臺灣博碩士論文加值系統

天麻中主要有效成分為天麻素(gastrodin)與其代謝物天麻苷元(p-Hydroxybenzyl alcohol, 4-HBA)，兩者均具有特殊生理活性。已有研究發現，天麻素具有抗癲癇、治療肢麻癱瘓、鎮靜、抗驚厥、抗炎、鎮痛、抗衰老及改善記憶學習等功效。氧化壓力會產生自由基，進而引起蛋白質、醣類及脂肪等成分進行連鎖反應，而破壞體內的細胞膜、蛋白質及核酸等，人體許多功能逐漸破壞，造成老化、癌症、動脈粥樣硬化(atherosclerosis)、發炎反應和免疫問題等疾病。本研究使用中藥天麻，利用不同溶劑(去離子水、甲醇或70%乙醇)之熱回流萃取天麻成分，測定萃取率、抗氧化活性試驗及細胞生存能力之測定。抗氧化活性測試包含α,α-diphenyl-β-picryl -hydrazyl (DPPH)自由基清除能力、還原力試驗和相對還原力、超氧歧化酶(superoxide dismutase) 活性、ABTS陽離子自由基清除活性及抑制硫代巴比妥酸鹽反應產物(thiobarbituric acid reactive substances, TBARS)生成之活性，並測定抗氧化物質總酚含量。結果顯示，天麻不同溶劑萃取率分別為：去離子水萃取物40.9%、甲醇萃取物20.6% 和乙醇萃取物9.77%；總酚含量之測定以甲醇萃取物最高，達6.48 mg/g；DPPH自由基清除能力以甲醇萃取物最佳，在濃度0.4 mg/mL時達到97.3%；在還原力測定中，去離子水萃取物有最高還原力，其相對還原力在0.8 mg/mL達23.3%；超氧歧化酶活性測定中，乙醇萃取物有最高活性，在濃度1 mg/mL時達到44.9%；ABTS陽離子自由基清除活性中，乙醇萃取物有最高活性，在濃度0.1 mg/mL時達到26.8%；抑制硫代巴比妥酸鹽反應產物之活性測定，乙醇萃取物具有最高活性，在濃度1 mg/mL時達到93.4%。以缺氧再灌氧模式對PC-12細胞株存活度測定中，70%乙醇萃取物有最高保護性，在濃度100μg/mL時達到81.3%; 以過氧化氫模式對PC-12細胞株存活度測定中，標準品4-HBA 之細胞保護活性較高，在濃度50 μg/mL 時約達97.1%; 熱迴流法之70%乙醇萃取物較差，其最佳保護活性在濃度50 μg/mL 時，細胞存活度仍達94.3%。

Gastrodin and p-hydroxybenzyl alcohol (4-HBA) are the main active constituents of Gastrodia elata of which has numerous physiological functons, and has been used as an anti- convulsant, analgesic and sedative agent for the therapeutics of epilepsy, paralysis, inflammation, aging, and ameliorated memory impairment. The oxidative stress is caused by free radicals that increase the chain reactions of proteins, polysaccharides and lipids. The results are altered cellular oxidation and impaired cellular function in many neurodegenerative diseases including aging, cancer, atherosclerosis, inflammation and immune problems. In ths study, extracts of Tianma (G. elata), extracted with a variey of solvents via diferrent extraction, were applied to the cell viability and the measurement of the yield, total phenol content and the in vitro antioxidant activity, including the free radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ABTS cation, reducing power assay, superoxide dismutase activity and the inhibition activity of thiobarbituric reactive substances (TBARS); the PC-12 cell line was established to evaluate the protective function of G. elata extracts. As a result, the water extract obtained via hot reflux has the highest yield of 40.9%. For the antioxidant activity evaluation, the methanolic extract of G. elata has the highest DPPH scavenging activity (97.3%) at a concentrationf 0.4 mg/mL and the ethanolic extract of G. elata has the highest inhibition capacity of TBARS (93.4%) at a concentration of 1.0 mg/mL. The result of PC-12 cell viability in hypoxia-reoxygenation model suggested that the ethanolic extract of G. elata has the highest protective effect (about 81.3%) at a concentra-tion of 100 μg/mL. In the protective activity result of H2O2-induced cell death model, the highest protective activity (about 97.1%) standard 4-HBA had and followed the 70% ethanolic extract of G. elata (about 94.3%) at a concentration of 50 μg/mL.

目錄
封面內頁
簽名頁
中文摘要…………………………………………………iii
英文摘要…………………………………………………v
致謝………………………………………………………vii
目錄………………………………………………………viii
圖目錄……………………………………………………xiii
表目錄……………………………………………………xiv
1. 緒論……………………………………………………‥1
2. 文獻回顧………………………………………………‥2
2.1 天麻簡介 ……………………………………………‥2
2.2 天麻分類 ……………………………………………‥2
2.2.1天麻特徵……………………………………………‥2
2.2.2天麻的營養方式……………………………………‥3
2.2.3天麻之藥物使用與安全性…………………………‥3
2.3天麻目前研究…………………………………………‥7
2.3.1 天麻萃取物抑制多巴胺系統相關疾病機轉 ……‥7
2.3.2 天麻萃取物抑制GABA系統相關疾病機轉………‥8
2.3.3 天麻萃取物抑制一氧化氮合成酶(NOS)機轉…‥14
2.3.4 天麻萃取物調控乙二醛酶解毒系統機制………‥15
2.4 自由基……………………………………………‥‥19
2.4.1 自由基簡介……………………………………‥‥19
2.4.2 自由基之產生…………………………………‥‥19
2.5 抗氧化機制…………………………………‥‥‥‥23
2.5.1 抗氧化劑………………………………………‥‥23
2.5.2 抗促氧化反應之原理………………………………24
2.5.3 過氧化氫誘發細胞損傷………………………‥‥25
2.6 實驗目的…………………………………………‥‥26
3. 材料與方法……………………………………………‥27
3.1 實驗材料 ……………………………………………‥27
3.1.1 天麻萃取物製備……………………………………27
3.1.2培養PC-12細胞株…………………………………‥27
3.2 試驗材料……………………………………………‥ 27
3.2.1 試驗溶劑…………………………………………‥27
3.2.2 試驗藥品……………………………………………28
3.2.3 PC-12細胞株之培養基組成……………………‥29
3.2.4 PC-12細胞株之培養方法………………………‥30
3.3 試驗設備……………………………………………‥30
3.4 實驗架構……………………………………………‥31
3.5 天麻之抗氧化試驗…………………………………‥33
3.5.1 天麻苷元含量分析………………………………‥33
3.5.2抗氧化成分分析…………………………………‥ 33
3.5.2.1 總酚化合物含量分析…………………………‥33
3.5.2.2 類黃酮含量測定………………………………‥34
3.5.3抗氧化活性測試…………………………………‥ 36
3.5.3.1還原力測試……………………………………‥ 36
3.5.3.2清除超氧陰離子活性試驗……………………‥ 36
3.5.3.3清除α,α-Diphenyl-β-picrylhydrazyl(DPPH) 自由基之測定…………‥37
3.5.3.4螯合亞鐵離子之測定……………………………………………………………‥ 37
3.5.3.5硫代巴比妥酸鹽反應產物 (TBARS)之測定………………………………………37
3.5.3.6清除ABTS陽離子自由基能力………………………………………………………38
3.5.4細胞存活度(Cell viability)試驗……………………………………………… 39
4. 結果與討論……………………………………………………………………………… 41
4.1 天麻萃取率………………………………………………………………………………41
4.2 天麻苷元含量測定………………………………………………………………………41
4.3 抗氧化成分含量分析……………………………………………………………………42
4.3.1 總酚化合物含量測定…………………………………………………………………42
4.3.2 類黃酮含量測定………………………………………………………………………42
4.4 抗氧化活性測試…………………………………………………………………………45
4.4.1 還原力測試…………………………………………………………………………‥45
4.4.2 螯合亞鐵離子之測定………………………………………………………………‥45
4.4.3 清除α,α-Diphenyl-β-picrylhydrazyl(DPPH) 自由基之測定…………‥ 47
4.4.4 清除超氧陰離子活性測試…………………………………………………………‥50
4.4.5 硫代巴比妥酸鹽反應產物 (TBARS)之測定…………………………………………52
4.4.6 清除ABTS陽離子自由基能力…………………………………………………………54
4.5細胞存活度(Cell viability)試驗……………………………………………………57
4.5.1缺氧再灌氧模式…………………………………………………………………………57
4.5.2過氧化氫模式……………………………………………………………………………57
5. 結論…………………………………………………………………………………………62
5.1結論…………………………………………………………………………………………62
5.2展望…………………………………………………………………………………………63
參考文獻…………………………………………………………………………………………64
附錄………………………………………………………………………………………………73
圖目錄
圖2.1 a 天麻植株…………………………………………………………………………………5
圖2.1 b 天麻乾燥塊莖……………………………………………………………………………6
圖2.2 a 天麻主要活性成分之化學結構式………………………………………………………12
圖2.2 b GABA與其代謝物之交互作用……………………………………………………………13
圖2.3 動物體內乙二醛類化合物代謝途徑………………………………………………………18
圖2.4 a自由基連鎖反應簡式………………………………………………………………………21
圖2.4 b脂肪酸之自由基連鎖反應…………………………………………………………………21
圖 3.1 實驗架構……………………………………………………………………………………32
圖 3.2 類黃酮之分類………………………………………………………………………………35
圖 4.1 天麻萃取物之相對還原力…………………………………………………………………46
圖 4.2 天麻萃取物之清除DPPH自由基能力………………………………………………………49
圖 4.3 天麻萃取物之清除超氧陰離子活性………………………………………………………51
圖 4.4 天麻萃取物之抑制TBARS活性……………………………………………………………53
圖 4.5 天麻萃取物之清除ABTS活性………………………………………………………………56
圖 4.6 天麻萃取物對細胞生存能力之測定(缺氧再灌氧模式)....................59
圖 4.7 天麻萃取物對細胞生存能力之測定(過氧化氫模式)......................60
表目錄
表 1. 天麻萃取物之產率、總類黃酮及總酚含量表..........................43
表 2. 天麻苷元含量.................................................44

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