從紫蘇種籽蛋白水解物中篩選出具血管收縮素轉換酶抑制效果之胜肽

Screening of peptides containing angiotensin-converting enzyme inhibitory activities from Perilla frutescens seed protein hydrolysate

廖顯祐(Liao Hsien-Yu)

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/NPUST202000057

摘要

高血壓被視為是種慢性病，也已經是目前最常見的文明病。為了改善高血壓的情況，已經有一些化學合成的降血壓藥物被開發出來，但通常會伴隨一些副作用。一般認為從天然食材中取得的降血壓活性胜肽，其副作用會低於化學合成的藥物。
本研究主要是以天然食材紫蘇種籽萃取出蛋白質，並利用嗜熱菌蛋白酶(Thermolysin)進行水解，以超過濾膜(3kDa cut-off)進行過濾，得到的出短鏈胜肽混合物再利用高效能液相層析儀(High performance liquid chromatography，HPLC)將水解物分群，並進行血管收縮素轉換酶(Angiotensin converting enzyme，ACE)活性測試。活性最高的分液再利用液相層析-串聯式質譜(LC-MS/MS)搭配從頭定序法(de novo sequencing)進行定序。鑑定到的胜肽以合成的胜肽驗證其身分與活性。我們也進一步研究這些胜肽的半抑制濃度、抑制動力學及分子嫁接模擬。結果顯示，紫蘇種籽之嗜熱菌蛋白酶水解物，具有ACE酵素的抑制能力，其IC50值為141.1±5µg/mL，活性分液進行鑑定之2段胜肽，分別為TY-4、LY-4。其中以TY-4具有較好的ACE的抑制活性，其IC50值為32.93±2.9 µM，而後續進行胜肽的酵素動力學與分子對接模擬，結果顯示其為ACE之競爭型抑制劑。

關鍵字

血管收縮素轉換酶 ； 紫蘇種籽 ； 高血壓 ； 活性胜肽

並列摘要

Hypertension considered as a chronic disease and now becomes one of the most common civilized diseases. In order to improve the condition of hypertension, some chemically synthesized blood pressure lowering drugs have been developed, but usually accompanied by some side effects. In general, it considered that the side effects of the active peptides obtained from natural ingredients are lower than those derived from synthetic chemicals.
In this study, proteins extracted from the perilla (Perilla frutescens) seeds were hydrolyzed using thermolysin, and filtered with an ultrafiltration membrane (3kDa cut-off) to obtain a mixture of small peptides. The high-performance liquid chromatography (HPLC) was used to fractionate the hydrolysate, and the activity of each fraction was tested using angiotensin-I-converting enzyme inhibitory (ACEI) assay. The most active fraction was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with de novo sequencing. The peptide sequences identified by LC-MS/MS were further confirmed using the corresponding synthetic peptides. The studies of IC50, inhibition kinetics, and molecular docking of the identified peptides towards ACE were also carried out. The result indicated that Perilla seeds hydrolyzed by thermolysin showed significant inhibitory ability against ACE. The IC50 value was 141.1±5 µg/mL. Two peptides from the most active fraction were identified to be LY-4 and TY-4. Among them, TY-4 showed the best ACEI activity (IC50=32.93±2.9 µM). Further study of inhibition kinetics and molecular docking indicated that TY-4 and LY-4 are competitive inhibitor against ACE.

並列關鍵字

Angiotensin-I converting enzyme (ACE) ； Perilla frutescens seed ； hypertension ； active peptide

參考文獻

Aderinola, T. A., Fagbemi, T. N., Enujiugha, V. N., Alashi, A. M., Aluko, R. E. J. F. s., & nutrition. (2019). In vitro antihypertensive and antioxidative properties of trypsin‐derived Moringa oleifera seed globulin hydrolyzate and its membrane fractions. Food science & nutrition, 7(1), 132-138.

Ahmed, H. M. (2019). Ethnomedicinal, Phytochemical and Pharmacological Investigations of Perilla frutescens (L.) Britt. Molecules, 24(1), 102.

Jing, A., & Li, B. (2013). Stability and antioxidative activities of casein peptide fractions during simulated gastrointestinal digestion in vitro: Charge properties of peptides affect digestive stability. Food research international, 52(1), 334-341.

Giovanna, B., Scigliuolo, G. M., Resta, D., & Arnoldi, A. (2014). ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes. Food Chemistry, 145, 34-40.

Chaudhary, S. K., De, A., Bhadra, S., & Mukherjee, P. K. J. P. B. (2015). Angiotensin-converting enzyme (ACE) inhibitory potential of standardized Mucuna pruriens seed extract. 53(11), 1614-1620.