臺灣博碩士論文加值系統

甲魚 (soft-shelled turtle,SST) 為中國傳統藥材，具有多種生理功效。本實驗研究目的為，利用甲魚粉經木瓜酵素水解製備，具有調解血壓功效的甲魚粉酵素水解產物。甲魚粉經反應曲面法二因子 (酵素添加量、水解時間) 旋轉式中心組合設計，利用胜肽濃度、 ACE 抑制活性為評估條件，發現 2% 甲魚粉懸浮液，在 pH 5.5 、 60℃ 、 5% 木瓜酵素、 3 小時水解時間，為較佳水解條件。未經水解的甲魚粉懸浮液其 ACE 抑制活性低， IC50 為 16.2±1.6 mg/mL ，經木瓜酵素水解得到的甲魚粉酵素水解產物 ACE 抑制活性明顯提高，IC50 為2.87±0.32 mg/mL。
在短期動物實驗中，以甲魚粉酵素水解產物 500 mg/kg BW 劑量單次管餵自發性高血壓大鼠 (spontaneously hypertension rat, SHR) 後，於管餵後5小時，其收縮壓顯著性下降；長期動物實驗中，以含 0.6% 甲魚粉酵素水解產物的飼料餵食，於第 4、6 週時，收縮壓較控制組顯著性下降，於第8週時收縮壓則極顯著下降，餵食甲魚粉酵素水解產物的 SHR 血漿中 ACE 活性顯著性低於控制組。綜合上述，甲魚粉經木瓜酵素水解後的水解產物具有調解血壓功效。

Chinese soft-shelled turtle (SST) is a traditional Chinese medicine, and was also confirmed that possessed various kinds of physiological functions. The objectives of this study were to produce anti-hypertension effect of SST powder hydrolysate digested by papain. SST powder was hydrolyzed under various conditions of hydrolysis time and enzyme-to substrate ratio (E/S, %) according to a response surface methodology (RSM) 2 factors rotatable central composite design, and hydrolysate were evaluated by peptide concentration and ACE inhibitory activity. The better hydrolysate conditions of SST powder hydrolysate digested by papain are pH 5.5, 60℃, 5% E/S for 3 hours. The SST powder showed limited IC50 value of ACE inhibitory activity were 16.2±1.6 mg/mL. The SST powder hydrolysate derived from hydrolysis of papain obviously increased ACE inhibitory activity, and IC50 were 2.87±0.32 mg/mL.
In short term animal experiment, single oral administration of 500 mg/kg BW of SST powder hydrolysate to spontaneously hypertension rat (SHR) was significantly lowered systolic blood pressure at 5th hours. In long term experiment, the experiment group feed diet containing 0.6% SST powder hydrolysate was noticeable decreased systolic blood pressure compared to control group at 4th and 6th weeks (p<0.05) and significant suppression at 8th weeks (p<0.01). The ACE activity of SHR feed diet containing 0.6% SST powder hydrolysate were lower than control group. The results suggested that SST powder hydrolysate digested papain can modulate blood pressure.

目錄
摘要 i
Abstract ii
目錄 iii
表目錄 viii
圖目錄 x
第一章 前言 1
第二章 文獻整理 3
1 高血壓 3
1.1 高血壓定義 3
1.2 高血壓種類 4
2 血管收縮素轉換酶 5
2.1 血管收縮素轉換酶生化特性 5
2.2 血管收縮素轉換酶調解血壓機制 7
3 活性胜肽 8
3.1 活性胜肽簡介 8
3.2 活性胜肽之生理活性 9
3.3 血管收縮素轉換酶抑制劑 9
3.3.1 抑制機制 9
3.3.2 食品來源的血管收縮素轉換酶抑制劑 11
4 自發性高血壓大鼠簡介 11
5 甲魚 12
5.1 甲魚簡介 12
5.2 甲魚之組成 13
5.3 甲魚之生理功效 13
6 反應曲面法 17
6.1 反應曲面法簡介 17
6.2 反應曲面法原理 18
6.3 中心組合設計 19
第三章 研究目的與架構 22
1 研究目的 22
2 研究架構 22
第四章 材料與方法 23
1 實驗材料 23
2 實驗藥品 23
3 儀器設備 24
5 實驗方法 25
4.1 甲魚粉一般成分分析 25
4.2 甲魚粉水解液製備 27
4.3 胜肽濃度測定 27
4.4 血管收縮素轉換酶抑制活性測定 28
4.5 水解物純化分離 29
4.6 自發性高血壓大鼠動物實驗 30
4.6.1 實驗一：管灌甲魚粉酵素水解產物對 SHR 血壓影響 30
4.6.1.1 動物飼養 30
4.6.1.2 實驗流程 30
4.6.1.3 血壓測定 30
4.6.2 實驗二：飼料添加甲魚粉酵素水解產物對 SHR 影響 31
4.6.2.1 動物飼養 31
4.6.2.2 實驗流程 31
4.6.2.3 血壓測定 31
4.6.2.4 樣品收集與分析 31
4.6.2.4.1 血液分析 31
4.6.2.4.2 腎臟分析 34
4.6.2.4.3 肺臟分析 34
4.6.2.4.4 肝臟分析 34
4.7 統計分析 35
第五章 結果 36
1. 甲魚粉一般成分分析 36
2. 以反應曲面法選擇甲魚粉酵素水解產物水解條件 36
3. 甲魚粉酵素水解產物純化分離 38
4. 自發性高血壓大鼠 (SHR) 實驗 38
4.1 實驗一：管灌甲魚粉酵素水解產物對 SHR 之血壓影響 38
4.2 實驗二：飼料添加甲魚粉酵素水解產物對 SHR 影響 39
第六章 討論 43
1. 甲魚粉一般成分分析 43
2. 以反應曲面法選擇甲魚粉酵素水解條件 43
3. 甲魚粉酵素水解產物純化分離 45
4. 自發性高血壓大鼠 (SHR) 實驗 46
4.1 管灌甲魚粉酵素水解產物對 SHR 之血壓影響 46
4.2 飼料添加甲魚粉酵素水解產物對 SHR 影響 47
第七章 結論 52
參考目錄 77
表目錄
表一、 美國國家聯席委員會對成人血壓分類標準及定義 4
表二、 食品中活性胜肽來源及功能性、胺基酸序列 10
表三、 甲魚不同組織部位的主要營養成分 14
表四、 甲魚不同組織部位胺基酸分析結果 15
表五、 甲魚粉之一般成分分析 54
表六、 反應曲面法實驗操作條件及各階層設計 55
表七、 反應曲面法試驗因子階層及結果表 56
表八、 胜肽濃度變方分析表 57
表九、 血管收縮素轉換酶抑制活性變方分析表 58
表十、 胜肽濃度、血管收縮素轉換酶抑制活性之反應曲面迴歸分析表 59
表十一、 甲魚粉水解條件 60
表十二、 餵食甲魚粉酵素水解產物飼料八週對自發性高血壓大鼠生長參數之影響 61
表十三、 餵食甲魚粉酵素水解產物飼料八週對自發性高血壓大鼠 ACE活性之影響 62
表十四、餵食甲魚粉酵素水解產物飼料八週對自發性高血壓大鼠之血漿總膽固醇、三酸甘油酯之影響 63
表十五、 餵食甲魚粉酵素水解產物飼料八週對自發性高血壓大鼠之血漿、肝臟 TBARS之影響 64
圖目錄
圖一、 ACE結構 6
圖二、 ACE調節血壓的機制 8
圖三、 食品蛋白質衍生活性胜肽的生理功效 10
圖四、 反應曲面法實驗流程 21
圖五、 OPA測定胜肽濃度機制 28
圖六、 胜肽濃度等高曲線圖 65
圖七、 ACE抑制活性百分比等高曲線圖 66
圖八、 甲魚粉酵素水解產物經Superdex Peptide 10/300 膠體管柱層析圖譜 67
圖九、24管區分物 ACE 抑制活性 68
圖十、自發性高血壓大鼠管餵甲魚粉酵素水解產物之收縮壓的變化情形 69
圖十一、自發性高血壓大鼠管餵甲魚粉酵素水解產物之舒張壓的變化情形 70
圖十二、 自發性高血壓大鼠管餵甲魚粉酵素水解產物之平均血壓的變化情形 71
圖十三、自發性高血壓大鼠餵食甲魚粉酵素水解產物之體重變化 72
圖十四、自發性高血壓大鼠餵食甲魚粉酵素水解產物之攝取量變化 73
圖十五、自發性高血壓大鼠餵食甲魚粉酵素水解產物之收縮壓的變化情形 74
圖十六、自發性高血壓大鼠餵食甲魚粉酵素水解產物之舒張壓的變化情形 75
圖十七. 自發性高血壓大鼠餵食甲魚粉酵素水解產物之平均血壓的變化情形 76

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