{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T14:54:20Z","timestamp":1791816860545,"version":"4.3.4"},"reference-count":48,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2020,10,12]],"date-time":"2020-10-12T00:00:00Z","timestamp":1602460800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004663","name":"Ministry of Science and Technology, Taiwan","doi-asserted-by":"publisher","award":["MOST 108-2113-M-020-001"],"award-info":[{"award-number":["MOST 108-2113-M-020-001"]}],"id":[{"id":"10.13039\/501100004663","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Ministry of Science and Technology","award":["MOST 109-2634-F-020-001"],"award-info":[{"award-number":["MOST 109-2634-F-020-001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Gac (Momordica cochinchinensis Spreng.) seed proteins (GSPs) hydrolysate was investigated for angiotensin I-converting enzyme (ACE) inhibitory activities. GSPs were hydrolyzed under simulated gastrointestinal digestion using a combination of enzymes, including pepsin, trypsin, and chymotrypsin. The screening of ACE inhibitory peptides from GSPs hydrolysate was performed using two sequential bioassay-guided fractionations, namely hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC). Then, the peptides in the fraction with the highest ACE inhibitory activity were identified by LC-MS\/MS. The flow-through (FT) fraction showed the most potent ACE inhibitory activity when HILIC fractionation was performed. This fraction was further separated using RP-HPLC, and the result indicated that fraction 8 (RP-F8) showed the highest ACE inhibitory activity. In the HILIC-FT\/RP-F8 fraction, 14 peptides were identified using LC-MS\/MS analysis coupled with de novo sequencing. These amino acid chains had not been recorded previously and their ACE inhibitory activities were analyzed in silico using the BIOPEP database. One fragment with the amino acid sequence of ALVY showed a significant ACE inhibitory activity (7.03 \u00b1 0.09 \u00b5M). The Lineweaver-Burk plot indicated that ALVY is a competitive inhibitor. The inhibition mechanism of ALVY against ACE was further rationalized through the molecular docking simulation, which revealed that the ACE inhibitory activities of ALVY is due to interaction with the S1 (Ala354, Tyr523) and the S2 (His353, His513) pockets of ACE. Bibliographic survey allowed the identification of similarities between peptides reported as in gac fruit and other proteins. These results suggest that gac seed proteins hydrolysate can be used as a potential nutraceutical with inhibitory activity against ACE.<\/jats:p>","DOI":"10.3390\/molecules25204635","type":"journal-article","created":{"date-parts":[[2020,10,17]],"date-time":"2020-10-17T07:23:22Z","timestamp":1602919402000},"page":"4635","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":23,"title":["ACE Inhibitory Activity and Molecular Docking of Gac Seed Protein Hydrolysate Purified by HILIC and RP-HPLC"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8185-4398","authenticated-orcid":false,"given":"Samuchaya","family":"Ngamsuk","sequence":"first","affiliation":[{"name":"Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tzou-Chi","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Biological Science and Technology, National Pingtung University of Science and Technology, 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6771-6690","authenticated-orcid":false,"given":"Jue-Liang","family":"Hsu","sequence":"additional","affiliation":[{"name":"Department of Biological Science and Technology, National Pingtung University of Science and Technology, 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan"},{"name":"International Master\u2019s Degree Program in Food Science, National Pingtung University of Science and Technology, 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan"},{"name":"Research Center for Animal Biologics, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan"},{"name":"Research Center for Tropic Agriculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,10,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"158","DOI":"10.4103\/2221-1691.256729","article-title":"Antioxidant compounds and capacities of gac (Momordica cochinchinensis Spreng) fruits","volume":"9","author":"Abdulqader","year":"2019","journal-title":"Asian Pac. J. Trop. Biomed."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1634","DOI":"10.1128\/CVI.00339-07","article-title":"Enhancement of serological immune responses to foot-and-mouth disease vaccine by a supplement made of extract of cochinchina momordica seeds","volume":"14","author":"Xiao","year":"2007","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1080\/10575630108041271","article-title":"A new multiflorane triterpenoid ester from Momordica cochinchinensis Spreng","volume":"15","author":"Hu","year":"2001","journal-title":"Nat. Prod. Lett."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"950","DOI":"10.3390\/ijms17060950","article-title":"A review of the latest advances in encrypted bioactive peptides from protein-rich waste","volume":"17","author":"Lemes","year":"2016","journal-title":"Int. J. Mol. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"32","DOI":"10.3390\/foods6050032","article-title":"Bioactive peptides","volume":"6","author":"Daliri","year":"2017","journal-title":"Foods"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"684","DOI":"10.1080\/10498850.2014.919049","article-title":"Optimization of hydrolysis conditions for production of angiotensin-converting enzyme inhibitory peptides from Basa fish skin using response surface methodology","volume":"25","author":"Zhang","year":"2016","journal-title":"J. Aquat. Food Prod. Technol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1297","DOI":"10.2174\/1381612033454892","article-title":"Food-derived bioactive peptides-opportunities for designing future foods","volume":"9","author":"Korhonen","year":"2003","journal-title":"Curr. Pharm. Des."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"975","DOI":"10.3390\/md11040975","article-title":"Evidence of anti-proliferative activities in blue mussel (Mytilus edulis) by-products","volume":"11","author":"Beaulieu","year":"2013","journal-title":"Mar. Drugs"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1038\/ajh.2011.180","article-title":"Treatment of hypertension with renin-angiotensin system inhibitors and renal dysfunction: A systematic review and meta-analysis","volume":"25","author":"Daien","year":"2012","journal-title":"Am. J. Hypertens."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1080\/080370500439146","article-title":"The renin-angiotensin system and cardiovascular disease","volume":"1","author":"Hollengerg","year":"2000","journal-title":"Blood Press."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1161\/01.RES.36.2.247","article-title":"Angiotensin I converting enzyme","volume":"36","author":"Erdos","year":"1975","journal-title":"Circ. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1321","DOI":"10.1016\/j.idairyj.2007.02.009","article-title":"ACE-inhibitory activity or probiotic yoghurt","volume":"17","author":"Donkor","year":"2007","journal-title":"Int. Dairy J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1517\/14740338.5.5.643","article-title":"Angiotensin-converting enzyme inhibitor-related angioedema: How to deal with it","volume":"5","author":"Beltrami","year":"2006","journal-title":"Expert Opin. Drug Saf."},{"key":"ref_14","unstructured":"Riding, G., Wang, Y., and Walker, P. (1992, January 25\u201327). A strategy for purification and peptide sequence analysis of bovine ephemeral fever virus structural proteins. Proceedings of the ACIAR PROCEEDINGS, Beijing, China."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1016\/S0165-9936(03)00605-8","article-title":"New trends in solid-phase extraction","volume":"22","author":"Poole","year":"2003","journal-title":"Trends Analyt. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1007\/s00216-011-5308-5","article-title":"Hydrophilic interaction liquid chromatography (HILIC)\u2014A powerful separation technique","volume":"402","author":"Buszewski","year":"2012","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1784","DOI":"10.1002\/jssc.200600199","article-title":"Hydrophilic interaction chromatography","volume":"29","author":"Irgum","year":"2006","journal-title":"J. Sep. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"965","DOI":"10.1093\/jaoac\/91.4.965","article-title":"BIOPEP database and other programs for processing bioactive peptide sequences","volume":"91","author":"Minkiewicz","year":"2008","journal-title":"J. AOAC Int."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Minkiewicz, P., Iwaniak, A., and Darewicz, M. (2019). BIOPEP-UWM database of bioactive peptides: Current opportunities. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20235978"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Zheng, Y., Wang, X., Zhuang, Y., Li, Y., Shi, P., Tian, H., Li, X., and Chen, X. (2020). Isolation of novel ACE-inhibitory peptide from naked oat globulin hydrolysates in silico approach. Molecular docking, in vivo antihypertension and effects on renin and intracellular endothelin-1. J. Food Sci.","DOI":"10.1111\/1750-3841.15115"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"190","DOI":"10.3390\/md17030190","article-title":"In silico analysis of relationship between protein from plastid genome of red alga Palamaria sp. (Japan) and angiotensin I converting enzyme inhibitory peptides","volume":"17","author":"Kumagai","year":"2019","journal-title":"Mar. Drugs"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Garcia-Vaquero, M., Mora, L., and Hayes, M. (2019). In vitro and in silico approaches to generating and identifying angiotensin-converting enzyme I inhibitory peptides from green macroalga Ulva lactuca. Mar. Drugs, 17.","DOI":"10.3390\/md17040204"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Sumikawa, K., Takei, K., Kumagi, Y., Shimizu, T., Yasui, T., and Kishimura, H. (2020). In silico analysis of ACE inhibitory peptides from chloroplats proteins of red alga Grateloupia asiatica. Mar. Biotechnol.","DOI":"10.1007\/s10126-020-09959-2"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e01747","DOI":"10.1016\/j.heliyon.2019.e01747","article-title":"Kinetics of angiotensin-1 converting enzyme inhibition and antioxidative properties of Azadirachta indica seed protein hydrolysates","volume":"5","author":"Arise","year":"2019","journal-title":"Heliyon"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1007\/s10068-018-0512-0","article-title":"Synergistic effect of fruit\u2013seed mixed juice on inhibition of angiotensin I-converting enzyme and activation of NO production in EA. hy926 cells","volume":"28","author":"Park","year":"2019","journal-title":"Food Sci. Biotechnol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"864","DOI":"10.1016\/j.phytochem.2009.04.006","article-title":"ACE inhibitory tetrapeptides from Amaranthus hypochondriacus 11S globulin","volume":"70","author":"Vecchi","year":"2009","journal-title":"Phytochemistry"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1080","DOI":"10.3390\/md8041080","article-title":"Angiotensin-I-converting enzyme (ACE) inhibitors from marine resources: Prospects in the pharmaceutical industry","volume":"8","author":"Wijesekara","year":"2010","journal-title":"Mar. Drugs"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.foodchem.2004.09.048","article-title":"Angiotensin I-converting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats","volume":"94","author":"Jung","year":"2006","journal-title":"Food Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1016\/j.foodres.2018.01.030","article-title":"High angiotensin-I converting enzyme (ACE) inhibitory activity of Alcalase-digested green soybean (Glycine max) hydrolysates","volume":"106","author":"Hanafi","year":"2018","journal-title":"Food Res. Int."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/j.chroma.2013.07.048","article-title":"On-line comprehensive two dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part II: Application to the separation of peptides","volume":"1306","author":"Heinisch","year":"2013","journal-title":"J. Chromatogr. A"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1125","DOI":"10.1016\/j.jff.2015.01.021","article-title":"Antioxidant and angiotensin-I converting enzyme (ACE) inhibitory activities of data seed protein hydrolysate prepared using alcalase, flavourzyme and thermolysin","volume":"18","author":"Ambigaipalan","year":"2015","journal-title":"J. Funct. Foods"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Saito, T. (2008). Antihypertensive peptides derived from bovine casein and whey proteins. Bioactive Components of Milk, Springer.","DOI":"10.1007\/978-0-387-74087-4_12"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1016\/j.jnutbio.2007.11.010","article-title":"The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease","volume":"19","author":"Erdmann","year":"2008","journal-title":"J. Nutr. Biochem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"484","DOI":"10.1016\/j.tifs.2005.07.003","article-title":"Quantitative structure activity relationship modelling of peptides and proteins as a tool in food science","volume":"16","author":"Pripp","year":"2005","journal-title":"Trends Food Sci. Technol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/j.biochi.2004.01.003","article-title":"A quantitative in silico analysis calculates the angiotensin I converting enzyme (ACE) inhibitory activity in pea and whey protein digests","volume":"86","author":"Vermeirssen","year":"2004","journal-title":"Biochimie"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"980S","DOI":"10.1093\/jn\/134.4.980S","article-title":"Hypotensive peptides from milk proteins","volume":"134","author":"FitzGerald","year":"2004","journal-title":"J. Nutr."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"S304","DOI":"10.1111\/j.1440-1681.1995.tb02929.x","article-title":"Anti-hypertensive effect of thermolysin digest of dried bonito in spontaneously hypertensive rat (SHR)","volume":"22","author":"Fujita","year":"1995","journal-title":"Clin. Exp. Pharmacol. Physiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"7810","DOI":"10.1021\/acs.jafc.9b01922","article-title":"Discovery and study of novel antihypertensive peptides derived from cassia obtusifolia seeds","volume":"67","author":"Shih","year":"2019","journal-title":"J. Agric. Food Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1016\/j.foodchem.2016.02.097","article-title":"In silico and in vitro analyses of the angiotensin-I converting enzyme inhibitory activity of hydrolysates generated from crude barley (Hordeum vulgare) protein concentrates","volume":"203","author":"Gangopadhyay","year":"2016","journal-title":"Food Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"424","DOI":"10.1016\/j.jprot.2015.08.018","article-title":"Screening, discovery, and characterization of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins","volume":"128","author":"Priyanto","year":"2015","journal-title":"J. Proteomics"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"10482","DOI":"10.1021\/acs.jafc.7b04522","article-title":"New ACE-inhibitory peptides from hemp seed (Cannabis sativa L.) proteins","volume":"65","author":"Orio","year":"2017","journal-title":"J. Agric. Food Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3598","DOI":"10.1021\/jf104998s","article-title":"Accumulation and identification of angiotensin-converting enzyme inhibitory peptides from wheat germ","volume":"59","author":"Yang","year":"2011","journal-title":"J. Agric. Food Chem."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Wang, R., Lu, X., Sun, Q., Gao, J., Ma, L., and Huang, J. (2020). Novel ACE Inhibitory Peptides Derived from Simulated Gastrointestinal Digestion in Vitro of Sesame (Sesamum indicum L.) Protein and Molecular Docking Study. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21031059"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1016\/j.foodchem.2009.02.062","article-title":"Angiotensin I-converting enzyme-inhibitory peptide fractions from albumin 1 and globulin as obtained of amaranth grain","volume":"116","year":"2009","journal-title":"Food Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1062","DOI":"10.1016\/j.peptides.2008.02.005","article-title":"The antihypertensive effect of peptides: A novel alternative to drugs?","volume":"29","author":"Hong","year":"2008","journal-title":"Peptides"},{"key":"ref_46","first-page":"239","article-title":"Structure and activity of angiotensin I converting enzyme inhibitory peptides derived from Alaskan pollack skin","volume":"35","author":"Byun","year":"2002","journal-title":"J. Biochem. Mol. Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"5484","DOI":"10.1021\/bi00644a014","article-title":"Design of potent competitive inhibitors of angiotensin-converting enzyme. Carboxyalkanoyl and mercaptoalkanoyl amino acids","volume":"16","author":"Cushman","year":"1977","journal-title":"Biochemistry"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1767","DOI":"10.1007\/s11746-011-1841-9","article-title":"Kinetics of enzyme inhibition and antihypertensive effects of hemp seed (Cannabis sativa L.) protein hydrolysates","volume":"88","author":"Girgih","year":"2011","journal-title":"J. Am. Oil Chem. Soc."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/25\/20\/4635\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:19:36Z","timestamp":1760177976000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/25\/20\/4635"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,10,12]]},"references-count":48,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2020,10]]}},"alternative-id":["molecules25204635"],"URL":"https:\/\/doi.org\/10.3390\/molecules25204635","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,10,12]]}}}