臺灣博碩士論文加值系統

近年來，隨著國人健康意識逐漸抬頭，中草藥植物的開發與運用，已成為重要的發展趨勢。本研究從數十種中草藥中，以總多酚含量測定及總黃酮含量測定篩選出富含多酚及類黃酮的材料，分別為女貞子、升麻、牛蒡子、地骨皮、辛夷、防風、知母、羌活、金銀花、荊芥、旋覆花、連翹、紫蘇、蒼耳子、蔓荊子等十五種實驗材料。本研究分為試管內與試管外試驗，第一部分探討其DPPH自由基清除率、TEAC總抗氧化能力、還原力、亞鐵離子螯合能力、微脂粒（liposome）氧化作用、Superoxide自由基清除能力、NO抑制能力、酪胺酸酶活性分析、總多酚含量、類黃酮含量測定等九個抗氧化平台找出較具潛力之藥材，進而建立此類藥材之HPLC化學成份指紋圖譜。第二部分為藉由巨噬細胞(RAW264.7)模式系統來評估中草藥萃取物對LPS誘導一氧化氮(nitric oxide, NO)生成率及分析胞內活性氧 (ROS)含量。
研究結果顯示，在抗氧化分析方面，發現連翹、旋覆花及蔓荊子有最佳的DPPH自由基清除率，連翹、旋覆花在濃度為100 ppm及蔓荊子在濃度為150 ppm時均超過90 %；TEAC值測定結果顯示連翹、旋覆花在濃度為75 ppm及蔓荊子在濃度為100 ppm時均超過90 %；亞鐵離子螫合能力結果顯示連翹、旋覆花在濃度為600 ppm及蔓荊子在濃度為700 ppm亞鐵離子螫合能力達80％以上；在還原能力測定結果顯示連翹、旋覆花在濃度為100 ppm及蔓荊子在濃度為150 ppm時其OD700值均超過0.477以上；在微脂粒氧化作用之抑制力試驗中，連翹、旋覆花於濃度為400 ppm時，抑制效果分別為93.3%、89.8%，蔓荊子在濃度600 ppm時抑制效果為95.9%；在清除superoxide方面連翹在濃度400 ppm，旋覆花、蔓荊子在濃度600 ppm時均超過80 %；在抑制NO方面連翹、旋覆花及蔓荊子在濃度250 ppm時抑制效果分別為94.3%、90.8%、88.6%；在總多酚定量試驗，連翹、旋覆花及蔓荊子每克萃取物含有相當於 226.5 mg、210.7 mg、139.9 mg之gallic acid。總類黃酮測定，連翹、旋覆花及蔓荊子之每克萃取物含有相當於126.4mg、125.2 mg、82.1mg之rutin。酪胺酸酶活性評估顯示，連翹、旋覆花及蔓荊子在濃度1500 ppm、2000 ppm與2000 ppm時，抑制率分別為87.0%、90.3% 與88.4%。連翹、旋覆花及蔓荊子HPLC的指紋圖譜，顯示含有quercitrin、rutin 、chlorogenic acid 、 kaempferol、 vanillic acid、 luteolin, ferulic acid等成份。在抗發炎試驗方面旋覆花在濃度100ppm 時有超過50%的抑制效果；連翹在100 ppm的時有超過50%的抑制效果；女貞子、防風、紫蘇葉、金銀花在濃度400ppm時抑制效果均超過50%。在ROS試驗方面，有四種樣品具有抑制ROS生成效果，分別是金銀花、羌活、知母以及連翹。在LPS誘導細胞產生ROS方面有七種樣品具有抑制生成效果，分別是金銀花、羌活、知母、升麻、蒼耳子、牛蒡子以及荊芥具有抑制ROS生成效果。

In recent years, people pay more attention on the health issues, therefore, the development and application of the Chinese herbals have become the world trend. In this study, 15 Chinese herbal medicine water extracts including Schizonepeta tenuifolia, Vitex trifolia, Saposhnikovia divaricata, Forsythia suspensa, Lonicera japonica, Inula britannica, Anemarrhena asphodeloides, Cimicifuga heracleifolia, Lycium chinense, Arctium lappa, Ligustrum lucidum, Perilla frutescens, Xanthium strumarium, Notopterygium incisum and Magnolia biondii were conducted to determine their antioxidant and anti-inflammatory effects. In Part 1, we focused on the studies of the antioxidant activities, include the scavenging effect on α,α-diphenyl-β-picrylhydrazyl(DPPH) radicals, the trolox equivalent antioxidant capacity (TEAC), reducing power, ferrous ion chelating power, inhibition NO capacity, scavenging superoxide radical capacity and inhibitory effect on lipid peroxidation total polyphenols and flavonoids. The HPLC-UV finger print chromatogram and antityrosinase effects of the potent herbs were also measured. In part two, we investigated the effects of Chinese herbals, on RAW 264.7 macrophages production NO induced by LPS.
The results show that all these herbal extracts demonstrate antioxidant abilities and in dose-dependent manner. Forsythia suspensa, Inula britannica, and Vitex trifolia have the greatest DPPH radical scavenging effect. Inula britannica, Vitex trifolia and Cuscuta chinessis have better antioxidative activity by TEAC. The ferrous ion chelating capacity of the Forsythia suspensa, Inula Britannica and Vitex trifolia have the best effect. The reducing power of the Forsythia suspensa, Inula Britannica and Vitex trifolia have the better effect. Inhibition NO capacity of Forsythia suspensa, Inula Britannica and Vitex trifolia have the better inhibition effect. Scavenging superoxide radical capacity of the Forsythia suspensa, Inula Britannica and Vitex trifolia have the greatest superoxide radical scavenging effect. Forsythia suspensa, Inula Britannica and Vitex trifolia have the better effect of inhibitory effect on lipid peroxidation. Forsythia suspensa, Inula Britannica and Vitex trifolia had total polyphenol contents (226.5 mg、210.7 mg and 139.9mg of gallic acid equivalent, respectively) and total flavonoid contents (126.4 mg、125.2mg and 82.1mg equivalent of rutin, respectively). Forsythia suspensa, Inula britannica and Vitex trifolia exhibit potent inhibitory effects on tyrosinase. A high performance liquid chromatography analysis revealed that phenolic acids and flavonoids, such as quercitrin, rutin, chlorogenic acid, kaempferol, vanillic acid, luteolin and ferulic acid . We found that six extracts of Chinese herbs could inhibit nitric oxide generation induced by LPS by 50% or more in RAW 264.7 cells at a concentration of 400µg/ml including Inula britannica, Forsythia suspensa, Saposhnikovia divaricata, Ligustrum lucidum, Perilla frutescens and Lonicera japonica. Inula britannica has the best inhibitory effect. We found that four extracts of Chinese herbs could inhibit ROS production of RAW 264.7 cell including Lonicera japonica, Notopterygium incisum, Anemarrhena asphodeloides and Forsythia suspense. That seven extracts of Chinese herbs could inhibit ROS production generations induced by LPS in RAW 264.7 cell including Schizonepeta tenuifolia, Anemarrhena asphodeloides, Cimicifuga heracleifolia, Arctium lappa, Lonicera japonica, Xanthium strumarium and Notopterygium incisum.

中文摘要 I
英文摘要 III
目錄 VI
圖目錄 X
表目錄 XII
縮寫表 XIII
第一章 前言 1
第二章 文獻回顧 2
第一節 中草藥文獻 2
第二節 自由基 6
一、 自由基(Freer radicals)之定義 6
二、 自由基來源及種類 6
第三節 抗氧化能力分析 8
一、 DPPH自由基清除能力之效應 8
二、 總抗氧化力（TEAC）之測定 9
三、 還原力之測定 9
四、 亞鐵離子螯合能力之測定 10
五、 微脂粒（liposome）氧化作用之抑制 10
六、 Superoxide自由基清除能力之效應 11
七、 NO抑制能力 11
八、 酪胺酸酶活性分析 12
第四節 抗氧化成份定量分析 12
一、 總酚含量測定 12
二、 類黃酮含量測定 13
第五節 高效能液相層析HPLC之分析 13
第六節 巨噬細胞 15
一、 免疫反應與巨噬細胞 15
二、 細菌內毒素與巨噬細胞 15
三、 發炎反應與巨噬細胞 16
第七節 一氧化氮 ( nitric oxide，NO ) 17
第八節 胞內活性氧 (ROS) 19
第三章 材料與方法 20
第一節 實驗材料 20
一、 實驗藥材 20
二、 實驗藥品 20
三、 實驗儀器 21
四、 細胞株 22
第二節 實驗架構 23
第三節 實驗方法 24
一、 樣品萃取 24
二、 樣品配置 24
三、 抗氧化能力試驗 24
四、 抗氧化成份定量分析 28
五、 細胞實驗 29
六、 高效能液相層析HPLC之分析 33
第四章 結果與討論 36
第一節 抗氧化能力試驗 36
一、 中藥萃取率 36
二、 DPPH自由基清除能力之效應 37
三、 總抗氧化力（TEAC）之測定 40
四、 還原力之測定 43
五、 亞鐵離子螯合能力之測定 46
六、 微脂粒（liposome）氧化作用之抑制 49
七、 Superoxide自由基清除能力之效應 52
八、 NO抑制能力 55
九、 酪胺酸酶活性試驗 58
第二節 抗氧化成份定量分析 61
一、 總酚含量測定 61
二、 類黃酮含量測定 63
第三節 細胞抗發炎能力分析 66
第四節 細胞存活率試驗(MTT Assay) 68
第五節 胞內活性氧 (ROS) 分析 70
第六節 HPLC成份分析 73
第五章 結論 74
第六章 文獻參考 74

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