薏苡籽實萃取物之抗氧化和抗發炎活性成分及其抗動脈硬化危險因子之效果

The Anti-oxidative and Anti-inflammatory Activities of Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) Seed Extract and Its Active Components and Effect on Anti-atherogenic Risk Factors

黃鼎文(Din-Wen Huang)

指導教授 ： 江文章

共同指導教授 ： 郭悅雄 ； 林雲蓮

國立臺灣大學/生物資源暨農學院/食品科技研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.00705

摘要

本論文第一部分之主要目的在探討薏苡籽實萃取物於延緩銅離子誘導低密度脂蛋白(low density lipoprotein, LDL)氧化與脂聚多糖(lipopolysaccharide, LPS)誘導RAW 264.7巨噬細胞之系統下的抗氧化與抗發炎效果。將薏苡殼經乙醇萃取後，可得薏苡殼乙醇萃取物(ethanolic extract of adlay hull, AHE)，AHE再利用溶劑分配之方法，將AHE區分為乙酸乙酯、正丁醇和水(AHE-Ea、AHE-Bu與AHE-H2O)三個不同區分層。AHE-Ea繼續再以管柱層析之方法區分為15個不同次區分物，AHE-Ea-A到AHE-Ea-O。結果顯示，AHE-Ea及其次區分物，AHE-Ea-J、AHE-Ea-K與AHE-Ea-M具經由調節一氧化氮合成酵素(inducible nitric oxide synthase, iNOS)與環氧化酵素-2 (cyclooxygenase-2, COX-2)蛋白質之表現抑制一氧化氮(nitric oxide, NO)和前列腺素(prostaglandin E2, PGE2)生成之抗發炎效果，經分離純化步驟，自上述有效次區分層中純化出eriodictyol (1)、一個ceramide類化合物(2)與p-coumaric acid (3)。且經實驗結果顯示，化合物(1)與(2)為首次自薏苡中分離並發現其為薏苡殼中主要提供抗發炎活性之成分。由此得知，AHE具有經由減少iNOS與COX-2蛋白質之表現減少發炎介質NO與PGE2生成以調控發炎反應之能力，且其效果有部分來自於分離鑑定出之化合物。
將薏苡種皮經乙醇萃取後(ethanolic extract of adlay testa, ATE)，利用溶劑分配之方法，將ATE區分為正己烷、乙酸乙酯、正丁醇和水(ATE-Hex、ATE-Ea、ATE-Bu與ATE-H2O)四個不同區分層。ATE-Ea及ATE-Bu分別繼續再以管柱層析之方法區分為ATE-Ea-a到ATE-Ea-h與ATE-Bu-A到ATE-Bu-F不同次區分物。結果顯示，ATE-Ea、ATE-Bu、ATE-Ea-e和ATE-Bu-C可經由延緩LDL氧化、清除DPPH自由基及抑制LPS誘導RAW 264.7巨噬細胞NO生成，展現抗氧化與抗發炎之效果，且上述之(次)區分物抑制發炎反應的效果應是經由調節iNOS與COX-2蛋白質之表現。具活性之區分物再經配合高效液相層析儀(high-performance liquid chromatogram, HPLC)，自ATE-Ea與ATE-Bu之有效次區分物中分析或純化出gallic acid (GA)、chlorogenic acid (CGA)、vanillic acid (VA)、caffeic acid (CA)、syringic acid (SA)、4-hydroxyacetophenone (4HA)、p-coumaric acid (PCA)、ferulic acid (FA)與2-O-β-glucopyranosyl-7-methoxy-4 (2H)-benzoxazin-3-one (GMBO)。將上述之成分以生理活性平台繼續探討其在ATE-Ea與ATE-Bu之生理活性所扮演之角色後發現，GA、CGA、CA與FA (10 micro-g/mL)是其中提供主要抗氧化與抗發炎能力之酚類化合物。經HPLC分析定量後得知，每克ATE-Bu-C次區分物中CGA之含量為30.3 mg，CA之含量則為9.02 mg，GMBO則含有189 mg；而每克ATE-Ea-e次區分物中VA之含量為1.31 mg，PCA之含量則為3.89 mg，FA則含有47.6 micro-g。綜合以上結果得知，ATE具有抗氧化及經由減少iNOS與COX-2蛋白質之表現調控發炎反應之能力，且其效果有部分來自於分離鑑定出之化合物。
將薏仁麩皮與精白薏仁經乙醇萃取後，可得薏仁麩皮乙醇萃取物(ethanolic extract of adlay bran, ABE)與精白薏仁乙醇萃取物(ethanolic extract of polished adlay, PAE)，ABE與PAE分別再利用溶劑分配之方法，區分為正己烷、乙酸乙酯、正丁醇和水(ABE-Hex、ABE-Ea、ABE-Bu與ABE-H2O；PAE-Hex、PAE -Ea、PAE -Bu與PAE-H2O)四個不同區分層。結果顯示，ABE-Ea與PAE-Ea具經由調節iNOS與COX-2蛋白質之表現抑制NO生成之抗發炎效果。而薏仁麩皮與精白薏仁萃取物之各區分物於保護LDL免於氧化之效果皆較種皮差。
本論文之第二部分，繼續以ATE、ATE-Ea或其中所含之酚酸類化合物並配合高膽固醇飼料餵予C57BL/6 apoE基因剔除小鼠四週，並與只吃高膽固醇飼料組之小鼠比較，探討ATE-Ea於小鼠體內防制動脈硬化危險因子之功效。發現所有給予樣品組之血清總膽固醇皆顯著下降。在肝臟抗氧化酵素方面，ATE-Ea隨著管餵劑量的上升，可增加榖胱甘肽還原酵素(glutathione reductase, GRd)、麩胺基硫轉移酵素(glutathione S-transferase, GST)與觸媒(catalase, CAT)之活性，降低脂質過氧化物的生成並提升榖胱甘肽(Glutathione)之含量，其中酚酸混合物組亦可顯著增加GRd之活性(p < 0.01) 。ATE-Ea並可抑制小鼠主動脈中發炎與氧化相關之蛋白質的表現，包括臍靜脈內皮細胞黏著分子(intercellular adhesion molecule-1, ICAM-1)、血管細胞黏著分子(vascular cell adhesion molecule-1, VCAM-1)、單核球趨化蛋白(monocyte chemoattractant protein-1, MCP-1)與NADPH oxidase之次單元(NOX4與p22phox)，並提升動脈內皮超氧歧化酵素(superoxide dismutase, SOD)，SOD-1及SOD-2的蛋白質表現達到保護動脈內皮之效果。而酚酸混合物組雖然在小鼠主動脈之發炎相關蛋白質無抑制之效果(ICAM-1與VCAM-1)，但可增加抗氧化酵素(SOD-1與SOD-2)與降低氧化壓力(NOX4與p22phox)相關蛋白質之表現。
綜合以上敘述後得知，ATE具有較佳之抑制LDL氧化的效果，且薏苡籽實各部位皆可經由調節iNOS與COX-2白質調控發炎反應，而薏苡殼與薏苡種皮之效果最少有部分來自於分離鑑定出之化合物。ATE-Ea亦可經由(1)降低血清總膽固醇；(2)提高肝臟抗氧化能力與(3)調節動脈內皮中發炎與氧化壓力之蛋白等危險因子達到減緩小鼠體內發生動脈硬化疾病之效果

關鍵字

薏苡 ； 抗氧化 ； 抗發炎 ； 低密度脂蛋白 ； DPPH ； 一氧化氮 ； 誘導型一氧化氮合成酵素 ； 環氧化酵素-2 ； 動脈硬化 ； C57BL/6 apoE基因剔除小鼠

並列摘要

The aims of the first part of this study were to investigate the effects of adlay seed extracts on the copper ion-mediated low density lipoprotein (LDL) oxidation and lipopolysaccharide (LPS)-induced inflammatory response in RAW 264.7 macrophages. Adlay hull ethanolic extract (AHE) was sequentially partitioned into ethyl acetate, n-butanol, and water-soluble fractions (AHE-Ea, AHE-Bu and AHE-H2O, respectively). AHE-Ea was further separated into AHE-Ea-A to AHE-Ea-O by column chromatography (CC). AHE-Ea and its subfractions, AHE-Ea-J, AHE-Ea-K, and AHE-Ea-M inhibited the nitric oxide (NO) and prostaglandin E2 (PGE2) formation through down-regulating inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. Eriodictyol (1), a ceramide, (2S,3S,4R)-2-[(2'R)-2'-hydroxytetracosanoyl amino]-1,3,4-octadecanetriol (2) and p-coumaric acid (3) were found in the subfractions, and the first two compounds appeared to be primarily responsible for the anti-inflammatory activity. This is the first time that eriodictyol and the ceramide have been found in adlay seed hulls, and the anti-inflammatory properties of the AHE-Ea fraction can be attributed, at least in part, to the presence of these two compounds.
The adlay testa ethanolic extract (ATE) was sequentially separated into n-hexane, ethyl acetate, n-butanol and water-soluble fractions (ATE-Hex, ATE-Ea, ATE-Bu and ATE-H2O, respectively). ATE-Ea and ATE-Bu were further separated into ATE-Ea-a to ATE-Ea-h and ATE-Bu-A to ATE-Bu-F respectively by CC. Results showed that ATE-Ea, ATE-Bu, ATE-Ea-e, and ATE-Bu-C expressed anti-oxidative and anti-inflammatory activities through the DPPH-radical scavenger, LDL oxidation inhibition, and NO inhibitory activity. Inflammation was further modulated by fractions of ATE-Ea and ATE-Bu, and their subfractions, ATE-Ea-e and ATE-Bu-C through downregulating the expression of iNOS and COX-2 proteins. Chlorogenic acid (CGA), vanillic acid (VA), caffeic acid (CA), syringic acid (SA), 4-hydroxyacetophenone (4HA), p-coumaric acid (PCA), ferulic acid (FA), and 2-O-β-glucopyranosyl-7-methoxy-4 (2H)-benzoxazin-3-one (GMBO), were analyzed or isolated from the active fractions of ATE by CC. Among them, CGA, CA, and FA were the major components responsible for the anti-oxidative and anti-inflammatory activities of ATE. Subsequently, HPLC analysis showed that each gram of ATE-Bu-C had 30.3 mg CGA, 9.02 mg CA, and 189 mg GMBO, while each gram of ATE-Ea-e had 1.31 mg VA, 3.89 mg PCA, and 47.6 micro-g FA. In brief, ATE had anti-oxidative and anti-inflammatory activities, and its effects were partially related to its phenolic components.
The adlay bran ethanolic extract (ABE) and polished adlay ethanolic extract (PAE) were sequentially separated into n-hexane, ethyl acetate, n-butanol and water-soluble fractions, respectively (ABE-Hex, ABE-Ea, ABE-Bu and ABE-H2O, respectively; PAE-Hex, PAE-Ea, PAE-Bu and PAE-H2O, respectively). Results showed that，ABE-Ea and PAE-Ea inhibited the NO formation through down-regulating iNOS and COX-2 protein expression. However, the effects of both fractions of ABE and PAE on prevention of LDL oxidation were worse than adlay testa.
The second part of this study, ATE, ATE-Ea, and the self-made mixture of phenolic acid components were further fed the C57BL/6 apo E-deficient (apoE-/-) mice with high cholesterol diet (HCD) for four weeks to investigate the anti-atherogenic risk factors effects of ATE-Ea in vivo and compared with wild type C57BL/6 mice. Results showed that when the HCD was supplied to the C57BL/6 apo EapoE-/- mice with or without the various samples for 4 weeks, the serum total cholesterol concentrations (TC) were all significantly decreased in test groups. The antioxidative enzymes in liver, glutathione reductase (GRd), glutathione S-transferase (GST) and catalase (CAT) were all up-regulated by high-dose ATE-Ea group resulting in the increasing of glutathione (GSH) and the decreasing of TBARS. The liver GRd is also significantly raised by self-made mixture of phenolic acid components group (p < 0.01). The expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), and the subunit of NADPH oxidase (NOX4 and p22phox) in the aorta were all inhibited, however, the anti-oxidative enzyme superoxide dismutase-1 (SOD-1) and SOD-2 were both up-regulated by ATE-Ea. Although the expression of inflammatory-related proteins (ICAM-1 and VCAM-1) of knockout mice aorta were not influenced by self-made mixture of phenolic acid components, the anti-oxidative enzymes were up-regulated (SOD-1 and SOD-2) and the oxidative stress-related proteins (NOX4與p22phox) were down-regulated by self-made mixture of phenolic acid components.
In conclusion, ATE had anti-oxidative, and all of the adlay seed extracts had anti-inflammatory activities, and the effects of AHE and ATE were both partially related to its components, and in vivo observations, together with the functions of ATE-Ea: (1) To lower the cholesterol level in the serum, (2) to up-regulate the antioxidative enzymes in the liver, and (3) to modulate the expression of inflammation and oxidative stress-related protein of aorta, in the pathogenesis of HCD-induced atherosclerosis in C57BL/6 apoE-/- mice.

並列關鍵字

Adlay ； antioxidative ； anti-inflammatory ； LDL ； DPPH ； NO ； iNOS ； COX-2 ； atherosclerosis ； C57BL/6 apoE deficient mice

參考文獻

林靜慧。2007。薏苡籽實乙醇萃取物及其次區分層對人類癌細胞生長之影響。國立台灣大學食品科技研究所碩士論文。

李明怡。2008。薏仁麩皮中防癌及抗發炎活性成分之分離與鑑定。國立台灣大學食品科技研究所博士論文。

陳亞琪。2005。雌激素處理對apoE基因剔除母鼠活性氧分子的產生與動脈粥狀硬化進程之影響。國立成功大學細胞生物與解剖學研究所。

呂建興。2002。黃柏葉部之成分研究。國立成功大學化學研究所碩士論文。

夏詩閔。2001。薏苡籽實萃取物對大鼠卵巢性類固醇激素、腦下腺黃體促素和濾泡刺激素分泌之效應。國立台灣大學食品科技研究所碩士論文。