發芽米萃取物之免疫調節活性與抗氧化性之研究 王秀育、張基郁

發芽米萃取物之免疫調節活性與抗氧化性之研究 王秀育、張基郁

E-mail: [email protected]

摘 要

發芽米(GBR)含有各種人體所需的營養素，具有多種機能性功效，例如: GABA、維生素E、食物纖維、抗氧化物質，以及 肌醇、長鏈脂肪酸等多種具有生物活性之物質。本研究以不同溶劑(磷酸緩衝溶液、鹼溶液及乙醇)進行萃取，探討不同溶 劑萃取所得之發芽米萃取物之抗氧化活性、對人類白血病細胞U937生長之抑制能力及免疫調節活性。 在抗氧化性部分，

利用磷酸緩衝溶液萃取所得之發芽米萃取物(PGBR)在DPPH自由基清除能力、還原力及超氧陰離子清除能力方面顯著優於 利用鹼溶液(AGBR)和乙醇(EGBR)溶液萃取所得之發芽米萃取物；而在Trolox當量抗氧化能力與亞鐵離子螯合能力方面則是 以EGBR之效果為最佳。 在細胞與免疫調節活性部分，以PGBR、AGBR及EGBR刺激條件培養液1天，在濃度為800 μg/mL，各樣品對U937細胞生長抑制率依序為49.83、57.39及51.23 %，相同濃度刺激條件培養液3天，其抑制率依序 為58.51、62.23及60.77 %，以AGBR抑制效果為最高。 以PGBR、AGBR及EGBR分別與單核細胞條件培養液共同培養1天 及3天，利用細胞激素分泌的測定探討發芽米萃取物之免疫活性，在濃度1000 μg/mL下，培養1天之條件培養液中所 含IL-1β分別為1293.17、1572.93及2094.71 pg/mL、培養3天分別為1797.56、1861.63及2613.95 pg/mL，由結果得知，IL-1 β之分泌量有隨著培養天數的增加而上升，樣品刺激單核細胞條件培養液分泌IL-1β之效果，以EGBR為最佳。利用不同 樣品刺激條件培養液後之TNF-α含量，刺激1天且在濃度為1000 μg/mL下，其條件培養液中TNF-α含量分別為1950.38

、546.30及1365.02 pg/mL，培養3天後之TNF-α含量分別為2346.54、914.82及1881.10 pg/mL，結果可知，經由PGBR刺激 後之條件培養液所含的TNF-α為最多。利用不同樣品刺激條件培養液後之IFN-γ含量，在分別經由PGBR、AGBR 及EGBR刺激1天，且在濃度為1000μg/mL下，其IFN-γ含量分別為853.30、911.75及893.46 pg/mL，培養3天分別

為1461.14、1135.47及907.91 pg/mL，由結果可知，經由PGBR刺激後之條件培養液所含的IFN-γ為最多。以不同發芽米萃 取物刺激單核細胞1天及3天後，在樣品濃度為100 μg/mL下，以PGBR、AGBR及EGBR刺激單核細胞1天後，條件培養液 中其NO含量分別為0.54、0.46及0.52 μM/mL；培養3天後，NO含量依序為0.80、0.68及0.66 μM/mL。 不同發芽米萃取 物與單核細胞分別培養1天及3天後，利用MTT法分析單核細胞生長比率，以PGBR、AGBR及EGBR與單核細胞培養1天後

，在濃度為100 μg/mL時，皆發現其具有促進單核細胞生長之效果，其細胞生長率約為1.02~1.04；將發芽米萃取物與單 核細胞培養3天後，在濃度為100 μg/mL時，PGBR及AGBR卻有抑制單核球細胞增生之效果，在500 μg/mL濃度下，其萃 取物更具有毒殺單核細胞生長之影響，其中以PGBR毒殺效果最為顯著。

關鍵詞 : 發芽米、免疫調節活性、抗氧化、細胞激素、人類白血血病細胞U937、人類單核球細胞 目錄

封面內頁 簽名頁 中文摘要 iii 英文摘要 v 誌謝 vii 目錄 ix 圖目錄 xiii 表目錄 xv 1. 緒論 1 2. 研究目的 4 3. 文獻回顧 5 3.1 米之 種類 5 3.1.1 稻米 5 3.1.2 糙米 5 3.1.3 糙米的機能成分及功用 6 3.1.4 發芽糙米及機能成分 7 3.1.5 糙米之一般組成分 9 3.2自 由基與抗氧化物質 9 3.3 免疫系統 10 3.3.1 先天或非專一性免疫防禦機制 10 3.3.2 後天或專一性免疫防禦機制 11 3.3.3 淋巴 類的細胞 12 3.3.3.1 T細胞 12 3.3.3.2 B細胞 12 3.3.3.3 單核球/巨噬細胞 13 3.3.3.4 自然殺手細胞 14 3.4 細胞激素 14 3.4.1腫 瘤壞死因子(Tumor necrosis factors, TNFs) 15 3.4.2 介白素(Interleukine, ILs) 16 3.4.3 干擾素(Interferons, IFNs) 17 3.4.4 一氧化 氮(Nitric oxide, NO) 18 3.5人類白血病細胞株U937 18 4. 材料與方法 22 4.1 實驗材料 22 4.1.1 發芽米 22 4.1.2 實驗細胞 22 4.1.3 實驗藥品 22 4.1.4 儀器設備 24 4.2 實驗方法 24 4.2.1 實驗流程 24 4.2.2 發芽米萃取物之萃取法 24 4.2.3 基本成分分析 25 4.3 發芽米萃取物之抗氧化能力測定 27 4.4 膠體過濾層析法 29 4.5 經由間接模式對U937細胞抑制作用之實驗 30 4.5.1 U937細胞之冷凍保存方法 30 4.5.2 U937細胞之解凍方法 30 4.5.3 U937細胞之培養方法 31 4.5.4 活細胞計數法 31 4.5.5 欲測 試樣品之前處理 31 4.5.6 人類單核細胞條件培養液 (MNC-CM）之製備 32 4.5.7 Polymyxin B (PMB) 試驗 33 4.5.8 樣品-單核 細胞條件培養液（MNC-CM）間接抑制U937細胞 33 4.6 評估U937細胞株之分化 -細胞表面抗原CD11b及CD14之測 34 4.7 樣品-單核細胞條件培養液細胞激素之含量測定 35 4.8 測定經由樣品刺激後人類單核細胞之生長比例 37 5. 實驗結果 39 5.1 發芽米組成分分析 39 5.2 發芽米萃取物之電泳分析 39 5.3 發芽米萃取物之抗氧化分析 42 5.3.1 亞鐵離子螯合能力 42 5.3.2 DPPH自由基清除能力 44 5.3.3 Trolox當量抗氧化能力 46 5.3.3 Trolox當量抗氧化能力 46 5.3.4 超氧陰離子清除能力 46 5.3.5 還原力 49 5.4 發芽米不同萃取物間接抑制U937細胞生長之實驗 52 5.5 U937細胞表面抗原CD11b及CD14之測定 55 5.6 條件 培養液中細胞激素含量之測定 57 5.6.1 條件培養液中細胞激素Interleukin-1β (IL-1β)之含量 57 5.6.2 條件培養液中細胞激 素Tumor necrosis factor-α (TNF-α)之含 60 5.6.3 條件培養液中細胞激素Interferon-γ (IFN-γ)之含量 60 5.6.4 條件培養液中 一氧化氮 (NO)之含量 65 5.7 不同發芽米萃取物促進人類單核細胞之生長效果 68 5.8 發芽米萃取物(PGBR) 經膠體過濾層析

法所得之區分物之抗 68 5.9 發芽米萃取物(PGBR)經膠體過濾層析法所得之區分物對單核 72 5.10 結果討論 77 5.10.1發芽米 萃取物之抗氧化分析 77 5.10.2 發芽米萃取物抑制U937細胞生長及免疫調節之效果 78 5.10.3發芽米萃取物(PGBR)經膠體過 濾層析後劃分物之抗氧化活 80 5.11 結論 82 參考文獻 83 圖目錄 圖3. 1本研究之實驗總流程圖 21 圖5. 1發芽米萃取物之電泳 分析 41 圖5. 2發芽米萃取物(PGBR、AGBR及EGBR)之亞鐵離子螯合能力 43 圖5. 3 發芽米萃取物(AGBR、AGBR及EGBR) 之DPPH自由基清能力 45 圖5. 4 Trolox 之標準曲線及線性迴歸之R-square ?(0.9935) 47 圖5. 5發芽米萃取物(AGBR、AGBR 及EGBR)之Trolox當量抗氧能力 48 圖5. 6發芽米萃取物(AGBR、AGBR及EGBR)之還原力 51 圖5. 7 U937細胞經間接作用後 細胞之CD11b與 56 圖5. 8不同發芽米萃取物刺激人類單核細胞條件培養液1天所產生之IL-1β含量變化 58 圖5. 9不同發芽米 萃取物刺激人類單核細胞條件培養液3天所產生之IL-1β含量變化 59 圖5. 10不同發芽米萃取物刺激人類單核細胞條件培養 液1天所產生之TNF-α含量變化 61 圖5. 11不同發芽米萃取物刺激人類單核細胞條件培養液3天所產生之TNF-α含量變化 62 圖5. 12不同發芽米萃取物刺激人類單核細胞條件培養液1天所產生之IFN-γ含量變化 63 圖5. 13不同發芽米萃取物刺激人 類單核細胞條件培養液3天所產生之IFN-γ含量變化 64 圖5. 14不同發芽米萃取物刺激人類單胞1天後細胞一氧化氮產出之 影響 66 圖5. 15不同發芽米萃取物刺激人類單胞3天後細胞一氧化氮產出之影響 67 圖5. 16不同發芽米萃取物刺激人類單核 球細胞1天後之細胞 69 不同發圖5. 17芽米萃取物刺激人類單核球細胞3天後之細胞 70 圖5. 18發芽米萃取物PGBR

之Sephadex G-25膠體過濾層析圖 71 表目錄 表5. 1發芽米一般組成分 40 表5. 2發芽米萃取物(PGBR、AGBR及EGBR)之超氧 陰離子清除能力 50 表5. 3發芽米不同萃取物刺激人類單核細胞條件培養液1天後 53 表5. 4發芽米不同萃取物刺激人類單核 細胞條件培養液3天後 54 表5. 5 PGBR萃取物經膠體過濾層析所得之區分物之抗氧化性及刺激人類單核細胞條件培養液1天 和3天後對U937細胞生長之抑制率 73 表5. 6以PGBR區分物刺激單核球細胞1天對其細胞生長率、NO及細胞激素分泌之影 響 74 表5. 7以PGBR區分物刺激單核球細胞3天對其細胞生長率、NO及細胞激素分泌之影響 75

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