自長莖葡萄蕨藻篩選製備具血管收縮素轉化酶抑制活性之胜肽

Screening of Angiotensin-I Converting Enzyme Inhibitory Peptides Derived from Caulerpa lentillifera

胡喬伊(Cesarea Hulda Joel)

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(2018年)

https://doi.org/10.6346/THE.NPUST.BST.005.2018.D01

摘要

具血管收縮素轉化梅(Angiotensin Converting Enzyme，ACE)抑制活性之胜肽，由於其具備抗高血壓之潛力及消費者對於合成藥物安全性之疑慮，使其成為現今食品工業中極具吸引力之研究主題。本研究之目的為利用不同蛋白酶水解長莖葡萄蕨藻 Caulerpa lentillifera，俗稱：海葡萄)，並由其水解物中篩選並製備具ACE抑制活性之胜肽。藉由不同酵素水解16小時獲得其水解物，並透過3kDa超過濾膜過濾，進而得到其短鏈胜肽。結果顯示，經由嗜熱菌蛋白酶水解之水解物，對ACE具有最高的抑制活性，其IC50值為11.86±0.98μg/ mL。將水解物利用逆相高效能液相層析儀進行生物活性指引分群，結果顯示，分液9具有最高的ACE抑制活性。將分液9利用液相層析-串聯式質譜儀進行胜肽鑑定分析，並透過從頭定序法(de novo)計算得到二段胜肽，序列分別為Phe-Asp-Gly-Ile-Pro (FP-5) 和Ala-Ile-Asp-Pro-Val-Arg-Ala (AA-7)，其IC50分別為58.89±0.68μM和65.76±0.92μM，且酵素動力學結果顯示，FP-5和AA-7皆為競爭型抑制劑。此研究首度報導自長莖葡萄蕨藻之水解物中篩選出具有抑制ACE之活性肽，將有益於高血壓之預防且具做為功能性食品開發之潛力。

關鍵字

ACE抑制胜肽 ； 生物活性指引分群 ； 海葡萄 ； 抗高血壓作用

並列摘要

Peptides with Angiotensin Converting Enzyme (ACE, EC 3.4.15.1) inhibitory activity have received considerable interest from the food industry due to their potential as antihypertensive and the consumer’s concern over the safety of the synthetic counterparts. The objectives of this study were to isolate ACE inhibitory peptide from Caulerpa lentillifera (known commonly as sea grape) protein hydrolysate using different proteases. Short-chain peptides were obtained after 16 hours of hydrolysis by various enzymes and followed by ultrafiltration using 3 kDa molecular weight cut off (MWCO) membrane. The results indicated that thermolysin hydrolysate had the highest ACE inhibitory activity with the IC50 value of 11.86±0.98μg/mL. Bioassay-guided fractionation was performed using reversed-phase high performance liquid chromatography (RP-HPLC). The fraction 9 showed the highest ACE inhibitory activity. Peptides in fraction 9 were sequenced by liquid chromatography tandem mass Spectrometry (LC-MS/MS). LC-MS/MS analysis of fraction 9 gave the peptides sequence as Phe-Asp-Gly-Ile-Pro (FP-5) and Ala-Ile-Asp-Pro-Val-Arg-Ala (AA-7) identified using de novo method. Furthermore, their IC50 values were determined as 58.89±0.68μM and 65.76±0.92μM, respectively. The kinetic study revealed that FP-5 and AA-7 are competitive inhibitors. Therefore, we can conclude that it is the first report about ACE inhibitory peptides derived from Caulerpa lentillifera and it may be beneficial for preventing hypertension and functional food development.

並列關鍵字

ACE inhibitory peptide ； antihypertensive effect ； sea grapes ； bioassay-guided fractionation

參考文獻

Ahluwali M, Bangalore S. Management of hypertension in 2017 targets and therapies. Current Opinion in Cardiology 2017; 32(4):413–421.

Ambigailapan, Priyatharini, Abdulrahaman S, Al-Khalifa, Fereidoon S. Antioxidant and angiotensin-I converting enzyme (ACE) inhibitory activities of date seed protein hydrolysates prepared using alcalase, flavourzyme and thermolysin. Journal of Functional Foods 2015; 18:1125-1137.

American Heart Association. Guideline: Understanding and Managing High Blood Pressure 2014. http://www.heart.org. Accessed at October 2017.

Anderson C, Arnolda L, Cowley D. Guideline for the diagnosis and management of hypertension in adults. National Heart Foundation of Australia 2016; ABN 98 008 419 761.

Andrews PR, Carson JM, Caselli A, Spark MJ, Woods R. Conformational analysis and active site modeling of angiotensin converting enzyme inhibitors. Journal Medical Chemistry 1985; 28:393-399.