An in vivo study of the antihypertensive effects of the umami peptide AHSVRFY from Parma ham and its intestinal digestion

Abstract

AHSVRFY, an umami peptide derived from Parma ham with reported in vitro angiotensin-converting enzyme (ACE) inhibitory activity (IC₅₀ = 16.3 ± 0.16 μM), lacks comprehensive in vivo validation. To address this, we established an “digestion–activation–delivery” hypothesis and systematically validated it. In spontaneously hypertensive rats (SHRs), a single oral dose of AHSVRFY induced a significant reduction in systolic blood pressure (SBP) (13.9 ± 2.7 mmHg, p < 0.01). UPLC-MS/MS analysis revealed its degradation into six peptides in the intestine, and four of them were identified as novel ACE inhibitors. The most potent dipeptide, FY (IC₅₀ = 45.11 ± 15.34 μM), was absorbed intact into the systemic circulation of SHRs. Crucially, the antihypertensive effect was mechanistically linked to a significant improvement in vascular endothelial function, as evidenced by increased plasma nitric oxide (NO) and decreased endothelin-1 (ET-1) levels. Furthermore, FY showed a protective effect on human umbilical vein endothelial cells (HUVECs) through the modulation of NO/ET-1. Additionally, its stable binding to the ACE active site was confirmed via molecular docking and dynamics simulations. This work shows that AHSVRFY's antihypertensive effect originates from GI-derived bioactive peptides, establishing a “digestion–activation–delivery” framework for the development of multifunctional food ingredients.

---

1. Introduction

Hypertension, a multifactorial disease, involves the renin–angiotensin system (RAS) and the kallikrein–kinin system (KKS) in blood pressure regulation, with ACE playing an essential role in both systems.¹ ACE has been identified as a key target for the treatment of hypertension. Long-term use of ACE inhibitors such as captopril, lisinopril, and enalapril can lead to side effects including taste disturbance, dry cough, headache, angioedema, and hypotension.² Therefore, bioactive peptides from natural proteins are gaining attention as a potential alternative or adjunct therapy for managing hypertension.³

A substantial body of research indicates that naturally occurring bioactive peptides with ACE inhibitory activity are derived from a wide array of sources, including soybean,⁴ sea cucumber viscera,⁵ Antarctic krill,⁶Sardina pilchardus,⁷Ulva,⁸ saury,⁹ black sesame seeds¹⁰ and Torreya grandis¹¹ among others. These peptides not only exhibited significant antihypertensive effects but also offered advantages such as good food safety, ease of absorption, and low toxicity. Furthermore, the therapeutic potential of bioactive peptides extends beyond hypertension, as evidenced by their remarkable capabilities in promoting angiogenesis and tissue repair, highlighting their multifaceted role in regulating vascular function.^12–14 Additionally, dry-cured ham is recognized as a rich source of various bioactive peptides. Nan et al. identified a novel antioxidant peptide, DWPDARGIWHND, in dry-cured ham,¹⁵ and studies showed that regular consumption can effectively reduce systolic/diastolic blood pressure, contributing to cardiac metabolic health through multiple mechanisms grounded in specific bioactive peptides.¹⁶

Prior to this, our research isolated and characterized the umami peptide AHSVRFY from ham, and the peptide showed a strong saltiness enhancement effect. The use of 0.02% umami peptide in 0.3% NaCl could be increased to 0.4%–0.6% NaCl.¹⁷ At the same time, the peptide exhibited strong ACE inhibitory activity (IC₅₀ = 16.3 ± 0.16 μM) and potent ABTS radical scavenging activity (446.71 ± 32.68 μM Trolox equivalent). In vitro simulated digestion experiments revealed that AHSVRFY had a digestion stability of 1.3%, with its C-terminal Tyr being readily cleaved by enzymes to generate AHSVRF (IC₅₀ = 18.30 ± 0.98 μM), exhibiting competitive inhibition.¹⁸ It is noteworthy that several food-derived ACE inhibitory peptides with even higher potency have been reported, such as the peptide NLFRP (IC₅₀ = 3.33 μM) from quinoa seeds and the peptide IY (IC₅₀ = 0.53 μM) from soybean protein isolates.^19,20 The pursuit of novel ACE inhibitory peptides is driven not only by their potency but also by other advantageous properties, such as unique precursor sources and multi-functionality. In this context, the umami peptide AHSVRFY, isolated from dry-cured ham, presents a compelling case. Although its IC₅₀ value is moderate compared to the most potent peptides, its ACE inhibitory potency is nearly 5 times greater than that of the novel ACE-inhibitory peptide VGLFPSRSF (IC₅₀ = 61.43 μM) from tilapia skin²¹ and 3 times more potent than IIPNEVY and ITPPVMLPP (IC₅₀ values of 57.54 and 40.37 μM, respectively) from green coffee.²² More importantly, it possesses a strong saltiness enhancement effect, offering a dual-functionality approach for developing antihypertensive and salt-reducing functional foods. However, the complex biological factors in real in vivo digestion, including gastrointestinal proteases, plasma peptidase degradation, and intestinal barrier effects, can significantly impact the antihypertensive activity of ACE-inhibitory peptides.²³ While numerous reports exist on novel food-derived ACE inhibitors, there is a significant paucity of information concerning the subsequent metabolic processes of these peptides following their ingestion.

To address this research gap, a novel “digestion–activation–delivery” hypothesis was proposed, positing that the in vivo efficacy of dietary peptides may depend not on the intact parent molecule but on bioactive fragments liberated during digestion. The present study systematically investigated whether the antihypertensive effect of AHSVRFY originates from such gastrointestinal-derived metabolites. The investigation verified the interplay among in vivo antihypertensive efficacy, the generation and systemic absorption of digestive peptides, and their impact on vascular function. This work establishes a “digestion–activation–delivery” framework, underscoring the importance of gastrointestinal metabolism in shaping the bioactivity of food-derived peptides and offering a new paradigm for developing oral peptide therapeutics and functional food ingredients.

2. Materials and methods

2.1 Materials and reagents

ACE (from rabbit lungs), captopril, and hippuric acid (Ha) were obtained from Sigma-Aldrich (St Louis, MO, USA). Hippuryl-histidyl-leucine (HHL) was acquired from Shyuanye Co. Ltd (purity >98%, Shanghai, China). HUVECs, Nexell Cell culture medium, fetal bovine serum (FBS), and penicillin/streptomycin (P/S) were acquired from Yuchun Biology Co. Ltd (Shanghai, China). The NO assay kit was supplied by Share-bio (Shanghai, China). The ET-1 assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Trifluoroacetic acid (TFA), formic acid (FA), and acetonitrile (ACN) were sourced from Thermo Fisher Scientific Co., Ltd (Waltham, MA, USA). All other reagents were of analytical grade.

2.2 Antihypertensive effects on SHRs

In this study, we followed the method described by Gao et al.²⁴ with minor modifications. Twenty-four male SHRs (8 week-old, weighing 230 ± 15 g, specific pathogen-free), characterized by a tail SBP exceeding 160 mmHg, were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd (China). All animal procedures adhered to the guidelines of the Chinese Council on Animal Care and were approved by the Institutional Animal Care and Use Committee of Hangzhou Medical College (approval no. ZJCLA-IACUC-20010014).

The 24 SHRs were randomly divided into four groups, with six rats in each group, and were acclimated for one week under standard laboratory conditions (temperature 22 ± 2 °C, relative humidity 55 ± 10% and 12 h light/12 h dark cycle, with ad libitum access to standard laboratory feed and drinking water). The experimental groups received AHSVRFY peptide solutions at concentrations of 10 and 30 mg kg⁻¹ in physiological saline, while the positive control group was administered captopril at 10 mg kg⁻¹, and the negative control group received an equivalent volume of physiological saline. After drug administration, the SHRs were pre-warmed at 37 °C for 10 min, and their SBP was measured using a Softron BP system (Softron BP-2000, Tokyo, Japan) at 0, 2, 4, 6, and 8 h post dosing. Five readings were taken at each time point, and the average value was recorded for analysis.

2.3 Peptide identification

2.3.1 Collection of intestinal contents and sample preparation. Prior to the experiment, the rats were fasted for 12 h (with free access to water). Twelve rats were randomly divided into two groups. A 10 mg mL⁻¹ solution of the peptide AHSVRFY (dissolved in sterile physiological saline) was administered via oral gavage using a standard stainless steel gavage needle at a dose of 50 mg per kg body weight. The control group received an equal volume of physiological saline. 1 h after administration, the rats were euthanized by intraperitoneal injection of sodium pentobarbital (200 mg kg⁻¹). The entire small intestine was immediately dissected and removed. Intestinal contents were gently flushed with 5 mL of ice-cold phosphate-buffered saline (PBS, pH 7.4) and collected into pre-chilled 15 mL centrifuge tubes. The collected intestinal lavage fluid was mixed with a four-fold volume of ice-cold acetonitrile (1 : 4, v/v) to precipitate proteins and terminate enzymatic activity. The mixture was vortexed vigorously for 3 min, followed by sonication in an ice-water bath for 15 min. Subsequently, it was centrifuged at 12 000g for 20 min at 4 °C. The supernatant was collected, passed through a 0.22 μm microfiber membrane filter, and the filtrate was stored at −80 °C for further analysis.

2.3.2 Peptide identification using UPLC-MS/MS. The intestinal metabolites were analyzed using a method consistent with that established in our previous work.¹⁸ Briefly, separation was performed on an ACQUITY UPLC H-Class system (Waters, Milford, MA, USA) coupled with a G2-XS QTOF mass spectrometer. Chromatography was carried out on a BEH300 C18 column (2.1 × 100 mm, 1.7 μm; Waters, USA) at a flow rate of 0.2 mL min⁻¹ with an injection volume of 10 μL. The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The gradient elution program was applied as follows: 5% B (0 min), 5%–30% B (2–30 min), 30% B (30–40 min), 30%–80% B (40–45 min), 80% B (45–55 min), 80%–5% B (55–60 min), and 5% B (60–70 min).

Mass spectrometric detection was operated in the positive ionization mode with data-dependent acquisition. Key source parameters included a capillary voltage of 3.0 kV, source temperature of 120 °C, and desolvation temperature of 350 °C. Nitrogen and argon were used as the cone and collision gases, respectively. Spectra were acquired over m/z 100–1000 with a scan time of 0.2 s and an inter-scan delay of 0.1 s, using a collision energy ramp of 15–30 V.

Data acquisition and processing were conducted using MassLynx v4.1 (Waters Corp.). Peptide identification was performed via de novo sequencing using the PepSeq tool within Biolynx software (Waters Corp.), with a parent ion tolerance of 5 ppm and a fragment ion tolerance of 0.02 Da.

2.4 Systemic exposure and vascular effects of FY in SHRs

After 12 SHRs were acclimated for one week under standard feeding conditions, they were fasted for 24 h with free access to water. Then, these 12 SHRs were randomly divided into two groups. The drug group received a single oral administration of 100 mg kg⁻¹ of the heptapeptide AHSVRFY dissolved in normal saline, whereas the control group was given the same volume of normal saline. Blood samples (500 μL) were collected via the retro-orbital plexus at pre-dose (0 h) and 0.5, 1, and 2 h post administration. At the end of the experiment, all rats were euthanized by an intraperitoneal injection of sodium pentobarbital (200 mg kg⁻¹). Whole blood was immediately transferred to heparin sodium-coated tubes pre-treated with a protease inhibitor cocktail (1 : 100 v/v, containing EDTA, aprotinin, and leupeptin) and maintained on ice. Plasma was separated by centrifugation at 3000 rpm for 10 min at 4 °C. The supernatant was mixed with 0.1% formic acid–acetonitrile solution (1 : 3 v/v) for protein precipitation during a 10 min ice bath, followed by centrifugation at 12 000 rpm for 15 min (4 °C). The resulting supernatant was aliquoted into 100 μL portions and stored at −80 °C until analysis. The analytical procedure was performed using UPLC-MS/MS, with chromatographic and mass spectrometric parameters identical to those described in section 2.3.2. The levels of NO and ET-1 in the plasma were determined using specific detection kits.

2.5 Peptide synthesis

The sequences of all the identified peptides were submitted to Apeptide Co., Ltd (Shanghai, China). These peptides were synthesized using solid-phase peptide synthesis methods, with a purity exceeding 98%; the purity reports are provided in SI S1.

2.6 In silico analysis of the identified peptides

Innovagen (https://www.innovagen.com/) was used to predict the peptides’ isoelectric point and net charge. Additionally, ADMETlab 3.0 (https://admetlab3.scbdd.com) was utilized for the predictive analysis of the peptides’ pharmacokinetic properties as well as the absorption, distribution, metabolism, and excretion characteristics.²⁵

2.7 Determination of ACE inhibitory activity

The inhibitory activity of the peptides against ACE was determined using a method adapted from Wu et al.²⁶ Peptides were dissolved in distilled water and mixed with HHL and incubated at 37 °C for 5 min. ACE was then added and the mixture was incubated for 60 min. The reaction was stopped with HCl, diluted with water, filtered, and analyzed using an Agilent 1260 HPLC system with a CAPCELL PAK C18 column.

The samples were eluted with mobile phase A (water containing 0.1% TFA) and mobile phase B (ACN containing 0.1% TFA). Captopril (10 ng ml⁻¹) was used as the positive control. The inhibition rate of ACE was calculated as follows:

where Ha_negative, Ha_sample, and Ha_blank are the relative areas of the Ha peak.

2.8 Effects of AHSVRFY on HUVECs

2.8.1 Cell culture. Primary HUVECs were seeded in an endothelial cell complete medium (supplemented with 5% FBS, 1% endothelial cell growth supplement, 1% streptomycin, and 1% penicillin solution). The cells were cultured in a humidified incubator at 37 °C with 5% carbon dioxide.

2.8.2 Determination of cell viability. The viability of HUVECs treated with ACE inhibitory peptides (HSVRF, SVRFY, and FY) or captopril for 24 and 48 hours was assessed using a MTT assay. Cells were seeded in 96-well plates at a density of 5 × 10³ cells per well. After treatment, MTT (5 mg mL⁻¹) was added to each well and incubated for 4 hours. The resulting formazan crystals were dissolved using DMSO, and the absorbance was measured at 490 nm. Cell viability was calculated as a percentage relative to that of the untreated control group.²⁷

2.8.3 Determination of NO and ET-1 contents. Peptide concentrations were selected based on preliminary cytotoxicity assays (MTT, >90% cell viability at 100 μg mL⁻¹). Cells were seeded in 24-well plates at 1 × 10⁵ cells per well and exposed to peptides at 40 and 80 μg mL⁻¹ for 24 h. A control group with only the medium and cells was included. The cell culture medium was collected and NO and ET-1 levels were measured using specific assay kits.

2.9 Molecular docking peptides with ACE

The human ACE structure (PDB ID: 1O8A) with a resolution of 2.00 Å was obtained from the RCSB Protein Data Bank (https://www.rcsb.org), and all water molecules and co-crystallized compounds were removed before hydrogen atoms were added to the protein. The 3D structures of the peptides were constructed using the RDKit library in Python, and energy minimization was performed using the MMFF94 force field. Molecular docking between the target peptides and ACE was conducted using AutoDock Vina 1.2.5, with the active site coordinates defined as X: 43.817, Y: 38.308, and Z: 46.652. The best docking poses of the peptides within the ACE active site were ranked based on the scores and binding energy values obtained from the docking analysis.²⁸

2.10 Molecular dynamics simulation

The molecular dynamics simulations were conducted using GROMACS 2020.06 with protocol modifications adapted from Dang et al.²⁹ The optimal FY–ACE complex conformation derived from molecular docking was parameterized with the CHARMM36 force field and solvated in a TIP3P water-filled truncated octahedron box. To neutralize system charge, Na⁺ and Cl⁻ counterions were added prior to energy minimization through the steepest descent algorithm (5000 steps), resolving steric clashes and optimizing the molecular geometry. Subsequent system equilibration employed the V-rescale thermostat (NVT ensemble, 300 K, 100 ps) and Berendsen barostat (NPT ensemble, 1 bar, 100 ps). Production simulations were then performed for 100 ns under periodic boundary conditions. Throughout simulations, hydrogen bond constraints were implemented via the LINCS algorithm, while long-range electrostatic interactions were calculated using the Particle Mesh Ewald (PME) method with a 1.2 nm cutoff. Post-simulation analyses including trajectory processing and visualization were executed using GROMACS utilities and VMD 1.9.3.

2.11 Statistical analysis

All experiments were performed in triplicate. Data are presented as mean ± standard deviation and were analyzed using one-way analysis of variance (ANOVA) with SPSS Statistics 27 (IBM), and the significance of differences in the data was determined using Duncan's test with a 95% confidence interval. For a visual representation of the results, Origin 2022 was employed. IC₅₀ values were determined by fitting dose–response curves using the Logistic function within the Basic Functions category of Origin software.

3. Results and discussion

3.1 Antihypertensive effects of AHSVRFY on SHRs

SHRs are recognized internationally as the most representative animal model for human essential hypertension with blood pressure exceeding 160 mmHg, and are widely used for evaluating antihypertensive effects.³⁰ To minimize the impact of initial SBP variations among the experimental rats on the results, we analyzed the changes in SBP rather than absolute values.

As shown in Fig. 1, there were significant changes in SBP within the treatment and positive control groups over the 0–8 h period. In SHRs treated with captopril for 2 h, SBP rapidly decreased, showing a highly significant difference from the control group, with a reduction of 13.4 ± 1.9 mmHg (p < 0.01). The SBP continued to decrease from 2 to 6 h, reaching its maximum antihypertensive effect at 6 h, where SBP was reduced by 23.0 ± 4.2 mmHg (p < 0.01). After 6 h, SBP began to recover. In the treatment group, the SBP of 10 mg kg⁻¹ and 30 mg kg⁻¹ groups decreased rapidly within 4 h after administration, with a decrease of 10.5 ± 1.9 and 10.8 ± 2.1 (p < 0.01), respectively. Throughout the recorded experimental period of 0–8 h post administration, the SBP of the treated groups consistently decreased and remained lower than that of the control group, indicating that the antihypertensive effect of the peptide AHSVRFY in SHRs is genuine and effective. Similarly, a daily dose of 97.2 mg of the peptide AHSVRFY would be sufficient for a 60 kg adult to achieve a comparable antihypertensive effect. This peptide can be obtained from Parma ham through a water-extraction process.

Fig. 1 The effects of normal saline (10 mg kg⁻¹), captopril (10 mg kg⁻¹), AHSVRFY (10 mg kg⁻¹), and AHSVRFY (30 mg kg⁻¹) on SBP of SHRs. The control group was compared with other groups. All data are presented as the mean ± SD of 6 results. * indicates p < 0.05 and ** indicates p < 0.01.

As expected, AHSVRFY alleviated hypertension in SHRs. Similarly, the peptide IIVFGRQLL derived from yeast extract significantly lowered SHRs’ blood pressure at a dose of 50 mg kg⁻¹.³¹ The experimental results indicated that AHSVRFY exhibited excellent antihypertensive effects in SHRs, with a recovery rate notably lower than that of captopril, suggesting a gentler and more sustained action. This phenomenon may be attributed to the degradation of AHSVRFY into shorter peptides with ACE inhibitory activity, which were absorbed at varying rates in the body, leading to a mild and prolonged antihypertensive effect. Such a relatively gentle mode of action could be more beneficial for the treatment of cardiovascular diseases.³² AHSVRFY, as a novel ACE-inhibitory peptide, not only exhibited superior ACE inhibitory activity in vitro but also showed effective antihypertensive action in vivo, encouraging further in-depth research into its potential therapeutic applications.

3.2 AHSVRFY's gastrointestinal digestion in vivo

Through our previous study, AHSVRFY has been confirmed to possess potent ACE inhibitory activity both in vitro and in vivo.¹⁸ For bioactive peptides to exert their effects, they must survive gastrointestinal (GI) digestion and maintain their structure and activity upon reaching their target.³³ However, AHSVRFY showed only 1.3% digestive stability in simulated GI digestion experiments, indicating that it does not reliably enter the bloodstream to elicit antihypertensive effects.¹⁸ To investigate the dynamic changes of AHSVRFY's antihypertensive action, we performed dissections on rats post administration of AHSVRFY and used non-administered rats as controls. Six peptides were identified in the intestinal contents by UPLC-MS/MS, and physicochemical and ADMET properties were predicted by silico analysis.

As shown in Table 1, following in vivo digestion, these peptides AHSVRFY, AHSVRF, HSVRF, SVRFY, SVRF and FY were found in relatively high abundances. In vitro, simulated digestion revealed that the peptide AHSVRFY was enzymatically cleaved into fragments AHSV and AHSVRF. Due to the complex biological environment within the gastrointestinal tract, the fragment AHSV was not detected in the intestinal contents. CYP3A4 is one of the most prominent isoforms within the cytochrome P-450 (CYP) enzyme system, playing a critical role in drug metabolism. These identified peptides exhibited no inhibitory effect on CYP3A4 activity. The results indicated that these peptides did not lead to any harmful accumulation in vivo. Additionally, these peptides were evaluated for their interaction with P-glycoprotein (P-gp), Madin–Darby Canine Kidney (MDCK) permeability, human Caco-2 cell permeability, and human intestinal absorption (HIA). Due to the large molecular weight of polypeptides, the human intestinal absorption rate was low, but its powerful antihypertensive function could not be denied.³⁴

Table 1 The HPLC-MS detection results of the gut contents and the prediction of the physicochemical and ADMET properties of the detected peptides

		AHSVRFY	AHSVRF	HSVRF	SVRFY	SVRF	FY
MW: molecular weight; HIA: human intestinal absorption capacity, classified as high (HIA^+) or low (HIA^−) absorption based on clinical absorption thresholds; BBB: blood–brain barrier permeability, dichotomized into penetrable (BBB+) and non-penetrable (BBB−) categories; PPB: plasma protein binding affinity; CYP3A4: cytochrome P450 3A4 metabolic interactions (non-substrate/non-inhibitor status); Pgp-substrate: P-glycoprotein transport activity, with MDCK permeability values >−5.15, indicating favorable transmembrane absorption potential.
Mass	Observed (m/z)	879.45	716.38	645.35	671.35	508.28	329.15
	RT (min)	5.52	4.16	3.96	6.56	5.32	4.98
	Height	4850	27 300	3480	20 300	34 700	6260
Physicochemical properties	MW (Da)	878.97	715.8	644.72	670.76	507.58	328.36
	Iso-electric point	9.57	10.59	10.59	9.57	10.59	3.41
	Net charge at pH 7	1.1	1.1	1.1	1	1	0
	Estimated solubility	Poor	Good	Good	Poor	Good	Poor
ADMET	BBB	0	0	0	0	0	0.003
	PPB	44.70%	33%	35.60%	42.20%	29.30%	61.10%
	CYP3A4 inhibitor	0	0	0	0	0	0.817
	CYP3A4 substrate	0	0	0.007	0.002	0.171	0
	Lipinski's rule	Rejected	Rejected	Rejected	Rejected	Rejected	Accepted
	Pfizer's rule	Accepted	Accepted	Accepted	Accepted	Accepted	Accepted
	Caco-2 permeability	−5.969	−5.853	−6.18	−6.45	−6.332	−6.325
	MDCK permeability	−5.375	−5.341	−5.283	−5.141	−5.153	−5.005
	Pgp inhibitor	0.151	0.051	0.001	0.011	0.003	0.001
	HIA	0.983	0.971	0.87	0.782	0.69	0.001

---

The mass spectra of these identified peptides are shown in Fig. 2; these six peptides reflected the synergistic action of various proteases in the digestive system.^35,36 Trypsin, which typically cleaves at the C-terminal side of lysine (Lys, K) and arginine (Arg, R) residues,³⁷ is likely responsible for the generation of the digested peptide fragment FY. Chymotrypsin, one of the principal proteases, preferentially cleaved at the carboxyl-terminal side of aromatic amino acids such as phenylalanine (F) and tyrosine (Y) of AHSVRFY,³⁸ leading to the formation of AHSVRF and FY. Subsequently, elastase may have participated in the cleavage at the carboxyl-terminal side of glycine (G),³⁹ further degrading AHSVRF into HSVRF and A. Additionally, chymotrypsin continued to act on the carboxyl-terminal side of nonpolar serine (S),⁴⁰ generating SVRFY and SVRF. This outcome illustrated the partial digestion of AHSVRFY by proteases in the GI tract of SHRs, resulting in multiple short peptide fragments. The existence of these short peptides suggested that AHSVRFY, along with its degradation products, may exhibit a variety of different biological effects within the intestine. Further research could explore the specific functions of these short peptides in vivo, providing deeper insights into their potential therapeutic applications.

Fig. 2 MS spectra of the peptide AHSVRFY after digestion. (A) AHSVRFY, (B) AHSVRF, (C) HSVRF, (D) SVRFY, (E) SVRF and (F) FY.

3.3 ACE inhibitory activities of the peptides

As shown in Fig. 3, three peptides (AHSVRF, HSVRF, and SVRFY) derived from gastrointestinal digestion exhibited potential ACE inhibitory activities, corresponding to the IC₅₀ values of 82.69 ± 5.33 μM, 113.73 ± 2.53 μM, and 92.25 ± 30.11 μM, respectively. Specially, the IC₅₀ values of the peptide FY were the lowest (IC₅₀ = 45.11 ± 15.34 μM). Research has indicated that the peptides with a low molecular weight are more likely to exhibit high ACE inhibitory activity, possibly due to steric hindrance preventing larger peptides from accessing the active site of ACE.⁴¹

Fig. 3 Fitting curve of the IC₅₀ value for the ACE-inhibitory peptides. (A) AHSVRF. (B) HSVRF. (C) SVRFY. (D) FY.

Earlier studies have shown that ACE-inhibitory peptides typically consist of nonpolar, hydrophobic amino acids such as Ala, Val, Phe, Trp, Met, Leu, and Ile, which facilitate the inhibition of ACE activity. Hydrophobic amino acids tend to be preferentially located at either the C- or N-terminal ends of peptides with high ACE inhibitory activity.⁴² The hydrophobic amino acid residues of Ala, Val, and Phe within the four peptide sequences conferred a potent ACE inhibitory activity for these peptides. Furthermore, those peptides with aromatic amino acids (Trp, Tyr, and Phe) at the C-terminal and hydrophobic amino acids at the N-terminal tend to exhibit higher ACE inhibitory activity.⁴¹ Thus, the high ACE inhibitory activity of the four peptides was primarily attributed to the Phe and Tyr residues at their C-terminal.

3.4 Effect of AHSVRFY on plasma NO and ET-1 levels in SHRs

Hypertension is closely associated with vascular endothelial cell (VEC) impairment, which is characterized by the dysregulation of key vasoactive factors such as ET-1 and NO.⁴³ ET-1 functions as a potent vasoconstrictor that contributes to vascular dysfunction, whereas NO serves as the primary vasodilator that regulates vascular tone and blood flow.^44,45 Importantly, ACE inhibitors exert antihypertensive effects partly through endothelium-dependent vasodilation. This is achieved by modulating these critical factors: suppressing ET-1 secretion and enhancing NO production in endothelial cells to restore vascular homeostasis.⁴⁶ Thus, the mechanism of antihypertensive drugs can be explained by the trends of NO and ET-1 in blood vessels.

The results showed that the administration of AHSVRFY induced significant and favorable changes in these key vasoactive factors. As shown in Fig. 4, the plasma NO concentration in the AHSVRFY-treated group increased rapidly and significantly, increasing from a baseline of 3.02 ± 0.26 μmol L⁻¹ to a peak of 14.72 ± 0.83 μmol L⁻¹ within 2 hours after administration (p < 0.001). This represents an approximately 4.9-fold increase in the circulating level of this crucial vasodilator. Conversely, the plasma concentration of the potent vasoconstrictor ET-1 decreased markedly in the treatment group, decreasing from 67.3 ± 2.25 pg mL⁻¹ to 54.75 ± 1.29 pg mL⁻¹ over the same period (p < 0.01). In sharp contrast, both NO and ET-1 levels remained stable and showed no significant changes in the saline control group throughout the experiment.

Fig. 4 Effects of AHSVRFY on the plasma levels of NO and ET-1 in SHRs.

The simultaneous sharp increase in NO and significant decrease in ET-1, observed only in the treated SHRs, provides compelling in vivo evidence that the bioactive peptide metabolites act to restore endothelial homeostasis. The surge in NO suggests a potent activation of the vasodilatory pathway by this vasoactive component, while the reduction in ET-1 indicates a successful mitigation of the vasoconstrictive tone typical of hypertension. This coordinated rebalancing of opposing vascular regulators constitutes a powerful mechanistic explanation for the observed blood-pressure-lowering effect and shows that the active principle derived from AHSVRHY digestion exerts its action through endothelium-dependent mechanisms.

3.5 Protective effects of the peptides on HUVECs

The observed restoration of the NO/ET-1 balance in SHR plasma strongly suggests a protective effect on the vascular endothelium. To investigate whether this effect is directly mediated by the bioactive peptide metabolites on endothelial cells, we subsequently employed HUVECs for further exploration.

3.5.1 Effects of HSVRF, SVRFY, and FY on HUVEC viability. The cytotoxicity of the peptides HSVRF, SVRFY, and FY in HUVECs was assessed via the MTT assay, as shown in Fig. 5(A). All three peptides exhibited excellent biocompatibility, with no significant cytotoxicity observed at 100 μg mL⁻¹ after 24 h of treatment. Extending the incubation period to 48 h or increasing the concentration to 200 μg mL⁻¹ resulted in a gradual decline in cell viability; nevertheless, the values remained above 80%, a finding that meets the cytotoxicity threshold criteria specified in ISO 10993-5.⁴⁷

Fig. 5 (A) Effects of ACE inhibitory peptides (HSVRF, SVRFY and FY) and captopril on the viability of HUVECs (n = 6). (B) Effects of the peptides on the production of ET-1 in the HUVECs. (C) Effects of the peptides on the production of NO in the HUVECs. The cell group treated with 10 μM captopril was designed as a positive control.

3.5.2 Evaluation of the ET-1 level. The ET-1 content in HUVECs was measured after 24 h, and the results are presented in Fig. 5(B). Compared to the control group (257.18 ± 8.23 pg mL⁻¹), the captopril group exhibited a significantly lower ET-1 level (221.50 ± 21.77 pg mL⁻¹) (p < 0.05). At concentrations of 40 μg mL⁻¹ and 80 μg mL⁻¹, the peptides HSVRF, SVRFY, and FY all significantly decreased the cellular production of ET-1 (p < 0.01) in a concentration-dependent manner. In particular, HSVRF displayed the strongest inhibitory effect on the ET-1 level, which reduced 33.16% and 35.44%, respectively, compared with the control group. Notably, the inhibitory effect of HSVRF on ET-1 was significantly superior to that of AHSVRF from our previous studies.¹⁸ The ET-1-lowering capabilities of SVRFY and FY were comparable to those of captopril, where the ET-1 levels in the SVRFY (40 μg mL⁻¹), SVRFY (80 μg mL⁻¹), FY (40 μg mL⁻¹), and FY (80 μg mL⁻¹) groups were 76.11%, 74.26%, 83.83%, and 75.53% compared with the control group, respectively. These results suggest that these four peptides contributed to the regulation of vascular dysfunction associated with cardiovascular disease.

3.5.3 Evaluation of the NO level. NO production in HUVECs following treatment with the three peptides was evaluated and is presented in Fig. 5(C). All the treated groups showed significantly increased NO levels (p < 0.01) compared with the control group. Similar to AHSVRF from our previous studies,¹⁸ HSVRF, SVRFY, and FY showed a concentration-dependent increase in the NO level. Notably, the increase in the NO level for HSVRF exceeded that for AHSVRF from our previous studies. At concentrations of 40 μg mL⁻¹ and 80 μg mL⁻¹, the levels of NO for HSVRF were 168.51% and 188.93% compared with the control group, respectively. The abilities of SVRFY and FY to increase the NO level were comparable to that of captopril. At the same concentration, the levels of NO in the SVRFY and FY groups were 127.68%, 156.40%, 129.07%, and 162.63% compared with the control group, respectively. It was evident that AHSVRF, HSVRF, SVRFY, and FY significantly reduced the cellular ET-1 production while concurrently increasing the NO level. These results indicated that the four peptides generated from AHSVRFY after in vivo digestion not only contributed to the protection of the vascular endothelial function but also exhibited antihypertensive effects comparable to those of captopril.

The consistent up-regulation of NO and concomitant down-regulation of ET-1 observed in HUVECs directly recapitulated the favorable shift detected in the plasma of AHSVRFY-treated SHRs. This high degree of concordance between the isolated cell system and the complex mammalian model shows that the endothelial protective effects of the digested peptides are not merely cell culture artifacts but are translationally relevant. Therefore, the HUVEC model proved to be a robust and predictive system for identifying peptides with genuine vasoprotective efficacy, as was ultimately confirmed within the pathophysiological context of hypertension in SHRs.

3.6 Molecular docking of ACE and the peptides

Molecular docking is an effective analytical tool for studying ligand–protein interactions to understand structure–activity relationships.⁴⁸ The interactions between ligands and receptors include hydrophobic interactions, van der Waals forces, hydrogen bonds, π–π interactions, and electrostatic interactions, with hydrogen bonds being the strongest non-covalent interaction between proteins and ligands.⁴⁹ The binding energies and hydrogen bond formation sites between the peptides and ACE are summarized in Table 2. Generally, if the binding energy is less than −5.0 kcal mol⁻¹, it suggests strong affinity between the active compound and the target site.⁵⁰ In our study, the peptides AHSVRF (−10.5 kcal mol⁻¹), HSVRF (−10.1 kcal mol⁻¹), and SVRFY (−9.7 kcal mol⁻¹) exhibited lower binding energies compared to that of the reported captopril (−8.72 kcal mol⁻¹).²⁵ However, FY (−7.7 kcal mol⁻¹) showed a similar binding energy to captopril, indicating that all four peptides formed stable complexes with ACE.

Table 2 The residues that formed hydrogen bonds with the peptides

Ligand	Binding energy (kcal mol^−1)	Residues that formed hydrogen bonds with the ligand
AHSVRF	−10.5	Gln281, His353, Ala354, Arg402, His410, Lys511, His513
HSVRF	−10.1	Glu162, Gln281, His353, Ala354, Ala356, His387, Lys511, His513, Arg522
SVRFY	−9.7	Glu162, Gln281, His353, Ala354, Ala356, His387, Glu411, Lys511, His513
FY	−7.7	Gln281, Asp415, Tyr520

---

It is important to note that the ACE inhibitory activity of these inhibitors not only depends on binding energy but also the critical binding sites within the ACE active site. Research has shown that the S1, S1′, and S2′ pockets constitute the main binding site of ACE. The S1′ pocket contains only the Glu162 residue, which is the most crucial for peptide–ACE interactions. The S1 pocket includes residues Ala354, Glu384, and Tyr523, while the S2′ pocket comprises Gln281, His353, Lys511, His513, and Tyr520.⁵¹ The three-dimensional binding modes and intermolecular interactions of the peptides with ACE are depicted in Fig. 6. Notably, hydrogen bonds were formed between all amino acid residues in the S2′ pocket of ACE and the peptides AHSVRF, HSVRF, and SVRFY. The peptide FY formed hydrogen bonds with three amino acid residues of ACE, two of which were located within the S2′ pocket (Gln281 and Tyr520). These interactions suggested that AHSVRFY digestion products could alter the conformation of ACE by binding to the S2′ pocket, thereby inhibiting ACE activity. Our results were consistent with previous studies.⁵² Additionally, both HSVRF and SVRFY formed hydrogen bonds with the Glu162 residue in the S1′ pocket, contributing to their lower binding energies. The molecular docking results elucidated that the hydrogen bond was the primary driving force between the peptides and partial amino acid residues in the S1′ and S2′ pockets, thus conferring high ACE inhibitory activity to these peptides. ACE can effectively regulate blood pressure in both RAS and KKS.¹ Thus, these four peptides with high ACE inhibitory activity exerted strong antihypertensive activity.

Fig. 6 Three-dimensional and two-dimensional diagrams of AHSVRF (A), HSVRF (B), SVRFY (C) and FY (D) docking with ACE (PDB: 1O8A).

3.7 Molecular dynamics simulation of the FY–ACE interaction

MD simulations enable the construction of protein–solvent models that closely mimic physiological conditions. This computational approach facilitates the investigation of relationships between dynamically generated experimental data and static molecular conformations while providing critical insights into structural transitions that remain experimentally undetectable.⁴⁸ To comprehensively evaluate the binding stability of the digestive peptides with ACE and elucidate its interaction mechanisms, we selected this top-performing peptide FY identified from in vitro assays for MD simulations. A ligand-free ACE system served as the control. Two independent systems were constructed, and molecular dynamics simulations were performed for 100 ns using GROMACS. Trajectory files obtained from the simulations were subsequently analyzed for multiple structural parameters: root-mean-square deviation (RMSD) to assess global conformational stability, root mean square fluctuation (RMSF) for residue-specific flexibility, radius of gyration (Rg) measuring molecular compactness, solvent accessible surface area (SASA) evaluating surface hydrophobicity, and time-dependent hydrogen bond formation patterns.

RMSD analysis (Fig. 7A) showed that the ACE–FY complex exhibited minor fluctuations between 20 and 60 ns and stabilized at 0.38 nm after 80 ns, reflecting gradual conformational equilibration. In contrast, the ligand-free ACE system stabilized at an RMSD of 0.47 nm during 80–100 ns, indicating structural loosening due to ligand absence.⁵³ As shown in Fig. 7(B), the RMSD of the ligand-free ACE post stabilization was significantly higher than that of the complex, confirming that the presence of the FY ligand enhances ACE structural stability. RMSF analysis (Fig. 7C) further revealed reduced fluctuation values for key residues in the ACE active center (Gln281, Asp415 and Tyr520) within the complex, consistent with molecular docking results. Additionally, the FY peptide reinforced structural rigidity by suppressing localized motions in the catalytic zinc-coordination region and substrate-binding loop (residues 150–200). Conversely, the C-terminal domain (residues 500–600) of the ligand-free system displayed higher flexibility.⁵⁴

Fig. 7 Molecular dynamics simulation of the ACE–FY complex. Representative structural snapshots of the complex at (A1) 0 ns and (A2) 100 ns. (B) RMSD of ligand-free ACE and the ACE–FY complex (0–100 ns). (C) RMSD during the stable phase (80–100 ns). (D) RMSF of ligand-free ACE and the ACE–FY complex (80–100 ns). (E) SASA. (F) Rg. (G) Hydrogen bonds between ACE and FY.

Rg and SASA analyses (Fig. 7D and E) further corroborate the aforementioned conclusions: the Rg of the complex stabilized within 2.40–2.45 nm, and the SASA remained at 260–270 nm², indicating a compact overall structure with limited exposure of hydrophobic cores. In contrast, the Rg of the ligand-free ACE stabilized at 2.50 nm during 80–100 ns, accompanied by an elevated SASA, attributed to structural unfolding and reduced stability.⁵⁵ Hydrogen bond interaction analysis (Fig. 7F) revealed that 6–8 persistent hydrogen bonds (for example, Tyr-Glu384) formed between the FY peptide and ACE, serving as the primary driving force for complex stability.

In summary, the FY peptide significantly enhances the dynamic stability of the ACE complex by restricting flexibility in the active center, maintaining structural compactness, and forming a stable hydrogen bond network. These findings provide dynamic structural evidence supporting the molecular mechanism of the FY peptide as a potent ACE inhibitor.

3.8 Exposure–response relationship of the ACE inhibitory peptide FY

To confirm the in vivo absorption and systemic exposure of the bioactive peptides resulting from the oral bioactivation of AHSVRFY, we performed UPLC-MS/MS analysis on plasma samples from SHRs (Fig. 8A: MS/MS). The key dipeptide metabolite, FY, was successfully identified. The chromatographic response of FY exhibited a time-dependent escalation (Fig. 8B), with peak areas progressing from 24 016 at 30 min to 81 733 at 60 min and 140 950 at 120 min, indicative of sustained systemic bioavailability. This ascending trajectory starkly contrasts with the rapid elimination kinetics characteristic of conventional small peptides, underscoring FY's unique pharmacokinetic behavior. To further confirm the stability of FY throughout the digestive process, an in vitro simulated digestion assay was performed according to the method established by Brodkorb et al.⁵⁶ As summarized in Table 3, the results showed that FY exhibited a high digestion stability of 98.5%, confirming its robustness under simulated gastrointestinal conditions. The corresponding chromatographic and mass spectrometric profiles are provided in SI S2.

Fig. 8 (A) MS/MS spectra of FY. (B) Peak area results of FY at four time points (0, 30, 60, and 120 min).

Table 3 Stability of the peptide FY during in vitro simulated gastrointestinal digestion (n = 3)

	References	Samples
Area (×10^9)	1.36 ± 0.01	1.35 ± 0.01

---

The observed plasma accumulation aligns with FY's previously established dose-dependent ACE inhibition, exhibiting potent activity with an IC₅₀ of 45.11 ± 15.34 μM. The delayed clearance kinetics may be attributed to enterohepatic recirculation or plasma protein binding, as documented for structurally analogous ACE inhibitors.⁵⁷ Such mechanisms are concordant with FY's inherent structural stability, which is likely mediated by the aromatic Phe–Tyr motif, a feature known to confer proteolytic resistance and prolong circulatory half-life.⁵⁸

Molecular docking simulations further validated FY's robust interaction with the S2′ pocket of ACE, a critical catalytic domain for angiotensin I conversion. This binding affinity, coupled with FY's resistance to enzymatic degradation, substantiates its potential for sustained therapeutic action. While the sampling regimen was restricted to three intervals, the data unequivocally show FY's capacity to attain and sustain bioactive plasma concentrations, addressing a pivotal challenge in peptide-based antihypertensive development.

4. Conclusion

This study establishes that the antihypertensive efficacy of the umami peptide AHSVRFY is fundamentally governed by its gastrointestinal bioactivation into smaller, systemically available fragments. The dipeptide FY emerged as the most potent ACE-inhibitory metabolite, whose absorption into circulation and subsequent modulation of vascular function—evidenced by elevated NO and suppressed ET-1 in SHRs—directly links its bioavailability to the observed physiological effects. Molecular simulations further delineate that FY stabilizes within the ACE active site via specific hydrogen bonds with Gln281 and His513, providing a structural basis for its inhibitory activity. Collectively, these findings position FY not merely as a digestive metabolite, but as the key effector mediating the parent peptide's in vivo action. By shifting the focus from intact peptide delivery to the liberation, absorption, and activity of bioactive fragments, this work establishes a “digestion–activation–delivery” framework with broad implications for the rational design of oral peptide therapeutics and functional foods.

Author contributions

Qian Ding: methodology, investigation, data curation, and writing – original draft. Tianyu Wang: methodology, investigation, and writing – original draft. Kuo Dang: software, methodology and data curation. Yanli Wang: formal analysis and review & editing. Daodong Pan: resources and supervision. Qiang Xia: review & editing. Xinchang Gao: conceptualization, supervision and project administration. Yali Dang: funding acquisition, supervision and project administration.

Conflicts of interest

There are no conflicts to declare.

Data availability

Data supporting this article are available within the manuscript and its Supplementary Information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5fo03304g.

Acknowledgements

This work was supported by the “Department of Agriculture and Rural Affairs of Zhejiang Province” (2025SNJF038), the “Science and Technology Department of Zhejiang Province” (2024C01248(SD2)), the “Ningbo Top Talent Project” (215-432094250), the “Innovation Yongjiang 2035 Key R&D Programme-International Sci-tech Cooperation Projects” (2024H001) and the “Quzhou City Science and Technology Plan Project” (2023K100).

References

1. Z. Lin, H. Wu and M. Zhang, Isolation, identification, and structure-activity relationship of novel ACE inhibitory peptides from earthworm protein in vitro gastrointestinal digestion product, Food Biosci., 2023, 55, 103010.
2. S. Y. Lee and S. J. Hur, Antihypertensive peptides from animal products, marine organisms, and plants, Food Chem., 2017, 228, 506–517.
3. Q. Zhou, D. Liao, L. Wang, L. Sun, Z. Tong, J. Sun, X. Lan and G. Zhou, Biophysical and simulation analysis of ACE tripeptide inhibitors derived from milk, Food Chem., 2025, 145633.
4. S. Tang, D. Chen, H. Shen, Z. Yuan, H. Wei, Y. Feng, L. Li, J. Dong and L. Zhang, Discovery of two novel ACE inhibitory peptides from soybeans: Stability, molecular interactions, and in vivo antihypertensive effects, Int. J. Biol. Macromol., 2025, 308, 142247.
5. Q. Liang, W. Zhou, S. Peng, Z. Liang, Z. Liu, L. Wang, H. Mukhtar and H. Mou, Exploration of ACE inhibitory peptides from sea cucumber viscera with DPP-IV inhibitory activity: Virtual screening, characterization, and potential mechanism investigation, Int. J. Biol. Macromol., 2025, 143843.
6. W.-Y. Zhu, Y.-M. Wang, X.-M. Dong, G.-X. Zhao, C.-F. Chi and B. Wang, Antioxidant peptides from Antarctic krill (Euphausia superba) hydrolysate: Stability, ACE inhibitory activity, and endothelial cells protection by regulating Keap1/Nrf2 pathway, J. Agric. Food Res., 2025, 20, 101745.
7. M. Shao, H. Wu, B. Wang, X. Zhang, X. Gao, M. Jiang, R. Su and X. Shen, Identification and characterization of novel ACE inhibitory and antioxidant peptides from Sardina pilchardus hydrolysate, Foods, 2023, 12, 2216.
8. Z. Li, Y. He, H. He, W. Zhou, M. Li, A. Lu, T. Che and S. Shen, Purification identification and function analysis of ACE inhibitory peptide from Ulva prolifera protein, Food Chem., 2023, 401, 134127.
9. S. Wang, L. Zhang, H. Wang, Z. Hu, X. Xie, H. Chen and Z. Tu, Identification of novel angiotensin converting enzyme (ACE) inhibitory peptides from Pacific saury: In vivo antihypertensive effect and transport route, Int. J. Biol. Macromol., 2024, 254, 127196.
10. T. Du, Y. Xu, X. Xu, S. Xiong, L. Zhang, B. Dong, J. Huang, T. Huang, M. Xiao and T. Xiong, ACE inhibitory peptides from enzymatic hydrolysate of fermented black sesame seed: Random forest-based optimization, screening, and molecular docking analysis, Food Chem., 2024, 437, 137921.
11. F. Wu, X. Luo, Y. Zhang, P. Wang, Y. Chang, Z. He and X. Liu, Purification, identification, and inhibitory mechanisms of a novel ACE inhibitory peptide from Torreya grandis, Nutrients, 2023, 15, 2374.
12. A. Adak, V. Castelletto, I. W. Hamley, J. Seitsonen, A. Jana, S. Ghosh, N. Mukherjee and S. Ghosh, Self-assembly and wound healing activity of biomimetic cycloalkane-based lipopeptides, ACS Appl. Mater. Interfaces, 2024, 16, 58417–58426.
13. I. W. Hamley, A. Adak and V. Castelletto, Influence of chirality and sequence in lysine-rich lipopeptide biosurfactants and micellar model colloid systems, Nat. Commun., 2024, 15, 6785.
14. A. Adak, G. Das, V. Gupta, J. Khan, N. Mukherjee, P. Mondal, R. Roy, S. Barman, P. K. Gharai and S. Ghosh, Evolution of potential antimitotic stapled peptides from multiple helical peptide stretches of the tubulin heterodimer interface: helix-mimicking stapled peptide tubulin inhibitors, J. Med. Chem., 2022, 65, 13866–13878.
15. Y.-d. Nan, C.-x. Ge, S.-q. Chen, M.-x. Cui, H.-m. Li, C.-c. Zhao, J. Wang, C.-x. Piao and G.-h. Li, Exploring the novel antioxidant peptides in low-salt dry-cured ham: Preparation, purification, identification and molecular docking, Food Chem., 2024, 446, 138697.
16. Y.-y. Hu, S. Xiao, G.-c. Zhou, X. Chen, B. Wang and J.-h. Wang, Bioactive peptides in dry-cured ham: A comprehensive review of preparation methods, metabolic stability, safety, health benefits, and regulatory frameworks, Food Res. Int., 2024, 114367.
17. X. Xie, Y. Dang, D. Pan, Y. Sun, C. Zhou, J. He and X. Gao, The enhancement and mechanism of the perception of saltiness by umami peptide from Ruditapes philippinarum and ham, Food Chem., 2023, 405, 134886.
18. L. Hao, X. Gao, T. Zhou, J. Cao, Y. Sun, Y. Dang and D. Pan, Angiotensin I-converting enzyme (ACE) inhibitory and antioxidant activity of umami peptides after in vitro gastrointestinal digestion, J. Agric. Food Chem., 2020, 68, 8232–8241.
19. Y. He, Z. Deng, Y. Lyu, Y. Yan, J. Liu and H. Liu, Integration of pre-trained GRU and molecular docking for virtual screening of quinoa seed derived ACE inhibitory peptides: An innovative prediction strategy, Food Chem., 2025, 145591.
20. Z. Xu, C. Wu, D. Sun-Waterhouse, T. Zhao, G. I. Waterhouse, M. Zhao and G. Su, Identification of post-digestion angiotensin-I converting enzyme (ACE) inhibitory peptides from soybean protein Isolate: Their production conditions and in silico molecular docking with ACE, Food Chem., 2021, 345, 128855.
21. Y. Dong, W. Yan, Y.-Q. Zhang and Z.-Y. Dai, A novel angiotensin-converting enzyme (ACE) inhibitory peptide from tilapia skin: Preparation, identification and its potential antihypertensive mechanism, Food Chem., 2024, 430, 137074.
22. H. Dai, M. He, G. Hu, Z. Li, A. Al-Romaima, Z. Wu, X. Liu and M. Qiu, Discovery of ACE Inhibitory Peptides Derived from Green Coffee Using In Silico and In Vitro Methods, Foods, 2023, 12, 3480.
23. Y. Wang, S. Chen, W. Shi, S. Liu, X. Chen, N. Pan, X. Wang, Y. Su and Z. Liu, Targeted Affinity Purification and Mechanism of Action of Angiotensin-Converting Enzyme (ACE) Inhibitory Peptides from Sea Cucumber Gonads, Mar. Drugs, 2024, 22, 90.
24. X. Gao, F. Bu, D. Yi, H. Liu, Z. Hou, C. Zhang, C. Wang, J.-M. Lin, Y. Dang and Y. Zhao, Molecular docking and antihypertensive effects of a novel angiotensin-I converting enzyme inhibitory peptide from yak bone, Front. Nutr., 2022, 9, 993744.
25. X.-Y. Zu, Y.-N. Zhao, Y. Liang, Y.-Q. Li, C.-Y. Wang, X.-Z. Zhao and H. Wang, Research on the screening and inhibition mechanism of angiotensin I-converting enzyme (ACE) inhibitory peptides from tuna dark muscle, Food Biosci., 2024, 59, 103956.
26. Q. Wu, H. Ma, L. Luo and S. Wu, Determination of angiotensin converting enzyme inhibitor activity by high performance liquid chromatography, Se P'u Chin. J. Chromatogr., 2005, 23, 79–81.
27. T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods, 1983, 65, 55–63.
28. J. Zhang, L. Liang, L. Zhang, X. Zhou, B. Sun and Y. Zhang, ACE inhibitory activity and salt-reduction properties of umami peptides from chicken soup, Food Chem., 2023, 425, 136480.
29. K. Dang, J. Lan, Y. Wang, D. Pan, L. Du, S. Suo, Y. Dang and X. Gao, Screening and evaluation of novel DPP-IV inhibitory peptides in goat milk based on molecular docking and molecular dynamics simulation, Food Chem.: X, 2025, 102217.
30. S. M. Auwal, S. B. M. Ghanisma and N. Saari, Improved systolic blood pressure (SBP)-lowering effect of stone fish-derived angiotensin-I converting enzyme (ACE)-inhibitory peptide entrapped in chitosan-coated alginate nanocomposites, Food Biosci., 2024, 60, 104349.
31. L. Liang, J. Zhang, B. Sun, M. Zhang, Y. Zhang, K. Li and Y. Zhang, ACE inhibitory activity and gastrointestinal digestion stability of umami peptides IIVFGRQLL from yeast extract, LWT, 2024, 116308.
32. J. Xie, S. Chen, P. Huan, S. Wang and Y. Zhuang, A novel angiotensin I-converting enzyme inhibitory peptide from walnut (Juglans sigillata) protein hydrolysates and its evaluation in Ang II-induced HUVECs and hypertensive rats, Int. J. Biol. Macromol., 2024, 266, 131152.
33. Z. Li, W. Zhang, M. A. Abubaker, Q. Shu and Y. Liu, In silico identification and experimental validation of two types of angiotensin-converting enzyme (ACE) and xanthine oxidase (XO) milk inhibitory peptides, Food Chem., 2025, 464, 141864.
34. A. Alvíz-Amador, N. Contreras-Puentes and J. Márquez-Lázaro, Bioactive peptides against angiotensin–converting enzyme I: An in silico study, Pept. Sci., 2024, 116, e24332.
35. A. Mehmood, A. Iftikhar and X. Chen, Food-derived bioactive peptides with anti-hyperuricemic activity: A comprehensive review, Food Chem., 2024, 139444.
36. S. Lee, K. Jo, Y.-S. Choi and S. Jung, Tracking bioactive peptides and their origin proteins during the in vitro digestion of meat and meat products, Food Chem., 2024, 139845.
37. J. Sánchez-García, S. Muñoz-Pina, J. García-Hernández, A. Tárrega, A. Heredia and A. Andrés, Protein digestibility and ACE inhibitory activity of fermented flours in older adults and standard gastrointestinal simulation, Food Res. Int., 2024, 180, 114080.
38. P. Chanajon, A. Hamzeh, F. Tian, S. Roytrakul, O. A. Oluwagunwa, D. Kadam, R. E. Aluko, S. Aueviriyavit, R. Wongwanakul and J. Yongsawatdigul, Hypotensive effect of potent angiotensin-I-converting enzyme inhibitory peptides from corn gluten meal hydrolysate: Gastrointestinal digestion and transepithelial transportation modifications, Food Chem., 2025, 462, 140953.
39. B. G. K. Madhavi, A. M. Wijethunga, O. D. Okagu and X. Sun, Defatted Wheat Germ Protein-Derived Peptides Showed Multiple Biological Activities from the Stomach to Small Intestine: In Silico and In Vitro Approaches, J. Agric. Food Chem., 2024, 72, 20527–20536.
40. P. Luasiri, P. Sangsawad, J. Pongsetkul, P. Paengkoum, C. Nakharuthai, S. Suwanangul, S. Katemala, N. Sujinda, J. Pinyo and J. Chainam, Exploration of nutritional and bioactive peptide properties in goat meat from various primal cuts during in vitro gastrointestinal digestion and absorption, Anim. Biosci., 2024, 37, 1096.
41. J. Li, H. Hu, X. Chen, H. Zhu, W. Zhang, Z. Tai, X. Yu and Q. He, A novel ACE inhibitory peptide from Douchi hydrolysate: Stability, inhibition mechanism, and antihypertensive potential in spontaneously hypertensive rats, Food Chem., 2024, 460, 140734.
42. T. Song, T. Zhang, Q. Cai, Y.-Y. Ding and Z. Gu, A novel angiotensin I-converting enzyme inhibitory peptide APPLRP from Grifola frondosa ameliorated the Ang II-induced vascular modeling in zebrafish model by mediating smooth muscle cells, Int. J. Biol. Macromol., 2024, 278, 134998.
43. Q. Li, K. Liu, Z. Gao and M. T. Rashid, An ACE-inhibitory peptide derived from maize germ antagonizes the Angiotensin II-induced dysfunction of HUVECs via the PI3K/Akt/eNOS signaling pathway, J. Funct. Foods, 2024, 112, 105967.
44. Y.-Q. Zhao, L. Zhang, J. Tao, C.-F. Chi and B. Wang, Eight antihypertensive peptides from the protein hydrolysate of Antarctic krill (Euphausia superba): Isolation, identification, and activity evaluation on human umbilical vein endothelial cells (HUVECs), Food Res. Int., 2019, 121, 197–204.
45. M. B. O'Keeffe and R. J. FitzGerald, Whey protein hydrolysate induced modulation of endothelial cell gene expression, J. Funct. Foods, 2018, 40, 102–109.
46. S.-K. Suo, Y.-Q. Zhao, Y.-M. Wang, X.-Y. Pan, C.-F. Chi and B. Wang, Seventeen novel angiotensin converting enzyme (ACE) inhibitory peptides from the protein hydrolysate of Mytilus edulis: Isolation, identification, molecular docking study, and protective function on HUVECs, Food Funct., 2022, 13, 7831–7846.
47. I. Kovrlija, K. Menshikh, H. Abreu, A. Cochis, L. Rimondini, O. Marsan, C. Rey, C. Combes, J. Locs and D. Loca, Challenging applicability of ISO 10993–5 for calcium phosphate biomaterials evaluation: Towards more accurate in vitro cytotoxicity assessment, Biomater. Adv., 2024, 160, 213866.
48. J. Liu, W. Song, X. Gao, J. Sun, C. Liu, L. Fang, J. Wang, J. Shi, Y. Leng and X. Liu, A combined in vitro and in silico study of the inhibitory mechanism of angiotensin-converting enzyme with peanut peptides, Int. J. Biol. Macromol., 2024, 268, 131901.
49. H. Zhang, L. Liang, B. Sun, R. Yang, Z. Liu, X. Mao and Y. Zhang, ACE inhibitory effect and saltiness-enhancing properties of chicken-derived umami peptides: Digestive stability, inhibition kinetics, multiple ligand docking and central composite design, Food Chem., 2025, 464, 141634.
50. J. Wang, Z. Wang, M. Zhang, J. Li, C. Zhao, C. Ma and D. Ma, Impact of Lactiplantibacillus plantarum and casein fortification on angiotensin converting enzyme inhibitory peptides in yogurt: identification and in silico analysis, Food Funct., 2024, 15, 3824–3837.
51. Z. Wang, M. Zhang, C. Zhao, J. Li, J. Wang, C. Ma and D. Ma, ACE-inhibitory peptides in sodium-reduced cheese with probiotic: Identification, in silico prediction and molecular interaction, LWT, 2024, 200, 116198.
52. Q. Wu, J. Du, J. Jia and C. Kuang, Production of ACE inhibitory peptides from sweet sorghum grain protein using alcalase: Hydrolysis kinetic, purification and molecular docking study, Food Chem., 2016, 199, 140–149.
53. N. F. Bras, P. A. Fernandes and M. J. Ramos, QM/MM study and MD simulations on the hypertension regulator angiotensin-converting enzyme, ACS Catal., 2014, 4, 2587–2597.
54. X. Bao, Y. Zhang, L. Wang, Z. Dai, Y. Zhu, M. Huo, R. Li, Y. Hu, Q. Shen and Y. Xue, Machine learning discovery of novel antihypertensive peptides from highland barley protein inhibiting angiotensin I-converting enzyme (ACE), Food Res. Int., 2025, 202, 115689.
55. T. Du, J. Yang, Y. Qin, X. Huang, J. Li, S. Xiong, X. Xu, L. Zhang, M. Zhao and H. Li, Transport and action of sesame protein-derived ACE inhibitory peptides ITAPHW and IRPNGL, Food Chem., 2025, 142965.
56. A. Brodkorb, L. Egger, M. Alminger, P. Alvito, R. Assunção, S. Ballance, T. Bohn, C. Bourlieu-Lacanal, R. Boutrou and F. Carrière, INFOGEST static in vitro simulation of gastrointestinal food digestion, Nat. Protoc., 2019, 14, 991–1014.
57. K. Liu, S. Lin, Y. Liu, S. Wang, Q. Liu, K. Sun and N. Sun, Mechanism of the reduced allergenicity of shrimp (Macrobrachium nipponense) by combined thermal/pressure processing: Insight into variations in protein structure, gastrointestinal digestion and immunodominant linear epitopes, Food Chem., 2023, 405, 134829.
58. C. C. Song, B. W. Qiao, Q. Zhang, C. X. Wang, Y. H. Fu and B. W. Zhu, Study on the domain selective inhibition of angiotensin–converting enzyme (ACE) by food–derived tyrosine–containing dipeptides, J. Food Biochem., 2021, 45, e13779.

---