DOPK was provided by the Three Rivers Palm Garden (Haikou, China). Trypsin (1 × 10⁴ U/g, derived from porcine pancreas), papain (3 × 10⁴ U/g), and pepsin (1 × 10⁵ U/g, derived from bovine stomach) were purchased from Kangfukuai Biotechnology Co. (Nanning, China). ACE, 4-(2-Pyridinazo)-resorcinol (PAR), and N-hippuryl-l-histidyl-l-leucine (HHL) were purchased from Sigma (St. Louis, MO, United States). Dithiothreitol, HEPES-KOH buffer, and other chemicals were purchased from Jinyangkeji Co. (Linfen, China).

Globulin was extracted from DOPK following the procedure described by Zheng et al. (2021), with 0.25 mol/L NaCl as the extraction solvent. Then the obtained DOPKG was hydrolyzed with papain to produce peptides of high zinc-chelating capacity. Briefly, DOPKG (2 g) was thoroughly dispersed in 100 mL of dH₂O, and then adjusted to pH 7.5 with 0.1 mol/L of HCl or 0.1 mol/L of NaOH. Papain (75 mg) was added, and the reaction solution was held at 55°C in a shaking water bath (XBZ-2; Shangyu Co., Hangzhou, China) for 95 min. Subsequently, the reaction solution was heated at 100°C for 8 min. The reaction mixture was cooled and centrifuged at 12,000 × g for 12 min. The supernatant was collected and freeze-dried to obtain DOPKGH. In addition, the trinitrobenzenesulfonic acid method was used to determine the hydrolysis degree (Adler-Nissen, 1979).

DOPKGH (dissolved in ultrapure water, 1 mg/mL) were passed through a W-45 ultrafiltration membrane with a filter diameter of 0.45 μm (Jieneng Co., Wuxi, China) (Zheng et al., 2021). The filtrate was lyophilized using an LGJ-10N freeze dryer (Keya Instrument Co., Beijing, China). The obtained powder was resolved in ultrapure water (1 mg/mL) and separated with Sephadex G-15 gel chromatography on a column (Ф1.2 × 80 cm). The elution rate was 2.6 mL dH₂O/min and monitored at 220 nm. The effluent fractions were collected and lyophilized, and their ACE inhibitory activity and Zn-chelating ability were determined (Udechukwu et al., 2018; Zaharuddin et al., 2022). The subfraction with the highest ACE-inhibitory activity was further separated using reverse-phase high performance liquid chromatography (RP-HPLC) on a Kromasil 100-5 C₁₈ column (4.6 × 250 mm, 5 μm; Eka Chemicails, Sweden) with deionized water containing 0.1% (v/v) trifluoroacetate as the elution solution A. Moreover, a linear gradient of acetonitrile containing 0.1% TFA (1%–22%, in 15 min) was used as the elution solution B. The flow velocity was maintained at 1.0 mL/min and the monitored wavenumber was 220 nm. The subfractions were separately collected and lyophilized, and the ACE-inhibitory and Zn-chelating activities were determined. The subfraction with the highest capacity was used for amino acid sequence identification.

ACE inhibitory activity was determined using the procedure described by Zaharuddin et al. (2022). Briefly, ACE (25 mU) was pre-incubated at 37°C for 10 min. Then ACE (75 µL), 225 µL of HHL (8.3 mmol/L), and the peptide solution (75 µL) were mixed and stirred at 75 rpm and 37°C for 60 min. The reaction was stopped by adding 375 µL of HCl (1 mol/L). Afterward 2.1 mL of ethyl acetate was added to extract the hippuric acid produced. The mixture was centrifuged at 14,000 g for 150 s. Subsequently, 1 mL of the upper solution (ethyl acetate extraction) was transferred to a glass test tube and heated at 120°C for 32 min. The tube was cooled to 25°C, deionized water (1 mL) was added, and the absorbance was measured at 228 nm. The control group was subjected to the same procedure, but without samples. The ACE inhibition ability of the samples was defined as the percentage of the difference in absorbance at 228 nm between the sample and control as compared to the absorbance at 228 nm of the control. The concentration of peptides required to inhibit half of the ACE activity was defined as the IC₅₀.

Moreover, the ACE inhibition kinetics of samples on ACE was analyzed based on the Lineweaver-Burk plot of ACE with the addition of peptides (0–60 μmol/L) identified in DOPKGH, following the same procedures as Urbizo-Reyes et al. (2022). The ACE substrate (HHL) concentration ranged from 0 to 7.60 mmol/L.

DOPKGH peptides (350 μg), 0.5 mL zinc sulfate solution (0.25 mmol/L), 0.5 mL DTT (8 mmol/L), 1 mL HEPES-KOH buffer (100 μmol/L), and 8 mL dH₂O were mixed thoroughly (Li et al., 2023). After stirring at 175 r/min and 37°C for 12 min, the zinc concentration of the reaction solution was measured using the 4-(2-Pyridinazo)-resorcinol method (Udechukwu et al., 2018). The standard regression curve of the zinc concentration (x, μg/mL) with absorbance at 500 nm (y) was y = 0.0901n(x) + 0.1012, and R² = 0.9802 (Li et al., 2023). The zinc chelation rate was defined as the reduction of zinc ions in the reaction solution per unit mass of sample (mg/g).

Amino acid sequence identification was conducted using a Q Exactive hybrid quadrupole orbitrap mass spectrometer (Thermo Fisher, Bremen, Germany) coupled with Peak-Studio-7.5-De-Novo™ software (Bioinformatics Solutions, Inc., Waterloo, Canada) according to the method described by Li et al. (2022). The DOPKGH sequences were verified by matching with the sequences of Elaeis guineensis recorded in the Biotechnology Information Database (Bethesda, MD, United States). The Zn-chelating ability of the obtained sequences was measured using the 4-(2-Pyridinazo)-resorcinol method (Udechukwu et al., 2018). Potential antihypertensive effects were predicted using both the BIOPEP and AHTPDB databases (Kumar et al., 2015). The predicted average local confidence (ALC) of antihypertensive peptides should be above 85%, and their vector machine software scores (SVMS) should be higher than zero (Zaharuddin et al., 2022). Chemical synthesis of the selected sequence was performed using the solid-phase synthesis method at Dingxiang Peptide Co. (Shaoxing, China). In addition, the physicochemical characteristics of the screened DOPKGH sequences were analyzed using the AHTPDB database.

The potential toxicity and allergenicity of the peptides identified in DOPKGH were predicted using ToxinPred (http://www.imtech.res.cn/raghava/toxinpred/) and AlgPred (http://www.imtech.res.cn/raghava/algpred/) databases, respectively (Sudheer et al., 2013).

The SYBLY-X.2.0.1 Murflex-Docking Tool (Tripos Int. Co., Saint Louis, MO, United States) was used to perform visual molecular simulations of the coordination between the screened DOPKGH peptides and the ACE crystal structure (Li et al., 2022). The ACE structure with the code PDB-108A, downloaded from the Protein Data Bank (http://www.rcsb.org), was used as a molecular docking template. The coordination patterns of DOPKGH peptides with ACE were selected mainly based on the predicted T-scores (the acceptable threshold was 6.0), C-scores, and the number and length of hydrogen bonds. The hydrophobic interactions of the screened DOPKGH peptides with ACE were studied using the LigPlot (Zaharuddin et al., 2022).

The simulation intestinal digest mucus (pH 6.80 ± 0.10) was composited of 6 g bile salt, 0.07 g pancreatin, 12.5 g NaHCO₃, and 200 mL ultrapure water. The simulated gastric digested mucus (pH 2.00 ± 0.10) contained 0.15 mol/L of NaCl and 0.35 mg/mL of pepsin (Wang et al., 2021). Peptides (5 g) were first hydrolyzed with simulated gastric digest mucus (30 mL). The digest was stirred at 135 rpm and 37°C for 80 min. Subsequently, the pH value of the digest was adjusted to pH 6.8 and 50 mL of simulated intestinal digest mucus was added. After stirring at 135 rpm and 37°C for 150 min, the digest was placed at 100°C for 10 min. The ACE inhibitory activity of the DOPKGH peptide was determined using untreated DOPKGH peptides for comparison.

Simultaneously, 5 mg of the DOPKGH peptide-Zn complexes was mixed with 50 mL of simulated gastric digested mucus (pH 2.00 ± 0.10) (Sun et al., 2021). After shaking at 37°C and 135 rpm for 90 min, Na₂HPO₄ (0.5 mol/L) was added and the mixture was quickly stirred until the pH of the digest increased to 6.80 ± 0.10. The simulated intestinal mucus digest (50 mL) was then added, and the digest was continuously stirred at 37°C and 135 rpm for 150 min. At 30 min intervals, an aliquot of the digest (0.8 mL) was removed for zinc concentration determination using the 4-(2-Pyridinazo)-resorcinol method (Udechukwu et al., 2018). The stability of DOPKGH peptide-Zn complexes was expressed as the residual zinc concentration after digestion and as a percentage of the zinc concentration before digestion. Zinc gluconate (100 g/mL) and ZnSO₄ (100 g/mL) were used for comparison.

After a week of environmental adaptation, thirty-two spontaneous hypertensive rats (SHR, 12 weeks old, 240 ± 15 g body weight, male; Beijing Medicine YaoLi Biotechnology Co., Ltd., China) were randomly divided into eight groups: negative, positive, DOPKGH peptide low-, middle-, and high-groups, and DOPKGH peptide-Zn complex low-, middle-, and high-groups (Kaprasob et al., 2022). Each group consisted of four rats. The rats in the negative control and positive groups were gastrically intubated with NaCl (0.9%) and captopril (10 mg/kg body weight once daily), respectively, while the rats in the high-, middle-, and low-dosage groups were orally administered 150, 100, and 50 mg of DOPKGH peptides (or peptide-Zn complexes)/kg/body weight once daily, respectively. The oral administration continued for 5 weeks. The diastolic and systolic blood pressures of the rats were measured every 7 days using the tail cuff method with a ZL-620-F non-invasive blood pressure apparatus (Anhui Yaokun Biotechnology Co., Ltd., Hefei, China). This experimental study was approved by the Institutional Animal Care and Use Committee of Shanxi Normal University. All animals received human care according to the Guideline Manual for the Care and Use of Laboratory Animals.

The results of all tests are expressed as mean ± standard deviation, and each experiment was repeated at least three times. Differences among data were analyzed by one-way analysis of variance coupled with Duncan’s multiple range tests using IBM SPSS Statistics software (Version 16, Chicago, IL, United States). Differences were considered statistically significant at p < 0.05.

The degree of hydrolysis of the DOPKG was 31.67%, which is consistent with the results of Tapal et al. (2016). The ACE-inhibitory capacity and zinc-chelating ability of DOPKGH were 19.71% ± 1.62% and 11.67 ± 1.83 mg/g, respectively. The profile in Figure 1A shows that DOPKGH was divided into three major subfractions (DOPKGH-1, DOPKGH-2, and DOPKGH-3) after purification using G-15 gel chromatography. Among them, the DOPKGH-2 exhibited the highest ACE-inhibitory capacity (39.11% ± 0.34%) and zinc-chelating capacity (19.31 ± 0.74 mg/g, Figure 1B). Therefore, it was further purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on an analytical C₁₈ column. As shown in Figure 2, only one main subfraction (DOPKGH-2) was obtained, and it was collected and used for amino acid sequence identification.

As shown in Table 1, there were five representative oligopeptides including Gln-Arg-Leu-Asp-Arg-Cys-Lys (QRLERCK), Leu-Leu-Leu-Gly-Val-Ala-Asn-Tyr-Arg (LLLGVANYR), Arg-Ala-Asp-Val-Phe-Asn-Pro-Arg (RADVFNPR), Arg-Val-Ile-Lys-Tyr-Asn-Gly-Gly-Gly-Ser-Gly (RVIKYNGGGSG), and Glu-Val-Pro-Gln-Ala-Tyr-Ile-Pro (EVPQAYIP) identified in DOPKGH-2-C based on the results of UPLC-ESI-MS/MS analysis. The in silico screening results showed that only EVPQAYIP had both potential ACE-inhibition and antihypertensive effects. As shown in Figure 3A, the relationship between synthesized EVPQAYIP ACE-inhibition ability (y) with its concentration (x) was y = 10.985, ln(x) ‒ 0.8859, and R² = 0.9458, from which its IC₅₀ value was calculated as 102.75 μmol/L. Moreover, the ACE-inhibition ability of EVPQAYIP was higher than that of peptides with similar mass such as GNPLSP derived from Arthrospira platensis (IC₅₀: 264.24 μmol/L; Wang et al., 2021) and PHQPLPP identified in rubing cheese (IC₅₀: 0.821 mg/mL, Wei et al., 2022) (p < 0.05), suggesting that EVPQAYIP is a relatively effective ACE-inhibitor.

It has been demonstrated that hydrophobic amino acids in C-terminal tripeptide, especially Phe and Trp (aromatic amino acids), Leu, Ile, Val (branched amino acids), and Pro residues, play a crucial role in the inhibitory effect of peptides on ACE, as these hydrophobic amino acids have a relatively high binding affinity for the active sites of ACE (Chen et al., 2022). Moreover, Lys or Arg residues in C-terminal tripeptide are instrumental in the ACE-inhibitory ability of peptides (Urbizo-Reyes et al., 2022). Additionally, previous studies have also found that the Val residue near the N-terminal is helpful for the coordination of peptides with ACE (Zaharuddin et al., 2022). The Val residue at the N-terminal of VSWNVLQEP, identified in King Bolete mushrooms, was demonstrated to have strong binding energy (−9.1 kcal/mol) with ACE (Kaprasob et al., 2022). Therefore, the hydrophobic amino acids, especially Pro, Ile, Val, and Tyr residues, in the C-terminal tripeptide or near the N-terminal, mainly contributed to the high inhibitory capacity of EVPQAYIP against ACE.

Moreover, EVPQAYIP exhibited considerable Zn-chelating ability (11.69 ± 0.38 mg/g, Table 1). A study on the structure-activity relationship of peptide-Zn chelates showed that peptides with terminal amino and/or carbonyl groups had a high chelating ability with Zn ions (Wang et al., 2020). The nitrogen atoms of the second and third amide residues at N-terminal can take part in the coordination with zinc ions (Udechukwu et al., 2018). Glu or Asp with γ-carboxyl group can increase the negative polarity of peptides and improve the binding force between zinc ions and peptides. Additionally, Pro residues can be used as a negative bridge ligand for zinc ions (Pina and Roque, 2009; Lin et al., 2023). Hence, amino acid residues, especially Pro and Glu, were predominantly responsible for the high binding affinity of EVPQAYIP for zinc ions. Figure 4 shows the ESI-MS/MS spectrum of EVPQAYIP.

As shown in Table 1, EVPQAYIP had a high content of hydrophobic amino acids (62.50%) and relatively high hydrophobicity (0.01), which was another reason for its high ACE-inhibitory capacity. Hydrophobic residues can increase the binding affinity of peptides to active sites of ACE (Pina and Roque, 2009). Moreover, QRLERCK exhibited the highest hydrophilicity (1.34), corresponding to its high Zn-chelating ability (17.30 ± 1.46 mg/g), because hydrophilic groups of peptides have more polar charge for binding with zinc ions (Wei et al., 2022). EVPQAYIP also showed considerable binding affinity with zinc ions (11.69 ± 0.38 mg/g), which was mainly attributed to the Glu residue in N-terminal. A previous study found that the-carboxyl group is a good chelating site for Zn ions (Sun et al., 2021).

Additionally, the isoelectric point (pI) of EVPQAYIP was 4.00. Peptides should avoid being used in solutions with pH values near their isoelectric points. At isoelectric points, the polarity and solubility of peptides dramatically decease, leading to an adverse effect on the coordination of peptides with ACE or zinc ions (Zheng et al., 2021).

As shown in Table 1, the in silico prediction results using the ToxinPred database showed that QRLERCK, LLLGVANYR, RADVFNPR, RVIKYNGGGSG, and EVPQAYIP were peptides without any toxicity. Furthermore, these peptides did not match any allergenic peptide sequences recorded in the AlgPred database. Additionally, short oligopeptides are less likely to have complete epitopes than those with larger masses (Piovesana et al., 2018). These results indicate that these four peptides have no potential allergenicity. However, further security studies, including cellular tests and in vivo assays, are required.

Bioactive peptides should be able to resist gastrointestinal digestion to enter the blood and exhibit health benefits (Magouz et al., 2023). As shown in Figure 3B, the relationship between the ACE inhibition ability (y) of EVPQAYIP and its concentration (x) conformed to the following regression equation: y = 13.536ln(x) ‒ 17.297, R² = 0.9528. Therefore, the IC₅₀ value of EVPQAYIP against ACE was 144.27 μmol/L after gastrointestinal digestion, which was higher than that of the untreated EVPQAYIP (102.75 μmol/L, Figure 3A), evidencing the inhibition ability of EVPQAYIP on ACE was reduced after gastrointestinal hydrolysis. However, EVPQAYIP retained 71.23% of its ACE-inhibitory activity against gastrointestinal digestion.

Moreover, as shown in Figure 8, during digestion with gastric digestive mucus (0–90 min), the EVPQAYIP-Zn complexes exhibited relatively stable Zn-solubility (66.15%–71.47%). However, Zn solubility dramatically decreased during digestion with intestinal digestive mucus (10–240 min) (p < 0.05). ZnSO₄ and zinc gluconate exhibit similar trends. The main reason for this was that the pH of the digestive fluid increased dramatically from 2.0 to 7.0. Most Zn ions in the digestive fluid form insoluble Zn salts as the pH increases (Wang et al., 2020). From 120 to 240 min, both the EVPQAYIP-Zn complexes and zinc gluconate showed higher zinc solubility than that of zinc sulfate (p < 0.05), indicating that EVPQAYIP can improve zinc stability in the intestine. In peptide-zinc complexes, zinc ions are generally located inside the peptide chain structure, protecting zinc ions from pH interference (Sun et al., 2021).

It has been shown that enzymes present in gastric or intestinal tissues can cleave polypeptides, especially peptide chains with Phe, Tyr, Lys, and Arg residues, resulting in a decrease in the bioactivity of peptides (Lee and Hur, 2017). Although EVPQAYIP contains a Tyr residue, it exhibits relatively stable ACE-inhibitory activity and Zn-chelating capacity under gastrointestinal hydrolysis, which is probably attributed to the Pro residues (with a rigid ring structure) present in EVPQAYIP (Zhang et al., 2022). Peptides containing Pro residues, such as KPVPR, QPHQPLPP, and INPPSTTN, identified in canary seed and kenaf proteins, respectively, are stable during gastrointestinal digestion (Urbizo-Reyes U et al., 2022; Zaharuddin et al., 2022). However, further studies are needed to investigate the effects of gastrointestinal hydrolysis on the structures of EVPQAYIP and EVPQAYIP-Zn complexes.

The effects of oral administration of EVPQAYIP and EVPQAYIP-Zn on diastolic blood pressure (DBP) and systolic blood pressure (SBP) of spontaneously hypertensive rats (SHR) are shown in Figures 9A,B, respectively. As shown in Figure 9B, both EVPQAYIP and EVPQAYIP-Zn complexes (50–150 mg/kg body weight) significantly decreased the systolic blood pressure of SHR from the first week of oral administration (p < 0.05). From the second week, oral administration of EVPQAYIP (100–150 mg/kg body weight) remarkably reduced the diastolic blood pressure of SHR (p < 0.05) (Figure 9A). EVPQAYIP-Zn complexes at 100–150 mg/kg body weight significantly lowered the diastolic blood pressure of SHR from the third week (p < 0.05) (Figure 9A). These results suggest that both EVPQAYIP and EVPQAYIP-Zn complexes have potential antihypertensive effects in vivo, although their efficiency was lower than that of captopril (p < 0.05). Moreover, EVPQAYIP and EVPQAYIP-Zn reduced the diastolic and systolic blood pressure of SHR in a dose-dependent manner. Additionally, at the same dose, the DBP (or SBP) of SHR orally administered EVPQAYIP was not significantly different from that of SHR orally administered EVPQAYIP-Zn complexes (p > 0.05), suggesting that chelation with zinc ions did not affect the potential antihypertensive effect of EVPQAYIP.