臺灣博碩士論文加值系統

甲魚（soft-shelled turtle, SST）是屬於高經濟的養殖產業，具多種生理功能。本研究主要探討去脂之甲魚粉以胃蛋白酶（pepsin）、胰蛋白酶（trypsin）和凝乳胰蛋白酶（chymotrypsin）水解後之水解物（PCT-SST），及其分子量大於或小於5000 Da的區分，對抑制血管收縮素轉化酶（ACE）之能力以及自發性高血壓大鼠（SHR）血壓的影響。未經酵素水解之甲魚肉，對ACE的抑制能力非常低，其IC50 為16.7 mg/mL，而經酵素水解之甲魚肉水解物，其抑制ACE能力顯著增加（PCT-SST之IC50 = 3.2 mg/mL），且分子量愈小之胜肽溶液對於抑制ACE活性之效果愈佳（Mr＜1000 Da之IC50 = 2.8 mg/mL）。
將抑制ACE能力最佳之胜肽溶液（Mr＜1000），利用膠體過濾層析，可得到六個主要劃分物，其中區分四、五、六這三個區分具有ACE抑制活性，而又以分子量介於1064 Da以及295 Da 之區分四以及區分五抑制ACE活性之效果最佳。
將PCT-SST溶液給予SHR，當單次管餵500 mg/kg BW或1000 mg/kg BW之劑量時，在管餵第6以及第8小時均可顯著降低SHR之收縮壓（p＜0.05），尤其以高劑量組降收縮壓效果更佳。舒張壓部分，同樣單次給予SHR 500 mg/kg BW 以及1000 mg/kg BW之劑量，在管餵第6以及第8小時之舒張壓顯著低於管餵前。而管餵分子量大於5000 Da之區分，給予高劑量（500 mg/kg BW）之SHR在管餵第10小時，其收縮壓顯著低於控制組；分子量小於5000 Da之胜肽溶液，則以管餵50 mg/kg BW之劑量，SHR在管餵第八小時與控制組者相較，收縮壓降低12 mmHg。以上結果顯示，甲魚之胃腸道酵素水解物對於SHR具有降血壓之功效。

Chinese soft-shelled turtle (SST) is one of highly economic products in cultivation in Taiwan, it have many kinds of physiological functions. This study was to investigate the antihypertensive effect and inhibition of angiotensin converting enzyme (ACE) activity of hydrolysate of SST powder (PCT-SST) derived from hydrolysis of pepsin, trypsin and chymotrypsin. The SST powder showed limited inhibition of ACE (IC50 = 16.7 mg/mL). The inhibitory activity of ACE in hydrolysate derived from hydrolysis of gastrointestinal enzyme increased markedly (IC50 = 3.2 mg/mL). The smaller molecular weight was performed better inhibition of ACE activity (IC50 of molecular weight less than 1000 Da was 2.8 mg/mL).
The fraction of molecular weight less than 1000 Da by ultrafiltration was separated into 6 fractions by gel filtration eluted with deionized water, in which 3 fractions showed ACE inhibition. The fraction 4 and fraction 5 of molecular weight between 1063 Da and 205 Da, has the best inhibition of ACE.
Single oral administration with the dose of 500 mg/kg BW and 1000 mg/kg BW from PCT-SST solution to spontaneously hypertension rat (SHR) showed noticeable decrease of systolic blood pressure (SBP) at the 6th, 8th hour (p<0.05). In addition, same dose of PCT-SST solution also significantly decreased of diastolic blood pressure (DBP) at the 6th, 8th hour. PCT-SST solution with molecular weight more than 5000 Da, showed lower SBP at the 10th hour after signal oral administration at dose of 500 mg/kg BW in SHR, when compared with that control group. On the other hand, molecular weight less than 5000 Da had lower SBP at the 8th hour after signal oral administration at dose 50 mg/kg BW than that control group. The results suggested that hydrolysate of SST powder derived from gastrointestinal enzyme have ACEI activity and antihypertensive effect to SHR.

目錄
頁次
中文摘要………………………………………………………………I
英文摘要………………………………………………………………..III
致謝……………………………………………………………………...V
目錄…………………………………………………………………VI
圖目錄………………………………………………………………IX
表目錄………………………………………………………………X
附圖目錄………………………………………………………………XI
附表目錄………………………………………………………………XII
壹、前言…………………………………………………………………..1
貳、文獻回顧…………………………………………………………..…3
一、高血壓簡介……………………………………………….………3
（一）高血壓的形成…………………………………………………4 （二）高血壓的種類…………………………………………………..4
二、自發性高血壓老鼠簡介……………………………..……………7
三、血管收縮素轉化酶之作用…………………………………….8
（一）血管收縮素轉化酶之生化特性………………………………8 （二）腎素-血管收縮系統之作用……………………………………8（三）血管收縮素轉化酶抑制劑………………………………..……9 （四）具ACEI之胜肽序列……………………………………..…10 （五）食品中ACE抑制劑之開發……………………………….…10
四、甲魚之相關研究………………………………………………16
（一）一般性質………………………………………………………16（二）甲魚之功能……………………………………………….…17
參、實驗材料與方法………………………………………………21
一、藥品與器材………………………………………………………21
（一）原料…………………………………………...………………..21（二）分析用試藥……………………………………………….…21 （三）儀器設備……………………………………………….……22
二、實驗方法…………………………………………………………23
（一）甲魚肉之酵素水解產物之製備…………….…………………23 （二）粗蛋白含量（Kjeldahl）測定………………………………23（三）水解物中可溶性蛋白質含量的測定………………………..24（四）水解率（degree of hydrolysis）之測定………………………24（五）胜肽濃度之測定……………………………………………25（六）ACEI之活性測定……………………………………………26（七）半抑制濃度（IC50）測定……………………………………27（八）水解物中具ACEI胜肽之分離與收集………………………27（九）劃分收集物對ACE抑制活性之測定………………………28（十） 具ACE抑制能力胜肽之純化……………………………...…28（十一）胺基酸的組成…………………………………………..…28（十二）自發性高血壓鼠（SHR）活體試驗………..………………29（十三）統計分析………………………………………………….30
肆、結果與討論…………………………………………………………31
一、水解物可溶性蛋白質含量及胜肽濃度……………………31
二、甲魚粉之水解物對抑制ACE之IC50…………………………33
三、甲魚粉之水解物對原發性高血壓大鼠血壓的影響…………35
（一）不同劑量之之酵素水解甲魚液對SHR收縮壓以及舒張壓之影 響………………………………………………….……………………36
（二）不同分子量甲魚水解物對SHR收縮壓以及舒張壓之影響……………………………………………………………………….38
四、各劃分物…………………………………………………………...40
五、劃分收集物對ACE抑制活性之測定……………………………..41
六、劃分收集物之胺基酸組成………………………………………...41
伍、結論…………………………………………………………………42
陸、參考文獻……………………………………………………………60
圖目錄
頁次
圖一、甲魚粉經胃蛋白酶、胰蛋白酶、凝乳胰蛋白酶水解之蛋白質及 胜肽濃度含量…………………………………………………..43
圖二、甲魚粉經胃蛋白酶、胰蛋白酶、凝乳胰蛋白酶處理之不同時間 水解率…………………………………………………….……44
圖三、單次灌食不同劑量甲魚肉粉末之酵素水解物對於自發性高血壓大鼠收縮壓之變化…………………………..…………………45
圖四、單次灌食不同分子量甲魚粉末之酵素水解物對於自發性高血壓大鼠收縮壓之變化……………………………………………..46
圖五、單次灌食不同劑量甲魚粉末之酵素水解物對於自發性高血壓大鼠舒張壓之變化……………………………………………..…47
圖六、單次灌食不同分子量甲魚粉末之酵素水解物對於自發性高血壓大鼠舒張壓之變化………………………………………..……48
圖七、甲魚水解液（分子量＜1000）之膠體層析圖譜………………..49
圖八、由Sephdex G-15劃分物之第4區分所得的高效液相層析圖…50
圖九、由Sephdex G-15劃分物之第5區分所得的高效液相層析圖………………………………………………………………………..51
圖十、由Sephdex G-15劃分物之第6區分所得的高效液相層析圖………………………………………………………………………52




表目錄
頁次
表一、國家聯席委員會對18歲以上成人之血壓分類標準與定義…….3
表二、未水解或經胃腸道酵素水解之甲魚粉其ACEI活性及相對活性
之改變…………………………………………………………53
表三、單次灌食不同劑量甲魚肉粉末之酵素水解物對於自發性高血壓 大鼠收縮壓之影響…………………………………………….54
表四、單次灌食不同劑量及分子量之甲魚肉粉末酵素水解物對於自發 性高血壓大鼠收縮壓之影響………………………..…………55
表五、單次灌食不同劑量甲魚肉粉末之酵素水解物對於自發性高血壓大鼠舒張壓之影響……………………………………………56
表六、單次灌食不同劑量及分子量之甲魚肉粉末酵素水解物對於自發性高血壓大鼠舒張壓之影響…………………………………57
表七、由SephdexG15劃分物之ACE抑制百分比……………………..58
表八、分子量小於1000之區分四其胺基酸組成…………………59











附圖目錄
頁次
附圖一：單次灌食不同劑量甲魚粉末之酵素水解物對於自發性高血壓大鼠收縮壓之影響…………………………………………..71
附圖二：單次灌食不同分子量甲魚粉末之酵素水解物對於自發性高血壓大鼠收縮壓之影響………………………………………..72
附圖三、單次灌食不同劑量甲魚粉末之酵素水解物對於自發性高血壓大鼠舒張壓之影響…………………………………………73
附圖四、單次灌食不同分子量甲魚粉末之酵素水解物對於自發性高血壓大鼠舒張壓之影響………………………………………..74
附圖五、單次灌食不同劑量及分子量之甲魚肉粉末酵素水解物對於自發性高血壓大鼠收縮壓之變化……………………………75
附圖六、單次灌食不同劑量及分子量甲魚粉末之酵素水解物對於自發性高血壓大鼠舒張壓之變化………………………………..76
附圖七、單次灌食不同劑量及分子量之甲魚粉末之酵素水解物對於自發性高血壓大鼠收縮壓之影響……………………………77
附圖八、單次灌食不同劑量及分子量甲魚粉末之酵素水解物對於自發性高血壓大鼠舒張壓之影響………………………………78









附表目錄
頁次
附表一、本實驗所使用蛋白酶的性質…………………………………79
附表二、單次灌食不同劑量甲魚肉粉末之酵素水解物對於自發性高血壓大鼠收縮壓之變化………………………………………80
附表三、單次灌食不同劑量及分子量之甲魚肉粉末酵素水解物對於自發性高血壓大鼠收縮壓之變化……………………………81
附表四、單次灌食不同劑量甲魚肉粉末之酵素水解物對於自發性高血壓大鼠舒張壓之變化………………………………………82
附表五、單次灌食不同劑量及分子量之甲魚肉粉末酵素水解物對於自發性高血壓大鼠舒張壓之變化……………………………83
附表六、動物與人體的每公斤體重劑量折算係數表............................84

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