Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.
Department of Chemistry Education, Faculty of Mathematics and Natural Sciences, Yogyakarta State University, Sleman 55281, Indonesia.
Peptides. 2023 Sep;167:171046. doi: 10.1016/j.peptides.2023.171046. Epub 2023 Jun 15.
The pearl garlic (Allium sativum L.) protein (PGP) was digested using pepsin, trypsin, α-chymotrypsin, thermolysin, and simulated gastrointestinal digestion. The α-chymotrypsin hydrolysate showed the highest angiotensin-I-converting enzyme inhibitory (ACEI) activity, with an IC value of 190.9 ± 11 µg/mL. A reversed-phase C solid-phase extraction (RP-SPE) cartridge was used for the first fractionation, and the S4 fraction from RP-SPE showed the most potent ACEI activity (IC =124.1 ± 11 3 µg/mL). The S4 fraction was further fractionated using a hydrophilic interaction liquid chromatography SPE (HILIC-SPE). The H4 fraction from HILIC-SPE showed the highest ACEI activity (IC =57.7 ± 3 µg/mL). Four ACEI peptides (DHSTAVW, KLAKVF, KLSTAASF, and KETPEAHVF) were identified from the H4 fraction using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and their biological activities were appraised in silico. Among the identified α-chymotryptic peptides, DHSTAVW (DW7), derived from I lectin partial protein, exhibited the most potent ACEI activity (IC value of 2.8 ± 0.1 µM). DW7 was resistant to simulated gastrointestinal digestion, and it was classified as a prodrug-type inhibitor according to the preincubation experiment. The inhibition kinetics indicated that DW7 was a competitive inhibitor, which was rationalized by the molecular docking simulation. The quantities of DW7 in 1 mg of hydrolysate, S4 fraction, and H4 fraction were quantified using LC-MS/MS to give 3.1 ± 0.1, 4.2 ± 0.1, and 13.2 ± 0.1 µg, respectively. The amount of DW7 was significantly increased by 4.2-fold compared with the hydrolysate, which suggested that this method is efficient for active peptide screening.
珍珠蒜（Allium sativum L.）蛋白（PGP）经胃蛋白酶、胰蛋白酶、α-糜蛋白酶、耐热蛋白酶和模拟胃肠道消化酶消化。α-糜蛋白酶水解物表现出最高的血管紧张素转化酶抑制（ACEI）活性，IC 值为 190.9 ± 11 µg/mL。反相 C 固相萃取（RP-SPE）柱用于第一次分级，RP-SPE 的 S4 级分显示出最强的 ACEI 活性（IC = 124.1 ± 11 µg/mL）。S4 级分进一步用亲水相互作用液相色谱 SPE（HILIC-SPE）分级。HILIC-SPE 的 H4 级分显示出最高的 ACEI 活性（IC = 57.7 ± 3 µg/mL）。通过液相色谱-串联质谱（LC-MS/MS）从 H4 级分中鉴定出 4 个 ACEI 肽（DHSTAVW、KLAKVF、KLSTAASF 和 KETPEAHVF），并对其进行了计算机评估。在鉴定的 α-糜蛋白酶肽中，来自 I 凝集素部分蛋白的 DHSTAVW（DW7）表现出最强的 ACEI 活性（IC 值为 2.8 ± 0.1 µM）。DW7 抵抗模拟胃肠道消化，根据预孵育实验将其归类为前药型抑制剂。抑制动力学表明 DW7 是一种竞争性抑制剂，这通过分子对接模拟得到了合理化。通过 LC-MS/MS 定量水解物、S4 级分和 H4 级分中 DW7 的含量，分别为 3.1 ± 0.1、4.2 ± 0.1 和 13.2 ± 0.1 µg。与水解物相比，DW7 的含量增加了 4.2 倍，这表明该方法对于筛选活性肽是有效的。
