薏仁麩皮萃取物乙酸乙酯區分層之抗發炎效果

Anti-inflammatory effects of ethyl acetate fractions from adlay bran extracts

劉家余(Chia-Yu Liu)

指導教授 ： 江文章

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00528

摘要

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。

關鍵字

薏仁麩皮 ； 脂多醣 ； RAW 264.7 鼠巨噬細胞 ； 抗發炎 ； 酚類化合物

並列摘要

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.

並列關鍵字

Adlay bran ； lipopolysaccharide ； RAW 264.7 murine macrophage cell line ； anti-inflammation ； phenolic compounds

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