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研究生:翁義宗
研究生(外文):I-Zong Weng
論文名稱:桑椹之抗氧化及對脂多醣所誘導RAW264.7巨噬細胞產生PGE2及NO之影響
論文名稱(外文):Effects of mulberry on antioxidant and on production of PGE2 and NO in LPS-induced RAW 264.7 macrophages.
指導教授:顏國欽顏國欽引用關係
指導教授(外文):Gow-Chin Yen, Ph. D.
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:132
中文關鍵詞:桑椹抗氧化抗發炎花青素C-3-GM-3-GCOX-2iNOS
外文關鍵詞:mulberryantioxidantanti-inflammatoryanthocyaninscyanidin-3-glucosidemalvidin-3-glucosidecyclooxygenase-2inducible nitric oxide synthase
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中文摘要
第一部份欲探討桑椹之抗氧化特性。第一部份乃將桑椹區分為之甲醇萃取物 (MEM)、水萃取物 (WEM)及富含花青素萃取物 (ARME),進行抗氧化及自由基清除能力之探討,並分析各萃取物中可能之抗氧化成分含量,以期找出具有抗氧化能力之活性成分。ARME顯示具有最佳之抗氧化及清除自由基能力,且抑制能力隨著其濃度增加有上升。ARME (125μg/ml) 具有較佳之清除過氧化氫能力 (49 %),其清除能力優於同濃度的Trolox,但其還原力卻不如gallic acid。在125μg/ml濃度下,ARME除了在清除超氧陰離子 (38.5 %) 外,在清除DPPH (91.2 %)、NO (44 %) 及ABTS+‧ (93.1%) 等自由基方面,均顯示有較強的自由基清除能力。由活性成分分析顯示,總多酚類、類黃酮及花青素含量均是以ARME含量為最多,推測三種桑椹萃取物之抗氧化活性與其所含總多酚類、類黃酮及花青素含量有關,尤其是三者之花青素含量與總抗氧化能力具有很高的相關性 (r2=0.81),由此可知,花青素可能於桑椹的生理活性功能中,扮演一重要角色;由花青素含量分析結果可知,Cyanidin-3-glucoside (C3-G) 為桑椹中含量較多之花青素。ARME可能具有開發為植物性來源之預防自由基相關疾病之抗氧劑潛力,值得更進一探討。
第二部份本研究藉由巨噬細胞模式系統來探討桑椹之富含花青素萃取物 (Anthocyanins-rich mulberry extract; ARME)、甲醇萃取物 (Methanol extract of mulberry; MEM),及桑椹含量最多之花青素種類C-3-G (Cyanidin-3-glucoside) 對於LPS (Lipopolysaccharide) 誘導RAW 264.7巨噬細胞產生PGE2 (Prostaglandin E2) 及NO (Nitric oxide) 之影響,並以紅酒及紅葡萄中含量最多之花青素種類M-3-G (Malvidin-3-glucoside; M-3G) 與C-3-G做一比較。結果顯示於實驗條件下,所有的樣品均不會對於細胞造成傷害,細胞存活率均於90 %以上。ARME及MEM具有較強之清除LPS (1μg/ml) 與RAW 264.7巨噬細胞作用2小時後所產生之胞內ROS,其能力分別為66 %及45 %。以1 μg/ml之LPS與RAW 264.7巨噬細胞反應24小時,會產生最多的NO及PGE2,而ARME與MEM (250 μg/ml)及C-3-G與M-3-G (500μM)分別具有53 % 與36 % 及83 % 與74 % 抑制NO之能力及51 % 與30 % 及73 % 與73 % 之抑制PGE2能力。另外以西方點墨法檢驗iNOS及COX-2蛋白質生成情形時發現,與只有加入LPS的結果相比,ARME、C-3-G及M-3-G對於iNOS及COX-2蛋白質生成皆具有影響力,其中以二種花青素單體較具效果,而MEM對於iNOS及COX-2蛋白質生成均不具影響,蛋白質生成均沒有明顯變化。在抑制mRNA表現上,三者皆具有抑制現象,以C-3-G最有效果,M-3-G次之,ARME則最差。
由ARME具有較高之抗氧化能力,及抑制NO及PGE2生成之生理活性功能,可知花青素於桑椹之生理功能特性上扮演一重要角色。推測花青素對於桑椹之抗氧化及有關發炎之酵素調節具有影響。而C-3-G在抑制iNOS方面較紅酒中含量較多之花青素M-3-G佳,而在抑制COX-2蛋白質方面則否。ARME、C-3-G及M-3-G抑制PGE2及NO生成之原因,除了可能直接清除NO外,亦可能是經由抑制COX-2及iNOS酵素蛋白質生成所造成,並降低iNOS mRNA的轉錄量,以達到影響NO及PGE2之生成。因此,桑椹中所含花青素、C-3-G及M-3-G具有成為抗發炎物質之潛力。
關鍵字:桑椹、抗氧化、抗發炎、花青素、C-3-G、M-3-G、COX-2、iNOS
Abstract
In Part 1, we focused on the studies of the antioxidant activities and free radical scavenging effects of three mulberry extracts, including methanol extract of mulberry (MEM), water extract of mulberry (WEM), and anthocyanins-rich mulberry extract (ARME). ARME possessed the highest activeities of antioxidation as well as scavenging free radicals in a concentration-dependent manner. ARME at the concentration of 125 μg/ml showed the best effect (49 %) in removing hydrogen peroxide in relation to Trolox. But, gallic acid was found to have a stronger reducing power than that of ARME. Moreover ARME (125μg/ml) possessed the highest activities on scavenging DPPH (91.2 %), NO (44 %), and ABTS+․(93.1 %), while it could scavenge 38.5 % superoxide anion. According to the analysis of biological active compounds, ARME contained the highest amount of polyphenols, flavonoids, and anthocyanins. It was inferred that the antioxidant activities of these three mulberry extracts were closely associated with the contents of total polyphenolic acids, flavonoids, and anthocyanins, while there was a high concentration between contents of the anthocyanins of the three extracts and their TEAC (R2=0.81). Therefore, anthocyanins were likely to play a significant role in the biological activities of mulberry. We may confirm that cyanidin-3-glucoside is the major anthocyanins in mulberry in terms of quantity. ARME is likely to be developed as a potential antioxidant to prevent the disease caused by free radicals, and is also worth a further discucssion.
In part two, we investigated the effects of ARME, MEM, and C-3-G, on RAW 264.7 macrophages production PGE2 and NO induced by LPS. And Malvidin-3-glucoside (M-3G), the major of anthocyanins in red wine, was used as a contract. The results showed there was no cytotoxic to RAW 264.7 macrophages in all experiments and conditions, and cell viability was larger than 90 %. After RAW 264.7 macrophages produced the highest amount of ROS by reacting with 1μg/ml LPS in two hours, ARME and MEM showed a stronger ability to scavenge ROS within cells, 66 and 45 %, respectively. Thus, we took ARME and MEM for further studies of the NO and PGE2 production. RAW 264.7 macrophages would produce the highest amount of NO and PGE2 after reacted with 1μg/ml LPS for 24 hours. ARME, MEM (250μg/ml), C-3-G, and M-3-G (500μM) could inhibit the NO production by 53, 36, 83, and 74 % ,respectively, and inhibit PGE2 production by 51, 30, 73, and 73 %, respectively. When we examine iNOS and COX-2 protein production with Western Blotting, we found that ARME, C-3-G, and M-3-G would affect the iNOS and COX-2 protein production, particularly C-3-G and M-3-G. MEM did not affect the production of iNOS and COX-2 protein, and there was no apparent change in protein production. For the inhibition of the expression of iNOS mRNA, all three anthocyanins possessed inhibitory activities in an order of C-3-G > M-3-G > ARME.
We understood that anthocyanins play an important role in the physiological activities of mulberry, because ARME had a better antioxidant ability. We have inferred that the antioxidant abilities of the anthocyanins in the mulberry could be a mediator of the enzymes of inflammation. C-3-G has higher effect than M-3-G in inhibiting the iNOS protein production, but not for the COX-2 protein. The reason why ARME, C-3-G, and M-3-G could inhibit PGE2 and NO by scavenging NO directly as well as inhibiting the production of COX-2 and iNOS protein leading to the lowered expression of iNOS mRNA. Therefore, we infer that anthocyanins in mulberry, such as C-3-G, and M-3-G could be developed as the potential to anti-inflammatory substances.
Key words: mulberry, antioxidant, anti-inflammatory, anthocyanins, C-3-G, M-3-G, COX-2, iNOS
目 錄
全文摘要(中).................................................I
全文摘要(英)................................................III
前言...........................................................1
文獻整理.......................................................3
一、前列腺素E2 (Prostaglandin E2;PGE2) 介紹……………………………3
1. 花生四醯酸 (Arachidonic acid) 代謝…………………………………...3
2. 環氧化酵素 (Cyclooxygenase;COX) 介紹……………………………3
二、一氧化氮 (Nitric oxygen;NO) 之介紹…………………………………8
1. 一氧化氮合成酵素 (Nitric oxygen synthase;NOS) 介紹……………..8
2. NO於生理及病理中之介紹………………………………………………9
三、脂多醣 (Lipopolysaccharide;LPS) 巨噬細胞活化的傳訊途徑………..12
1. LPS結構………………………………………………………………….12
2.巨噬細胞的活化………………………………………………………12
3. COX-2及iNOS蛋白質酵素的活化…………………………………….13
四、活性氧 (reactive oxygen species;ROS) 之介紹……………………….16
1. ROS之重要性……………………………………………………………16
2. ROS的傷害性……………………………………………………………16
五、抗氧化之重要性………………………………………………………….19
1. 生物體之抗氧化防禦系統……………………...………………………19
2. 抗氧化劑的作用原理…………………………………………………...19
3. 天然抗氧化劑…………………………………………………………...20
六、桑椹及其中花青素介紹…………………………………………………20
1. 桑椹介紹………………………………………………………………..20
2. 桑椹之現代研究………………………………………………………..21
3. 花青素之介紹…………………………………………………………..22
七、研究動機……............................................................................................25
八、本論文之研究架構……………………………………………………….26
第一部份:桑椹之抗氧化性
摘要……………………………………………………………………………28
前言……………………………………………………………………………29
實驗流程………………………………………………………………………32
材料與方法……………………………………………………………………33
結果……………………………………………………………………………40
一、桑椹萃取物之製備……………………………………………………40
二、清除過氧化氫之能力…………………………………………………40
三、α,α-Diphenyl-β-picrylhydrazyl (DPPH)自由基清除能力測定……40
四、清除超氧陰離子能力之測定………………………………………….41
五、清除Sodium nitroprusside (SNP)生成NO之試驗…………………42
六、總抗氧化能力 (TEAC)……………………………………………….42
七、還原力測定…………………………………………………………….43
八、抗氧化物質測定……………………………………………………….43
討論……………………………………………………………………………45
第二部份:桑椹對LPS所誘導RAW 264.7巨噬細胞產生PGE2及NO之影響
摘要……………………………………………………………………………59
前言……………………………………………………………………………61
實驗流程………………………………………………………………………66
材料與方法……………………………………………………………………67
結果……………………………………………………………………………74
一、桑椹萃取物、C-3-G及M-3-G對RAW 264.7巨噬細胞之細胞存活率
試驗……………………………………………………...…………...74
二、清除胞內活性氧………………………………………………………74
三、LPS刺激NO生成的時間與劑量條件建立…………………….……76
四、ARME及MEM對LPS誘導生成NO之影響………………………76
五、C-3-G及M-3-G對LPS誘導生成NO之影響………………………76
六、ARME及MEM對LPS誘導iNOS蛋白質生成之影響…………….77
七、C-3-G及M-3-G對LPS誘導iNOS蛋白質生成之影響…………….77
八、LPS刺激PGE2生成的時間與劑量條件建立………………………..78
九、ARME及MEM對LPS誘導生成PGE2之影響……………………78
十、C-3-G及M-3-G對LPS誘導生成PGE2之影響……………..………78
十一、ARME及MEM對LPS誘導生成COX-2蛋白質表現的影響……79
十二、C-3-G及M-3-G對LPS誘導生成COX-2蛋白質表現的影響……79
十三、ARME、C-3-G及M-3-G對LPS誘導生成iNOS mRNA合成的影響……………………………………………………………………79
討論…………………………………………………………………………81
總結論………………………………………………………………………..108
參考文獻…………………………………………………………………...109
表 次
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