臺灣博碩士論文加值系統

研究報告指出紫蘇子油具有預防動脈硬化、化學物質誘發之癌症以及改善免疫功能等。近年來，脫脂紫蘇子中所含酚類化合物已被指出具有抗菌及抗脂氧化酶活性之能力，且報告也指出其中含有多酚類化合物的物質，具有抗氧化、抗發炎、抗過敏、抗病毒以及抗癌之能力。雖然脫脂紫蘇子殘渣為一種工業廢棄物，其中之多酚類化合物或許可發展為保健功能產品原料之來源。本研究探討紫蘇子不同溶劑萃取物多酚含量及其抗氧化力與調控發炎反應之能力。紫蘇子萃取物分為以甲醇 (ME)、水萃取 (WE) 及分別以正己烷 (HM)、超臨界二氧化碳流體 (5000 psi, 40, 60, 80℃)(SM40, 60, 80) 脫脂後之殘渣，再以甲醇萃取等六部分。紫蘇子萃取物中酚類化合物，分別以高效能液相層析儀與液相層析-離子阱質譜儀分離確認其主要酚類成分為咖啡酸、迷迭香酸、迷迭香酸糖苷化合物、木犀草素與芹菜素。
體外抗氧化活性方面，以不同化學檢測方法分析其抗氧化能力，包括還原力、HRP-H2O2-luminol 發光系統、清除DPPH與ABTS•+ 陽離子自由基，結果顯示大多以ME與HM效果為最佳。另外對於細胞內抗氧化酵素活性之影響方面，以巨噬細胞 RAW264.7進行超氧陰離子歧化酶 (SOD) 及觸酶 (CAT) 活性之探討。在SOD活性實驗中，加入ME (400 μg/mL) 或HM (400 μg/mL) 可以顯著增加SOD之活性 (P < 0.05) 且巨噬細胞先經脂多醣誘發發炎反應後，添加ME (200 μg/mL) 或HM (400 μg/mL) 仍可顯著增加其活性。另外CAT之活性，較低濃度之ME (100 μg/mL) 或HM (200 μg/mL)，即能顯著提升 (P < 0.01) 其活性，若先以脂多醣誘發發炎反應後，添加ME (400 μg/mL) 或HM (400 μg/mL) 仍可顯著增加CAT活性，顯示ME或HM在巨噬細胞具有清除自由基之能力。
在抗發炎方面，探討以脂多醣誘發巨噬細胞RAW 264.7生成一氧化氮 (NO)、腫瘤壞死因子-α (TNF-α)、前列腺素E2 (PGE2)、誘發型一氧化氮合成酶 (iNOS)、II型環氧合酶 (COX-2) 及抑制蛋白 (IκB-α) 之表現。紫蘇子酚類成分於濃度250 μM (咖啡酸、迷迭香酸) 與25 μM (木犀草素、芹菜素) 皆能夠顯著抑制NO、PGE2、TNF-α、iNOS及COX-2的生成。ME、HM及SM40於濃度200 μg/mL以上，即可顯著抑制NO的產生，且呈劑量依隨效應；此三種萃取物在濃度400 μg/mL時，亦能顯著抑制TNF-α的產生，而WE皆不具抑制效果。另外ME、HM及WE皆對PGE2的產生具有抑制作用。綜合結果顯示萃取物中尤以ME、HM皆能抑制NO、PGE2及TNF-α的生成，而ME與HM在濃度400 μg/mL時，皆能顯著降低iNOS、COX-2表現 (P <0.01) 及阻斷IκB-α的降解。因此本研究之結論指出，紫蘇子多酚萃取物具有良好抗氧化效果，且可藉由阻斷IκB-α蛋白質降解，使NF-κB的活性下降，進而抑制細胞激素、iNOS及COX-2蛋白質的產生，進而達到抗發炎之功效。

Several reports have suggested that perilla seed oil has some functions for preventing atherosclerosis and chemically induced cancer and improving immune activity. The antioxidant, anti-inflammatory, anti-allergy, anti-viral and anti-cancer effects of phenolic compounds from perilla seed also have been reported in the past few years. Recently, phenolic compounds which existed in defatted perilla seed have been indicated with antimicrobial and antilipoxygenase activities. Although the defatted residue from perilla seed might be a kind of waste in oil and lipid industries, the existed polyphenols might be possible as one of good sources for developing a healthy food. In this study, we aimed to investigate the polyphenol contents of different solvent extracts from perilla seed and/or defatted seed, their antioxidant activity and the ability on inflammatory regulation in the lipopolysaccharide (LPS)-induced macrophage inflammatory response also would be studied. The preliminary crude extracts were prepared from perilla seed by using boiling water or methanol. After that, the defatted perilla seed from methanol extraction was re-extracted with n-hexane (HM). In addition, supercritical fluid extraction (carbon dioxide under 5000 psi pressure, at 40, 60 and 80 C and expressed as S40, S60, S80, respectively) was applied in the defat procedures in replace of the traditional used hexane solvent. The defatted meal was then extracted by using methanol (extract expressed as SM). In chemical composition analysis, phenolic components of different solvent extracts from perilla seed were isolated and identified by high performance liquid chromatography (HPLC) with photodiode array detection (PDA) and ion trap mass spectrometry with negative electron spray ionization mode. Four compounds, i.e. caffeic acid、rosmarinic acid、rosmarinic acid glucoside (Ra-G)、luteolin and apigenin were identified.
In the antioxidant activities assays in vitro, including reducing power, horseradish peroxidase (HRP)-H2O2-luminol system, scavenging activity on DPPH free radical and ABTS•+ cation free radical, were evaluated and compared. Results showed that the better effects of extracts on free radicals scavenging were from ME and HM. In addition, the antioxidant enzymes of superoxide dismutase (SOD) and catalase (CAT) were further compared in RAW 264.7 cell assays. In which, SOD activities were obviously up-regulated in the doses of 400 μg/mL from ME and HM, while the activities were also obviously elevated after adding ME (200 μg/mL) or HM (400 μg/mL) into the LPS-induced macrophages. CAT activities were obviously up-regulated in the doses of 100 and 200 μg/mL from ME and HM, respectively. The obviously elevated effects of ME and HM on the activities of antioxidant enzymes in LPS-stimulated cells were shown in ME and HM at 200, 400 μg/mL, respectively, in which the activities of SOD and CAT were elevated at 400 μg/mL concentrations significantly elevated the activities of CAT.
In anti-inflammatory experiments, murine macrophage RAW 264.7 cells were conducted for studying the inhibition abilities of extracts on LPS-induced inflammation markers, i.e. tumor necrosis factor-α (TNF-α), nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and inhibitor κB-α (IκB-α) proteins. Results showed that NO, PGE2, TNF-α, iNOS and COX-2 production were significantly inhibited by those of phenolic components found in perilla seed, including caffeic acid or rosmarinic acid in the doses of 250 μM and apigenin or luteolin in the doses of 25 μM. NO production was significantly inhibited at 200 μg/mL in ME, HM and SM40, respectively, and in a concentration-dependent manner. Moreover, the production of TNF-α was obviously (P < 0.05) inhibited at the concentration of 400 μg/mL in each extract as above. Contrarily, water extract showed no obviously effect on the productions of NO and TNF-α. PGE2 production was significantly inhibited at 400 μg/mL in ME, HM and WE, respectively. Western blot analysis revealed the suppression of iNOS, COX-2 expressions and IκB-α degradation were in the doses of 400 μg/mL from HM and SM, respectively. Our results suggested that extracts prepared from defatted perilla seed had antioxidant activities and the inhibition activities on the production of cytokines. Moreover, the production of iNOS and COX-2 proteins induced by LPS could be inhibited by extracts through blocking IκB-α degradation and therefore the inactivation of NF-κB.

中文摘要.................................................................................................Ⅴ
英文摘要...............................................................................................VII
圖次.........................................................................................................X
表次......................................................................................................XIV
第一章 序論............................................................................................1
第二章 文獻整理…................................................................................2
第一節 紫蘇…....................................................................……………2
第二節 超臨界二氧化碳..............................…………………..………8
第三節 化學冷光…..............................………………………………..9
第四節 自由基與抗氧化防禦系統..............................…………….....13
第五節 巨噬細胞與發炎反應..............................…………………….33
第三章 研究目的與實驗架構. ..............……………………………...46
第四章 材料與方法…..............……………………………………….48
第一節 材料來源..............…………………………………………….48
一、樣品..............……………………………………………………...48
二、實驗試藥.........................................................................................48
三、實驗設備…..............……………………………………………...51
第二節 實驗方法..............…………………………………………….52
一、紫蘇子萃取物之製備….....……………………………………....52
二、一般組成成分分析.........................................................................53
三、紫蘇子酚類化合物之含量分析….....…………………………....55
(一) 總酚類化合物 (Total polyphenols)含量之測定….......................55
(二) 總類黃酮 (Total flavonoids)含量之測定…..................................55
(三) 縮合型單寧 (Condensed tannins)含量之測定..............................55
四、抗氧化性檢測方法….....................................................................56
(一) DPPH自由基清除能力測定...........................................................56
(二) 還原力(Reducing power)測定…...................................................56
(三) Trolox抗氧化當量活性..................................................................57
(四) HRP-luminol-H2O2 system..............................................................57
五、HPLC及LC-MS分析…...................................................................58
(一) HPLC分析.......................................................................................58
(二) LC-MS分析.....................................................................................58
六、細胞培養.........................................................................................59
七、細胞存活率測定 (MTT assay)…..........................................……60
八、細胞內抗氧化酵素活性分析….....................................................61
九、細胞內發炎物質分析.....................................................................62
十、統計分析….....................................................................................66
第五章 結果與討論…...........................................................................67
第ㄧ節 紫蘇子成份分析.......................................................................67
ㄧ、紫蘇子之一般組成.........................................................................67
二、紫蘇子萃取物之產率與酚類化合物之組成.................................67
三、紫蘇子萃取物之主要酚類成分定性定量分析.............................70
第二節 化學性抗氧化活性測定...........................................................76
一、DPPH自由基清除能力...................................................................76
二、還原力.............................................................................................78
三、總抗氧化能力.................................................................................80
四、HRP-H2O2-luminol system..............................................................82
第三節 細胞內抗氧化酵素系統................................ ..........................88
一、紫蘇子多酚粗萃物對巨噬細胞RAW 264.7細胞存活率之影響..88
二、超氧陰離子歧化酶 (SOD)活性................. ..................................90
三、觸酶 (CAT)活性............................................................................90
第四節 抗發炎活性...............................................................................93
一、ㄧ氧化氮 (NO)生成......................................................................93
二、腫瘤壞死因子-α (Tumor necrosis factor-alpha；TNF-α)生成.....96
三、前列腺素E2 (Prostaglandins E2；PGE2)生成................................98
四、紫蘇子萃取物對誘發型一氧化氮合成酶 (iNOS)表現之影響..100
五、紫蘇子萃取物對II型環氧合酶 (COX-2)表現之影響.................103
六、紫蘇子萃取物對κB抑制蛋白 (IκB-α)表現之影響.....................106
第六章 結論.....……………………………………………………….110
第七章 參考文獻……………………………………………………..111

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