Lemons are an important agriculture produce in Pingtung, mainly produced in four rural areas: Gaoshu, Ligang, Jiuru and Yanpu. In the Ligang Farmers association, lemon production has immensely contributed to improving health in the rural areas.
Lemons are mainly used for lemon juice production and have a large number of byproducts (peels, seeds, residue) that can pose environmental disposal problems. There is a need for the conversion of the byproducts into useful products such as therapeutic agents, and lemons have been used in folk medicine for the treatment of several ailments. Lemons have also been found to possess several health properties. This study will investigate the cytotoxic activity and chemical constitutes of lemon seeds extract in order to identify the compounds that are responsible for their medicinal properties.
The main aim of the study was to investigate the chemical ingredients of lemon seeds and their cytotoxic properties. The air-dried lemon seeds were extracted using methanol. The methanol extract was sequentially partitioned with n-hexane (1:1), ethyl acetate (1:1), n-butanol (1:1) to obtain the hexane, ethyl acetate, butanol and water layer. The soluble layers were evaluated for cytotoxic activities using SAS, OCEM-1 and NOK cells. The hexane and ethyl acetate layer had significant cytotoxic properties. Therefore, the two layers were further fractionated using the column chromatography and HPLC to obtain three triphenol. The compounds isolated were identified as obacunone (T1), limonin (T2) and nomilin (T3).
In cytotoxic assays, the hexane layer had significant cytotoxic effect in both the cancer cell and the normal epithelial cells. Compound 1 has the highest activity amongst the three compounds. The result of the in vitro assays indicate that the extracts are not selectively specific to tumor or cancer cells because they also affected the epithelial cells.

目錄
摘要 …………………………………………………………………...I Abstract…………………………………………………………………... III 誌謝 ……………………………………………………………………V
目錄 ……………………………………………………………………VI
表目錄………………………………………………………………….. VIII
圖目錄…………………………………………………………………. IX
壹、前言………………………………………………………………......1
一、緒言………………………………………………………………..1
二、植物簡介………………………………………………………… ..2
三、文獻回顧…………………………………………………………..4
四、研究動機…………………………………………………………14
貳、材料與方法…………………………………………………………15
一、檸檬種子化學成分之分離與純化 ................................................15
(一) 實驗藥品與儀器………………………………………………15
(二) 實驗方法 ……………………………………………………..17
二、探討檸檬種子萃取物抑制口腔癌細胞株生長能力…………….20 (一) 細胞培養材料………………………………………………….20
1. 細胞培養……………………………………………………..20
2. 細胞生長測試藥品…………………………………………..20
(二)細胞株…………………………………………………………..20
1. SAS…………………………………………………………...20
2. OCEM-1………………………………………………………20
3. NOK…………………………………………………………..21
(三) 儀器 …………………………………………….……………21
(四) 檸檬種子萃取物抑制口腔癌細胞株存活試驗 ……………21
參、結果與討論………………………………………………………….22
一、化合物結構摘要 …………………………………………………23
二、化合物結構解析………………………………………………….23
三、探討檸檬種子萃取物抑制口腔癌細胞株生長能力…………….36
(一) 檸檬種子萃取物對於口腔癌細胞生長抑制效果…………36
(二) 檸檬種子化合物 T1、T2 及 T3 抑制口腔癌細胞株結果..36
肆、結論………………………………………………………………….41
伍、化合物數據………………………………………………………….44
陸、參考文獻 ………………………………………………………….46
作者簡介……………………………………………………………….…49


表目錄
表一、檸檬葉之化學成分 ……………………………………………4
表二、檸檬種子之化學成分……………………………………………7
表三、檸檬果皮之化學成分……………………………………………8
表四、檸檬種子各分層之含量………………………...………………17
表五、化合物 1 之氫和碳核磁共振光譜數據…………………….…25
表六、化合物 2 之氫和碳核磁共振光譜數據…………………….…29
表七、化合物 3 之氫和碳核磁共振光譜數據…………..……………33
表八、檸檬種子萃取物對於口腔癌細胞毒殺效果……………………37
表九、檸檬種子化合物對於口腔癌細胞毒殺效果……………………39
表十、檸檬種子萃取物與化合物對於口腔癌細胞毒殺效果總結……41



IX

圖目錄
圖一、檸檬 ………………………………………………………………3
圖二、檸檬種子…………………………………………………………3
圖三、檸檬種子甲醇粗萃物之分配萃取流程…………………………18
圖四、檸檬種子成分分離圖……………………………………………22
圖五、化合物 1 之氫核磁共振光譜圖…………………………………26
圖六、 化合物 1 之碳核磁共振光譜圖 ………………………………26
圖七、化合物 1 之質譜圖………………………………………………27
圖八、化合物 2 之氫核磁共振光譜圖…………………………………30
圖九、化合物 2 之碳核磁共振光譜圖…………………………………30
圖十、化合物 2 之質譜圖………………………………………………31
圖十一、化合物 3 之氫核磁共振光譜圖………………………………34
圖十二、化合物 3 之碳核磁共振光譜圖………………………………34
圖十三、化合物 3 之質譜圖……………………………………………35
圖十四、檸檬種子萃取物對於口腔癌細胞毒殺效果…………………38
圖十五、檸檬種子化合物對於口腔癌細胞毒殺效果…………………40
圖十六、檸檬種子萃取物及化合物對於口腔癌細胞毒殺效果………43

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