臺灣博碩士論文加值系統

牛樟芝在台灣是一種傳統的中國草藥，已經被利用來治療多種疾病。不過，牛樟芝香氣成分的免疫活化功能目前還未有相關的研究。本研究的目的在於分離及鑑定牛樟芝精油的主要香氣成分及探討此成分的化學結構及生物功能之間的關連性。利用氣相層析/質譜 (GC/MS) 分析鑑定出牛樟芝精油主要的香氣成份為gamma-dodecalactone (gamma-DDL)。透過氣相層析和流式細胞儀分析 (flow cytomertry)，發現gamma-DDL可以進入細胞並誘導臍帶血單核球細胞表徵改變及活化自然殺手細胞(NK cells)。同時我們也發現gamma-DDL可以轉變成開環形式的4-hydroxydodecanoic acid後進而活化自然殺手細胞。利用對掌性衍生劑 (chiral derivatising agents) 及高壓液體層析 (HPLC) 分離具有活性結構的gamma-DDL。結果只有R結構的gamma-DDL具有活化自然殺手細胞的能力。此外，也鑑定出存在於牛樟芝中的gamma-DDL是屬於R結構的。最後，我們觀察到在自然殺手細胞中gamma-DDL可以快速的活化介白素-2 (interferon-2) 基因的表現並且透過介白素-2自體分泌循環機制 (autocrine) 活化自然殺手細胞。除了自然殺手細胞外，gamma-DDL也可能透過不同的活化機制去誘導T細胞、B細胞、單核球細胞、樹突狀細胞的活化。

In Taiwan, Antrodia camphorata (A. camphorata) is a traditional Chinese medicine and it has been utilized to treat a wide variety of diseases. However, the immuno-activating function of the flavor components from A. camphorata had not yet been investigated. In the present work, the volatile oil of A. camphorata was analyzed with the aim of isolating and identifying the major flavor compound and investigating the relationship between the chemical structure and biological functions of compound. The major component of A. camphorata volatile oil was identified as gamma-dodecalactone (gamma-DDL) by GC/MS analysis. By GC and flow cytomertry analysis, suggested that gamma-DDL entered the cells for the induction of human umbilical cord blood mononuclear cells (hUCB-MNCs) phenotypic changes and activation of NK cells. gamma-DDL was found to convert its open ring form: 4-hydroxydodecanoic acid for the activation of NK cells. The active configuration of gamma-DDL was deduced with the help of chiral derivatising agents (CDAs) and HPLC analysis. Only the R-configurated form of gamma-DDL was able to mediate NK cell activation. Furthermore, the configuration of gamma-DDL was identified in A. camphorata also as R-form. Finally, gamma-DDL induced rapid activation of interferon-2 (IL-2) gene in NK cells and the IL-2 autocrine system to activate NK cells. gamma-DDL also induced the activation of T cells, B cells, monocytes and DCs, but there might be different mechanisms of activation.

謝 誌 i
中 文 摘 要 ⅳ
Abstract ⅴ
Abbreviations ⅹ
List of Figures xi ⅹⅱ
List of Tables ⅹⅳ
1. Introduction 1
1-1.1 Antodia camphorata 1
1-1.2 The components of A. camphorata 2
1-1.3 The components of volatile oil from plants and herbs 5
1-2.1 Isomers 9
1-2.2 Enantiomers : chiral compounds 11
1-2.3 gamma-Dodoecalactone (gamma-DDL) 13
1-3.1 Screening platform of immune system 15
1-3.2 The activation of immune cells 17
1-3.3 NK cells 21
1-3.4 IL-2 and IL-2 receptor 23
2. Materials and methods 25
2.1 Materials 25
2.2 Methods 27
2.2-1.Analysis of gamma-DDL from the volatile oil by FID-GC 27
2.2-2 Identification of gamma-DDL from volatile oil by GC/MS 27
2.2-3 Measurement of the hUCB and hPB phenotypic changes by flow cytometric analysis 28
2.2-4. Determination of cell viability by WST assay 29
2.2-5. Detection of NK cells activation after treatment of g-DDL 29
2.2-6. Determination of gamma-DDL concentration in medium and cells by GC analysis 30
2.2-7 Determination of immune cells activation by Q-PCR assay 31
2.2-8 Determination of IL-2 concentration in medium by cytokine ELISA 33
2.2-9 Synthesis of 4-hydroxydodecanoic acid (gamma-DDL(OR)) 33
2.2-10 Chiral separation by chiral derivatising agents (CDAs) method 34
2.2-11 Identification gamma-DDL and gamma-DDL(OR) extracted from A. camphorata by HPLC 36
3. Rationale and study aim 37
4. Results 39
4-1.Identification of the major compound (gamma-DDL) from volatile oil of A. camphorata 39
4-2. Effect of pure gamma-DDL and volatile oil of A. camphorata on the phenotypic changes of hUCB 43
4-3. Stimulation of CD56 marker expression of hUCB-NK cells 47
4-4. Induction of CD69 gene expression of CD3, CD14, CD19, CD56 and CD83 cell populations by gamma-DDL 49
4-5. Comparison of the CD69 marker activation between hUCB-NK cell and hPB-NK cells treated with gamma-DDL 51
4-6. Intracellular or extracellular activation of gamma-DDL on hPBMCs and hPB-NK cells 55
4-7. Conversion of gamma-DDL into 4-hydroxydodecanoic acid (gamma-DDL(OR)) in NK cells 59
4-8 Deduction of major configuration of gamma-DDL to activate NK cells 64
4-9 Determination of the configuration of gamma-DDL from A.camphorata 67
4-10. Induction of gene expression of NK cells activation by gamma-DDL 69
4-11. gamma-DDL enhanced CD69, FasL and granzyme B gene expression through IL-2 autocrine system 72
4-12. gamma-DDL or gamma-DDL(OR) can activate different immune cell populations through the induction of CD69 expression. 74
5. Discussions 76
6. Conclusion 81
References 82
Research publications………………………………………………….90

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