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研究生:廖恬瑤
研究生(外文):T'ien-Yao Liao
論文名稱:蜂王蛹蛋白及水解物對細胞DNA氧化性傷害及LDL氧化之抑制研究
論文名稱(外文):Studies on the Inhibition of Cell DNA Oxidative Damage and LDL Oxidation by Bee Pupal Protein and Its Hydrolysates
指導教授:張基郁張基郁引用關係
指導教授(外文):Chi-Yü Chang
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:80
中文關鍵詞:蜂王蛹抗氧化性氧化傷害低密度脂蛋白
外文關鍵詞:bee pupal proteinantioxidant activityOxidative damagelow density lipoprotein (LDL)
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本研究以蜂王蛹蛋白為材料,利用alcalase及flavourzyme進行一階段與兩階段水解,探討蜂王蛹蛋白經酵素水解前後對細胞DNA氧化性傷害及LDL氧化之抑制作用。
在蜂王蛹蛋白水解研究方面,蜂王蛹蛋白以1、1.5及2 % alcalase進行水解20小時,水解率分別為8.89、8.99及9.19 %;以1、1.5及2 % flavourzyme進行水解16小時,水解率分別為9.57、8.59及9.91 %;蜂王蛹蛋白以1.5% alcalase水解4小時後,再以1、1.5及2 % flavourzyme水解12小時,水解率分別為9.03、9.49及8.98 %。
在Fenton reaction誘導DNA氧化傷害之抑制研究方面,蜂王蛹蛋白、一階段(alcalase、flavourzyme)及兩階段水解物皆有抑制去氧核糖氧化傷害的效果,其中以蜂王蛹蛋白效果最大,於1 mg/mL濃度時,分別可抑制47.06、33.70、24.19及43.09 %之氧化傷害。蜂王蛹蛋白及其水解物皆有降低8-OH-2'-dG之生成,其抑制能力大小順序為蜂王蛹蛋白<flavourzyme一階段水解物<alcalase一階段水解物≒兩階段水解物。在Bleomycin-Fe3+ 誘導DNA氧化傷害研究方面,蜂王蛹蛋白及水解物皆無明顯的促氧化效果。
在LDL的氧化修飾研究方面,對Cu2+誘導LDL氧化生成TBARS之影響,以1 mg/mL之兩階段水解物之效果最大,其抑制能力大小順序為兩階段水解物>alcalase一階段水解物>蜂王蛹蛋白>flavourzyme一階段水解物。在共軛雙烯的生成量方面,蜂王蛹蛋白及水解物皆可延滯共軛雙烯的生成。在濃度1 mg/mL時,蜂王蛹蛋白及水解物皆有明顯的抑制效果,其lag time約為240 min,為控制組的2倍。
The bee pupal protein isolated from bee pupa was used as materials in this study, and was hydrolyzed by alcalase and flavourzyme through one- and two-stage processes. The inhibition of cell DNA oxidative damage and LDL oxidation.by the hydrolysates was investigated.
In the aspects of the hydrolysis of bee pupal protein, the degree of hydrolysis (DH) of the hydrolysates by 1.0, 1.5, and 2.0 % alcalase were 8.89, 8.99 and 9.19 %, respectively. The DH of the hydrolysates by 1.0, 1.5, and 2.0 % flavourzyme were 9.57, 8.59, and 9.91 %, respectively. The DH of the hdrolysates by alcalase for 4 hrs and followed by 1.0, 1.5, and 2.0 % flavourzyme for 12 hrs were 9.03, 9.49, and 8.98 %, respectively.
In the aspects of the inhibitory effect of bee pupal protein and its hydrolysates on Fenton reaction induced oxidative damage of DNA molecules, the results showed that the bee pupal protein, one-stage hydrolysates by alcalase or flavourzyme, and two-stage hydrolysates by alcalase and flavourzyme had an inhibitory effect on the oxidative damage of deoxyribose. At a concentration of 1 mg/mL, the bee pupal protein, one-stage hydrolysates (alcalase or flavourzyme), and two-stage hydrolysates exhibited 47.06, 33.70, 24.19, and 43.09 % inhibition, respectively. The bee pupal protein had the highest inhibitory activity. All the samples could reduce the formation of 8-OH-2'-dG. The quanttiy reduced by the samples was in an order of bee pupal protein<one-stage hydrolysate by flavourzyme<one stage hydrolysate by alcalase ≒ two-stage hydrolysate. The bee pupal protein and its hydrolysates had no significant pro-oxidant effect on DNA oxidative damage induced by bleomycin-Fe3+.
In the aspects of LDL oxidation induced by Cu2+, the results showed that two-stage hydrolysate exhibited the highest inhibitory acitivity on the TBARS formation, at a concentration of 1 mg/mL. The inhibitory activities of the samples were in an order of two-stage hydrolysate>one-stage hydrolysate by alcalase>bee pupal protein>one-stage hydrolysate by flavourzyme. All the samples could reduce the formation of conjugated dienes. At a concentrations of 1 mg/mL, the lag time of conjugated diene formation for all the samples was around 240 min, 2 times of the control.
封面內頁
簽名頁
授權書iii
中文摘要iv
英文摘要vi
誌謝viii
目錄ix
圖目錄xiii
表目錄xv
1.前言1
2.文獻回顧3
2.1蜂蛹之簡介3
2.1.1蜂蛹之成份3
2.1.2蜂王蛹的研究及應用4
2.2蛋白質水解7
2.2.1蛋白質水解酵素7
2.2.2水解方式及條件7
2.2.3蛋白質水解特性及應用8
2.2.4 酵素水解之影響因子9
2.3蛋白質及水解物之機能性11
2.3.1血管緊縮素轉化酶抑制胜肽11
2.3.2抗氧化胜肽12
2.3.3類鴉片胜肽(opiod peptides)12
2.3.4免疫活性胜肽(immunopeptides)13
2.3.5礦物質結合胜肽-酪蛋白磷酸胜肽13
2.3.6抗菌活性14
2.4氧化作用14
2.4.1自由基14
2.4.2自由基的來源與種類15
2.4.3氧化壓力18
2.4.4抗氧化防禦系統19
2.4.5費頓反應(Fenton reaction)21
2.4.6DNA氧化傷害23
2.5人類低密度脂蛋白24
2.5.1氧化修飾低密度脂蛋白(OxLDL)26
2.5.2丙二醛(Malondialdehyde, MDA)27
2.5.3硫代巴比妥酸反應物質(TBARS)27
3.材料與方法29
3.1實驗材料29
3.1.1原料29
3.1.2藥品29
3.1.3儀器設備30
3.1.4蛋白質分解酵素32
3.2實驗方法與分析項目32
3.2.1本實驗流程32
3.2.2基本組成分析32
3.2.3蜂王蛹蛋白試液製備35
3.2.4水解物之製備35
3.2.4.1一階段水解35
3.2.4.2兩階段水解36
3.2.5蜂王蛹蛋白及水解物對生物分子氧化傷害 之抗氧化性37
3.2.6蜂王蛹蛋白及水解物於抑制LDL氧化之探討39
3.2.6.1LDL製備39
3.2.6.2蜂王蛹蛋白及水解物對銅離子誘導LDL氧化之影響40
3.2.7統計分析41
4.結果與討論42
4.1蜂王蛹之基本成分分析42
4.2蜂王蛹蛋白之酵素水解43
4.2.1以不同濃度alcalase水解蜂王蛹蛋白之水解率變化43
4.2.2以不同濃度flavourzyme水解蜂王蛹蛋白之水解率變化47
4.2.3兩階段酵素水解蜂王蛹蛋白之水解率變化49
4.3對生物分子氧化傷害之抗氧化性49
4.3.1蜂王蛹蛋白及水解物對Fenton reaction誘導的deoxyribose氧化傷害之影響49
4.3.2蜂王蛹蛋白及水解物對Fenton reaction誘導2'-deoxyguanosine(2'-dG)氧化形成8-hydroxy-2'-deoxy-guanosine(8-OH-2'-dG)之影響51
4.3.3蜂王蛹蛋白及水解物對bleomycin-Fe3+ 誘導DNA氧化傷害之影響56
4.4蜂王蛹蛋白及水解物對銅離子誘導LDL氧化修飾之影響58
4.4.1蜂王蛹蛋白及水解物對Cu2+誘導LDL氧化生成TBARS之影響58
4.4.2蜂王蛹蛋白及水解物對Cu2+誘導LDL氧化生成共軛雙烯(conjugated diene, CD)之影響63
5.結論69
參考文獻71
圖3.1實驗流程圖33
圖4.1以alcalase水解蜂王蛹蛋白之水解率44
圖4.2以flavourzyme水解蜂王蛹蛋白之水解率46
圖4.3使用酵素(alcalase和flavourzyme)水解蜂王蛹蛋白48
圖4.4蜂王蛹蛋白及水解物對Fe3+-EDTA/H2O2/Asc誘導去氧核糖氧化傷害之影響50
圖4.5蜂王蛹蛋白及水解物對bleomycin-Fe3+誘導DNA傷害之影響57
圖4.6蜂王蛹蛋白對Cu2+誘導LDL氧化生成TBARS之影響59
圖4.7蜂王蛹蛋白水解物(Alcalase)對Cu2+誘導LDL氧化生成TBARS之影響60
圖4.8蜂王蛹蛋白水解物(Flavourzyme)對Cu2+誘導LDL氧化生成TBARS之影響61
圖4.9蜂王蛹蛋白水解物(Alcalase 和Flavourzyme)對Cu2+誘導LDL氧化生成TBARS之影響62
圖4.10蜂王蛹蛋白對銅離子誘導LDL形成共軛雙烯之影響65
圖4.11蜂王蛹蛋白水解物(Alcalase)對銅離子誘導LDL形成共軛雙烯之影響66
圖4.12蜂王蛹蛋白水解物(Flavourzyme)對銅離子誘導LDL形成共軛雙烯之影響67
圖4.13蜂王蛹蛋白水解物(Alcalase 和Flavourzyme)對銅離子誘導LDL形成共軛雙烯之影響68
表2.1蜂王蛹所含的營養素5
表2.2蜂蛹及蜂王漿胺基酸含量6
表2.3內源性及外源性的抗氧化系統22
表2.4健康人類低密度脂蛋白的組成25
表4.1蜂王蛹之一般組成分42
表4.2蜂王蛹蛋白對Fenton reaction所誘導2'-dG形成8-OH-2'-dG之影響52
表4.3蜂王蛹蛋白水解物(Alcalase)對Fenton reaction所誘導2'-dG形成8-OH-2'-dG之影響53
表4.4蜂王蛹蛋白水解物(Flavourzyme)對Fenton reaction所誘導2'-dG形成8-OH-2'-dG之影響54
表4.5蜂王蛹蛋白水解物(Flavourzyme和Alcalase)對Fenton reaction所誘導2'-dG形成8-OH-2'-dG之影響55
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