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研究生:吳漢強
研究生(外文):WU, HAN-CHIANG
論文名稱:固態培養北蟲草之抗氧化、抗腫瘤與抑制血管張力素I型轉化酶作用
論文名稱(外文):Antioxidant, Anti-tumor and ACEI Activities of Cordyceps militaris by Solid State Fermentation
指導教授:梁志欽梁志欽引用關係
指導教授(外文):LIANG, ZENG-CHIN
口試委員:張世良黃冠中宋祖瑩梁志弘
口試委員(外文):CHANG, SHIH-LIANGHUANG, GUAN-JHONGSONG, TUZZ-YINGLIANG, CHIH-HUN
口試日期:2019-06-27
學位類別:博士
校院名稱:大葉大學
系所名稱:生物科技與產業博士學位學程
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:英文
論文頁數:142
中文關鍵詞:北蟲草蟲草素固態米類發酵乳腺癌抗增殖作用血管收縮素轉換酶抑制劑細胞凋亡作用抗氧化作用
外文關鍵詞:Cordyceps militarisCordycepinFermented riceBreast cancerAnti-proliferation,Angiotensin-converting-enzyme inhibitorApoptosisAnti-oxidation
DOI:10.1615/IntJMedMushrooms.2019031138
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北蟲草(Cordyceps militaris)萃取物功效常受不同菌株、其萃取部位與培養方式等影響,導致其有效成分含量多所變異。有鑑於此,本研究比較採自昆明(Kunming strain, K)及漳洲(Zhangzhou strain, Z)北蟲草菌株,經固態培養後的子實體(FBE)及其發酵米基(FRE),以乙醇萃取後的萃取液,探討其對人體乳腺癌細胞株(MCF-7及MDA-MB-23)之抗增殖作用(anti-proliferation)、細胞凋亡 (apoptosis)、細胞抗氧化酵素表現及體外抗氧化活性之影響。
結果顯示,漳州菌株發酵米基(FRE-Z)顯較高含量的蟲草素 (Cordycepin),同時其對清除:1,1-二苯基-2-三硝基苯肼(DPPH)、超氧自由基(Superoxide)、羥基(Hydroxyls)和抑制低密度脂蛋白氧化 (Low- density lipoprotein oxidation)具顯著效果。此外,FRE-Z萃取物相較其他萃取物,培養24小時即可顯著抑制MCF-7 (0.7 mg/ mL) 與MDA-MB-231 (1 mg/ mL)乳腺癌細胞增生,透過Annexin V和Propidiam iodide染色證實,其機轉可能與其於以0.5 mg/ mL濃度處理4小時處理後可顯著誘發MCF-7細胞凋亡細胞有關,研究並進一步證實其細胞凋亡途徑主要透過促進p53腫瘤抑制蛋白磷酸化。另一方面, FRE-Z萃取物處理2小時後,亦可短暫誘發MCF-7細胞中Cu / Zn超氧化物歧化酶酵素表現。因此,本研究證實FRE-Z萃取物,相較於其子實體萃取物與昆明菌株萃取物包含子實體與米基,具有顯著治療抗乳腺癌之潛力。
另一部分研究,比較金針菇(Flammulina velutipes)、小白靈菇 (Pleurotus ostreatus)、杏鲍菇(Pleurotus eryngii)、北蟲草米基 (Cordyceps militaris/ fermented brown rice base),以不同溶劑萃取和不同萃取條件所得到的萃取物,對血管張力素I型轉化酶抑制劑 (ACEI)活性表現調控分析。血管張力素I型轉化酶抑制劑活性以金针菇粉末經乙醇萃取1小時為最高,達86.9 %;萃取時間6小時,則以小白靈菇粉末經乙醇萃取對血管張力素I型轉化酶抑制劑活性為最高,達76.1 %。
根據我們的結論,北蟲草漳州菌株發酵米基萃取物可得到較高濃度的蟲草素到達14.36 mg / g,並具有更高的抗氧化作用。在抗癌作用中,漳州菌株發酵米基萃取物在非侵襲性MCF-7和侵襲性MDA-MB-231乳腺癌細胞系中顯示出更高的細胞毒活性。該機制與誘導酶促抗氧化劑銅和鋅超氧化物歧化酶1 (SOD1)的漳州菌株發酵米基萃取物表達以及p53介導的凋亡途徑的激活有關。具有高產漳州菌株發酵米基萃取物已成為乳腺癌治療蟲草素的有效來源。
本研究結果顯示,以粉末方式透過乙醇萃取,增加萃取液反應面,並缩短萃取時間,可獲得較高血管張力素I型轉化酶抑制劑活性。

Production and efficacy of bioactive compounds form Cordyceps militaris (C. militaris) fruiting bodies and its fermentation grain was usually variants due to different strains. Through comparison of the anti-proliferative, apoptotic, and antioxidative properties of ethanolic extracts of fruiting bodies (FBE) and its fermented rice (FRE) from two strains of C. militaris applied to human breast cancer cell lines (MCF-7 or MDA-MB-231).
We revealed that FRE of the Zhangzhou strain (FRE-Z) produced a high level of cordycepin and exhibited comprehensive in vitro antioxidant activity against DPPH, superoxide radicals, hydroxyls, and low-density lipoprotein oxidation. Only FRE-Z showed dose-dependent inhibition of cell proliferation, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-di phenyl tetrazolium bromide assay in MCF-7 (0.7 mg/mL) and MDA-MB-231 cells (1 mg/mL) after 24-h culture. The anti-proliferative effect of FRE-Z were associated with an early stage of apoptosis induction at 4 h of treatment with 0.5 mg/mL FRE-Z in MCF-7 cells, as determined using annexin V and propidium iodide staining of apoptotic cells. The anti-proliferative effect was determined to occur through p53 activation but not through the release of mitochondrial apoptosis-inducing factor or caspase-9 activation for an initial culture period of 16 h. In addition to transient increase in cellular antioxidant enzyme, Cu/Zn superoxide dismutase was found in MCF-7 cells after 2-h treatment with FRE-Z. Therefore, FRE-Z exhibited various dose-dependent and exposure-time-dependent activities and has potential for application in breast cancer chemoprevention.
On the other part of study related to ACEI activity in edible mushroom, four edible or medicinal mushrooms: Flammulina velutipes, Pleurotus ostreatus, Pleurotus eryngii, Cordyceps militaris/ fermented brown rice base were treated in difference condition and extraction medium. We found the best ACEI activity in F. velutipes in powder form and under ethanol extraction for 1 h was 86.9 %. In 6 h extraction time, the higher ACEI activity was noticed in P. ostreatus in powder form and ethanol extraction was 76.1 %.
On our conclusion, strain of C. militaris had had a high concentration of cordycepin in the FRE-Z (14.36 mg/g) and had a higher anti-oxidation effect. In anti-cancer effect, FRE-Z was showed a higher cytotoxic activity in both noninvasive MCF-7 and aggressive MDA-MB-231 breast cancer cell lines. The mechanism was related to induce expression in FRE-Z of the enzymatic antioxidants copper and zinc superoxide dismutase 1 (SOD1) and activation of the p53-mediated apoptosis pathway. Strain of C. militaris Zhangzhou with high-producing medium of fermented rice had served as an efficient source of cordycepin for breast cancer therapy.
On the other hand, increasing reaction surface (powder from) and shorten extraction time (1 h) would get a better ACEI activity.

封面內頁
簽名頁
ABSTRACT iii
中文摘要 v
ACKNOWLEDGEMENTS vii
CONTENTS viii
LIST OF FIGURES xi
LIST OF TABLES xii
ABBREVIATION xiii
Chapter I. Introduction 1
I-1 General information about Cordyceps militaris 2
I-2 Life cycle of nature C. militaris 3
I-3 Cultivation of Cordyceps military 4
I-4 Bioactivities of Cordyceps militaris acted as the Chinese medicine 7
I-5 Recent research on bioactivities of Cordyceps militaris 8
I-6 Bio-medicinal functional components isolated in Cordyceps militaris 13
I-7 Introduction and bio-medical function of adenosine 15
I-8 Introduction and bio-medicinal function in cordycepin 17
I-9 Cordycepin and adenosine in anti-oxidation effect 24
I-10 Role of cordycepin in anti-cancer effect 24
I-11 Edible mushrooms included C. militaris to an effect as anti-hypertension 31
I-12 Summaries of research purposes of this dissertation 38
Chapter II. Materials and methods 40
II-1 Mushrooms used 40
II-2 Drugs and chemicals 40
II-3 Equipment & software 41
II-4 Mushroom and inoculum preparation 41
II-5 Grain substrate preparation and cultivation 42
II-6 Sample preparation for extraction 42
II-7 Extracts preparation for determined ACEI activity 43
II-8 Cell line and cell culture 43
II-9 HPLC analysis of cordycepin and adenosine in C. militaris 43
II-10 Determination of the bioactive components of cordycepin and adenosine 44
II-11 Antioxidant activities assays 44
II-11-1 DPPH radical-scavenging activity 44
II-11-2 Superoxide radical scavenging assay 45
II-11-3 Hydroxyl radical scavenging assay 46
II-11-4 Low-density lipoprotein peroxidation inhibition assay 46
II-12 Cell viability on MCF-7 and MDA-MB-231 with C. militaris strains 47
II-13 Annexin V/propidium iodide staining 48
II-14 Determination of caspase activity 48
II-15 Western blotting 49
II-16 Assay of ACE inhibitory activity 49
II-17 Statistical analysis 50
Chapter III. Results 51
III-1 Concentration of cordycepin and adenosine in ethanol extracts C. militaris. 51
III-2 In vitro evaluation of antioxidant activity 52
III-3 Cytotoxicity of breast cancer cell lines 55
III-4 Cell-death pathway in treated breast cancer cell line 56
III-5 Regulation of cellular antioxidant enzymes 57
III-6 The effect of ACEI activities in the different types of edible mushroom 59
Chapter IV. Discussion 64
Chapter V. Conclusion 71
Chapter VI. References 72
Appendix 108
I. Acceptation Letter 108
II. PublicatioN 109
III. Patent 123


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