山藥黏質多醣抗氧化與免疫調節活性之研究

Study on the Activities of the Antioxidant and Immune Regulations of Yam Tuber Mucilage

劉皓宇(Hao-Yu Liu)

指導教授 ： 侯文琪 ； 商惠芳

臺北醫學大學/藥學院/生藥學研究所/碩士(2004年)

摘要

中文摘要 山藥自古以來，無論在東、西方均被視為有益健康的食品或藥品，所以山藥的各種營養成分非常值得被深入研究與開發。本研究以省產三種不同品種山藥 (Dioscorea spp.)為材料，包含台農一號（TN1）、台農二號（TN2）、名間長紅山藥（MJ），抽取並經過部分純化（包括SDS沉澱及鈣離子沉澱去除）後所得黏質多醣（Mucilage）來作抗氧化及免疫調節活性之研究。在評估山藥黏質多醣抗氧化活性時，所測試的項目包括DPPH自由基清除、氫氧自由基清除、超氧自由基清除、抗低密度脂蛋白氧化，並以ESR進行氫氧自由基清除實驗。實驗結果顯示，不同品種山藥之黏質多醣均具有抗氧化能力，而純化過後黏質多醣之抗氧化能力較佳。評估山藥黏質多醣的免疫調節功效時，則使用動物模式（BALB/c小白鼠）進行測試，分別給予三種純化過之黏質多醣和香菇多醣（作陽性對照）來作比較。本研究以流式細胞儀（flow cytometry）作為測試非特異性免疫活性（innate immunity）之技術平台，其中包含淋巴細胞亞群的分佈、顆粒性白血球和單核球細胞的吞噬作用、自然殺手細胞活性的分析。同時亦測試山藥黏質多醣對老鼠腹腔巨噬細胞與巨噬細胞株（RAW264.7）體外吞噬作用之影響。而特異性免疫活性之評估則以OVA抗原（ovalbumin）免疫老鼠後，利用ELISA分析專一性抗體產生濃度的高低；並以MTT定量法測試老鼠脾臟細胞受OVA刺激後的分裂能力。結果顯示在第一週時，TN2及MJ組老鼠周邊血液中T cell（CD3＋）總數顯著增加（p＜0.01），而其中增加的又以輔助T細胞（T helper cell, CD4＋）（p＜0.05）為最顯著，Cytotoxic T cell (CD8＋)的比率則無顯著性變化。TN2及MJ組的B cell (CD19＋)比率則相對顯著減少（p＜0.01）。而第三週時僅MJ組的T cell（CD3＋）總數有顯著性增加（p＜0.01），B cell (CD19＋)比率有相對顯著減少（p＜0.01）。而BALB/c小白鼠周邊血液中單核球細胞的吞噬能力在第一週時MJ組（p＜0.05）、TN1和TN2組（p＜0.01）有顯著性增加，而顆粒性白血球的吞噬能力則皆無明顯變化。但至第三週時，四種樣品皆顯著促進顆粒性白血球之吞噬能力；而單核球細胞之吞噬能力除TN2組外其餘三種樣品皆呈顯著增加。而四種樣品在試管中亦均能促進老鼠腹腔巨噬細胞與巨噬細胞株（RAW264.7）之吞噬能力。自然殺手細胞毒殺能力的測定結果發現：Effector cell和Target cell比例從5：1至40：1時，MJ組自然殺手細胞毒殺能力均比對照組有顯著增加（p＜0.01），而其他二組（TN1、TN2）和對照組比較，自然殺手細胞毒殺能力雖有增加但較不明顯。以OVA抗原免疫BALB/c小白鼠後，結果發現香菇多醣和三種山藥黏質多醣皆可促進專一性抗OVA抗體IgG和IgM之生成，而其中以TN1和香菇多醣效果較顯著。比較OVA免疫後老鼠的脾臟細胞在OVA抗原刺激後之分裂能力，亦發現香菇多醣和三種山藥黏質多醣組老鼠脾臟細胞增生的能力均較對照組強。

關鍵字

黏質多醣 ； 台農一號山藥 ； 台農二號山藥 ； 名間長紅山藥 ； 抗氧化 ； 免疫調節 ； 流式細胞儀

並列摘要

Abstract The yam（Dioscorea spp）has been widely used as food and remedy in China and Western countries. It has been reported to have a lot of bioactive substances for promotion of health and longevity, and is worthy to be further studied. In this study, the crude extracts of yam tuber of Dioscorea alata L.cv.Tainong 1 (Tainong no.1 yam；TN1)、Dioscorea alata L.cv.Tainong 2 (Tainong no.2 yam；TN2) and D. alata L.var. purpurea (Roxb.) Ming.Jen（MJ）were precipitated with SDS and removal of CaCl2 precipitates to get the partially purified mucilage polysaccharides. For evaluating the antioxidant effects of the mucilage polysaccharide, a series of in vitro tests, including DPPH radical scavenging activity assay, hydroxyl radical scavenging activity assay, superoxide radicals scavenging activity, anti-human low density lipoprotein (LDL) oxidation test, and hydroxyl radical scavenging activity assay by electron spin resonance（ESR）spectrometry were detected. The results demonstrated that different species of yams exhibited different antioxidant ability, and the purification process could increase the antioxidant activity of the mucilage polysaccharide. Taken together, our results suggest that mucilage polysaccharides of the yam tuber might play an important role on antiradicals and antioxidants. For determining and comparing the immunomodulating effects of the yam mucilage polysaccharide of the three species, the innate and adaptive immunity were evaluated on the BALB/c mice. BALB/c mice were assigned to five groups：group TN1, TN2 and MJ were daily, orally administrated with 0.5 ml mucilage polysaccharide （10 mg / ml） separately for 5 weeks. Group Lentinan（Len） was the positive control group and fed with purified Lentinan（0.05 mg / ml）. Blood samples were drawn from the retroorbital sinus on day 7 and 21, and the flow cytometry was used to analyze the lymphocyte subpopulation, phagocytosis of granulocyte and monocyte. In addition, the stimulating indexes of mucilage polysaccharide on the phagocytosis of peritoneal macrophages and the RAW264.7 cell line were also determined by in vitro assay. Our results demonstrated that the ratio of T helper cells in the peripheral blood of groups TN1, TN2 and MJ increased significantly, especially Group MJ. All three kinds of mucilage polysaccharide could enhance the phagocytic activity of granulocyte, monocytes and macrophages both in vivo and in vitro tests. On day 36, the mice were sacrificed, and the splenocytes were harvested for analysis of Nature killer cell cytotoxicity. The natural killer cells of Group MJ had better cytotoxic activity than the other samples. In the evaluating of specific immunity, we found that the production of specific anti-OVA antibody and proliferation of splenocytes after specifically stimulating with OVA were also enhanced on mucilage groups. In summary, oral administration of yam tuber mucilage for 5 weeks may enhance the innate and adaptive immunity.

並列關鍵字

Mucilage ； TN1 ； TN2 ； MJ ； ANTIOXIDANT ； IMMUNEMODULATION ； flow cytometry

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