Allergic Immune-regulatory Effects of Adlay Bran and Isolation and Identification of Bioactive Components

過敏性疾病是免疫失調所造成的過敏反應，包括有異位性皮膚炎、過敏性鼻炎及氣喘…等。在台灣地區過敏症狀的盛行率逐年增高，除了以藥物立即性的遏止不良過敏反應發生，如何藉由日常飲食養生上來調節免疫力，減少過敏症狀以避免藥物資源的浪費是重要的課題。過敏反應的發生包括早期肥大細胞釋放組織胺等過敏介質，晚期肥大細胞分泌細胞激素及後續巨噬細胞的發炎反應，因此本研究首先探討薏苡籽實乙醇萃取物對老鼠嗜鹼性白血球細胞株RBL-2H3及老鼠巨噬細胞RAW 264.7其抗過敏及抗發炎之功效。 以1 μM A23187誘導肥大細胞RBL-2H3產生過敏反應的實驗中，除了精白薏仁外，薏苡籽實各部位乙醇萃取物皆有效抑制A23187誘導細胞去顆粒化釋放β-hexosaminidase的程度，以薏仁麩皮乙醇萃取物乙酸乙酯層其EC50 為40.8 μg/mL最佳。進一步實驗發現薏仁麩皮乙醇萃取物乙酸乙酯層40~100% EtOAc/Hex極性區分物抑制組織胺釋放的EC50在71~87 μg/mL之間，此外亦可有效抑制細胞分泌IL-4、IL-6及TNF-α，顯示薏仁麩皮不僅抑制細胞去顆粒化，也同時抑制了細胞激素的分泌。薏仁中含有palmitic acid、4-hydroxybenzaldehyde、6-methoxy-3H-benzoxazol、4-hydroxyacetophenone、p-coumaric acid及quercetin等抗過敏物質，可藉由抑制胞內ROS生成，抑制Akt及ERK的磷酸化，降低PKC蛋白質的表現，進而影響肥大細胞的訊息傳遞而達到抗過敏的功效。以1 μg/mL LPS誘導巨噬細胞RAW 264.7產生發炎反應的實驗中，薏仁麩皮乙醇萃取物乙酸乙酯層40~100% EtOAc/Hex極性區分物在50 μg/mL濃度下不影響細胞生存率，可抑制細胞釋放一氧化氮。當濃度在6~30 μg/mL之間，更可有效降低細胞分泌IL-6及TNF-α等發炎性細胞激素的含量，顯示薏仁麩皮具有抗發炎的功效。 利用體外初代細胞培養探究薏仁麩皮萃取物對動物體的免疫調節作用，實驗結果發現薏仁麩皮萃取物可藉由降低IL-6及TNF-α的含量來抑制小鼠腹腔細胞發炎反應。對於小鼠脾臟細胞，薏仁麩皮萃取物則可顯著增加IL-2的含量，並降低IL-4及IL-5的細胞激素分泌，推測薏仁麩皮萃取物可調節Th1/Th2免疫反應。進一步確認薏仁麩皮萃取物的抗過敏活性，實驗利用OVA致敏鼠的模式，以管餵薏仁麩皮乙醇萃取物乙酸乙酯層區分物進行動物實驗。結果顯示，薏仁麩皮樣品可顯著降低動物體內特異性IgE抗體的生成，並藉由提升IFN-γ且顯著減少IL-4及IL-5的分泌，達到調節Th1/Th2免疫反應之功效，進而減緩動物體內過敏症狀。 為探究薏仁麩皮中可能之抗過敏成分，進一步針對薏仁麩皮乙醇萃取物乙酸乙酯層40~100% EtOAc/Hex沖提物進行分離純化，結果由分離純化出22個化合物，其中有16個化合物為首次自薏苡分離純化而得，包括12個類黃酮tangeretin (FL1)、new flavonoid (FL2)、formononetin (FL3)、davidigenin (FL4)、homoeriodictyol (FL5)、hesperetin (FL6)、isoliquiritigenin (FL8)、liquiritigenin (FL9)、3,5,6,7,8,3'',4''-heptamethoxyflavone (FL10)、chrysoeriol (FL11)、3,5,7,3'',4''-pentamethoxyflavone (FL12)和3,5,7,4''-tetramethoxyflavone (FL13)；2個色酮類5-hydroxy-7-methoxychromone (C1)、5,7-dihydroxychromone (C2)、1個酚酸酯類ethyl ferulate (PE1)及1個固醇類6α-hydroxystigmast-4-en-3-one (S1)。更進一步建立HPLC/MS的系統，以利於分析薏仁中類黃酮類成分。 綜上所述，薏仁麩皮中含有多樣性的類黃酮成分，推測這類化合物在過敏免疫反應的調節，抑制早期肥大細胞的過敏反應，減緩晚期巨噬細胞的發炎反應，及調節動物體內Th1/Th2免疫反應上可能扮演著功能性成分。綜合以上結果，薏仁麩皮乙醇萃取物具有調節免疫並減緩過敏的保健功效。Allergy is an immune dysfunction, including atopic dermatitis, allergic rhinitis, asthma, etc. In Taiwan, the allergic disease is more and more prevalent. In addition to the usage of drugs to stop the allergic responses, taking advantage of dietary regimen is more important to regulate the immune system, to reduce the allergic symptom and avoid the waste of drugs. The inflammatory response is caused by allergic mediators such as histamine released and cytokine secretion by the mast cell, and the inflammatory response activated by macrophage. Therefore, we first investigated the anti-allergic and anti-inflammatory effects of ethanolic extracts of adlay on the RBL-2H3 cells and RAW 264.7 cells. Except the polished adlay, the ethanolic extracts from different parts of adlay can suppress the β-hexosaminidase released from RBL-2H3 cells stimulated with 1 μM A23187. The ethyl-acetate fraction from ethanolic extracts of adlay bran (ABE-EtOAc) presents more effective inhibition with an EC50 at 40.8 μg/mL. The 40~100% EtOAc/Hex sub-fractions of ABE-EtOAc can significantly inhibit the release of histamine with EC50 between 71~87 μg/mL. Moreover, ABE-EtOAc suppresses IL-4, IL-6 and TNF-α secreted from RBL-2H3 cells indicates that the adlay bran can inhibit not only the cell degranulation but also the cytokine secretion. The alday contains anti-allergic compounds, such as palmitic acid, 4-hydroxybenzaldehyde, 6-methoxy-3H-benzoxazol, 4-hydroxyacetophenone, p-coumaric acid and quercetin, can reduce ROS production, inhibit the phosphorylation of Akt and ERK, and decrease the PKC protein expression. These combined effects influence the signaling transduction in RBL-2H3 cells, and showing the anti-allergic effect. The 40~100 % EtOAc/Hex sub-fractions of ABE-EtOAc at 50 μg/mL has no toxicity on the RAW 264.7 cells and inhibit the NO release. Furthermore, ABE-EtOAc suppresses the IL-6 and TNF-α secretion in LPS (1 μg/mL)-stimulated RAW 264.7 cells in concentration dependent manner at 6~30 μg/mL. From above results demonstrate that the adlay bran has an anti-inflammatory effect. In the primary cells isolated from mice study, ABE-EtOAc suppresses the IL-6 and TNF-α secretion from peritoneal macrophages revealing the anti-inflammatory effects ex vivo. Furthermore, ABE-EtOAc can regulate the Th1/Th2 immune reaction via up-regulating IL-2 and down-regulating IL-4 and IL-5. Moreover, ABE-EtOAc reduces the anti-OVA IgE production, increased IFN-γ secretion and decreased IL-4 and IL-5 secretion in OVA immunized BALB/c mice. The ABE-EtOAc affects allergic reaction through balances of Th1/Th2 immune responses. To explore the anti-allergic components, the ABE-EtOAc-40~100% EtOAc/Hex fraction was isolated. There are 22 compounds isolated from adlay bran. Among them, 16 compounds were first isolated from the adlay including 12 flavonoids, tangeretin (FL1)、new flavonoid (FL2)、formononetin (FL3)、davidigenin (FL4)、homoeriodictyol (FL5)、hesperetin (FL6)、isoliquiritigenin (FL8)、liquiritigenin (FL9)、3,5,6,7,8,3'',4''-heptamethoxyflavone (FL10)、chrysoeriol (FL11)、3,5,7,3'',4''-pentamethoxyflavone (FL12)和3,5,7,4''-tetramethoxyflavone (FL13), and two chromones, 5-hydroxy-7-methoxychromone (C1), 5,7-dihydroxychromone (C2) and a phenolic ester, ethyl ferulate (PE1), and one steroids, 6α-hydroxystigmast-4-en-3-one (S1). Furthermore, a high performance liquid chromatography - mass spectrometer (HPLC/MS) method was successfully applied to screen flavonoids in adlay extracts. In conclusion, various flavonoids are found in the active fractions of adlay bran. They are suggested to correlate with the allergic response of mast cell in early phase and the inflammatory reaction of macrophage in late phase. Moreover, ABE-EtOAc can balance the Th1/Th2 immune responses. As a consequence the ethanolic extracts of adlay bran strengthen the modulation of immune system and alleviate allergic symptoms