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研究生:呂欣怡
研究生(外文):Hsin-Yi
論文名稱:蛹蟲草水萃取物之抗氧化、抗發炎與抗血管新生之研究
論文名稱(外文):The effects of antioxidant, anti-inflammation and anti-angiogenesis of the water extracts from Cordyceps militaris
指導教授:李宗貴黃效民黃效民引用關係
指導教授(外文):Chong-Kuei Lii, Ph. D.
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:營養學研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:54
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蛹蟲草(Cordyceps militaris)又名北蟲草或北冬蟲夏草,與冬蟲夏草同屬於真菌之蟲草屬,其藥理、藥效及病理作用與冬蟲夏草大致相同。目前蛹蟲草已可用人工培育的方式大量生產,且在許多文獻中被證實具有抗腫瘤、調節免疫力和改善疲勞等功效,顯示蛹蟲草可能具有多樣之藥理活性。本實驗研究目的是測試經由熱水萃取的蛹蟲草子實體萃取物(CME)是否具有抗氧化、抗發炎與抗血管新生的能力。在總抗氧化能力測試(trolox equivalent antioxidant capacity assay)中,CME表現出良好的抗氧化能力;而在發炎反應的試驗上,結果顯示CME具有抑制LPS及IFN-γ所活化的巨噬細胞(RAW 264.7)產生一氧化氮(NO)及iNOS基因表現的功能;在血管新生試驗中,首先藉由人類臍帶靜脈內皮細胞(HUVEC)的增生、移動和類血管生成試驗,觀察CME在抑制血管新生上的活性,結果發現CME可抑制HUVEC的增生、移動與類血管形成之功能,其次,在雞胚尿囊絨毛膜的血管新生試驗中,CME也具有抑制血管內皮生長因子(VEGF)刺激雞胚尿囊絨毛膜產生的新生血管的功能。本實驗結果顯示:蛹蟲草熱水萃取物在抗氧化、抗發炎與抗血管新生上確實有良好的效果,此一結果對於未來應用蛹蟲草於保健食品或醫藥用品之開發,可提供一個良好的參考指標。


Cordyceps militaris, a parasitical fungus in moth pupae, is very close to Cordyceps sinensis both in morphology and phylogenesis. It has been reported that Cordyceps militaris and Cordyceps sinensis have similar pharmacokinetics. Recently, it was reported that the artifical cultivation of Cordyceps militaris is successfully developed in industrial large scale. The purpose of this study was to investigate the antioxidant, anti-inflammatory and anti-angiogenic effects of hot water extracts from Cordyceps militaris (CME). In the trolox equivalent antioxidant capacity (TEAC) assay, CME revealed a high level of antioxidant capacity. In anti-inflammatory test, CME inhibited the NO production and iNOS expression in LPS and IFN-γ-stimulated RAW 264.7 macrophage cells. In anti-angiogenic assay, CME inhibited the proliferation, migration and tube-like formation in human umbilical vein endothelial cell (HUVEC). Moreover, CME showed a potent inhibitory activity upon VEGF-induced angiogenesis on the chick embryo chorioallantoic membrane vascularisation (CAM) assay. These functional studies provide a beneficial evidence on the development of health food and drug from Cordyceps militaris.

綱目

目錄 Ⅰ
圖次 Ⅲ
表次 Ⅳ
中文摘要 1
英文摘要 2
文獻回顧
一、蛹蟲草簡介
(一) 分類 3
(二) 特性 3
(三) 人工培育 3
(四) 藥用活性 4
二、抗氧化
(一) 自由基 5
(二) 活性氧 5
(三) 自由基與活性氧之形成 6
(四) 自由基與疾病 6
三、發炎反應
(一) 脂多醣類(Lipopolysaccharides, LPS) 7
(二) 一氧化氮(NO) 8
(三) Nuclear factor (NF)-κB 9
四、血管新生
(一) 血管新生的步驟 10
(二) 血管內皮生長因子(Vascular Endothelial Ggrowth Factor, VEGF)
(1) 命名 11
(2) VEGF的生成 11
(3) VEGF的結構與分類 12
(4) VEGF接受體 12
研究動機 19
實驗模式 20
實驗材料 21
實驗方法
一、細胞株培養 24
二、細胞株冷凍保存 24
三、細胞株解凍 24
四、樣品製備 25
五、總抗氧化能力測試(Trolox Equivalent Antioxidant Capacity assay,
TEAC assay) 25
六、細胞存活率分析(MTT Assay) 25
七、NO Assay 26
八、反轉錄酶鏈鎖反應(Reverse Transcription Polymerase Chain Reaction, RT-PCR)
(一) 總RNA抽取(Total RNA Isolation) 26
(二) 反轉錄酶鏈鎖反應(Reverse Transcription-Polymerase Chain
Reaction, RT-PCR) 26
九、細胞增生試驗(Cell Proliferation Assay) 27
十、細胞移動試驗(Cell Migration Assay) 27
十一、類血管生成試驗(Tubule Formation Assay) 28
十二、雞胚尿囊絨毛膜血管增生試驗(Chick Embryo Chorioallantoic
Membrane Vascularization Assay) 28
統計分析 29
實驗結果 30
討論 33
結論 37
結果圖表 38
參考文獻 46

圖次

文獻回顧
圖一、乾燥蛹蟲草子實體 14
圖二、Brucella spp.之LPS結構概要圖 14
結果
圖一、蛹蟲草水萃取物(CME)在TEAC assay (Trolox Equivalent Antioxidant
Capacity assay)中總抗氧化能力的表現 38
圖二、不同濃度蛹蟲草水萃取物(CME)對巨噬細胞(RAW 264.7)存活率的影響(MTT assay) 39
圖三、蛹蟲草水萃取物(CME)對由LPS及IFN-γ活化巨噬細胞(RAW 264.7)
產生NO的影響 40
圖四、蛹蟲草水萃取物(CME)對由LPS及IFN-γ活化巨噬細胞(RAW 264.7)
iNOS mRNA表現的影響 41
圖五、蛹蟲草水萃取物(CME)對人類臍帶靜脈內皮細胞(HUVEC)增生的影響 42
圖六、蛹蟲草水萃取物(CME)對人類臍帶靜脈內皮細胞(HUVEC)移動的影響 43
圖七、蛹蟲草水萃取物(CME)對於VEGF刺激人類臍帶靜脈內皮細胞(HUVEC)所形成網狀結構的影響 44
圖八、蛹蟲草水萃取物(CME)在雞胚尿囊絨毛膜血管增生試驗對血管新生的影
響 45

表次

文獻回顧
表一、自由基與活性氧的種類 15
表二、自由基的來源 16
表三、氧自由基對生物體分子的傷害與可能導致的病變 17
表四、酚類物質及抗氧化維生素之總抗氧化活性及膳食來源 18


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