紫色狼尾草抗發炎試驗及有效成分分析

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標題:	紫色狼尾草抗發炎試驗及有效成分分析 Evaluation of anti-inflammatory effects and effective fractionation of purple napiergrass (Pennisetum purpureum Schumach) 本研究主要探討紫色狼尾草（Pennisetum purpureum Schumach）之抗氧化及抗發炎的保健功效。本論文係以不同條件萃取的紫色狼尾草萃取物對小鼠巨噬細胞株RAW264.7發炎介質生成之影響，來測試其體外抗發炎效果與機制，並進一步確認其有效成分。將紫色狼尾草乾燥粉末分別以沸水萃取10分鐘所得的萃取液以及水、50%乙醇和95%乙醇室溫振盪萃取1小時並重複三次後之萃取液，經減壓濃縮或凍結乾燥製備成萃取物來進行試驗，其萃取率分別為16.0%、18.2%、19.4%、18.9%。使用高效液相層析儀（high performance liquid chromatography, HPLC）分析，發現萃取物中含有矢車菊素-3-葡萄糖?（cyanidin-3-glucoside）、沒食子酸（gallic acid）、綠原酸（chlorogenic acid）、咖啡酸（caffeic acid）及鞣花酸（ellagic acid）等酚類物質。在抗氧化物質測定上，紫色狼尾草沸水萃取物在總酚物質及類黃酮物質含量比其他萃取條件為佳，每克萃取物分別含有149.1 mg 沒食子酸當量（gallic acid equivalents, GAE）之酚類物質及112.9 mg 芸香?當量（rutin equivalents, RE）之類黃酮物質。不同條件的萃取物進行抗氧化能力測試，由實驗結果得知，紫色狼尾草沸水萃取物較其他條件萃取物具有較佳的1,1-diphenyl-2-picrylhydrazyl（DPPH）自由基清除能力，其IC50數值為6.20 mg/mL。沸水萃取物具有最佳的鐵螯合能力，在20 mg/mL下可螯合91%之亞鐵離子。50%乙醇萃取物則具最佳Trolox當量的抗氧化能力（Trolox equivalent antioxidant capacity, TEAC）及還原力（Ferric reducing antioxidant capacity, FRAC），其TEAC為312.8 mM Trolox equivalents (TE)/g，而FRAC為20.0 mg 抗壞血酸當量（ascorbic acid equivalents, AAE）/g。沸水萃取物及50%乙醇萃取物具有較高的氧自由基吸收能力（Oxygen radical absorbance capacity, ORAC），分別為187.6及189.0 mM TE/g。為了解紫色狼尾草萃取物是否具有抗發炎的功效，以小鼠巨噬細胞株RAW264.7建立體外抗發炎模式，藉由脂多醣（Lipopolysaccharide, LPS）誘導活化RAW264.7細胞，觀察紫色狼尾草萃取物處理後對其一氧化氮（nitric oxide, NO）、腫瘤壞死因子-α（tumor necrosis factor-α, TNF-α）、介白素-6（interleukin-6, IL-6）和介白素-1β（interleukin-1β, IL-1β）發炎介質生成的影響。分別以LPS（1 μg/ml）及不同條件萃取的紫色狼尾草萃取物（12.5~250 μg/ml）處理RAW264.7細胞，收集細胞及其上清培養液分析其發炎介質含量。結果顯示，不同條件萃取之紫色狼尾草萃取物處理的RAW264.7細胞未觀察到細胞毒性作用，且對LPS誘發的RAW264.7細胞之TNF-α、IL-6和IL-1β等發炎介質具有顯著抑制效果，其中以95%乙醇萃取物之抑制效果最佳，在250 μg/mL的濃度下具有最高的抑制率可抑制54.5% TNF-α、52.3% IL-6及68.1% IL-1β。從西方墨點法的結果中發現，紫色狼尾草萃取物可減少誘導型一氧化氮合?（inducible nitric oxide synthase, iNOS）和環氧合?-2（cyclooxygenase-2, COX-2）的表現量，其中是以95%乙醇萃取物具有最佳的抑制效果，在250 μg/mL的濃度下可抑制51.3% iNOS及59.6% COX-2的表現量。為確認紫色狼尾草萃取物中有效的抗發炎成分，利用半製備型HPLC將紫色狼尾草95%乙醇萃取物進行區段分離。依時間間隔分離出七個區段（區段A~G），在分別收集其析出液進行抗發炎試驗，結果顯示區段E能夠有效抑制發炎介質的生成，且區段E與95%乙醇萃取物對於iNOS及COX-2表現的抑制程度在統計上表現一致。藉由HPLC的波長掃描結果推測區段E的成分可能為黃酮類物質，其結構中可能帶有甲基或糖基以及數個羥基。綜合上述結果，以含有最多酚類物質的紫色狼尾草沸水萃取物之抗氧化能力最佳，而95%乙醇萃取物之抗發炎效果最佳，其透過抑制iNOS及COX-2之表現來降低TNF-α等發炎介質的生成，進而達到抗發炎之效果。 This study was to investigate the antioxidant capacities and anti-inflammatory activities of purple napiergrass (Pennisetum purpureum Schumach) that were extracted under different conditions. To further clarify the possible mechanisms underlying the anti-inflammatory effects of purple napiergrass, utilized there was the purple napiergrass extracts to analyze its chemical composition and evaluated its potential anti-inflammatory effects in LPS-stimulated RAW264.7 macrophages in vitro treatment.The extracts of the dried powder of purple napiergrass were extracted with boiling water for 10 minutes and the extracts of water, 50% ethanol and 95% ethanol were shaken at room temperature for 1 hour and repeated three times. It was concentrated under reduced pressure or further lyophilized. The extracts extraction rates were 16.0%, 18.2%, 19.4%, and 18.9%, respectively. The phenol compounds would be identified, contained cyanidin-3-glucoside, gallic acid, chlorogenic acid, caffeic acid and ellagic acid by HPLC technique.Among the extracts, boiling water extract of purple napiergrass showed that of the highest total phenol and flavonoid content of 149.1 mg gallic acid equivalents (GAE)/g and 112.9 mg rutin equivalents (RE)/g, respectively. Based on DPPH free radical scavenging activity, the boiling water extract exhibited the best capacity of other extracts, with a better IC50 value of 6.20 mg/mL. The boiling water extract has the best iron chelating ability (91.0%), when all the extracts at the same concentration (20 mg/mL). The 50% ethanol extract has the best Trolox equivalent antioxidant capacity (312.8 mM Trolox equivalents/g) and Ferric reducing antioxidant capacity (20.0 mg ascorbic acid equivalents/g), which is significantly higher than other extracts. The oxygen radical absorbance capacity (ORAC) indicates higher values for boiling water and 50% ethanol extracts compared to that of others extracts of equivalent to 937.9 and 945.1 μM Trolox equivalents/mg, respectively. To assess the anti-inflammatory activity of purple napiergrass extracts, cytokines production by RAW264.7 cells was evaluated after culture of RAW264.7 cells with different concentration of extracts in the presence of LPS. Cytokines contained nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β). MTT analysis results showed that of the extracts did not significantly affect cell viability. And the extract has a significant inhibitory effect on TNF-α, IL-6 and IL-1β of RAW264.7 cells, among which 95% ethanol extract has the highest inhibition rates of 54.5%, 52.3% and 66.5% at a concentration of 250 μg/mL.In order to identify the compounds likely to provide the greatest anti-inflammatory, the semi-preparative HPLC was used to separate the 95% ethanol extract, and seven fractions (fractions A to G) were separated according to the time interval, of which fraction E could effectively inhibit inflammatory mediators and the inhibition rate of 95% ethanol extract is similar. It is estimated by wavelength scanning that fraction E is flavonoids, and the structure may have methyl or sugar groups and several hydroxyl groups. Based on the above results, the polyphenol enriched boiling water extracts from purple napiergrass are a good source of natural antioxidants, and the best anti-inflammatory effect of 95% ethanol extract from purple napiergrass is associated with the inhibition cytokines production through the downregulation of iNOS and COX-2 expression in LPS-stimulated RAW264.7 macrophages.
作者:	楊尚緯
貢獻者:	保健營養系王明雄
關鍵字:	紫色狼尾草抗氧化抗發炎小鼠巨噬細胞株RAW264.7 Purple napiergrass Antioxidant Anti-inflammation RAW264.7 macrophages
日期:	2020
上傳時間:	2022-10-21 10:31:13 (UTC+8)
關聯:	學年度：108， 124頁
顯示於類別:	[保健營養系(所) ] 博碩士論文

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