Biological Activity of Polysaccharides of Pleurotus djamor Extracts

蕈類不僅可當作食物，它獨特的內含物在醫學上更是一種極具有價值的成分，這些內含物包含許多蕈類的二級代謝物，例如酚類化合物、多肽化合物、萜類物質、類固醇、外源凝集素、多醣體、多醣體生肽和多醣蛋白複合物。蕈類含有豐富的多醣體，近年來的研究將多醣體利用於許多生物試驗上，例如糖尿病、降低膽固醇、抗老化、抑制惡性腫瘤等等。粉紅菇(Pleurotus djamor)是一種在市場上新興的蕈類，屬於蠔菇類(oyster mushroom)的側耳菇，其多醣體的含量和生物活性目前還未被完整的發現，但從前人文獻中發現蠔菇類之熱水及醇類萃取物具有抗菌作用、抗病毒作用、抗人類後天免疫缺陷病毒、抗肉瘤作用、抗誘變作用、抗低血壓作用、保肝作用、降膽固醇作用、抗腫瘤作用、抗氧化作用、降血糖作用、抗發炎反應、免疫調節、抗老化作用等等多種生物活性潛力。本試驗特別針對粉紅菇熱水萃取物之多醣體進行試驗，分別測定了粉紅菇多醣體萃取物之還原能力、清除DPPH能力及清除H2O2之能力等等多種抗氧化力性質，也同時測定了粉紅菇多醣體萃取物之總醣量、分子量分布。粉紅菇多醣體萃取物之對於生物活性之試驗，探討生物活性的方式是透過子宮頸癌細胞(Hela)的MTT細胞存活率試驗來觀察粉紅菇的生物活性並觀察粉紅菇多醣體對於人類肝癌細胞株(HA22T)、人類乳癌細胞株(MCF7)、子宮頸癌細胞株的形態是否發生改變。 Mushrooms have been not only used as food materials with their unique flavor and texture, but also recognized as an important source of biologically active compound of medicinal value. Mushrooms have a variety of accumulated secondary metabolites such as phenolic compounds, polypeptides, terpenes, and steroids. Mushrooms also have lectins, polysaccharides, polysaccharide-peptides, and polysaccharide-protein complexes which are known to have immunomodulatory and anticancer activities. Mushroom is rich in polysaccharides, and the polysaccharides has been recently researched for many biological and medical tests, such as treating diabetes, lowering the cholesterol, anti-aging, cancer-inhibiting and so on. Pink oyster mushroom (Pleurotus djamor) is a kind of mushrooms emerging on the market, and it is one of the oyster mushroom in Pleurotus. Its polysaccharide content and the biological activity has yet to be fully discovered, so that this test through the MTT cell viability test by the cervix cancer cells (Hela) to observe the biological activity of pink oyster mushroom and also we observe the morphology change of different cancer cell lines through adding the polysaccharides.口試委員審定書...............................................................................................................# 謝誌....................................................................................................................................i 中文摘要...........................................................................................................................ii ABSTRACT.....................................................................................................................iii CONTENTS.....................................................................................................................iv 圖目錄...........................................................................................................................viii 表目錄.............................................................................................................................ix Chapter 1 簡介................................................................................................................1 Chapter 2 文獻回顧........................................................................................................3 2.1抗菌作用(Antimicrobial).....................................................................................3 2.2抗病毒作用(Antiviral).........................................................................................3 2.3抗人類免疫缺陷病毒(Anti-Human Immunodeficiency Virus, HIV).................4 2.4抗肉瘤作用(Antineoplasticism)..........................................................................4 2.5抗腫瘤作用(Antitumor)......................................................................................5 2.6抗誘變作用(Antimutagenesis)............................................................................5 2.7抗氧化作用(Antioxidation).................................................................................6 2.7.1自氧化反應...............................................................................................7 2.7.1.1自氧化反應機構....................................................................................8 2.7.1.2抗氧化劑................................................................................................9 2.7.1.3抗氧化物................................................................................................9 2.7.2自由基致癌、促癌作用及其發生機制...................................................10 2.7.2.1自由基在化學致癌過程中的作用..............................................10 2.7.2.2自由基與促癌作用......................................................................11 2.7.2.3自由基致癌和促癌可能發生的機制..........................................13 2.8降血脂作用(Antilipidemia)...............................................................................15 2.9降血糖作用(Anti-Hyperglycemia)....................................................................15 2.10抗低血壓作用(Anti-Hypotension)..................................................................16 2.11抗發炎反應(Anti-Inflammation).....................................................................16 2.12保肝作用(Hepatoprotection)...........................................................................17 2.13降膽固醇作用(Hypocholesterolemia).............................................................17 2.14免疫調節(Immunomodulatory).......................................................................18 2.15抗老化作用(Anti-Ageing)...............................................................................18 Chapter 3 材料與方法..................................................................................................19 3.1材料與藥品........................................................................................................19 3.1.1粉紅菇菌種.............................................................................................19 3.1.2藥品.........................................................................................................19 3.1.3實驗儀器.................................................................................................21 3.2 粉紅菇多醣體萃取..........................................................................................22 3.2.1一次純化粗多醣.....................................................................................23 3.2.2二次純化粗多醣.....................................................................................24 3.2.3去除帶電分子.........................................................................................24 3.2.4去除聚醣蛋白複合體.............................................................................24 3.2.5去除蛋白質.............................................................................................24 3.3總醣測定............................................................................................................25 3.4分子量測定........................................................................................................26 3.5抗氧化性質分析................................................................................................26 3.5.1還原能力測定.........................................................................................27 3.5.2清除DPPH自由基能力測定..................................................................27 3.5.3清除 H2O2的測定..................................................................................28 3.6解凍冷凍細胞....................................................................................................28 3.7細胞繼代培養....................................................................................................30 3.8細胞計數............................................................................................................31 3.9細胞冷凍保存....................................................................................................32 3.10 細胞實驗操作................................................................................................33 3.11磷酸鹽緩衝溶液配製......................................................................................34 3.12 胎牛血清配製................................................................................................34 3.13 培養基配製....................................................................................................35 3.14 MTT assay 細胞存活率分析.........................................................................35 3.15細胞免疫螢光染色..........................................................................................36 Chapter 4 結果與討論..................................................................................................38 4.1 總醣含量分析..................................................................................................38 4.2 分子量之測定..................................................................................................40 4.3 粉紅菇抗氧化性質分析..................................................................................43 4.3.1 還原能力測定.......................................................................................43 4.3.2 清除DPPH自由基能力測定................................................................49 4.3.3 清除H2O2能力測定..............................................................................53 4.4 多醣抑制腫瘤細胞分析..................................................................................59 Chapter 5 結論..............................................................................................................66 Chapter 6 參考文獻......................................................................................................6