臺灣博碩士論文加值系統

本研究分為兩個部分，以體外實驗探討海參內臟之酵素水解液中是否具有抑制血管收縮素轉換酶及抗氧化之胜肽形成；另在短期動物實驗中，餵食自發性高血壓大鼠 (spontaneously hypertension rat, SHR) 不同劑量及不同分子量的海參內臟之酵素水解液，能否具有降低血壓的效果。實驗中將海參內臟粉末，分別加入胃蛋白酶與凝乳胰蛋白酶 (PC) 或Prozyme 6與Flavourzyme (P6F) 進行水解得到之水解產物。體外實驗中，P6F水解液具較佳之血管收縮素轉換酶 (ACE) 抑制能力，且總抗氧化能力、還原能力及清除DPPH自由基之能力之抗氧化活性皆較PC水解液顯著地佳。P6F水解液再經由超過濾劃分，分子量小於1 kD之區分具有最佳之ACE抑制作用及抗氧化活性；進一步將分子量 1 kD以下的劃分物利用膠體層析分離後，分子量約在430 Da之區分具最佳之ACE抑制能力而約在990 Da之區分最具抗氧化活性。動物實驗中，以單次不同濃度之P6F水解液管餵 SHR，結果顯示當給予 900 mg/kg BW之劑量時，於3小時後即可顯著降低收縮壓 (p<0.05)，並可持續至6小時。在給予分子量小於1 kDa之胜肽溶液，於SHR體內可有效降低血壓。綜合上述，經酵素水解之海參內臟能生成具有降血壓及抗氧化物質，並對SHR有降低血壓之效果。

The study was divided into two parts. In vitro, the enzymatic hydrolysis of the organs of sea cucumbers (OSC) were used to elucidate if there’s any antioxidative and angiotensin converting enzyme inhibition (ACEI) materials generated. In vivo, spontaneously hypertensive rats (SHR) were gavaged and feed with the hydrolysate or the different fraction of hydrolysate for a short-term to find out whether it have any antihypertensive effects .The hydrolysates obtained by proteolized with pepsin and chymotrypsin (PC) or Prozyme 6 and Flavourzyme (P6F). In vitro studies, results indicated that P6F hydrolysate had higher ACEI as well as higher activity in Trolox equivalent antioxidant capacity, 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity, and reducing power. The P6F hydrolysates were separated by ultrafiltration membranes, the fraction of MW<1 kDa was found having the highest ACEI and antioxidativity activity. The fraction of MW<1 kDa was further separated by gel filtration chromatography. The peptide with molecular weight of approximately 430 and 990 Da possessed the strongest activity of ACEI and antioxidation respectively. In animal studies, oral administration of P6F hydrolysate with the dose of 900 mg/kg bw to SHR exerted a significant lowering effect (p<0.05) on SBP after 3 h and lasted to 6 h. In addition, the fraction of MW<1 kDa of the hydrolysate had an antihypertensive effect significantly in SHR. In conclusion, the enzymatic hydrolysis of OSC may generate the hydrolysate with ACEI and antioxidative capacities, and exert an antihypertensive effect in SHR.

中文摘要...................................................Ⅰ
英文摘要..................................................Ⅱ
致謝.....................................................Ⅲ
目錄......................................................Ⅳ
表目錄....................................................Ⅶ
圖目錄....................................................Ⅷ
附圖目錄...................................................Ⅸ
壹、 緒言...............................................1
貳、 文獻回顧...........................................2
一、 高血壓簡介.........................................2
(一) 定義.............................................2
(二) 高血壓之分類.......................................3
(三) 造成高血壓相關因素................................5
(四) 控制及預防高血壓..................................5
二、 血管收縮素轉化酶....................................6
(一) ACE之生化特性......................................6
(二) ACE對血壓的影響.....................................8
(三) 血管收縮素轉化酶抑制劑..............................10
三、 自由基與氧化壓力....................................12
(一) 自由基和活性氧.....................................12
(二) 氧化壓力與氧化傷害.................................13
四、 海參與海參內臟.....................................15
(一) 海參簡介...........................................15
(二) 海參的生理功能.....................................16
(三) 海參內臟之生理功能.................................16
五、 蛋白質水解物之簡介..................................17
(一) 降血壓活性胜肽.....................................18
(二) 抗氧化活性胜肽.....................................24
六、 高血壓自發性大鼠簡介................................28
七、實驗目的...............................................29
參、 材料與方法.........................................30
一、 藥品與器材.........................................30
(一) 實驗材料..........................................30
(二) 分析用試藥........................................30
(三) 儀器設備..........................................31
(四) 實驗流程圖........................................33
1. 酵素水解物之體外實驗..................................33
2. 體內實驗之流程........................................34
二、實驗方法............................................36
(一) 海參內臟之前處理......................................36
(二) 水解條件..............................................36
(三) 水解物可溶性蛋白質含量測定.............................36
(四) 胜肽濃度之測定.........................................37
(五) 水解液機能性之測定.....................................38
(六) 機能性胜肽之分離與收集................................40
(七) 自發性高血壓鼠活體實驗.................................42
(八) 統計分析..............................................43
肆、結果與討論.............................................44
一、水解物可溶性蛋白質含量及胜肽濃度........................44
二、海參內臟酵素水解液之機能性比較............................46
(一) 酵素水解液抑制血管收縮素轉化酶 (ACE) 之能力.............46
(二) 酵素水解液之抗氧化能力..................................47
三、機能性胜肽之分離與收集..................................50
(一) 不同分子量區分之機能性.............................50
(二) 分子量小於 1000 Da 之膠體層析......................51
(三) 劃分收集物中主要抑制 ACE 之胜肽的分離純化...........53
四、酵素水解液對自發性高血壓大鼠 (SHR) 血壓短期作用之影響.....54
(一) 不同劑量之酵素水解液對 SHR 心跳、收縮壓及舒張壓之影響..54
(二) 不同分子量之酵素水解液對SHR心跳、收縮壓及舒張壓之影響..56
伍、結論..................................................58
陸、參考文獻...............................................76

表目錄

表一、高血壓的定義.................................................3
表二、海參內臟之酵素水解液對血管收縮素轉換酶 (ACE) 之抑制能力...59
表三、海參內臟之酵素水解液總抗氧化力及清除 DPPH 自由基之能力比較60
表四、海參內臟之酵素水解液還原力比較..........................61
表五、海參內臟之蛋白酵素水解液中不同區分抑制ACE 之能力.........62
表六、海參內臟之蛋白酵素水解液中不同區分之抗氧化性比較..........63
表七、Sephadex G-15 各劃分物的 ACE 有效抑制百分比............64
表八、 Sephadex G-15 各劃分物之抗氧化性比較..................65
表九、單次灌食不同劑量海參內臟之蛋白酶水解液對自發性高血壓大鼠心跳
之影響..................................................66
表十、單次灌食不同劑量海參內臟之蛋白酶水解液對自發性高血壓大鼠收縮壓
之影響...................................................67
表十一、單次灌食不同劑量海參內臟之蛋白酶水解液對自發性高血壓大鼠舒張壓之影響..............................................68
表十二、單次灌食不同分子量之海參內臟之蛋白酶水解液對自發性高血壓大鼠心跳之影響..............................................69
表十三、單次灌食不同分子量之海參內臟之蛋白酶水解液對自發性高血壓大鼠收縮壓之影響...............................................70
表十四、單次灌食不同分子量之海參內臟之蛋白酶水解液對自發性高血壓大鼠舒張壓之影響...............................................71

圖目錄

圖一、血管收縮素轉化酶 (ACE) 之活化位置與 ACEI 胜肽之結合模擬圖…7
圖二、RAS及KKS作用途徑........................................11
圖三、Captopril 作用原理...........................................12
圖四、海參內臟粉末經胃蛋白酶、凝乳胰蛋白酶水解之可溶性蛋白質及
胜肽含量變化......................................................72
圖五、海參內臟粉末經 Prozyme 6、Flavourzyme 水解之可溶性蛋白質及
胜肽含量變化......................................................73
圖六、海參內臟水解液 (分子量<1,000之區分) 之膠體層析圖譜...........74
圖七、由Sephadex G-15 劃分物之 E 區分所得的高效液相層析圖.........75

附圖目錄

附圖一、單次管灌不同劑量海參內臟之蛋白酶水解液對自發性高血壓大鼠
收縮壓之變化.....................................................93
附圖二、單次管灌不同劑量海參內臟之蛋白酶水解液對自發性高血壓大鼠
舒張壓之變化.....................................................94
附圖三、單次管灌不同分子量之海參內臟之蛋白酶水解液對自發性高血壓
大鼠收縮壓之變化.................................................95
附圖四、單次管灌不同分子量之海參內臟之蛋白酶水解液對自發性高血壓
大鼠收縮壓之變化.................................................96
附圖五、海參內臟之酵素水解液還原力比較...........................97

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