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研究生:胡少卜
研究生(外文):Shao-Pu Hu
論文名稱:朱紅栓菌子實體萃取物抗發炎及促進吞噬作用之體內及體外功能性研究
論文名稱(外文):The Bioactivities of Pycnoporus cinnabarinus Carpophores Extract on Anti-inflammation and Phagocytosis in vitro and in vivo Study
指導教授:陳啟楨陳啟楨引用關係
指導教授(外文):Chi-Jhen Chen
學位類別:碩士
校院名稱:南台科技大學
系所名稱:生物科技系
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:2008
畢業學年度:96
語文別:中文
論文頁數:142
中文關鍵詞:朱紅栓菌清除能力抗發炎吞噬作用普魯士藍還原力酚類化合物血管新生雞胚胎
外文關鍵詞:Pycnoporus cinnabarinusMTT AssayNitrite AssayGriess reactionPhagocytosis AssayChick Chorioallantoic Membrane AssayMay-Grunwald Giemsa StainDPPHReduction of Prussian Blue AssayTotal Phenolic Compound AnalysisRT-PCR
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朱紅栓菌(Pycnoporus cinnabarinus)在傳統中醫學文獻上記載,具有消炎解熱、止血及抗菌等效用。本研究中製備三種樣品,分別將人工栽培之朱紅栓菌子實體用95%酒精萃取、10%酒精萃取及兩者1:1混合,針對其抗炎能力及增強免疫能力進行研究。利用MTT Assay,以CHO-K1正常中國倉鼠卵巢細胞株進行實驗得到IC50為濃度4mg/ml;Nitrite Assay實驗中,以RAW264.7細胞株進行實驗,在4mg/mL濃度得到皆具有明顯抑制NO釋放約80%的效果;Phagocytosis Assay實驗中,以E. coli做為抗原給予RAW264.7細胞株吞噬,在濃度4mg/ml發現可有效增加細胞吞噬的能力,其中又以1:1混合的樣品最佳,可達到三倍吞噬量;以CAM Assay試驗72小時,三種樣品皆無抑制血管新生及毒殺胚胎現象發生;使用8mg/kg LPS對SD rat進行i.p.誘導發炎反應對給予20mg/kg PCAE對大鼠治療得到相當好的療效,減輕大鼠腹瀉及眼角出血的症狀;抽取腹水中的離體Leukocytes於體外進行吞噬實驗發現可有效降低發炎反應造成過高的吞噬作用(% of phagocytosis cells)。本研究結果證實朱紅栓菌子實體酒精萃取物具有降低NO釋放的抗發炎效果,卻又同時可以增強Macrophage的吞噬能力並推測PCAE可使LPS誘導發炎的大鼠回復正常的生理狀態,此外萃取物於1mg/mL時DPPH清除能力達到約80%及約26μg/mL維他命C的普魯士藍還原力。本研究發現Nitrite assay、Phagocytosis assay、DPPH清除率、普魯士藍還原力與酚類化合物含量成正相關,因此推測萃取物的有效成分可能來自酚類化合物。
Pycnoporus cinnabarinus with the functions of relieve internal heat, fever, hemostasis, anti-bacteria and anti-inflammation was recorded in traditional Chinese pharmacology. Three kinds of samples extracted by 95% ethanol, 10% ethanol and the 1:1 mixture of both were studied in anti-inflammation and enhance phagocytosis activity. The concentration of 4mg/mL was treating with ChoK1 for IC50 through MTT Assay, decreased about 80% NO production by Nitrite Assay, and enhanced three folds of phagocytosis activity on RAW264.7. All samples had no anti-angiogenesis activity and no damage on chicken embryo by CAM Assay. The Pycnoporus cinnabarinus alcohol extract (PCAE) could reduce inflammation symptoms of diarrhea and eyes bleeding caused by LPS, and the phagocytosis activity of peripheral leukocytes can be reverted to usual as non-inflammation rat. In this study we proved P. cinnabarinus carpophores ethanol extract had anti-inflammation activity by decreasing NO production and enhanced phagocytosis activity with macrophage in vitro. The results indicate that PCAE had anti-inflammation activity to reply physiological state. The PCAE has also great anti-oxidant activity, about 80% DPPH clearance ratio and equal to 26μg/mL of vitamin C reduction of prussian blue activity in 1mg/mL treatment. Eventually the nitrite, Phagocytosis, DPPH clearance, as well as reduction of prussian blue assays suggesting the effect elements are polyphenolic compounds.
目錄
博碩士論文授權書......................................................i
博碩士論文電子檔案上網授權書...........................................ii
碩士論文口試及格證明書...............................................iii
摘要................................................................iv
誌謝................................................................vi
目次...............................................................vii
圖目錄.............................................................xii
表目錄............................................................xiii
附圖目錄...........................................................xiv
附表目錄............................................................xv
第一章文獻回顧........................................................1
1.1 菇類簡介.........................................................1
1.2 菇類的生態特性....................................................1
1.3 菇類機能性........................................................2
1.3 菇類的多醣體功能...................................................3
1.4 菇類生物活性物質...................................................4
1.5.1 抗生素.........................................................4
1.5.2 抗腫瘤物質......................................................5
1.5.3 降血壓物質......................................................6
1.6 朱紅栓菌分類......................................................6
1.7 朱紅栓菌相關研究...................................................7
1.7.1 抗菌活性........................................................7
1.7.2 抗癌細胞活性....................................................7
1.7.3 分解木質素活性..................................................7
1.7.4 生成Vanillin活性................................................8
1.8 免疫系統..........................................................8
1.8.1 發炎反應........................................................9
1.8.2 巨噬細胞與吞噬作用..............................................10
1.8.3 免疫系統與傷口癒合..............................................10
1.8.4 發炎因子.......................................................11
1.9 Lipopolysaccharide..............................................12
1.10 血管新生........................................................13
1.11 氧化作用........................................................14
1.11.1 自由基.......................................................14
1.11.2 自由基之來源..................................................14
1.11.3 自由基對生物分子的傷害.........................................15
1.12 生物抗氧化系統..................................................16
1.13 多酚類.........................................................16
1.13.1 多酚類介紹....................................................16
1.13.2 多酚類抗氧化功效...............................................19
1.13.3 多酚類抗發炎功效...............................................20
第二章研究方法.......................................................25
2.1 實驗儀器與藥品...................................................25
2.1.1 實驗材料.......................................................25
2.1.2 實驗設備.......................................................26
2.1.3 實驗藥品.......................................................27
2.2 實驗方法.........................................................28
2.2.1 樣品製備.......................................................28
2.2.2 細胞培養.......................................................28
2.2.2.1 細胞繼代培養.................................................28
2.2.2.2 細胞株保存...................................................29
2.2.2.3 細胞株解凍...................................................29
2.2.2.4 細胞計數與存活測試............................................29
2.2.3 MTT Assay.....................................................29
2.2.4 Nitrite Assay.................................................30
2.2.4.1 Griess reaction試劑製備......................................30
2.2.4.2 RAW 264.7培養...............................................30
2.2.4.3 Nitrite測定.................................................30
2.2.4.4 MTT Assay...................................................30
2.2.5 Phagocytosis Assay............................................31
2.2.5.1 吞噬E. coli.................................................31
2.2.5.2 吞噬Latex Beads.............................................31
2.2.6 Chick Chorioallantoic Membrane(CAM)Assay....................31
2.2.6.1 CAM Assay原理...............................................31
2.2.6.2 CAM Assay步驟...............................................32
2.2.7 SD Rat試驗....................................................32
2.2.7.1 SD Rat Phagocytosis Assay...................................32
2.2.7.2 May-Grunwald Giemsa Stain...................................33
2.2.8 2,2-diphenyl-1-picrylhydrazyl(DPPH)Clearance Experiment.....33
2.2.8.1 DPPH清除原理.................................................33
2.2.8.2 DPPH製備....................................................33
2.2.8.3 清除DPPH實驗步驟.............................................34
2.2.9 Reduction of Prussian Blue Assay..............................34
2.2.9.1 Prussian Blue還原力原理......................................34
2.2.9.2 藥品配備.....................................................34
2.2.9.3 還原力實驗步驟...............................................35
2.2.10 Total Phenolic Compound Analysis.............................35
2.2.10.1 原理.......................................................35
2.2.10.2 藥品配備....................................................35
2.2.10.3 實驗步驟....................................................35
2.2.11 Purification of RNA from Cells and Tissue by Phenol-Guanidium Thiocyanate-Chloroform Extraction...................................36
2.2.11.1 RNA萃取原理.................................................36
2.2.11.2 試劑配備....................................................36
2.2.11.3 實驗步驟....................................................36
2.2.12 Reverse Transcription Polymerase Chain Reaction(RT-PCR)....37
2.2.12.1 RT-PCR原理.................................................37
2.2.12.2 實驗試劑....................................................37
2.2.12.3 實驗步驟....................................................38
2.2.13 Polymerase Chain Reaction(PCR).............................38
2.2.13.1 PCR原理....................................................38
2.2.13.2 實驗試劑....................................................38
2.2.13.3 實驗步驟....................................................39
2.2.14 統計分析......................................................39
第三章 實驗結果......................................................40
3.1 MTT Assay.......................................................40
3.1.1 PCAE對CHO-K1 cell line毒殺能力.................................40
3.1.2 95% PCAE對HeLa cell line 具有專一性抗癌能力.....................40
3.1.3 10% PCAE對MCF-7 cell line具有專一性抗癌能力.....................41
3.2 Nitrite Assay...................................................41
3.2.1 PCAE具明顯抑制NO產生功效........................................41
3.2.2 PCAE對RAW 26.7 cell line不具有明顯毒性..........................42
3.2.3 PCAE抑制NO Index..............................................42
3.3 Phagocytosis Assay..............................................42
3.3.1 PCAE促進RAW 264.7 cell line吞噬E. coli.........................43
3.3.2 PCAE促進RAW 264.7 cell line吞噬Latex Beads.....................43
3.3.3 PCAE抑制SD Rat Leukocyte吞噬能力...............................43
3.4 SD Rat Pathology................................................44
3.4.1 SD Rat Viability Ratio........................................44
3.4.2 PCAE治療LPS誘導SD Rat Diarrhea症狀.............................44
3.4.3 PCAE治療LPS誘導SD Rat Eyes Blooding症狀........................45
3.4.4 PCAE使SD Rat整體健康率回復......................................45
3.5 CAM Assay.......................................................45
3.6 抗氧化試驗.......................................................46
3.6.1 PCAE對DPPH Clearance Ratio ....................................46
3.6.2 PCAE對普魯士藍還原力............................................46
3.6.3 總多酚含量測試.................................................47
3.7 發炎因子基因表現..................................................48
第四章 結果討論......................................................49
第五章 參考文獻.....................................................104
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