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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李昆達 | |
dc.contributor.author | Cheng-Han Chung | en |
dc.contributor.author | 鍾承翰 | zh_TW |
dc.date.accessioned | 2021-06-08T00:54:29Z | - |
dc.date.copyright | 2015-08-11 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-03-25 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18196 | - |
dc.description.abstract | 牛樟芝 (Antrodia cinnanomea) 是台灣特有的藥用真菌,由於其子實體是寄生在台灣特有樹種牛樟樹 (Cinnamomum kanehirai) 樹幹內壁而命名。本研究同時針對牛樟芝子實體抗癌活性與代謝物進行探討,進而建立生物與化學品管之方法,並確認液態醱酵培養法中具有抗癌效果的活性成份及其抑癌機制,尋找可能的品管指標與抗癌新藥成分。為了研究不同批次牛樟芝子實體的抗癌活性並建立生物活性品管標準,我們收集三批次之牛樟芝子實體進行分析。結果顯示,三批次之牛樟芝子實體酒萃物對於肺癌 (A549)、肝癌 (HepG2) 與乳癌 (MCF-7) 皆有抑癌活性,我們建議可根據牛樟芝子實體酒萃物對於癌細胞株IC50 值做為品管之標準。本研究進一步探討牛樟芝子實體酒精萃取物的抑癌機制,利用牛樟芝酒精萃取物處理A549細胞後,經由Annexin V/PI檢測、caspase-3和caspase-9的活性分析與cyclin D1蛋白質分析,證明牛樟芝子實體酒精萃取物能誘導癌細胞的凋亡,我們建議caspase-3和caspase-9活性上升與cyclin D1蛋白質表現量下降做為蛋白質品管指標。我們進一步進行JK細胞蛋白質體反應分析,發現不同批次之牛樟芝子實體生物反應相似度均大於0.9,我們建議Jurkat細胞蛋白質反應相似度可做為生物品管標準。此外,我們根據不同來源牛樟芝子實體的代謝物指紋圖譜,運用新方法建立不同來源牛樟芝的子實體化學品管方法:我們利用phytomics similarity index (PSI)方法,分析牛樟芝不同菌株與培養於不同基質的子實體其代謝物的差異。結果顯示,不同菌株與培養於不同基質的牛樟芝子實體均含有8個主要的指標成份,難以僅根據這些指標成份存在分辨其培養方式的差異。在我們的研究中使用PSI分數來評估牛樟芝子實體的代謝物相似度,不同的牛樟芝菌株與生長在不同的培養基質之子實體,其PSI分數有所差異。我們的結論是,PSI分析對不同來源的牛樟芝子實體具有良好的辦識度。最後,我們研究牛樟芝液態醱酵中的抗癌活性成份,結果發現,醱酵濾液中的2,3-dimethoxy-5 -methyl-1,4-benzoquinone (Coenzyme Q 0; CoQ0) 具有抗癌活性。 CoQ0能抑制 A549、HepG2和SW480三株癌細胞生長。此外,CoQ0誘發A549細胞產生reactive oxygen species(ROS)與細胞凋亡,此作用可被抗氧化劑抗壞血酸所抑制。我們推論,牛樟芝液態醱酵濾液中CoQ0可做為牛樟芝醱酵濾液抗癌活性的指標成份,並能誘導肺癌A549細胞產生ROS導致細胞凋亡而達到抑癌效果。 | zh_TW |
dc.description.abstract | Antrodia cinnanomea is a unique medical mushroom in Taiwan. The fruiting body of A. cinnamomea is parasite on the inner cavity of the endemic species Cinnamomum kanehirai. In this study, we research on the anti-cancer activity and metabolites of A. cinnamomea fruiting bodies, and then, we setup the biological and chemical quality control methods. We also tried to find the anti-cancer compound and the mechanism in A. cinnamomea submerged culture. The anti-cancer compound might be the bioactive marker for quality control and novel chemotherapeutic agent for cancer treatment. To study the anti-cancer effect and establish the biological quality control method from different batches of A. cinnamomea fruiting bodies, we collected three batches of A. cinnamomea fruiting bodies in the study. The resulted indicated that three batches of fruiting bodies can inhibit the cell viability of lung cancer A549 cells, liver cancer HepG2 cells and breast cancer MCF-7 cells. We suggested that the IC50 values could be used in quality control. Furthermore, we investigated the apoptosis mechanism of the fruiting bodies ethanol extract. Treatment of A549 cells with A. cinnamomea ethanol extract induced apoptotic cell death, which was characterized by positive results of annexin V/PI assay, caspase-3, caspase-9 activation and inhibition of cyclin D1. We suggested that the increase of caspase-3, caspase-9 activity and the inhibition of cyclin D1 could be used in biological quality control of protein marker. Then, we established the Jurkat cell proteomic response analysis method. The similarity of Jurkat cell proteomic response can be use in quality control. Besides, the phytomics similarity index (PSI) analysis was employed for the chemical quality evaluation of the A. cinnamomea fruiting bodies from different strains and grown on various substrates. The results indicated that the different types of A. cinnamomea fruiting bodies contain eight index compounds, and that it was difficult to discriminate between them solely on the basis of those index compounds. In our research, we used PSI scores to assess the metabolite similarity of the fruiting bodies of A. cinnamomea. It was revealed that fruiting bodies from various A. cinnamomea strains and grown on different culture substrates produce distinct PSI scores. We concluded that the PSI analysis had good selectivity on the different types of A. cinnamomea fruiting bodies. Finally, we investigated the anti-cancer effects of on A549 human lung cancer cells using the ethyl acetate extract from submerged culture filtrates. Our results showed that 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q0; CoQ0) derived from A. cinnamomea submerged culture filtrates has anti-cancer activity. CoQ0 treatment reduced the viability of A549, HepG2, and SW480 cancer cell lines. Furthermore, CoQ0 induced reactive oxygen species (ROS) generation and apoptosis in A549 cells, which was inhibited by the antioxidant ascorbic acid. To our knowledge, these data demonstrate for the first time that CoQ0 derived from A. cinnamomea submerged culture filtrates exerts its anti-cancer effect through the induction of ROS-mediated apoptosis in A549 human lung cancer cells. We study the anticancer activity of A. cinnamomea fruiting bodies. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T00:54:29Z (GMT). No. of bitstreams: 1 ntu-104-D99b22002-1.pdf: 4691354 bytes, checksum: 1f02605afb74b25e76b55eb07c4bc81b (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 摘要...............................................................................................................................II
Abstract.....…………………………………………………………………………...IV Contents.......................................................................................................................VI Contents of table………………………………………….………………………….XI Contents of figure……………………………………………. ………………...…..XII Chapter I. Introduction and literature review…………..…………………….…….…1 1.1 Recent developments in medical mushrooms as anti-cancer therapeutics……..2 1.2 Antrodia cinnamomea……………………………………………….………....3 1.3 Pharmacological effects of A. cinnamomea…………………………………...5 1.4 Chemical constituents of A. cinnamomea…………………………….………11 1.5 Artificial fruiting bodies of A. cinnamomea…………………………………...13 1.6 Studies on the quality control of A. cinnamomea…………….……………..…14 1.7 A. cinnamomea submerged culture……………………………………...…….14 1.8 Aim of this study………………………………………………………………16 Chapter II. Study on the anti-cancer activity and biological quality control of A. cinnamomea fruiting bodies…………………………...…………………26 2.1 Introduction……………………………………………………………………27 2.2 Materials and methods………………………………………………………...29 2.2.1 A. cinnamomea fruiting bodies……………………………………………29 2.2.2 Polymerase chain reaction of internal transcribed spacer gene….………..29 2.2.3 Preparation of A. cinnamomea fruiting body ethanol extract……..………30 2.2.4 Cell viability analysis……………………………………………….…….31 2.2.5 Apoptosis analysis…………………………………………………..…….31 2.2.6 Caspase activity assay…………………………………………………….32 2.2.7 Preparation of cell extracts and immunoblot analysis…………………….33 2.3.8 Jurkat cell proteomic response analysis……………..……………………34 2.3.9 LC-MS/MS analysis…………………………………….………………..35 2.3.10 Statistical analysis……………………………………………………….36 2.3 Results………………..…………………………………………..……………38 2.3.1 Batch-to-batch comparison of anti-cancer activity of A. cinnamomea fruiting bodies……………………………………………………………38 2.3.2 Effect of A. cinnamomea extract on apoptosis induction…………………39 2.3.3 Biological quality evaluation by using Jurkat cell proteomic response analysis ……..……………………………………………………………39 2.4 Discussions…………………………………………………………………….40 Chpater III. Chemical quality evaluation of A. cinnamomea fruiting bodies using phytomics similarity index analysis ……………………………………..49 3.1 Introduction……………………………………………………………………50 3.2 Materials and methods ………………………………………………………..53 3.2.1 A. cinnamomea fruiting bodies……………………………………………53 3.2.2 Preparation of A. cinnamomea fruiting body ethanol extract……………..53 3.2.3 HPLC and electrospray ionization-mass spectrometry (ESI-MS) analysis.54 3.2.4 Phytomics similarity index method……………………………………….54 3.3 Results ……………….……………………………………………..…………56 3.3.1 HPLC-MS chromatography of index compounds in A. cinnamomea BCRC 35398 fruiting bodies…………………………………………….………..56 3.3.2 Phytomics similarity index analysis of ethanol extracts of the fruiting bodies of different A. cinnamomea strains ……………………..…………57 3.3.3 Phytomics similarity index analysis of ethanol extracts of A. cinnamomea fruiting bodies grown on different substrates……………………………..58 3.4 Discussion……………………………………………………………………..60 Chapter IV. Study on the anticancer activity of A. cinnamomea submerged culture..67 4.1 Introduction……………………………………………………………………68 4.2 Materials and methods. ……………………………………………………..71 4.2.1 Reagents and chemicals…………………………………………………...71 4.2.2 Organism and inoculums………………………………………………….71 4.2.3 Submerged fermentation………………………………………………….71 4.2.4 Preparation of broth extracts……………………………………………...72 4.2.5 Cell culture………………………………………………………………..72 4.2.6 Activity guided purification of anti-cancer compounds…………………..73 4.2.7 Cell viability analysis……………………………………………………..73 4.2.8 GC-MS analysis…………………………………………………………..74 4.2.9 1H NMR measurements at 400 MHz……………………………………...75 4.2.10 Apoptosis analysis……………………………………………………….75 4.2.11 Determination of the intracellular ROS level……………………………76 4.2.12 Statistical analysis……………………………………………………….76 4.3 Results…………………………………………………………………………..77 4.3.1 Ethyl acetate extract from A. cinnamomea submerged culture filtrates inhibits cancer cell survival……………………………………………….77 4.3.2 Isolation and identification of CoQ0 as the major anti-cancer compound in A. cinnamomea submerged culture extract……………………………..77 4.3.3 CoQ0 induced apoptosis of A549 lung cancer cells………………………78 4.3.4 CoQ0 triggered A549 cell apoptosis via inducing ROS generation………79 4.3.5 Production of CoQ0 by A. cinnamomea submerged culture in a 5L -fermenter………………………………………………………………….80 4.4 Discussions…………………………………………………………………….81 Chapter V. Conclusion…………………………………………………………...….93 References..…………………………………………………………………………98 Appendix……………………………………………………………………………109 | |
dc.language.iso | en | |
dc.title | 牛樟芝抗癌活性及其品質管控之研究 | zh_TW |
dc.title | Study on the anti-cancer activity and quality control of Antrodia cinnamomea | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 陳俊任,吳定峰,劉啟德,何士慶,賴宗賢 | |
dc.subject.keyword | 牛樟芝,液態深層培養,抗癌活性,品質管控, | zh_TW |
dc.subject.keyword | Antrodia cinnamomea,submerged culture,anti-cancer activity,quality control, | en |
dc.relation.page | 109 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2015-03-25 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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