臺灣博碩士論文加值系統

樟芝 (Antrodia camphorida) 生長於台灣特有之牛樟樹 (Cinnamomun kanehirai) 中。根據文獻指出樟芝具有解毒、保肝、降血糖、抗腫瘤、抗氧化、抗過敏、抗發炎等生物活性功能，近年來由於牛樟樹受到濫砍之影響，逐漸稀有野生樟芝生長不易，改以分離子實體之菌絲來培養大量培養菌絲體為主。因此，本研究以樟芝菌絲體為材料，利用不同溶媒系統萃取出5個多醣區分物，分別進行化學相關組成之分析，並探討各區分物對脂多醣 (lipopolysaccharide, LPS) 誘發老鼠巨噬細胞 (macrophage, RAW264.7) 所造成之發炎反應是否具有調節之作用。利用不同溶媒系統所萃取之AC-1、AC-2、AC-3、AC-4及AC-5五個區分物，其回收率分別為2.92%、10.38%、1.65%、0.34%、1.64%。於清除1,1-二苯基苦味肼基自由基團 ( 1,1-diphenyl-2-picryl hydrazyl，DPPH�P) radical能力方面，在濃度1 �嵱下，以AC-1清除率最高，可達74.53%。螯合亞鐵離子 (Fe2+) 能力方面，各區分物在濃度10 mg/mL 時，其螯合效果以AC-5最高，可達73.43%。此外，配合膠體透析層析法 ( gel permeation chromatography，GPC ) 分析所得各區分物之分子量，分別為AC-1 (152~354 kD)、AC-2 (4.8~386 kD)、AC-3 (5.2~677 kD)、AC-4 (28.7~911 kD)及AC-5 (124~411 kD)。於比較AC-1與AC-2之區分物對脂多醣誘發老鼠巨噬細胞RAW264.7發炎基因之調節研究中，結果顯示，在AC-2區分物中，隨著濃度增加 (50-200 �慊/ mL) 明顯降低inducible nitric oxide synthase (iNOS) mRNA、iNOS protein及其產物nitric oxide (NO)，且可同時抑制interleukin-6 (IL-6) mRNA、IL-10 mRNA之表現。於Cytokine chip中AC-2 可明顯抑制IL-6、IL-10、Monocyte chemoattractant-1 (MCP-1)、Monocyte chemoattractant-5 (MCP-5)與Regulated upon activation, normal T-cell expressed presumably (RANTES) 之蛋白質表現。然而，AC-1和AC-2皆無法抑制cyclooxygenase-2 (COX-2)基因之表現。綜合以上結果顯示，AC-2可藉由抑制脂多醣誘發老鼠巨噬細胞RAW264.7所造成發炎基因之表現，進而達到抗發炎之功效。

Abstract

Antrodia ca mphorate , uniquely grown and found in Taiwan, has been found only growing on the rotten and wet inner wall of the heart wood of the camphor trees Cinnamomum kanehirai Hay. It has long been used as a remedy for chemical antidote including food and drug intoxifications, diarrhea, abdominal pain, hypertension, itchy skin, liver disease and cancer. Recently, the population of the speies Cinnamomum kanehirai is almost extinguishing rapidly in Taiwan resulted in the scarcity of wild grown fruiting bodies of A. camphorate. Separating A. camphorate hyphae from fruiting bodies and inoculating them into a liquid culture medium is now a well-known and popular technique for the production of A. camphorata mycelia. In this present study, five fractionated polysaccharides isolated from mycelia of A. camphorate were prepared from systemic solvent extraction procedures and their proximate composition was investigated for characterizing the different structure. Furthermore, the major isolated fractions (AC-1 and AC-2) were investigated for the modulation effects on lipopolysaccharide-induced inflammation reactions in mouse macrophage cell line (RAW264.7). Five polysaccharide fractions AC-1, AC-2, AC-3, AC-4 and AC-5 were obtained with yield of 2.92 %, 10.38 %, 1.65 %, 0.34 % and 1.64 %, respectively. Gel permeation chromatography analysis showed the molecular weight range of each are AC-1 (152~354 kD), AC-2 (4.8~386 kD), AC-3 (5.2~677 kD), AC-4 (28.7~911 kD) and AC-5 (124~411 kD). In radicals scavenging acivities, AC-1 fraction showed greatest efficacy on DPPH radical scavenging activity around 74.53 % at 10 �嵱. The ferrous ion chelating power of each fraction showed highest efficacy at 10 mg/ mL, in which AC-5 elicited over 73.43 % inhibitory effect, much higher than that of citric acid. Furthermore, the effects of the extracted polysaccharide fractions on bacterial lipopolysaccharide (LPS) induce cytokines and NO production were in RAW264.7 cell line monitored. AC-2 but not AC-1 inhibited LPS-induced iNOS protein and mRNA expression, and dose-dependently (50-200�n�慊/ mL) suppressed NO production. Meanwhile, AC-2 was also found with inhibition activity on the production of IL-6 and IL-10 in LPS-induced mRNA expression. Using the cytokines array analysis, AC-2 was also shown with ablilty to inhibit LPS-induce protein expression of IL-6, IL-10, Monocyte chemoattractant-1 (MCP-1), Monocyte chemoattractant-1 (MCP-5) and Regulated upon activation, normal T-cell expressed presumably (RANTES). However, both AC-1 and AC-2 did not indicate the LPS-induced cyclooxygenase-2 (COX-2) gene expression. These results suggested that the A. camphorate polysaccharide AC-2 fraction exhibit its anti-inflammatory activity in mouse macrophage cells by inhibiting LPS-induced NO, IL-6, IL-10, MCP-1, MCP-5,and RANTES production.

目錄…………………………………………………………I
縮寫表…………………………………………………………… IV
中文摘要………………………………………………………VI
英文摘要………………………………………………………VIII
圖目錄……………………………………………………………X
表目錄……………………………………………………………XII

I. 緒論…………………………………………………1
II. 實驗材料……………………………………………………9
III. 實驗方法…………………………………………………15
III-1 一般成分分析……………………………………………15
III-2 多醣之區分………………………………………………17
III-3 抗氧化成分分析……………………………………………18
III-4 膠體透析層析……………………………………………18
III-5 單糖組成分析……………………………………………19
III-6 老鼠巨噬細胞株RAW264.7之培養…………………………20
III-7 細胞毒性試驗……………………………………………21
III-8細胞存活率………………………………………………21
III-9 一氧化氮試驗……………………………………………22
III-10 製備細胞溶解液…………………………………………23
III-11 蛋白質濃度測定………………………………………………23
III-12 西方點墨法………………………………………………24
III-13 抽取細胞全量RNA………………………………………28
III-14 反轉錄反應………………………………………………30
III-15 聚合酶連鎖反應…………………………………………31
III-6 蛋白質晶片試驗……………………………………………33
IV. 結果………………………………………………………36
IV-1 樟芝菌絲體之一般成分分析………………………………36
IV-2樟芝各多醣區分物之收率、蛋白質及碳水化合物之含量……36
IV-3樟芝各多醣區分物之平均分子量………………………37
IV-4 樟芝各多醣區分物之單糖組成……………………………37
IV-5 樟芝各多醣區分物對螯合亞鐵離子之能力………………37
IV-6 樟芝各多醣區分物對清除DPPH自由基之能力……………38
IV-7 分子量標準曲線圖……………………………………… 38
IV-8樟芝各多醣區分物藉由Sephadex G-100管柱之管柱溶離圖…38 IV-9樟芝各區分物對RAW 264.7 cells毒殺作用之影響……………39
IV-10樟芝各區分物影響RAW 264.7 cells之存活率分析………… 39
IV-11樟芝區分物對LPS誘導RAW 264.7 cells 其NO產生之影
響……………………………………………………40
IV-12 樟芝區分物對LPS誘導RAW 264.7 cells其 iNOS 蛋白質表現
之影響………………………………………………………… 40
IV-13 樟芝區分物對LPS誘導RAW 264.7 cells其iNOS mRNA表現之
影響……………………………………………………………41
IV-14 樟芝區分物對LPS誘導RAW 264.7 cells其COX-2 蛋白質表現
之影響…………………………………………………………41
IV-15 樟芝區分物對LPS誘導RAW 264.7 cells其COX-2 mRNA表現
之影響…………………………………………………………42
IV-16 樟芝區分物對LPS誘導RAW 264.7 cells其Cytokines蛋白質表
現之影響………………………………………………………42
IV-17 樟芝區分物對LPS誘導RAW 264.7 cells其IL-6 mRNA表現之
影響……………………………………………………………43
IV-18 樟芝區分物對LPS誘導RAW 264.7 cells其IL-10 mRNA表現之
影響……………………………………………………………43
V. 討論 ………………………………………………………44
VI. 參考文獻……………………………………………………47

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