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學校名稱輔仁大學
系所名稱食品營養學系
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學號493446136
研究生(中)陳慧芳
研究生(英)Huei-Fang Chen
論文名稱(中)米蛋白水解物胜肽對抑制血管收縮素轉化酶活性及降血壓之效果
論文名稱(英)The antihypertensive effect and angiotensin I-converting enzyme activity inhibition of rice protein isolate hydrolysate
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指導教授(中)王果行
指導教授(英)
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國圖全文開放日期.2007.01.01
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學位類別碩士
畢業學年度94
出版年
語文別中文
關鍵字(中)米蛋白水解物、降血壓胜肽、血管收縮素轉換酶
關鍵字(英)rice protein isolate hydrolysate, antihypertensive peptides, angiotensin converting enzyme
摘要(中)高血壓是常見的慢性疾病症狀,為目前已開發國家中引起心血管疾病的重要因素之一。腎素-血管收縮素系統 (Renin-angiotensin system, RAS) 主要在調節血壓、水分及電解質之恆定, RAS異常會造成高血壓的發生,其中血管收縮素轉換? (Angiotensin converting enzyme, ACE) 在血壓調控扮演重要角色。研究指出米發酵製成的清酒和酒渣水解物具有降血壓之作用。因此,本研究將含不同比率 (10、20、40%) 含少量糖分 (CHO : 35.4%) 的米分離蛋白粉 (rice protein isolate, RPI) 飼料餵食自發性高血壓大白鼠 ( Spontaneously hypertensive rat, SHR ),探討其對動物體內血壓及血脂的影響。另外,利用麴菌 (Aspergillus oryzae) 發酵產生的酵素分解米蛋白質,純化並鑑定米分離蛋白水解物 (Rice protein isolate hydrolysate, RPIH) 中具有降血壓功能之胜?,及其對於SHR大白鼠血壓短時間內的影響。結果顯示,飲食中添加不同比例的RPI,對血壓無顯著的影響,但確可降低血脂 。體外實驗中, RPIH在不同水解時間下 (0、10、30、60、120分鐘) ,以水解120分鐘後具最佳抑制ACE之能力 (Angiotensin converting enzyme inhibition, ACEI = 84.1 %, IC50= 5.83 mg/ml ),其水解率為29.4 ± 0.4%,蛋白質濃度為0.69 ± 0.04 (mg/ml)。將此水解液利用膠體過濾層析,可得五個主要劃分物,其中A2具有最強ACE抑制效果 (ACEI = 94.4%, IC50= 0.1 mg/ml),進一步以逆相高效液相層析純化,得A2-8之胜?其ACEI為91.9%。 動物實驗中,將抑制能力最佳的水解液 (RPIH),以單次不同劑量餵食SHR,當給予每公斤體重1克或2克,可在第2及4小時顯著降低收縮壓 (p < 0.05)。 降低管餵劑量 (0.5 g/kg),同樣可於第2及4小時顯著降低收縮壓 (p < 0.05),若給予0.1 g/kg則只在第4小時具有降低收縮壓 (p < 0.05) 之作用。綜合上述,RPI可顯著影響動物體內血脂濃度,且米蛋白質利用酵素水解可純化具有抑制血壓之胜?,對動物血壓有短期降血壓之效果,可利於開發其附加價值。
摘要(英)Hypertension is a common chronic symptom and a risk factor for cardiovascular diseases in the people of the developed countries. The renin-angiotensin system (RAS) is a blood volume and/or blood sodium content regulatory cascade that plays an essential role in the homeostasis of blood pressure, water and electrolytes. Inappropriate activation of the RAS may lead to elevation of blood pressure, in which angiotensin converting enzyme (ACE) plays a key role in blood pressure regulation. In recent studies, the peptide fractions of sake and hydrolysates of sake lee which are from rice fermentation have been found to be antihypertensive. Therefore, the purposes of this study are to determine the effect of rice protein isolate (RPI) which contained 35.4% CHO on blood pressure (BP) and lipid concentrations in spontaneously hypertensive rat (SHR). Meanwhile, antihypertensive peptides were purified and identified from rice protein isolate hydrolysates (RPIH) with Aspergillus oryzae fermented and the antihypertensive effect in SHR were also examined. Results indicated that there was no significant different of change in BP by different content of RPI in diet, but lower blood lipid concentrations were found. In addition, proteases from fermented Aspergillus oryzae reacted to rice protein isolate for 0, 10, 30, 60 or 120 min and the highest ACE inhibitory (ACEI = 84.1%, IC50= 5.83 mg/ml) activity was at 120 min (R120). The degree of hydrolysis of R120 was 29.4 ± 0.4%, and soluble protein content was 0.69 ± 0.04 mg/ml. The R120 was separated into 5 fractions by gel fractions, and the A2 fraction had the highest inhibition of ACE (94.4%, IC50= 0.1 mg/ml). This fraction was further purified by reversed-phase HPLC and the ACEI of A2-8 was 91.9%. Single oral dose of RPIH was given to SHR rats and the dose of 0.5, 1 or 2 g/kg b.wt. of RPIH exerted a significant lowering effect on systolic blood pressure (SBP) after 2 and 4 hour (p < 0.05). Furthermore, a lower dosage of oral administration 0.1 g/kg RPIH showed a reduced SBP at 4 hour (p < 0.05). In conclusions, the blood lipid concentrations could be reduced by adding RPI in the diet in an animal model and antihypertensive peptide was found in RPIH and added value of the rice protein isolate may be developed.
論文目次目 錄 中文摘要………………………………………………………………..…I 英文摘要…………………………………………………………………III 致謝……………………………………………………………………….V 壹、前言……………………………………….………………………… 1 貳、文獻回顧…………………………………………………………… .3 一、高血壓……………………………………………………………. 3 (一) 高血壓簡介………………………………………………… 3 (二) 高血壓之成因與分類……………………………………….3 (三) 高血壓的預防與治療…………………………………… …5 二、血壓的調控………………………………………………………. 7 (一) 腎素-血管收縮素系 (Renin-angiotensin system, RAS).….. 7 (二) 激?釋放?-激?系統 (kallikrein–kinin system, KKS)…...8 (三) RAS與KKS……………………………………………….…9 三、血管收縮素轉化?(Angiotensin converting enzyme)…….……...10 (一) ACE生化特性……………………………………………. .10 (二) ACE 結構特性…………………………………………..…11 (三) ACE抑制原理…………………………..…………………..11 四、食品蛋白質對血壓的影響……………………………………….14 (一) 動物性蛋白……………………………………………..… 14 (二) 植物性蛋白………………………………………………...16 (三) 發酵食品..………………………………………………….17 五、稻米………………………………………………………………..20 六、自發性高血壓鼠 (Spontaneously hypertension rat;SHR) ..…... 21 ?、材料與方法…………………………………………………….…… 23 第一部份 餵食不同劑量米分離蛋白粉 (Rice protein isolate, RPI) 對血壓及血脂的影響 (一) 米分離蛋白粉 (Rice protein isolate, RPI) 製備………….23 (二) 米分離蛋白食品成分分析………………………………...23 (三) 實驗動物及分組……………………………………….…..24 (四) 飼料配方……………………………………………….…..25 (五) 血壓測定方法…………………………………………...…25 (六) 血液脂質分析…………………………………………..….25 第二部份 利用體外實驗方式篩選米分離蛋白水解物 (Rice protein isolate hydrolyaste , RPIH) 中具有抑制血管收縮素轉化?之胜? 一、實驗流程....................................................................................28 二、實驗方法 (一) 米蛋白液之萃取與純化……………………………….......28 (二) 米分離蛋白水解物 (Rice protein isolate hydrolysate ,RPIH) 樣品製備................................................................................29 (三) 蛋白質含量測定...................................................................29 (四) 胜?濃度測定.......................................................................29 (五) 水解率的測定……………………………………...………30 (六) 米分離蛋白水解液對ACE抑制能力之測定.....................31 (七) 半抑制濃度 (IC50 ) 測定.................................................... 32 (八) 水解物中具ACEI胜?之分離與收集.................................32 (九) 劃分收集物對ACE抑制活性之測定..................................33 (十) 具ACE抑制能力胜?之純化..............................................33 第三部分 米分離蛋白水解物 (RPIH) 於活體中對血壓短期作用之影響 一、實驗流程....................................................................................34 二、實驗方法 (一) 實驗動物...............................................................................34 (二) 血壓測定...............................................................................35 (三) 血漿中ACE濃度測定…………………………………….35 肆、統計方法.............................................................................................37 伍、結果與討論..........................................................................................38 不同劑量米分離蛋白粉 (RPI) 對血壓及血脂的影響 一、米分離蛋白粉之組成份…………………………………….38 二、飼料攝取量與體重改變量…………………………………..38 三、血壓變化……………………………………………….…….39 四、血脂變化…………………………………………………......39 由米分離蛋白水解物 (Rice protein isolate hydrolyaste , RPIH) 中製備具有抑制血管收縮素轉化?之胜? 一、不同水解時間對RPIH中成份的影響…………………..…43 二、RPIH對血管收縮素轉換? (ACE) 活性之抑制效果…....44 米分離蛋白水解物 (RPIH) 對SHR血壓及血漿ACE活性之短期作用影響 一、血壓之影響…………………………………………………47 二、血漿ACE活性變化………………………………………..49 陸、結論……………………………………………...……………..…….50 柒、參考文獻..............................................................................................51 表 目 錄 表一、SHR飼料組成百分比……………………………………………60 表二、倉鼠飼料組成百分比…………………………………………….61 表三、米分離蛋白成分百分比………….………………………………62 表四、餵食米分離蛋白粉後對高血壓鼠每日攝食量、體重增加量及飼料效率之影響…………………………………………………63 表五、餵食米分離蛋白粉後對高血壓鼠血壓及心跳之影響……...….64 表六、餵食米分離蛋白粉後對高血壓鼠血液中三酸甘油酯之影響....65 表七、餵食米分離蛋白粉後對高血壓鼠血液中膽固醇之影響…....…66 表八、米蛋白水解液於不同水解時間下可溶性蛋白濃度、胜?濃度及水解率含量…………..…………………………………………..67 表九、米分離蛋白液經經不同水解時間抑制血管收縮素轉換?之能力…………………………………………………………………68 表十、米分離蛋白水解物之膠體層析劃分物其ACE抑制百分比及IC50……………………………………………………………….69 表十一、米分離蛋白水解物對高血壓鼠心跳短期影響………….……70 表十二、米分離蛋白水解物對高血壓鼠收縮壓短期影響…………..…71 表十三、米分離蛋白水解物對高血壓鼠平均血壓短期影響……….…72 表十四、米分離蛋白水解物對高血壓鼠舒張壓短期影響………….…73 表十五、管餵米分離蛋白水解物對高血壓鼠血液血管收縮素轉換?活 性之短期變化……………………………………………...….74 圖 目 錄 圖一 RAS及KKS作用途徑………………………………………..…9 圖二、 ACE 結構…………………….…………………………….......13 圖三、 Captopril、enalaprilat及lisinopril 的作用原理……………….13 圖四、 經水解120分鐘米分離蛋白水解物之膠體層析圖譜………..75 圖五、 經Superdex? 10/300 GL過濾後收集劃分物A2所得之高 效液相層析圖…..……………………………………………....76 圖六、 Captopril對SHR收縮壓短期影響…………….………………77 圖七、米分離蛋白水解物 (C0) 對SHR收縮壓之短期影響……...…78 圖八、 米分離蛋白水解物 (R120) 對SHR收縮壓之短期影響……....79 附 表 目 錄 附表一、米蛋白胺基酸組成……………………………………………..80 附表二、餵食米分離蛋白粉後對倉鼠血液中膽固醇變化之影響….…81 附 圖 目 錄 附圖一、餵食米分離蛋白粉造成高血壓鼠血液中三酸甘油酯之改變………………………………………………………………82 附圖二、餵食米分離蛋白粉造成高血壓鼠血液總膽固醇之改變…….83 附圖三、餵食米分離蛋白粉造成高血壓鼠血液低密度脂蛋白膽固醇之改變……………………………………………………………84 附圖四、 餵食米分離蛋白粉造成高血壓鼠血液高密度脂蛋白膽固醇之改變……………………………………………………………85 附圖五、餵食米分離蛋白水解物 (C0) 對SHR收縮壓改變量………86 附圖六、餵食米分離蛋白水解物 (R120) 對SHR收縮壓改變量…….87
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