Educational guide
ACE inhibitory casein peptide lowers blood pressure and ...
Hypertension is a major risk factor for cardiovascular diseases and all-cause mortality 1 . While antihypertensive medications are effective, they often come with side effects such as cough and edema, and may lead to drug dependence 2 . Therefore, lifestyle mo
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Hypertension is a major risk factor for cardiovascular diseases and all-cause mortality1. While antihypertensive medications are effective, they often come with side effects such as cough and edema, and may lead to drug dependence2. Therefore, lifestyle modifications, including dietary changes, have emerged as an alternative way to regulate blood pressure. Studies on substances derived from milk revealed that casein-derived peptides possess antihypertensive properties3,4,5.
Casein hydrolysates, produced through enzymatic hydrolysis of casein4, have been extensively studied for their blood pressure-lowering effects. Notably, peptides such as Ile-Pro-Pro and Val-Pro-Pro, derived from bovine casein, have been shown to reduce blood pressure in both humans and spontaneously hypertensive rats5,6,7. Similarly, oral administration of Met-Lys-Pro can potentially lower systolic blood pressure (SBP) in rat models as well3. The primary mechanism by which these bioactive peptides reduce blood pressure is most likely to be through angiotensin-converting enzyme (ACE) inhibition3,4,5. ACE plays a crucial role in the renin-angiotensin system, converting angiotensin I to angiotensin II, a potent vasoconstrictor that elevates blood pressure8. Additionally, ACE is also able to inactivate bradykinin, a vasodilatory peptide that helps lower blood pressure9. Therefore, the casein-derived peptide, as a potential angiotensin-converting enzyme inhibitor (ACEI), offers a promising approach to blood pressure management.
Inhibitors can deactivate ACE by either occupying its active site or inducing conformational changes in the enzyme10,11. However, the in vivo antihypertensive activity of milk-derived peptides does not always correlate with their in vitroACE inhibitory activity, likely due to limited bioavailability and the degradation of active peptide sequences. The bioactivity of peptides is determined by their inherent amino acid sequence and composition12. By studying the precise interactions between certain peptide sequences and specific binding site residues in ACE, molecular docking improves our knowledge of structure-function correlations and aids in the prediction of biological activity in vivo.
Emerging evidence suggests that gut microbiota and their metabolites are closely related to hypertension development. Dysbiosis, or an imbalance in gut microbiota, has been implicated in the pathogenesis of hypertension13,14,15. For instance, long-term butyrate intervention has been shown to attenuate hypertension, vascular oxidative stress, and inflammation in angiotensin II-induced mice16. Similarly, acetate and propionate also possess antihypertensive properties. These metabolites may be involved in blood pressure regulation17.
In recent years, many antihypertensive peptides from different food protein sources have been reported18. Although the antihypertensive effects of hydrolyzed casein peptides have been well-documented in animal studies and in vitro experiments, further clinical trials are needed to confirm their efficacy and safety in humans and for the physiological basis of its antihypertensive effects. In this study, the effects of hydrolyzed casein peptides containing GPFPIIV and FFVAPFPEVFGK peptide (HCP-C7C12) ingestion, on lowering blood pressure will be investigated in subjects with prehypertension or hypertension. Rojas-Ronquillo et al.19 showed that YQEPVLGPVRGPFPIIV, a long peptide containing GPFPIIV, had ACE inhibitory activity. When a peptide has a certain activity, some of the short peptides contained in this peptide or the long peptides containing this peptide may also have a similar biological function to the peptide. Relevant studies have shown that GPFPIIV has ACE inhibitory activity but with unknown IC50value20. Similarly, FFVAPFPEVFGK, first isolated from cow milk casein by Maruyama and Suzuki in 1982, has an IC50 value of 77 µM and has demonstrated antihypertensive effects in both animal and human studies21,22,23. However, the mechanisms underlying its blood pressure-lowering effects, particularly in relation to gut microbiota and serum metabolites, remain underexplored.
In this context, the molecular interactions involved with HCP-C7C12 induced ACE-inhibitory effects were elucidated. Meanwhile, the changes in the serum metabolites and intestinal microbiota of the participants will be analyzed to explore the possible mechanisms of antihypertensive effects. The aims of this present study were to: (1) investigate the blood pressure-lowering effect of HCP-C7C12 in vivo; and (2) elucidate the possible mechanisms of its antihypertensive effects. We hypothesized that both SBP and diastolic blood pressure (DBP) would decrease after regular HCP-C7C12 peptide tablet ingestion in Chinese adults with prehypertension/hypertension over an eight-week intervention period. To test our hypothesis, we carried out a randomized, double-blind, placebo-controlled, clinical trial in adults aged 30–65 years.
Results
ACE inhibitory activity of hydrolyzed casein peptide
The ACE inhibitory activities of hydrolyzed casein peptide complexes, HCP-C7 C12 and HCP002, were tested. As shown in Supplementary Fig. S1, both peptides had ACE inhibitory activities, with IC50 values of 1.354 mg/mL and 1.605 mg/mL, respectively. Hence, HCP-C7 C12, which demonstrated a higher ACE inhibitory activity, was selected for further analysis.
Identification of peptides in hydrolyzed casein peptide
The potential ACE inhibitory peptides derived from the HCP-C7 C12 were listed in Supplementary Table S1. The results have been arranged by PeptideRanker values from highest to lowest. According to the results, the three most potential bioactive peptides were LPFPRL, FFVAPFPEVFGK, and GPFPIIV. As LPFPRL was unable to complete molecular docking, the two most potential bioactive peptides, FFVAPFPEVFGK and GPFPIIV, were further analyzed in this study.
Molecular docking
The docking results were shown in Figs. 1 and 2. Both FFVAPFPEVFGK and GPFPIIV interacted strongly with ACE. According to Supplementary Table S2, the potential energy was − 159.445 kcal/moL and − 365.865 kcal/moL, respectively. The van der Waals were − 22.839 kcal/moL and − 28.952 kcal/moL, respectively. The electrostatic energies combined with ACE were − 194.172 kcal/moL and − 435.781 kcal/moL, respectively. The strength of affinity between the peptide and ACE was represented by the value of -CDOCKER energy. The affinity of FFVAPFPEVFGK (156.09 kcal/moL) was greater than that of GPFPIIV (101.216 kcal/moL). Therefore, FFVAPFPEVFGK might have a stronger ACE inhibition effect. Eight hydrogen bonds were observed with ACE-GPFPIIV interaction (SER-516, GLU-411, ALA-354, HIS-513, TYR-520, ALA-356, GLU-384, ARG-522), which was especially suitable for the carboxyl and amino groups of compounds to form salt bridges with LYS-511; and three (GLN-281, HIS 513, and TYR-360) for ACE-FFVAPFPEVFGK, which was especially suitable to form salt bridges with ARG-124, ARG-522, GLU-123, and GLU-376. These interactions could effectively promote the formation of stable complexes between small molecules and ACE, and further promote peptide-induced inhibition of ACE activity.
Fig. 1The binding mode of angiotensin-converting enzyme with GPFPIIV. (a) The 3D structure of complex. (b) The 3D detail binding mode of ligand with protein. (c) The 2D detail binding mode of ligand with protein.
Fig. 2The binding mode of angiotensin-converting enzyme with FFVAPFPEVFGK. (a) The 3D structure of complex. (b) The 3D detail binding mode of ligand with protein. (c) The 2D detail binding mode of ligand with protein.
Subjects for randomized double-blind placebo-controlled trial
Of the 147 people who initially signed up, 16 were excluded due to their non-adherence to all inclusion criteria. A total of 131 subjects were eventually enrolled (Fig. 3), of which 57 were male and 74 were female. Seventeen dropped out during the intervention. Of these twelve (3 males and 9 females) were from the test product group (HCP) and 5 (1 male and 4 females) were from the placebo group, and they were excluded from subsequent statistical calculations. The overall dropout rate was 13.0%. Of the 114 subjects who completed the study, 54 had ingested the test tablets, and 60 had ingested placebo tablets. According to Table 1, no significant difference in the baseline characteristics between the group receiving HCP-C7 C12 tablets and placebo groups was shown. Test session schedules were shown in Supplementary Fig. S2.
Fig. 3Flow chart of subject recruitment.
Table 1 Demographic characteristics of the subjects.Blood pressure and heart rate
The mean change rates of SBP, DBP, and heart rate of all subjects at each time point compared to the baseline during the intervention are shown in Fig. 4. The average baseline SBP and DBP were 146.00 and 84.46 mmHg, respectively, in the HCP group and 143.60 and 84.82 mmHg, respectively, in the placebo group. Compared to the placebo group, the reduction rate of SBP in the HCP group was more significant since the first week of starting to take the test tablets (P < 0.01). Similarly, the reduction rate of DBP was also more significant in the HCP group since the second week of the intervention (P < 0.01). Throughout the intervention period, there were no significant differences in both SBP and DBP change rates in the placebo group. Over an 8-week intervention, the mean differences of SBP and DBP levels in the HCP were (mean ± SD) 14.02 ± 2.94 mmHg and 8.11 ± 2.45 mmHg with the reduction rate of 9.41% and 9.53%, respectively, which were significant compared with those (1.72 ± 2.68 mmHg in SBP and 2.10 ± 2.14 mmHg in DBP with the reduction rate of 1.13% and 2.44%; P < 0.01) in the placebo group. The effects of oral administration of tablets on HR levels were not significant in both groups at the end of intervention, though occasionally significant differences in the rates of change between the two groups at week 4 and week 5. These results suggested that the HCP intervention has successfully regulated the blood pressure of hypertensive patients to within the normal range.
Fig. 4Effect of oral administration of the HCP-C7 C12 and placebo tablets on the change rate of (a) SBP, (b)DBP and (c) HR at one-week intervals during the intervention period among subjects in the test product (HCP; n = 54) and placebo (n = 60) groups. Results are expressed as mean ± standard deviation. ## significant at P < 0.01 compared to the baseline; * significant at P < 0.05 compared to the placebo group; ** significant at P < 0.01 compared to the placebo group by paired sample t-test. SBP: systolic blood pressure; DBP: diastolic blood pressure; HR: heart rate.
Efficacy indices
The mean values of blood rheology indicators of subjects in the HCP and placebo groups were present in Supplementary Table S3. The plasma viscosity, whole blood low shear viscosity, whole blood high shear viscosity, and fibrinogen concentration tended to decrease in the HCP over an 8-week intervention, but the difference was not significant, that is, none of the blood rheology indicators were significantly influenced by the HCP-C7 C12 tablet.
As shown in Supplementary Table S4, in both HCP and placebo groups, the cholesterol, triglyceride, low-density lipoprotein, and high-density lipoprotein levels were not significantly changed at the end of the study. Despite a decline in the lipoprotein phospholipase A2 (Lp-PLA2) level (229.05 ± 66.79 µg/L) at the end of the study in the HCP, but it was not significant when compared to the baseline (234.46 ± 53.39 µg/L). A significant increase was seen in the Lp-PLA2 level (266.84 ± 48.24 µg/L) in the placebo group compared to the baseline (213.58 ± 58.67 µg/L; P < 0.01). The Lp-PLA2 levels at the end of the intervention were significantly different between the two groups (P < 0.01).
In Supplementary Table S5, over the 8-week intervention, the endothelin-1 levels in both HCP and placebo groups exhibited a decreasing trend without statistically significant. The intercellular adhesion molecule 1 (ICAM-1) level in the HCP was non-significantly increased. According to the results from the color Doppler flow imaging metrics of subjects (Fig. 5a and b), there were significant increases in brachial artery basal internal diameter (D0) (from 4.22 ± 0.64 to 4.34 ± 0.61 mm for HCP; P < 0.05 and from 4.20 ± 0.54 to 4.51 ± 0.62 mm for placebo group; P < 0.01, respectively) and end-diastolic internal diameter of brachial artery (D1) (from 4.51 ± 0.58 to 4.91 ± 0.56 mm for HCP; P < 0.01 and from 4.52 ± 0.60 to 4.85 ± 0.67 mm for placebo group; P < 0.01, respectively) in both groups. The flow-mediated dilation of the brachial artery (FMD) in the HCP (13.58 ± 7.22%) were increased significantly when compared to the baseline (7.53 ± 6.76%; P < 0.01) and placebo group (8.19 ± 12.83%; P < 0.01).
Fig. 5Results from the color doppler flow imaging metrics of the subjects, including (a) IMT, D0, and D1 (mmHg) and (b) FMD (%) in the test product group (HCP; n = 54) and the placebo group (n = 60) before and after the intervention. Results are expressed as mean ± standard deviation. # significant at P < 0.05 compared to the baseline; ## significant at P < 0.01 compared to the baseline; * significant at P < 0.05 compared to the placebo group by two-way repeated measures ANOVA. IMT: intima-media thickness; D0: brachial artery basal internal diameter; D1: end-diastolic internal diameter of the brachial artery; FMD: flow-mediated dilation of the brachial artery.
All reported symptoms are listed in Supplementary Table S6. The treatment improved the recorded seven symptoms (headache, dizziness, palpitation, tinnitus, insomnia, dysphoria, and soreness and weakness of waist and knees) to a moderate extent in the HCP with each improvement rate of more than 50%. For the main symptom scores of subjects, a significant downward trend was observed in the HCP from (mean ± SD) 2.51 ± 0.89 to 1.94 ± 1.06 at 4 weeks, and to 1.26 ± 0.95 at 8 weeks after starting to take test tablets (P < 0.01), as shown in Supplementary Fig. S3. These results suggested that HCP-C7 C12 possesses a favorable safety profile, and may have a role in improving specific symptoms.
Serum differential metabolites
A serum non-targeted metabolomic analysis was performed to explain the potential molecular mechanisms underlying the blood pressure-improving effects of HCP-C7 C12 intervention. Orthogonal partial least squares discriminant analysis (OPLS-DA) analysis unveiled a significant demarcation in the HCP group before and after the intervention (Supplementary Fig. S4). The types of serum metabolites varied significantly before and after intervention. Out of the ten potential biomarkers that were found, three were found to be up-regulated and seven to be down-regulated (Table 2). The outcomes elucidated that HCP-C7 C12 intervention led to reductions in the relative levels of serum constituents including D-Ala-D-Ala, histidine, phenylalanine, epiandrosterone, all-trans-retinoic acid, methionine and leucine. Conversely, the relative levels of serum betaine, Vitamin K1 and phosphocholine witnessed increases.
Table 2 Differential serum metabolites and their alteration trends in the HCP group before and after the intervention by orthogonal partial least squares discriminant analysis (OPLS-DA).Gut microbiota diversity
We further analyzed the gut microbiota to explore whether undigested casein peptides in HCP-C7 C12 indirectly regulate blood pressure by modulating the gut microbiota. The composition of the gut microbiota was assessed in 39 participants (19 in the HCP, 20 in the placebo group) before and after the intervention. At the operational taxonomic unit (OTU) level, alpha-diversity of the gut microbiota was assessed using Shannon, Simpson, Chao1 and ACE indices. In the HCP, simpson, Chao1 and ACE indices all showed increasing trends, but did not reach statistical significance. At the end of the intervention, all alpha-diversity indices in the HCP were higher than those in the placebo group (Fig. 6a-d).
Fig. 6(a) Alpha diversity of intestinal microbiota including Shannon, Simpson, Chao1 and ACE of baseline and end in the placebo and intervention group. (b) The principal coordinate analysis (PCoA) performing on Bray-Curtis distances among intestinal microbiota before and after intervention in two groups. LEfSe multilevel discriminant analysis of species differences (LDA > 1.0) (c) in the HCP group before and after the intervention, and (d) between the two groups after the intervention. n = 19 in the test product group (HCP); n = 20 in the placebo group. Placebo_BL: the baseline of the placebo group; Placebo_END: the endpoint of the placebo group; HCP_BL: the baseline of the HCP group; HCP_END: the endpoint of the HCP group.
The similarities and distinctions in the composition of gut microbiota between the two groups were depicted through principal coordinate analyses (PCoA) utilizing unweighted Unifrac distance (Fig. 6e). The results indicated that the samples of the HCP and the placebo group were basically clustered in different quadrants, and no significant difference was observed in microbial diversity in both groups when self-compared before and after the intervention, but it was significant when compared between the two groups (R2 = 0.059, P < 0.05).
When it comes to the gut microbiota composition, the phylum Bacillota dominated the intestinal microbiota (Supplementary Fig. S5a). Faecalibacterium, Gemmiger, Escherichia, Blautia, and Bifidobacterium were the predominant genera (Supplementary Fig. S5b).
According to LEfSe (Fig. 6f-g), a total of 5 and 46 biomarkers (LDA > 1) that were significantly different in the HCP groups before and after the intervention, and between the HCP and placebo groups after the intervention were identified, respectively. Bacillota was the particular taxonomic group that has the greatest LDA score between the two groups. At the genus level, we identified that the level of Fodinicurvata was lowered after the intervention in the HCP group. When compared with the placebo group after the intervention, subjects in the HCP had higher levels of nine bacterial genera including Clostridium, Gemella, Roseburia, Parasutterella, Muribaculaceae, Adlercreutzia, Escherichia, Guopingia, and Dorea, while seven exhibited significantly decreased relative abundance, including Klebsiella, Haemophilus, RF39, Lawsonibacter, Alistipes, Faecalibacterium, Merdimonas.
As for the PICRUSt2-based gut microbiota function prediction (Supplementary Fig. S6), compared to the baseline, the HCP exhibited a significant increase in the mean proportion of two functional pathways, including 2-aminophenol degradation (P = 0.039) and L-tryptophan degradation XII (P = 0.046). 54 functions underwent significant changes when compared between the HCP and placebo groups after the intervention, with a notable increase in the mean proportion of L-valine degradation I (P = 0.024), pyruvate fermentation to propanoate I (P = 0.000741), tricarboxylic acid (TCA) cycle V (P = 0.00471), TCA cycle I (P = 0.024). Additionally, the mean proportion of various functions associated with the metabolism of blood pressure-related amino acids saw a significant change with a decrease of superpathway of biosynthesis (P = 0.018), superpathway of L-lysine, L-threonine and L-methionine biosynthesis I (P = 0.024) and an increase of L-arginine biosynthesis III increases (P = 0.018).
These results demonstrate that the supplementation of HCP-C7 C12 indeed exerted a prebiotic effect, activating the propionate and butyrate production pathway and increasing the abundance of gut microbes that produce short-chain fatty acids (SCFAs) or exhibit anti-inflammatory activity, such as Roseburia lenta, Agathobaculum butyriciproducens, and Adlercreutzia equolifaciens16,24,25,26.
Correlation analysis
The results presented in Fig. 7 unveil significant correlations. Specifically, vitamin K1 exhibited a negative correlation with Muribaculum gordoncarteri (r = −0.380, P = 0.007), Actinomyces graevenitzii (r = −0.383, P = 0.007), and Lachnoanaerobaculum gingivalis (r = −0.393, P = 0.005), but positively correlated with Guopingia tenuis (r = 0.294, P = 0.040) and Faecalibacterium prausnitzii (r = 0.447, P = 0.001). Histidine exhibited a negative correlation with Guopingia tenuis (r = −0.316, P = 0.027), Lawsonibacter asaccharolyticus (r = −0.377, P = 0.008), Adlercreutzia equolifaciens (r = −0.333, P = 0.020), and Eisenbergiella porci (r = −0.331, P = 0.020). Furthermore, phosphocholine was in a positive relation to Muribaculum gordoncarteri (r = 0.371, P = 0.009), Haemophilus aegyptius (r = 0.413, P = 0.003), and Fusobacterium varium (r = 0.369, P = 0.009), while the latter two were reversely correlated with epiandrosterone (r = −0.392, P = 0.005 for Haemophilus aegyptius; r = −0.328, P = 0.021 for Fusobacterium varium). Notably, phosphocholine was negatively correlated with SBP (r = −0.327, P = 0.022). In addition, betaine displayed a positive correlation with Klebsiella variicola (r = 0.339, P = 0.017) and Clostridium transplantifaecale (r = 0.297, P = 0.038). Methionine and leucine were in positive relation to Roseburia lenta (r = 0.314, P = 0.028) and Guopingia tenuis (r = 0.294, P = 0.020), respectively. Phenylalanine, all-trans retinoic acid, and D-Ala-D-Ala were all in negative relation to Clostridium porci (r = −0.302, P = 0.035), Agathobaculum butyriciproducens (r = −0.284, P = 0.048), and Fodinicurvata sediminis (r = −0.317, P = 0.027), respectively.
Fig. 7Heat map of Spearman correlations of blood pressure, differential gut bacterial species and the significant serum differential metabolites. * P < 0.05 obtained using Spearman rank correlation analysis. The color bar with numbers indicates the correlation coefficients. SBP: systolic blood pressure; DBP: diastolic blood pressure.
Safety
Most of the subjects maintained good mental status, sleep quality, dietary status as well as urinary and fecal conditions throughout the study, which indicated test tablets had no adverse effects on their general conditions (Supplementary Table S7). No significant differences of the blood, urine, stool routine, and blood biochemical parameters in both HCP and placebo groups were observed at the end of the intervention (Supplementary Table S8). No allergy or other adverse effects including nausea, flatulence, diarrhea, and abdominal pain were experienced by the subjects.
Discussion
Our study showed that oral administration of hydrolyzed casein hydrolysates for 8 weeks reduced both SBP and DBP in prehypertensive or hypertensive adults who were not taking medication during the whole intervention period. The reductions in SBP and DBP in the HCP were 14.02 mmHg and 8.11 mmHg, respectively, corresponding to reduction rates of 9.41% and 9.53%. These findings are in line with previous long-term intervention studies on casein hydrolysates27,28,29. The antihypertensive effects of HCP-C7 C12 can be attributed to the presence of ACEI peptides and the pronounced prebiotic effects exerted by certain peptides.
In our study, no subjects reported allergies or other adverse effects such as nausea, flatulence, diarrhea, or abdominal pain, and more than half of the HCP subjects with at least one of the recorded symptoms reported an improvement after the ingestion of casein hydrolysate tablets. This aligns with previous evidence that casein hydrolysates can alleviate insomnia, muscle soreness, and stress-related disorders30,31. This is particularly noteworthy, as some antihypertensive medications, especially ACE inhibitors, are associated with side effects such as cough and renal dysfunction32. Tuomilehto et al.33 clarified that the ACE inhibitory activity of any antihypertensive medications that primarily works by ACE inhibition is far higher than that of the food-derived peptides. This may explain why our test tablet showed a blood pressure-lowering effect without the characteristic side effects of antihypertensive medications. Thus, these results indicated the test tablets containing casein hydrolysate had not only no detrimental effects on the overall health of humans, but also certain amelioration effects on some common symptoms.
The antihypertensive effect of HCP-C7 C12 was likely mediated through multiple mechanisms. First, HCP-C7 C12 acted as a potent ACE inhibitor to reduce blood pressure. The composition and structure of amino acids contributed substantially to the ACE inhibitory activity. Interactions between multiple hydrogen bonds between ACE and peptides stabilize the enzyme-peptide complex, which in turn promote peptide-induced inhibition of ACE activity34. According to molecular docking results, the two casein peptides were relatively hydrophobic, as GPFPIIV can interact with HIS-383, VAL-518, PHE-512, VAL-380, PHE-527, PHE-457, TYR-523, HIS-387, while FFVAPFPEVFGK can interact with VAL-379, VAL-380, ALA-354, HIS-383, HIS-410, HIS-387, TRP-59, PHE-391. In particular, the benzene ring of FFVAPFPEVFGK can form pi-anion, pi-pi conjugated interaction with ASP453, HIS-410, HIS-387. It is noteworthy that the peptide bond of both two casein peptides can form coordination with zinc ion, which has an important contribution to the stability of small molecules. The formation of stable complexes between small molecules and ACE can be efficiently encouraged by interactions including hydrogen bond interactions, hydrophobic interactions, and electrostatic interactions. Based on the findings, it was possible that FFVAPFPEVFGK may have had more inhibitory effectiveness than GPFPIIV because of its greater interactions inside the ACE. Furthermore, both FFVAPFPEVFGK and GPFPIIV contain 2–12 amino acids, which is consistent with the finding that reported the number of amino acids found in the most ACE inhibitory peptides35. According to the literature, the types of amino acids located in three positions near the C-terminal of ACE inhibitor peptide are important for activity. The amino acid residues at the C-terminal position of the peptide have substantial impacts on the binding of these peptides to ACE. Generally, peptides containing hydrophobic, aromatic, positive, and basic residues at the C-terminal position are the most effective ACE inhibitors5,36,37. Peptides containing hydrophobic residues, taken proline and phenylalanine as examples, at the C-terminal position are the most effective ACE inhibitors9,36. Due to the unique chemical structure of proline, it is able to have an interaction with positively charged residues of the enzyme36. In addition, the presence of branched aliphatic residues, such as valine and isoleucine, at the N-terminal position has also been considered to improve ACE inhibitory activity35. Taken together, the presence of isoleucine and valine residues at N-terminus positions, respectively, and proline at the C-terminus position are considered to contribute to the ACE inhibitory activity. Furthermore, the positive charge on the ε-amino group of C-terminal lysine side-chains considerably adds to the ACE inhibitory activity35. In terms of the peptide FFVAPFPEVFGK, the third amino acid at the C-termina is phenylalanine (Phe, F), which is an aromatic amino acid, and the third amino acid at the N-terminal is valine (Val, V). However, the C-terminal 3 amino acids of peptide GPFPIIV do not contain aromatic, positive, or basic amino acids. Therefore, FFVAPFPEVFGK may have better activity than GPFPIIV partly because the composition of C-terminal amino acids of the former one is more conducive to inhibiting ACE activity.
Although both peptides have proven to be good ACE inhibitors in vitro, their ability to maintain bioactivity after gastrointestinal digestion is a concern. The structural changes will possibly perform when proteins are absorbed into the gastrointestinal tract, which could further interfere with the bioactivity of peptides36. The degree of enzymatic hydrolysis of bioactive peptides mainly depends on the degree of sensitivity to gastrointestinal digestive enzymes. The amino acid composition, peptide chain length and physicochemical properties of active peptides are different to the degree of sensitivity to enzymes. For example, active peptides containing proline and hydroxyproline residues are usually able to resist the degradation of digestive enzymes and maintain structural integrity38. Therefore, it is assumed that these peptides can stay bioactive during gastrointestinal digestion and reach the cardiovascular system to exert an antihypertensive effect as they contain proline residues.
The mechanism behind the hypotensive effect of casein hydrolysates cannot be explained just by the ACE inhibitory activity of the casein peptides because food-derived peptides have less powerful ACE inhibitory activity than ACE inhibitors. Yoshizawa et al.39 reported that lactotripeptides can result in an increase in FMD, which was consistent with our findings, suggesting that HCP-C7 C12 tablets can improve vascular endothelial function. Therefore, the improvement of vascular endothelial function may be also considered as a possible explanation for the blood pressure-lowering effect.
In our study, the level of serum betaine was significantly increased after the intervention (Table 2). The three metabolites with significantly increased levels were negative correlated with SBP and DBP (Fig. 7). Betaine is described as having an anti-inflammatory effect and acts as an important methyl donor in the transmethylation of homocysteine to methionine40,41. Homocysteine is a cardiovascular risk factor that promotes oxidative stress, inflammation, and endothelial dysfunction42. Inflammation is known to promote the development of hypertension by downregulating nitric oxide synthase activity and producing an excess of reactive oxygen species, which in turn produce endothelial dysfunction and oxidative stress, respectively43. Therefore, an increase in betaine levels may influence the blood pressure by improving endothelial function and arterial stiffness. Conversely, serum methionine level was significantly decreased in the HCP group after the 8-week intervention (Table 2). An increase in homocysteine levels appears to be indirect mechanism by which methionine affects blood pressure, which alters the endothelial function by stimulating the production of asymmetrical dimethylarginine, thereby limiting the synthesis of NO and ultimately elevating blood pressure44. In addition, HCP-C7 C12 intervention also caused a significant decrease in serum leucine and phenylalanine levels (Table 2). One study has found that leucine activated mTORC1 in the brain of rats, which would increase the activity of sympathetic nerves connecting the brain and kidneys, thereby causing an increase in blood pressure45. Hence, declining the level of serum leucine can significantly inhibit the increase of blood pressure. In addition, high levels of phenylalanine can be converted to tyrosine, which as a precursor of norepinephrine synthesis can activate the sympathetic nervous system, as well as damage to vascular endothelial cells, increase reactive oxygen species, and increase oxidative stress state, which further leads to vasoconstriction and ultimately induces hypertension46,47. Notably, the three serum metabolites with significantly decreased levels had strong positive correlations with SBP (Fig. 7).
This study also revealed that HCP-C7 C12 exerted prebiotic-like effects, modulating the gut microbiota in ways that may contribute to its antihypertensive effects. Studies have shown that a higher abundance of Klebsiella, Desulfovibrio and Alistipes had an association with higher blood pressure, while Clostridium and Roseburiawere confirmed to be deficient in hypertensive models48,49,50,51. According to our findings (Fig. 6c-d), the abundance of the above bacterial genera was significantly improved after the intervention. Furthermore, the Roseburia species and Agathobaculum butyricproducenshave been suggested as potential health markers because of their anti-inflammatory and butyrate-producing properties25,26. They produce SCFAs, particularly butyrate, which play an anti-inflammatory role by binding to several G-protein-coupled receptors and acting as histone deacetylase inhibitors52. In addition, Adlercreutzia equolifaciensalso possesses anti-inflammatory properties24. Notably, the metabolites with significantly reduced serum metabolite levels showed a significant negative correlation with beneficial bacteria. For example, histidine was negative correlated with Adlercreutzia equolifaciens and Lawsonibacter asaccharolyticus. In addition, phenylalanine, all-trans retinoic acid, and D-Ala-D-Ala all were negatively correlated with Agathobaculum butyriciproducens. Conversely, Vitamin K1 and betaine, the serum metabolites with significantly increased levels, were positively correlated with Faecalibacterium prausnitzii and Clostridium transplantifaecale. These results indicate that our HCP-C7 C12 is beneficial in promoting the production of SCFAs, as well as the anti-inflammatory effects of beneficial bacteria, thus achieving a hypotensive effect.
In terms of metabolic function, there were 54 different metabolic pathways between the HCP and placebo groups, which mainly involved in nucleotide degradation, amino acid synthesis, and glycolytic pathway. HCP-C7 C12 tablet ingestion enhanced the pathways involved in propionic acid production, thereby contributing to blood pressure reduction. As for the metabolic pathway of amino acids, studies have shown that amino acids influence blood pressure through a variety of pathways, including NO synthesis, vascular remodeling, and neurotransmitter production53. High L-valine concentrations have been proved to be associated oxidative stress54, thus promoting hypertension. L-Arginine acts as a precursor molecule for the synthesis of NO, resulting in blood vessel relaxation and improved blood flow in endothelial cells55. Conversely, L-lysine was found to be hypercholesterolemic, which further exacerbated the NO-dependent endothelial dysfunction55. The significant differences in the amino acid metabolic pathways caused by our study may explain the mechanism of lowering blood pressure by HCP-C7 C12.
One of the strengths of our study is its randomized double-blind placebo-controlled clinical design and this was the first time to elucidate the blood pressure-lowering effect of HCP-C7 C12 in clinical trial. Subjects appeared to follow the instructions well and data were analyzed based on the per-protocol set. As we took into account not only the blood pressure levels of the subjects after random grouping, but also the main factors affecting the outcome such as age, gender, course and severity of disease, and type of medication taken, as far as possible, the baseline characteristics, including the blood pressure levels at the initial, were similar between the HCP and placebo groups. Subjects were also allowed to maintain their living habits including physical activity and dietary patterns throughout the study. Therefore, similar results could be observed in different populations. It was shown that the HCP-C7 C12 tablet can reduce blood pressure without common side effects associated with antihypertensive drugs. In addition, molecular docking further suggested that the ACE inhibitory activity of HCP-C7 C12 might be linked to the creation of H-bonds and other interactions with active site residues. Furthermore, we investigated the possible mechanisms of lowering blood pressure in terms of serum differential metabolites and changes in intestinal microbiota. However, there were some limitations in our study. The results may be overestimated due to the high initial blood pressure levels of subjects. In addition, this trial only enrolled subjects who were not yet taking hypotensive medications, so whether HCP-C7 C12 is effective in subjects taking antihypertensive drugs needs further investigation. In the future, the levels of bradykinin, NO, and SCFAs should be measured to provide a more comprehensive understanding of the blood pressure-lowering mechanism.
To conclude, we confirmed that an eight-week intervention with oral administration of the HCP-C7 C12 tablets significantly improved both SBP and DBP compared to the placebo in adults with prehypertension or hypertension in a randomized, double-blind, placebo-controlled clinical trial. The antihypertensive mechanisms may be due to (1) HCP-C7 C12 acting as an ACE inhibitory peptide, inhibiting the production of angiotensin II, (2) intervening to induce changes in amino acid abundance, resulting in anti-inflammatory and antioxidant effects, as well as improving endothelial function. HCP-C7 C12 also exerted prebiotic-like effects and increased the levels of the plasma anti-inflammatory molecules. These recent study results suggest that long-term intakes of the tablets containing HCP-C7 C12 might be beneficial for the management of blood pressure, and thereby cardiovascular event protection in prehypertensive/hypertensive individuals.
Materials and methods
Study materials
Test tablets were supplied by Guangzhou Greencream Biotech Co., Ltd. The test product and the placebo were both manufactured as tablets with the same packaging, flavor, and appearance. Each tablet, with a net weight of 0.4 g, comprised 37.5% (150 mg) HCP-C7 C12. The primary ingredient of the test tablet was hydrolyzed casein peptide powder, as opposed to the maltitol and milk powder that mainly composed the placebo tablet. Thus, casein hydrolysate was not found in the placebo tablets. The HCP group ingested two tablets each time, twice a day, while the placebo group took the same dosage of placebo tablets instead.
HCP-C7 C12 and HCP002 were two hydrolyzed casein peptide complexes, supplied by Guangzhou Greencream Biotech Co., Ltd. The preparation process and the enzyme preparations used were roughly the same, but they differed in the post-treatment process of the enzymatic hydrolysis products. HCP-C7 C12 was mainly enriched in a certain molecular weight range of peptides through the combination of primary filtration and ultrafiltration, while HCP002 was enriched in the way of precipitation centrifugation of all the peptides obtained by hydrolysis.
Measurement of ACE-inhibitory activity
The ACE-inhibitory activity was measured using a previous published method of Shalaby et al.56, with minor modifications. A glass bottle of ACE with a 0.25U unit was gradually filled with 1 mL of distilled water. Mixed well and set aside for use. The following were the specific methods: the enzyme-labeled plate was filled with 10 µL of ACE aqueous solution (0.25 U/mL) and 10 µL of ACE inhibitory peptide sample with varying concentrations (0.25, 0.5, 1.0, 2.0, and 4.0 mg/mL) without being mixed for a while. Then 150 µL of preheated (37℃, 5 min) substrate (1.0 mmol/L Furanacroloyl-Phe-Glu-Glu dissolved in 50 mmol/L Tris-HCl buffer with pH 7.5 and NaCl 0.3 mol/L) was added to initiate the reaction. The microplate was quickly placed into the enzymoscope and the absorbance A1 at 340 nm was recorded. The absorbance A2 at 340 nm was measured again 30 min after the reaction. In the blank control, 10 µL Tris-HCl buffer was substituted for the ACE inhibitory peptide-like solution, and the initial absorbance of the blank was denoted as A01 and the post-reaction as A02. The absorbance change was used to compute the ACE inhibition rate. (△Ainhibitor = A1 - A2, △Ablank = A01 - A02).
$${\rm Relative \:ACE \:inhibitory \:activity} \:(\%) = (1-\frac{\Delta\: {\rm Ainhibitor}}{\Delta\:\:Ablank})\:\times\:100\%$$
Various concentrations of the samples were assayed to calculated the IC50, which represents the concentration of inhibitor required to 50% inhibitory activity.
Identification of peptides in hydrolyzed casein peptide complex
The hydrolyzed casein peptide complex with higher ACE inhibitory activity was diluted into 2 mg/mL solution with sterile water, and then filtered by disposable 0.22 μm needle filter. The permeate was subjected to High Performance Liquid Chromatography-electrospray tandem mass spectrometry (HPLC-MS/ESI) on an Agilent 6530B Quadrupole Time-of-Flight (Q-tof) mass spectrometer. ACQUITY UPLC HSS T3 Column (100 mm×2.1 mm with a particle size of 1.8 μm) was used with the mobile phase A of 0.1% (v/v) formic acid solution and the mobile phase B of acetonitrile by gradient elution. Elution was performed with a linear gradient of solvent in A from 0 to 5% B in 4 min, 5–10% in 2 min, 10–40% in 24 min and 40–90% in 4 min at 40℃ and a flow rate of 0.05 mL/min. The parameters of MS/MS were set as: positive ion mode; ESI ion source temperature was 500℃; spray voltage was 5500 V.
Prediction of potential biological activity of peptides
PeptideRanker website (http://distilldeep.ucd.ie/PeptideRanker/) was used to evaluate the potential target bioactive peptides. A unique PeptideRanker score, ranging from 0 to 1, was obtained by entering the peptide’s amino acid sequence into the website; higher scores indicate a higher likelihood of becoming a bioactive peptide. PeptideRanker was utilized in this study to forecast the potential biological activity of peptides and was applied in the screening process of active peptides.
Molecular Docking
Molecular docking was performed using the Discovery Studio 2019 software (DS 2019, Accelrys Software Inc., San Diego, USA). The molecular structures of GPFPIIV and FFVAPFPEVFG used for docking were generated according to their amino acid sequences with Discovery Studio 2019. The crystal structure of human ACE was downloaded from the Protein Data Bank (PDB, 1O8 A.pdb). Prior to docking, the ACE crystal structure was treated with water removal and hydrogenation. After that, Minimization module was used for optimization and energy minimization, and CHARMm was selected as the force field. The treated peptides served as ligands for molecular docking. The binding site with a radius of 16 Å and coordinates xyz were 39.32, 38.43 and 49.78, respectively. The interconnection mode was semi-flexible and the interconnection function was CDOCKER. According to the molecular docking results, the interactions between the molecular docking results were evaluated, such as the -CDOCKER_ENERGY value, interaction sites and interaction force types.
Randomized controlled trial design
The study was conducted in accordance with the principles of the Declaration of Helsinki. The trial has been formally registered in the China Clinical Trial Registry (No. ChiCTR2300075309). All subjects provided written informed permission before the study after the protocol was approved by the Institutional Review Board committee of Qingdao University (QDU-HEC-2023156).
The study was a randomized, double-blind, placebo-controlled clinical trial carried out at the Affiliated Hospital of Qingdao University, which started in December 2023 and was scheduled to last 8 weeks. Building upon the insights of preceding research57, we adopted a significance threshold of 0.05, an efficacy rate of 0.80, and anticipated a sample omission rate of 10%. Consequently, the recruitment target was set at 96 subjects. Subjects were randomly allocated into the test product group (HCP) and the placebo group following stratified randomization procedures. An allocation coordinator who was not involved with the researchers conducted the randomization according to a random-number table, and the randomization code was kept in a sealed envelope to ensure blinding. Therefore, the treatment assignments were kept blinded from the researchers, participants, and other study staff during the intervention period. Main factors affecting the results such as age, gender, course and severity of disease, and type of medication taken were considered so that a balance test could be conducted to ensure comparability between groups.
Subjects in the HCP were given two HCP-C7 C12 tablets each time, twice a day by oral administration for 8 consecutive weeks. Similarly, subjects in the placebo group took placebo tablets in the same dosing regimen. No food or exercise restrictions were imposed throughout the intervention.
Subjects would be quality-control followed up during the intervention period to inquire about and record any adverse product reactions. At the end of the intervention, safety and efficacy parameters were tested. The differences in the latter before and after the intervention were compared by data statistics.
Study population
Subjects from the Affiliated Hospital of Qingdao University were included in the study. Inclusion criteria included: (1) age range of 30 to 65 years; (2) higher than 120 mmHg SBP and/or 80 mmHg DBP, where blood pressure in the range of 120–139 mmHg systolic and/or 80–89 mmHg diastolic blood pressure was defined as prehypertension58. Criteria for exclusion were as follows: (1) diagnosis of severe systemic diseases such as liver, kidney, and hematopoietic disease; (2) pregnancy or breastfeeding; (3) an allergy to the test product; (4) reluctance or incapacity to complete the study. A total of 131 subjects were eventually enrolled, of which 57 were male and 74 were female.
Outcome measures
The primary outcome of the study was the change rate of blood pressure compared to the baseline. The secondary outcomes included the changes in the serum biomarkers, FMD, as well as adverse events and tolerability throughout the study, including gastrointestinal symptoms, allergic reactions, and other potential side effects.
Blood collection and analysis
Fasting (12 h overnight) blood samples (10 mL) were collected twice in total: at the baseline and the end of the study respectively. Serum was centrifuged for 15 min (3000 r/min) and instantly stored at −80 °C, which were further used to measure the following parameters: white blood cell count, platelet count, red blood cell count, haemoglobin, total protein, alanine aminotransferase, aspartate aminotransferase, albumin, creatinine, urea, glucose, plasma viscosity, whole blood high shear viscosity, whole blood low shear viscosity, fibrinogen, hematocrit, erythrocyte electrophoretic time, erythrocyte aggregation index, erythrocyte deformation index, the index of rigidity of erythrocyte, total cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein, Lp-PLA2, endothelin-1, and ICAM-1. An automated analyzer (Hitachi 7180, Hitachi Instrument, Suzhou, Jiangsu; LB-2 A, Hlife Technology, Shunyi, Beijing) and Elisa kits (Human ET-1 ELISA Kit & Human ICAM-1 ELISA Kit, Jingmei Biotechnology Co., Ltd., Jiangsu, China) were used to measure these parameters.
For non-targeted metabolomic analysis of serum, 50 µL of serum was mixed with 250 µL pre-cooled acetonitrile at room temperature and then incubated on ice for 15 min. Then, the supernatant was centrifuged at 15,000 r/min at 4 °C, passing through a 0.22 µM organic membrane before addition to the Sampling vial. Quality Control (QC) samples were created by mixing 2 µL of all the samples together and subsequently separated via ultra-performance liquid chromatography (UPLC) system (Agilent, Boston, USA). Furthermore, to assess the stability of the Liquid Chromatography-Mass Spectrometer (LC-MS) analysis, the QC sample was examined every 6 samples59. An Agilent 6530B Q-tof mass spectrometer fitted with an ion funnel (iFunnel) electrospray ionization source was used to acquire mass spectrometry (MS) data. Data were acquired in ESI (+) mode, with the following source parameters: the auxiliary gas flow rate was 13 µL, the sheath gas flow rate was 40 µl, the capillary temperature was 320℃, the atomizer temperature was 420℃, and the spray voltage was 3.5 kv. The data acquisition mode was Full scan + ddMS2, and the first-level scan range was m/z 100–1200. Reference masses of 121.050 and 922.009 were used to continuously calibrate the mass accuracy. The data were acquired in centroid mode, and the raw data files (.d) were converted to.abf format using the ABF converter (https://www.reifycs.com/AbfConverter/index.html). The MS-DIAL software executed peak detection, alignment, gap filling, and annotations. Compound annotations were conducted by comparing their MS/MS spectral similarity with the MoNA (https://mona.fiehnlab.ucdavis.edu) MS/MS libraries, MyCompoundID database (http://www.mycompoundid.org), and Metlin (https://xcmsonline.scripps.edu). OPLS-DA and peak table preprocessing were performed using integrated mass spectrometry-based untargeted metabolomics data mining (IP4M) software60. Based on the variable importance in the projection (VIP) threshold of 1 from the OPLS-DA model, differential metabolites in the metabolic profiles of the HCP at the end of intervention compared with baseline, and the HCP compared with the placebo group were obtained.
Stool collection and 16 S rRNA amplicon sequencing
Subjects were asked to collect 5–10 grams of feces in 30 mL sterile containers with screw caps and placed them in an ice box or −20°C refrigerator for storage. These samples were retrieved within 5 h and frozen at −80°C. Sequencing of the 16S rRNA-amplified fragments (v3-v4 from the fecal microbiota of the participants was performed by Beijing Biomarker Technologies Co. Ltd. Barcoded specific bacterial primers was used for PCR amplification: forward primer 5’- ACT CCT ACG GGA 210 GGC AGC A-3’ and reverse primer 5’-GGA CTA CHV GGG TWT CTA AT-3’). Raw sequencing data were deposited in the Sequence Read Archive Database of the National Center for Biotechnology Information (accession number: PRJNA1196614). Gut microbiota sequencing data were analyzed using EasyAmplicon v1.14 (https://github.com/YongxinLiu/EasyAmplicon). The NCBI 16 S Microbial database served as the basis for the BLCA algorithm, which was used to reclassify representative sequences. The 1. subset db acc.py from the BLCA package was used for processing61. Furthermore, analysis was also performed online using the MicrobiomeAnalyst (https://www.microbiomeanalyst.ca/MicrobiomeAnalyst)62. The functional content of microbial communities was predicted using PICURSt2 software package (v2.2.0-b) as MetaCyc ortholog profiles. The significance of species, genera, and MetaCyc pathways was further analyzed using STAMP software63.
Safety and efficacy indices
The general conditions of subjects, including mental status, sleep quality, dietary status, and urinary and fecal conditions, were recorded by face-to-face inquiry and graded as poor, average, and good. Electrocardiogram, X-ray, abdominal ultrasound, blood, urine, and stool routine examinations, and hepatic and renal functions tests were performed both before and after the intervention to determine study eligibility and to ensure no physiological illness was present as well as no significant changes in relevant parameters of these tests.
The main symptoms of subjects were recorded by inquiring each subject whether and how severely they had experienced the following: headache, dizziness, palpitation, tinnitus, insomnia, dysphoria, as well as soreness and weakness of waist and knee. There were three records in total: on the day subjects began to take the tablets, at week 4 and at the end of the intervention.
Blood pressure and heart rate were measured every week during the intervention period. Subjects were advised not to drink alcohol and coffee, not to eat and drink anything after 21:00, not to do vigorous activities, and not to smoke on the day before the measurement. Blood pressure was measured in the morning from the right arm using the same automatic sphygmomanometer (RBP-300, Raycome, Shenzhen, Guangdong) by the same experienced nurse during the whole intervention period. Subjects were required to keep a sitting position when they arrived at the hospital following 15 to 20 min of rest. Measurements of blood pressure were undertaken at 2-minute intervals of a total of 2 times, and the mean value of these two measurements was recorded as the blood pressure. If there was a difference of more than 5 mmHg between two values for the same subject, a third measurement was taken. Changes in blood pressure were compared by subtracting baseline blood pressure at the intervention nodes.
The endothelial function of the brachial artery, including intima-media thickness, D0, and D1, was measured by LOGIQ E9 color Doppler ultrasound diagnostic instrument twice in total: at the baseline and the end of the intervention. Subjects were placed in a supine position after resting for 5 min. The probe (7 MHz linear array probe) was placed in the brachial artery 2 cm above the elbow of the right upper arm, and the pulsed Doppler sampling volume was placed in the center of the vessel with an angle of intersection between the sound beam and the vessel < 60°. After D0 at rest was measured, the sphygmomanometer cuff was inflated and pressurized to 280 mmHg, which was maintained for 4 min before deflation. The end-diastolic internal diameter of the brachial artery at the same site was measured in succession within 60 to 90 s after deflation. D0 and D1 were each measured three times, and the average of these three readings was used to determine their respective values. The FMD of the brachial artery was calculated as follows: FMD (%) = (D1-D0)/D0 \(\:\times\:\) 100%.
Statistical analysis
The parameters analyzed in the study were expressed as the mean and standard deviation (SD). The efficacy analysis was conducted using the per-protocol analyses.
Depending on the distribution’s normality, either one-way analysis of variance (ANOVA) or the Kruskal-Wallis test was employed, while a paired sample t-test or the Wilcoxon signed-rank test was utilized as appropriate to compare pre- and post-intervention results. Two-way repeated measures ANOVA was used to compare the differences between groups. The significance level for each two-sided test was set at 5% or 1%.
The main symptom scores were graded by self-assessment of the subjects according to the severity: 3 for severe, 2 for moderate, and 1 for mild. The lower score the milder symptoms, while the higher score the more severe symptoms. At the end of the study, an improvement in symptom scores greater than or equal to 1 was considered effective.
Statistical analyses were performed using R software (R Foundation for Statistical Computing, Vienna, Austria) and SPSS26. Spearman correlation analysis was performed to determine the correlation among the blood pressure, differential gut bacterial species and serum metabolites using Spearman’s ranking, implemented through the R package ‘psych’. The R package pheatmap was used to visualize the data matrix. Differences in the beta-diversity were explored by bray_curtis distance based PCoA and tested through permutational multivariate analysis of variance (PERMANOVA). The linear discriminant analysis (LDA) effect size (LEfSe) was employed to conduct the quantitative analyses of biomarkers across groups. Bacteria with LDA score > 1 and P-value < 0.05 were screened for significant changes. To identify changes in microbial communities between the two groups, similarity analysis and permutational multivariate ANOVA were done using Bray-Curtis dissimilarity distance matrices. Bilateral P < 0.05 was regarded statistically significant, and the corrected P-value was determined using Storey’s False Discovery Rate (FDR) method to adjust for multiple comparisons.
Data availability
Raw sequence data are available in the Sequence Read Archive (SRA) under BioProject accession PRJNA1196614. It is accessible at: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1196614/.
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Funding
This research was supported by Qingdao University Joint Research Platform Foundations (BZ-KYZRKX-20211108-031).
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Authors and Affiliations
Institute of Nutrition and Health, Qingdao University, Qingdao , China
Kexin Li, Jin Sun & Ce Qi
Jiaozhou Maternal and Child Health Care Family Planning Service Center, Traditional Chinese Medicine, Hong Kong, China
Peng Jiang
Qingdao University-Fine Bio-Breast Milk Source Nutrients and Health Joint Research Platform, Qingdao, 266071, China
Shuangqi Li
Authors
- Kexin Li
- Peng Jiang
- Shuangqi Li
- Jin Sun
- Ce Qi
Contributions
K. L., P. J., S. L., J. S., and C. Q. conceived of and designed the experiments. K. L. and P. J. carried out the studies, participated in collecting data, and performed the statistical analysis. K. L., S. L., and J. S. participated in acquisition, analysis, or interpretation of data and draft the manuscript. All authors read and approved the final manuscript.
Corresponding author
Correspondence to Ce Qi.
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The authors declare no competing interests.
Ethics approval and consent to participate
This work has been carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. This research was approved by the Institutional Review Board committee of Qingdao University (Ethics number: QDU-HEC-2023156) and registered with the China Clinical Trial Registry (No. ChiCTR2300075309) on 01/09/2023. Informed consent was obtained from all participants.
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Li, K., Jiang, P., Li, S. et al. ACE inhibitory casein peptide lowers blood pressure and reshapes gut microbiota in a randomized double blind placebo controlled trial. Sci Rep 15, 13840 (2025). https://doi.org/10.1038/s41598-025-98446-6
Received: 16 January 2025
Accepted: 11 April 2025
Published: 22 April 2025
Version of record: 22 April 2025
DOI: https://doi.org/10.1038/s41598-025-98446-6