臺灣博碩士論文加值系統

薏仁 (Coix lachryma-jobi L.var. ma-yuen Stapf.) 麩皮具有調節免疫、抗發炎和抑制腫瘤細胞生長等生理活性。本研究室欲自薏仁麩皮中研發出具有抗發炎、抑制腫瘤以及減輕放射線治療副作用之保健食品，因此本研究以開發薏仁麩皮成保健食品為方向，利用脂多醣 (lipopolysaccharide, LPS) 誘導RAW 264.7 鼠巨噬細胞模式，比較薏仁麩皮乙醇萃取物 (adlay bran ethanolic extract, ABE) 與薏仁麩皮乙酸乙酯萃取物 (adlay bran ethyl acetate extract, ABEa) 分別經不同極性溶劑 (正己烷、乙酸乙酯、正丁醇和水) 區分後之抗發炎效果與產率差異。
結果顯示，ABE 和ABEa中乙酸乙酯區分層 (ethyl acetate fraction of ABE and ABEa, ABE-Ea and ABEa-Ea) 抑制一氧化氮 (nitric oxide, NO) 生成效果均最佳，其產率分別為0.91% 和1.16% (以新鮮薏仁麩皮濕重為基準)，ABEa-Ea 之產率較ABE-Ea 高27.5%。進一步將ABE-Ea 和ABEa-Ea 分別以30% 乙酸乙酯/正己烷 (ethyl acetate/n-hexane, Ea/Hex)、80% Ea/Hex、100% Ea 和95% EtOH 等溶劑經矽膠管柱層析得到四個次區分層。結果顯示，ABE-Ea之80% Ea/Hex、100% Ea 和95% EtOH 次區分層及ABEa-Ea 之80% Ea/Hex 和100% Ea 次區分層具有良好的抑制NO 生成效果。後續實驗證明，在50 μg/mL濃度下，上述五個次區分層具有抑制誘導型一氧化氮合成酶 (inducible nitric oxide synthase, iNOS) 和環氧化酶 (cyclooxygenase, COX)-2蛋白表現作用，其中ABE-Ea 之95% EtOH 次區分層可完全抑制iNOS 和COX-2 蛋白表現。除ABEa-Ea 之100% Ea 次區分層外，其他四個次區分層皆可顯著地抑制細胞分泌介白素 (interleukin, IL)-6，但只有ABE-Ea 之95% EtOH 次區分層和ABEa-Ea 之80% Ea/Hex 次區分層可顯著地抑制細胞分泌腫瘤壞死因子 (tumor necrosis factor, TNF)-α。經HPLC-MS定量後得知ABE-Ea 之95% EtOH 次區分層含量最多之酚類化合物為對羥基苯甲酸 (p-hydroxybenzoic acid, 2770 μg/g sample)、香豆酸 (p-coumaric acid, 750 μg/g sample) 和檞皮素 (quercetin, 433 μg/g sample)，而ABEa-Ea 之80% Ea/Hex 次區分層含量最多之酚類化合物為橘皮素 (tangeretin, 1579 μg/g sample)、川陳皮素 (nobiletin, 1423 μg/g sample) 和對羥基苯甲酸 (991 μg/g sample)。本研究結果顯示，不同極性次區分層中以ABE-Ea之95% EtOH 次區分層和ABEa-Ea之80% Ea/Hex 次區分層之抗發炎效果較佳，其抗發炎效果可能是由不同酚類化合物所造成。

Adlay (Coix lachryma-jobi L.var. ma-yuen Stapf.) bran has been reported that possesses many physiological activities such as immune modulation, anti-inflammation, and antitumor. Our laboratory desires to develope a product that possesses the effects of anti-inflammation, antitumor, and attenuating the side effects of radiotherapy. The aim of this study is to compare the adlay bran ethanolic extract (ABE) and adlay bran ethyl acetate extract (ABEa) that were respectively partitioned into n-hexane, ethyl acetate, n-butanol, and water fractions for the anti-inflammtory effects screening in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cell model and the differences of the yields.
The results showed that ethyl acetate fraction (ABE-Ea/ABEa-Ea) has the strongest inhibition effect on nitrite production in both extracts, and the yields of them were 0.91% and 1.16% of wet weight of fresh adlay bran, respectively. The yield of ABEa-Ea was 27.5% higher than that of ABE-Ea. ABE-Ea and ABEa-Ea were further subfractionated into 30% ethyl acetate/n-hexane (Ea/Hex), 80% Ea/Hex, 100% Ea and 95% ethanol (EtOH) by silica gel chromatography. The results showed that ABE-Ea-80% Ea/Hex, ABE-Ea-100% Ea, ABE-Ea-95% EtOH, ABEa-Ea-80% Ea/Hex, and ABEa-Ea-100% Ea could inhibit the nitrite production in LPS-stimulated RAW 264.7 murine macrophage cells. In addition, the above five subfractions could inhibit the inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 protein expressions at 50 μg/mL. Among of these five subfractions, the ABE-Ea-95% EtOH could completely inhibit iNOS and COX-2 protein expressions. Except for the ABEa-Ea-100% Ea, other four subfracions significantly inhibited the secretion of interleukin (IL)-6. However, only the ABE-Ea-95% EtOH and ABEa-Ea-80% Ea/Hex could significantly inhibit the secretion of tumor necrosis factor (TNF)-α. Based on the HPLC-MS analysis, the more abundant phenolic compounds in ABE-Ea-95% EtOH in orders are p-hydroxybenzoic acid (2770 μg/g sample), p-coumaric acid (750 μg/g sample), and quercetin (433 μg/g sample); those in ABEa-Ea-80% Ea/Hex are tangeretin (1579 μg/g sample), nobiletin (1423 μg/g sample), and p-hydroxybenzoic acid (991 μg/g sample). In conclusion, ABE-Ea-95% EtOH and ABEa-Ea-80% Ea/Hex had the most potential for anti-inflammation, and the different phenolic compounds might attribute to the anti-inflammatory activities.

摘要....................................................................І
Abstract..................................................................III
縮寫對照表........................................................... V
目錄................................................................VIII
圖目錄...............................................................XI
表目錄..............................................................XIII

壹、 前言...............................................................1
貳、 文獻整理...........................................................2
ㄧ、發炎反應與巨噬細胞..........................................2
（一）發炎反應 (inflammation)...................................2
（二）巨噬細胞 (macrophage) 及其功能.......................3
（三）LPS 誘導巨噬細胞產生發炎反應......................3
（四）發炎反應評估指標.............................6
1、iNOS 與NO..............................6
2、COX-2...............................7
3、促發炎細胞激素...........................9
二、 薏苡........................................10
（一）薏苡簡介...............................10
（二）薏苡籽實一般成分組成.......................10
（三）自薏仁麩皮中分離出來的植物化學成分...............13
（四）薏苡之抗發炎相關研究.......................29
參、 研究動機與實驗架構.................................32
（一）研究動機...............................32
（二）實驗架構...............................33
肆、 材料與方法.....................................35
一、實驗材料.....................................35
（一）薏仁麩皮原料............................35
（二）試驗藥品................................35
（三）儀器設備................................38
二、實驗方法.....................................39
（一）ABE、ABE-Ea 和ABE-Ea 之不同極性次區分層之製備.....39
（二）ABEa、ABEa 不同溶劑區分層和ABEa-Ea 之不同極性次區分層之製備...................................42
（三）RAW 264.7 鼠巨噬細胞培養.......................46
（四）細胞存活率測定 (MTT assay).....................46
（五）NO 生成測定............................46
（六）與發炎反應相關之COX-2 和iNOS 蛋白之檢測...........47
（七）總多酚含量測定............................49
（八）細胞激素分析 (IL-6 及TNF-α).....................49
（九）以HPLC-MS 定量酚類化合物.......................49
三、統計分析.....................................50
伍、 結果.........................................52
一、薏仁麩皮不同溶劑萃取物對LPS 誘導RAW 264.7鼠巨噬細胞株細胞存活率和NO 生成之影響...............................52
（一）ABE 之不同溶劑區分層及ABE-Ea 之不同極性次區分層.......52
1、ABE 之不同溶劑區分層對細胞存活率之影響.........52
2、ABE 之不同溶劑區分層對NO 生成之影響........52
3、ABE-Ea 之不同極性次區分層對細胞存活率之影響.......56
4、ABE-Ea 之不同極性次區分層對NO 生成之影響......56
（二）ABEa、ABEa 之不同溶劑區分層及ABEa-Ea 之不同極性次區分層....................................................59
1、ABEa對細胞存活率之影響....................59
2、ABEa對NO 生成之影響.......................59
3、ABEa 之不同溶劑區分層對細胞存活率之影響.......62
4、ABEa 之不同溶劑區分層對NO 生成之影響..........62
5、ABEa-Ea 之不同極性次區分層對細胞存活率之影響.......65
6、ABEa-Ea 之不同極性次區分層對NO 生成之影響.....65
二、較具抑制LPS 誘導NO 生成作用之次區分層對RAW 264.7 鼠巨噬細胞株iNOS 和COX-2 蛋白的影響.........................68
三、較具抑制LPS 誘導NO 生成作用之次區分層對RAW 264.7 鼠巨噬細胞株IL-6 細胞激素生成的影響..........................68
四、較具抑制LPS 誘導NO 生成作用之次區分層對RAW 264.7 鼠巨噬細胞株TNF-α 細胞激素生成的影響......................71
五、薏仁麩皮萃取物乙酸乙酯層之不同極性次區分層總多酚含量.........71
六、p-Hydroxybenzoic acid 和protocatechuic acid 對細胞存活率和NO 生成之影響.......................................74
七、標準品之滯留時間、游離態分子量與線性回歸方程式.............77
八、ABE-Ea 之四個次區分層酚類化合物含量.................77
九、ABEa-Ea 之四個次區分層酚類化合物含量...................77
陸、 討論.........................................81
ㄧ、薏仁麩皮萃取物乙酸乙酯區分層之產率與抗發炎效果比較........81
二、薏苡籽實不同部位萃取物之抗發炎效果比較................81
三、薏仁麩皮萃取物對巨噬細胞存活率之影響................82
四、薏仁麩皮萃取物乙酸乙酯層之不同極性次區分層對巨噬細胞NO 生成之影響.......................................83
五、薏仁麩皮萃取物乙酸乙酯層之不同極性次區分層對巨噬細胞iNOS 和COX-2 蛋白之影響.............................94
六、薏仁麩皮萃取物乙酸乙酯層之不同極性次區分層對巨噬細胞分泌細胞激素之影響...................................95
七、薏仁麩皮萃取物乙酸乙酯層之不同極性次區分層抗發炎成分之探討....96
柒、 結論.........................................98
捌、參考文獻.........................................99

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