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研究生:張裴文
研究生(外文):Pei-weng chang
論文名稱:肉桂中抗神經發炎成分的鑑定
論文名稱(外文):Identification of anti-neuroinflammatory components in cinnamon
指導教授:何素珍何素珍引用關係
指導教授(外文):Su-Chen Ho
學位類別:碩士
校院名稱:元培科技大學
系所名稱:食品科學研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
畢業學年度:101
語文別:中文
論文頁數:104
中文關鍵詞:阿茲海默症帕金森氏症肉桂肉桂醛BV2微膠細胞酯多醣
外文關鍵詞:Alzheimer diseaseParkinson's diseasecinnamoncinnamaldehydeBV2 microglialipopolysaccharides
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神經退化性疾病,如:阿茲海默症(Alzheimer disease)及帕金森氏症(Parkinson’s disease)的罹患率會隨著老化而增加,而高等糖化終產物(AGEs)的堆積及神經發炎反應被認為是此類疾病發展及惡化的重要因素。肉桂具有高度的抗氧化及抗發炎活性,能抑制週邊巨噬細胞的活化及發炎反應,但肉桂能否抑制微膠細胞活化所介導的神經發炎反應,以及肉桂中何種活性成分具有抗神經發炎能力目前並不清楚。因此,本研究探討肉桂萃取物的抗神經發炎能力,並鑑定其主要活性成分。
肉桂乙醇粗萃物依序以正己烷、乙酸乙酯、正丁醇和水等不同極性的溶劑進行萃取分劃,分別經真空濃縮及冷凍乾燥後,分析總多酚及類黃酮含量、抗氧化能力、抗糖化能力及抑制LPS誘導BV2微膠細胞分泌促發炎激素的抗神經發炎能力,並利用高效能液相層析儀(HPLC)及電噴霧離子質譜儀(ESI-MS)分析鑑定各分劃物的主要組成分,用以探求出肉桂中抗神經發炎的主要活性成分。
成分分析結果顯示:乙酸乙酯分劃物的總多酚(81.3mg GAE/g)及類黃酮(33.7mg CE/g)含量最豐富,正己烷分劃物,分別為16.7mg GAE/g及4.4mg CE/g。乙酸乙酯分劃物具有最強的鐵還原能力(0.2 mmole TE/g)及氧自由基吸收能力(1.1 mmole TE/g)。此外,各分劃物的抗氧化能力與其總多酚及類黃酮含量呈正相關。在體外BSA/glucose糖化系統中,2 mg/ml的正己烷、正丁醇、乙酸乙酯及水分劃物分別抑制了97.6%、94.3%、87.8%及71.8%的螢光AGEs的生成,顯示正己烷分劃物具有最高的抗糖化活性,反之,水分劃物的抗糖化效果最差。
抗神經發炎活性結果顯示:50 μg/mL正己烷分劃物幾乎能完全抑制發炎激素NO、TNF-α及IL-6的分泌,抑制率分別達99.4%、95.2%與99.5%,具有最佳的抗神經發炎活性。200 μg/mL乙酸乙酯分劃物分別能抑制98.9%、88.3%與92.6%的NO、TNF-α及IL-6的分泌;正丁醇及水分劃物在200 μg/mL濃度下雖能抑制發炎激素的分泌,但抑制率都不達50%,顯示正丁醇及水分劃物的抗神經發炎活性較差。此外,50 μg/mL正己烷分劃物及200 μg/mL乙酸乙酯分劃物能完全抑制iNOS蛋白質及iNOS、IL-1β、IL-6與TNF-α mRNA的表現。正己烷分劃物也顯著地降低細胞核中NF-κB的活性(72.7%),表示正己烷分劃物是藉由阻斷上游轉錄因子NF-κB的活化,進而抑制iNOS、IL-6、TNF-α等下游促發炎因子的表現,發揮其抗神經發炎作用。
以LC-MS分析鑑定肉桂各分劃物中主要成分,結果發現:相較於乙酸乙酯、正丁醇和水等分劃物,正己烷分劃物中含有較高的香豆素(37.8 mg/g)與肉桂醛(463.2 mg/g)。在100 μM濃度下,香豆素幾乎無法抑制發炎激素的分泌,反之,肉桂醛分別能抑制77.2%、80.8%及81.2%的NO、IL-6及TNF-α的分泌,具有很強的抗神經發炎活性。
綜合本論文研究結果推論,肉桂醛為肉桂中主要的抗神經發炎活性成分,期許肉桂及其主成分肉桂醛可作為預防神經退化性疾病的輔助食品。

The prevalence of neurodegenerative diseases including Alzheimer disease and Parkinson’s disease is increased with aging. Accumulation of advanced glycation end-products (AGEs) and neuroinflammation are considered as the critical causative factors of neurodegenerative diseases. Although the potent anti-inflammatory and antioxidant activity of cinnamon has been well documented in peripheral macrophages, the effect of cinnamon on microglia-mediated neuroinflammation is unclear. Thus, purpose of this study is to evaluate the anti-neuroinflammatory capacity of cinnamon and, furthermore, to identify the corresponding active components.
Cinnamon ethanolic extract was extracted, in sequence, with hexane, ethyl acetate, butanol and water and then vacuum-dried or freeze-dried to remove solvent. Each fraction solid was subjected to determine the total phenolic, total flavonoids, antioxidant capacity, anti-glycation capacity and anti-neuroinflammatory activity. The anti-neuroinflammatory activity of each fraction was evaluated based on their capacity to inhibit pro-inflammatory factor secretion in LPS-activated BV2 microglia. High-performance liquid chromatography (HPLC) and Electrospray ionization mass spectrometry (ESI-MS) was applied to analyze the composition of each fraction.
Among the four fractionations, ethyl acetate fraction contained the most abundant phenolics (81.3mg GAE/g) and flavonoids (33.7mg CE/g), hexane fraction contained 16.7mg GAE/g of phenolics and 4.4mg CE/g of flavonoids, respectively, was the poorest. Ethyl acetate fraction had the best iron-reducing ability (0.2 mmole TE/g) and oxygen radical absorbance capacity (1.1 mmole TE/g). Furthermore, the amount of phenolics and flavonoids of each fraction highly correlated with their respective antioxidant capacity. At a concentration of 2 mg/ml, hexane, butanol, ethyl acetate and water fraction inhibited the formation of fluorescence AGEs by 97.6%, 94.3%, 87.8% and 71.8%, respectively, in an in vitro glucose-mediated protein glycation system. This result indicated that hexane fraction had potent anti-glycation capacities, while water fraction was poor at anti-glycation.
Hexane fraction at a lower concentration, namely 50 μg/mL, could completely inhibit the production and expression of LPS-induced NO, TNF-α and IL-6, therefore, had the best anti-neuroinflammatory activity. Ethyl acetate fraction inhibited LPS-induced NO, TNF-α and IL-6 secretion by 98.9%, 88.3% and 92.6%, respectively, at a concentration of 200 μg/mL. In contrast, butanol and water fraction at a concentration of 200 μg/mL did not have any inhibitory effect on LPS-induced inflammatory factor secretion, was the poorest. Furthermore, 50 μg/mL hexane fraction and 200 μg/mL of ethyl acetate fraction significantly decreased the iNOS protein expression as well as iNOS, IL-1β, IL-6 and TNF-α mRNA levels. Hexane fraction also significantly reduced the LPS-induced NF-κB binding activity by 72.7%, implying that hexane fraction down-regulated the expression of these pro-inflammatory factors by through suppression the activation of the convergent upstream transcription factor NF-κB.
LC–MS results showed that hexane fraction contained abundant coumarin (37.8 mg/g) and cinnamaldehyde (463.2 mg/g). At a same concentration (100 μM), cinnamaldehyde inhibited LPS-induced NO, IL-6 and TNF-α secretion by 77.2%, 80.8% and 81.2%, respectively, in contrast, coumarin could not provide any inhibitory effect on these inflammatory factor secretion. This result indicated that cinnamaldehyde had potent anti-neuroinflammatory activity and might be the main active component responsible for the anti-neuroinflammatory capacity of cinnamon.
In sum, cinnamon has a potent anti-neuroinflammatory capacity which is primarily attributed to cinnamaldehyde. Additionally, based on their anti-neuroinflammatory capacity, cinnamon and cinnamaldehyde may be useful as a dietary adjuvant to prevent neurodegenerative diseases.

論文口試審定書 II
誌謝 III
中文摘要 IV
英文摘要 VI
目錄 VIII
圖目錄 X
表目錄 XII
縮寫對照表 XIII
前言 1
第一章 文獻回顧 3
1.1 阿茲海默症簡介 3
1.2 帕金森氏症簡介 4
1.3 自由基與氧化壓力對人體的影響 5
1.4 神經膠質細胞BV2介紹 8
1.5 酯多糖介紹 9
1.6 肉桂簡介 13
1.7 肉桂主要組成分介紹 16
研究架構 .19
第二章 材料與方法 20
2.1肉桂樣品來源與製備 21
2.2肉桂分劃物之成分分析 21
2.3抗氧化能力分析 23
2.4體外糖化分析 .24
2.5肉桂分劃物細胞實驗 25
2.6肉桂分劃物其主要活性成分之分析 33
2.7統計分析 35
第三章 結果與討論 37
3.1肉桂分劃物成分分析 37
3.2肉桂分劃物抗氧化活性之探討 37
3.3肉桂分劃物體外糖化之探討 38
3.4肉桂分劃物抑制BV2神經膠質細胞發炎活性之探討 39
3.5肉桂分劃物其主要組成份之分析結果 42
3.6肉桂主要組成分純品抗發炎活性分析 43
第四章結論 46
第五章 參考文獻 .82


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三、 網路資料:
美聯社-
Last Updated: Monday, June 11, 2007 | 12:55 PM ET The Associated Press
國際厚生健康園區-
http://www.24drs.com/special_report/Alzheimers_disease/about_0.asp
上海肯強儀器有限公司-
http://www.shkq17.com/xibaozhu/BV2.html
中華康網-
http://www.cnkang.com/zyzy/201212/915522.html
Chemical book -
http://www.chemicalbook.com/ProductIndex.aspx
Blue blood-
http://hermes.mbl.edu/marine_org/images/animals/Limulus/blood/lps.html

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