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研究生:高雅玲
研究生(外文):YA-LING KAO
論文名稱:市售靈芝產品品管指標之研究
論文名稱(外文):Study on Quality Control Markers in Commercial Ganoderma lucidum Products
指導教授:許輔許輔引用關係
口試委員:周志輝潘敏雄繆希椿
口試日期:2014-07-11
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:園藝暨景觀學系
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:139
中文關鍵詞:(13)-β-D-葡聚醣LZ-8靈芝多醣活性靈芝菌絲體 
外文關鍵詞:(13)-β-D-glucanLZ-8ImmunomodulationG. lucidum mycelia
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  本論文目的為釐清總糖、(1,3)-β-D-葡聚醣或 LZ-8 含量是否可做為代表產品活性之指標,並找到判別產品適當的活性檢測濃度範圍。第一部分測量各產品總糖含量,並對活性多醣 (1,3)-β-D-葡聚醣,以專一性之 aniline blue 螢光呈色法分析其含量。結果發現樣品間此兩種成分含量差異很大。第二部分進行活性試驗,調整樣品總糖濃度或 (1,3)-β-D-葡聚醣濃度一致時,各樣品活化小鼠腹腔巨噬細胞和巨噬細胞株 RAW 264.7 分泌 TNF-α 和 IL-6 的效果卻有顯著差異。因此,總糖與 (1,3)-β-D-葡聚醣含量不宜做為評估市售靈芝產品的活性指標。接著,將各市售靈芝產品以相同稀釋倍率與細胞共培養,結果顯示樣品能顯著活化小鼠腹腔巨噬細胞和 RAW 264.7 分泌 TNF-α 及 IL-6。進一步測試樣品稀釋倍率與活性的相關性,發現稀釋範圍介於 300-2500 μg/mL (30-250 mg 市售靈芝產品溶於 100 mL 磷酸鹽緩衝生理鹽水),可以有效辨別各樣品是否具有活性。
  在活化小鼠脾臟細胞的部分,市售靈芝產品皆無法顯著提升 TH1 和 TH2相關細胞激素 IFN-γ、IL-2 和 IL-4 的分泌量。此外,以膠體電泳及西方轉漬法分析,皆證實市售靈芝產品中靈芝蛋白 LZ-8 含量極低。第三部分欲以高效液相層析儀 (HPLC) 分析菌絲體中 LZ-8 含量。結果顯示,無論是否添加標準品 LZ-8 於菌絲體溶液中,皆無法偵測到 LZ-8 的存在。將菌絲體溶液通過玻璃纖維去除多醣,再添加標準品 LZ-8 後,以 HPLC 分析則可測到添加的標準品 LZ-8。因此,推測菌絲體中影響 HPLC 分析 LZ-8 含量的物質可能為多醣類。進一步以 exo-(1,3)-β-D-glucanase 和 endo-(1,3)-β-D-glucanase 切除 (1,3)-β-D-葡聚醣後,仍無法分析到菌絲體中的 LZ-8,所以僅能確定干擾分析的物質並非 (1,3)-β-D-葡聚醣,但該物質實際結構仍有待釐清。
  綜合上述實驗結果,因總糖、(1,3)-β-D-葡聚醣和 LZ-8 皆不適合做為靈芝產品品管指標,故建議應直接分析樣品活性,可選擇巨噬細胞株 RAW 264.7 與樣品共培養後,測量 TNF-α 和 IL-6 分泌量,評估產品之活性優劣,適當產品範圍為 30-250 mg 市售靈芝產品溶於 100 mL 磷酸鹽緩衝生理鹽水,此方法可用於靈芝產品製程品管,以確保產品品質一致性。

Ganoderma lucidum, a medicinal fungi, has been used in Traditional Chinese Medicine (TCM) for its health promoting constituents, including polysaccharides, proteins, triterpenes, sterols, and fatty acids. However, G. lucidum contains various and complex bioactive compounds. It was difficult to distinguish the bioactivity of commercial G. lucidum products from specific component. Most products were claimed on the basis of their total sugar or crude polysaccharides contents, but immunomodulatory protein (LZ-8) and (1,3)-β-D-glucan, were not shown in the nutrition facts. In this study, we decided to examine whether the contents of total sugar or (1,3)-β-D-glucan could be used as the quality and functional indicators, and what would be the appropriate concentrates for the evaluation in product activities. First, we determined content of total sugar and content of (1,3)-β-D-glucan in commercial products with phenol-sulfuric acid method and fluorometric method. Our results showed that there were significant differences on the contents of total sugar and (1,3)-β-D-glucan in the eight commercial products. Samples with the same contends of total sugar or (1,3)-β-D-glucan had different capability to induce secreation of TNF-α and IL-6 in murine macrophages and RAW 264.7. Therefore, the quantity of total sugar and (1,3)-β-D-glucan might not be an appropriate functional indicators. Our results also revealed that the approach of diluting the commercial product solutions which were cultured with murine macrophages to 30 to 250 mg commercial product in 100 mL PBS could be used to distinguish bioactivities of commercial products. Further, the eight commercial products could not increase production of cytokine (IFN-γ, IL-2, and IL-4) by murine splenocytes. In addition, SDS-PAGE and Western blotting analysis of these samples showed that commercial G. lucidum products could rarely include immunomodulatory protein LZ-8. In the third part, mycelia from G. lucidum were lysed by ultrasonic reactor and analyzed for the content of LZ-8 by HPLC. LZ-8 peak have been observed as lysis solution filtered through glass fiber membranes and added 100 μg/mL standard LZ-8 to the solution. As shown above, we assumed that the component which interfered the HPLC analysis of LZ-8 should be polysaccharide. Sample solution was further treated with exo-(1,3)-β-D-glucanase and endo-(1,3)-β-D-glucanase in order to remove the potented interfering substance, but no LZ-8 peak have been observed. As a result, it was inferred that the interfering substance might be polysaccharides but not (1,3)-β-D-glucans. In conclusion, the content of total sugar, (1,3)-β-D-glucan, and LZ-8 could not be the functional indicators. The appropriate dosages of sample cultured with murine macrophages were 30 to 250 mg of commercial product in 100 mL PBS and could be used to assess product activities. In the third part, we inferred that polysaccharids might be the main factor to interfer the HPLC analysis of the free form of LZ-8.

誌謝 II
摘要 III
Abstract V
總目錄 VII
圖目錄 X
表目錄 XIII
壹、前人研究 1
一、靈芝簡介 1
二、靈芝中的活性成分 2
2.1. 靈芝三萜類 3
2.2. 靈芝免疫調節蛋白 Ling Zhi-8 (LZ-8) 4
2.2.1 LZ-8 簡介 5
2.2.2 LZ-8 免疫之調節活性 6
2.2.3 重組 LZ-8 蛋白之免疫調節活性 8
2.3. 靈芝多醣 10
2.3.1 靈芝多醣簡介 11
2.3.2 靈芝多醣之免疫調節活性 13
2.4 靈芝蛋白與靈芝多醣免疫調節功能之差異 17
三、靈芝總糖和 (1,3)-β-D-葡聚醣含量的分析方法 19
四、研究動機與架構 22
貳、材料與方法 24
一、市售靈芝產品總糖和 (1,3)-β-D-葡聚醣含量測定 24
1.1. 總糖含量測定-酚硫酸法 (phenol-sulfuric acid method) 24
1.2. (1,3)-β-D-葡聚醣的定量法-aniline blue 螢光呈色法 24
二、細胞培養 26
2.1. 巨噬細胞株 RAW 264.7 細胞培養 26
2.2. BALB/c 小鼠腹腔巨噬細胞培養 28
2.3. BALB/c 小鼠脾臟細胞培養 30
三、酵素免疫連結分析 32
3.1. 細胞激素之測定 32
3.2. 抗體 IgM 之測定 34
四、小鼠腹腔巨噬細胞產生一氧化碳分析 36
五、變性膠體電泳分析 37
5.1. 鑄膠 37
5.2. 電泳操作方法 38
5.3. 膠片染色與褪色 39
六、醣蛋白染色分析 40
七、西方轉漬分析 41
八、重組 LZ-8 蛋白質之純化 43
九、菌絲體樣品製備 46
十、以酵素處理菌絲體破菌溶液 46
十一、以高效液相層析儀 (HPLC) 分析樣品 LZ-8 含量 47
十二、統計分析 48
參、研究結果 49
一、市售靈芝產品總糖及 (1,3)-β-D-葡聚醣的含量 49
二、市售靈芝產品的活性指標 50
2.1 以總糖作為市售靈芝產品的活性成分規格標準 50
2.1.1 市售靈芝產品活化小鼠腹腔巨噬細胞效果 50
2.1.2市售靈芝產品活化巨噬細胞株 RAW 264.7 效果 51
2.1.3 市售靈芝產品活化小鼠脾細胞效果 52
2.2 以 (1,3)-β-D-葡聚醣作為市售靈芝產品的活性成分規格標準 53
2.2.1 市售靈芝產品活化小鼠腹腔巨噬細胞效果 53
2.2.2 市售靈芝產品活化巨噬細胞株 RAW 264.7 效果 54
2.2.3市售靈芝產品活化小鼠脾細胞效果 55
2.3 以樣品活性作為市售靈芝產品之規格標準 56
2.3.1 市售靈芝產品活化小鼠腹腔巨噬細胞效果 56
2.3.2 市售靈芝產品活化巨噬細胞株 RAW 264.7 效果 57
2.3.3 市售靈芝產品活化小鼠脾細胞效果 58
2.4 市售靈芝產品 B、E 和 H 活化小鼠腹腔巨噬細胞趨勢 58
三、靈芝菌絲體中 LZ8 蛋白分析方法的研究 60
肆、討論 62
一、市售產品中總糖和 (1,3)-β-D-葡聚醣含量差異甚大且並無 LZ-8 存在 62
二、活性指標 63
2.1 總糖和 (1,3)-β-D-葡聚醣含量無法作為活化小鼠腹腔巨噬細胞之活性指標 63
2.2 總糖和 (1,3)-β-D-葡聚醣含量無法作為活化巨噬細胞株 RAW 264.7 之活性指標 66
2.3 總糖和 (1,3)-β-D-葡聚醣含量無法作為活化小鼠脾臟細胞之活性指標 67
2.4 建議檢測活性之市售靈芝產品稀釋濃度為 300-2,500 μg/mL 70
三、靈芝菌絲體中含有會干擾高效液相層析儀 (HPLC) 分析 LZ-8 含量之物質 71
伍、結論 73
陸、參考文獻 75
附錄 137


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