利用聯繼生物活性指引分群方法篩選台灣紅藜的血管收縮素轉換酶抑制肽

Screening of Angiotensin-I Converting Enzyme Inhibitory Peptide Derived from Taiwan Red Quinoa (Chenopodium formosanum) using Tandem Bioassay-Guided Fractionation

丁寧汝(Dininurilmi Putri Suleman)

指導教授 ： 徐睿良 ； -

國立屏東科技大學/農學院/生物科技系/碩士(2019年)

https://doi.org/10.6346/NPUST202000048

摘要

血管收縮素 I轉換酶 （Angiotensin-I Converting Enzyme, ACE）是
一種肽基二肽酶，可藉由去除羧基末端的二肽來催化十肽血管收縮素
I 轉化成八肽血管收缩劑—血管收縮素 II。長期以來，ACE 被認為是
調節血壓的腎素-血管收縮素系统的關鍵部分，ACE 抑制劑對於治療
高血壓非常重要。台灣红藜或杜鹃（Chenopodium formosanum）是台
灣傳統的穀類植物之一。它包含一種可能具有治療高血壓的生物活
性的蛋白質來源。其蛋白質含量高於其他普通穀類植物，例如小
麥，燕麥或大米。本研究旨在台灣紅藜蛋白水解物中篩選血管收縮素
I 轉換酶（ACE）的活性胜肽。 先從台灣紅藜中萃取出蛋白質，再以
多種蛋白酶水解，分别是嗜热菌素、胃蛋白酶、胰蛋白酶和α-胰凝
乳蛋白酶。使用體外 ACE 抑制測定法評估所得各水解產物的 ACE 抑
制活性。結果表明，嗜热菌蛋白酶之水解產物表現出最高的抑制活性，
高達 77.67％。使用強陽離子交換（SCX）和逆相高效液相層析(RP
HPLC）進行活性指引分群，以找出具有最高 ACE 抑制活性的最佳分
液。來自 SCX的分液 F4（抑制活性達 87.06％）和來自串聯 SCX-RP
HPLC 分離的 F4.3（抑制活性達 89.30％）。使用液相層析-串聯式質
譜(LC-MS/MS)搭配從頭定序法(de novo sequencing)對分液 F4.3進一步 測序，並鑑定出胜肽 Val-Tyr-Leu-Ala-Glu-Leu-His-Phe（VF-8）、Leu-Gly-Ala-Val-Pro-Pro-Arg-Tyr（LY-8）、Ile-Ala-Arg-Asp-Ser-Ala-Ala-Val-Phe（IF-9），其半抑制濃度(IC50)值分別為 24.26 ± 1.63 µM、30.48 ± 1.39 µM 和 72.57 ± 3.5 µM。酵素動力學研究和分子模擬對接 實驗表明 VF-8 是競爭型抑制劑。因此，我們可以得出結論，此為第 II 一個源自台灣紅藜的 ACE 抑制肽，可能有助於預防高血壓，具發展 為功能性食品的潛力。

關鍵字

血管收縮素I轉換酶 ； 生物活性肽 ； 台灣紅藜 ； 蛋白水解產物

並列摘要

Angiotensin I-converting enzyme (ACE) is a peptidyl dipeptidase that catalyzes the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II, the vasoconstrictor agent, by removing a carboxy-terminal dipeptide. ACE has long been known to be a crucial part of the renin-angiotensin system that regulates blood pressure, and ACE inhibitors are important for the treatment of hypertension. Taiwan Red Quinoa (Chenopodium formosanum) is one of the Taiwan traditional cereal plant. It contains a protein source that potentially has bioactivity for hypertension treatment. Its protein contained is higher than other general cereal plant such as wheat, oat or rice. This study aims to explore a biological activity to inhibit ACE from Red Quinoa protein hydrolysate.
Proteins extracted from Red Quinoa were hydrolyzed by various proteases, namely thermolysin, pepsin, trypsin, and α-chymotrypsin. The ACE inhibitory activities of the resulting hydrolysates were evaluated using in vitro ACE inhibitory assay. The result indicated that thermolysin hydrolysate showed the highest inhibitory activity, as high as 77.67%. Tandem bioassay-guided fractionations using Strong Cation Exchange (SCX) and Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) were performed to find out the best fractions with the highest ACE inhibitory activity. F4 (87.06%) from SCX and F4.3 (89.30%) from Tandem SCX-RPHPLC fractionations showed the best inhibition activity, respectively. F4.3 was further sequenced using liquid chromatography tandem mass spectrometry coupled with de novo sequencing to get some potential peptides. Peptide Val-Tyr-Leu-Ala-Glu-Leu-His-Phe (VF-8), Leu-Gly-Ala-Val-Pro-Pro-Arg-Tyr (LY-8) and Ile-Ala-Arg-Asp-Ser-Ala-Ala-Val-Phe (IF-9) were identified with high score and the IC50 values were determined as 24.26 ± 1.63 µM, 30.48 ± 1.39 µM, and 72.57 ± 3.5 µM, respectively. The kinetic study and the simulation molecular docking revealed that VF-8 is a competitive inhibitor. Therefore, we can conclude that this study is the first report about ACE inhibitory peptides derived from Taiwan Red Quinoa and it may be beneficial for preventing hypertension and functional food development.

並列關鍵字

Angiotensin I-converting enzyme ； hydrolysates ； hypertension ； peptide ； Taiwan Red Quinoa

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