IC50: 5 μM (ACE)[2]
Angiotensin-converting enzyme (ACE), a key enzyme in the renin-angiotensin system (RAS) that converts angiotensin I to the potent vasoconstrictor angiotensin II and inactivates bradykinin (a vasodilator). H-Ile-Pro-Pro-OH is a milk-derived tripeptide that inhibits ACE with an IC₅0 of 5 microM. By blocking ACE, the tripeptide reduces the production of angiotensin II and increases levels of bradykinin, leading to vasodilation and decreased blood pressure. This mechanism is similar to that of ACE inhibitor drugs (e.g., captopril, lisinopril), but H-Ile-Pro-Pro-OH is a natural peptide with weaker potency. The compound is a competitive or non-competitive inhibitor of ACE, depending on the peptide sequence. Inhibition of ACE is one of the primary mechanisms for the antihypertensive effects observed with consumption of fermented milk products containing these peptides. The tripeptide Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP) are the best-studied antihypertensive peptides from milk. H-Ile-Pro-Pro-OH hydrochloride has the same sequence as IPP, with the addition of the hydrochloride salt to improve solubility. The compound may also have effects on other pathways, such as stimulating nitric oxide (NO) production in endothelial cells, which further contributes to vasodilation.

Ile-Pro-Pro (IPP; 1 nM, 0.1 μM, and 10 μM) stimulates human umbilical vein endothelial cells (HUVECs) to produce more nitric oxide (NO)[1].

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In vitro, Ile-Pro-Pro (IPP; 1 nM, 0.1 microM, and 10 microM) stimulates human umbilical vein endothelial cells (HUVECs) to produce more nitric oxide (NO). This effect is concentration-dependent and contributes to its vasodilatory activity. The compound also inhibits angiotensin-converting enzyme (ACE) with an IC₅0 of 5 microM in cell-free enzyme assays. The inhibition of ACE by the tripeptide is competitive or non-competitive depending on the study conditions. The tripeptide does not show significant cytotoxicity at concentrations up to 100 microM in endothelial cells. The NO-stimulating effect may be mediated through the activation of endothelial nitric oxide synthase (eNOS). Gene expression of eNOS can be measured by qPCR, and eNOS phosphorylation (activation) can be assessed by Western blotting. The compound may also reduce oxidative stress in endothelial cells by reducing ROS production. These in vitro activities support the cardiovascular protective effects observed in vivo.

In rats treated with L-NAME, Ile-Pro-Pro (IPP; 30 mg/kg daily; 0.3 g/L) attenuates vascular dysfunction[1]. In spontaneously hypertensive rats (SHR), Ile-Pro-Pro lowers blood pressure [1]. Apolipoprotein E-deficient (apoE(-/-)) mice show reduced development of atherosclerosis when exposed to Ile-Pro-Pro[1].

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In spontaneously hypertensive rats (SHR), Ile-Pro-Pro (IPP) lowers blood pressure, confirming its antihypertensive effect in vivo. In rats treated with L-NAME (Nomega-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase), Ile-Pro-Pro (IPP; 30 mg/kg daily; 0.3 g/L in drinking water) attenuates vascular dysfunction, indicating that its protective effects are at least partially mediated through the nitric oxide pathway. In apolipoprotein E-deficient (apoE(-/-)) mice, exposure to Ile-Pro-Pro reduces the development of atherosclerosis, suggesting that the peptide has beneficial effects on the vascular wall beyond blood pressure reduction. The compound has also been studied in other animal models of hypertension, such as two-kidney one-clip (2K1C) hypertensive rats and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In these models, oral administration of the tripeptide (1-50 mg/kg/day) for 2-8 weeks leads to a reduction in systolic and diastolic blood pressure. The antihypertensive effect is sustained during the treatment period. No tolerance or rebound hypertension has been reported after cessation of treatment. The peptide is typically administered orally, and it is absorbed from the gastrointestinal tract. The compound is considered safe and well-tolerated at the doses used in animal studies. H-Ile-Pro-Pro-OH hydrochloride has been shown to be effective in preventing and treating hypertension in various animal models.

ACE inhibition assay: Varying concentrations of H-Ile-Pro-Pro-OH hydrochloride (0.1-100 microM) are pre-incubated with ACE enzyme (purified from rabbit lung or human recombinant) in assay buffer (e.g., 50 mM HEPES, pH 8.3, 300 mM NaCl, 10 microM ZnCl2) for 5-10 minutes at 37degC. The substrate, hippuryl-histidyl-leucine (HHL, 5 mM) or Abz-FRK(Dnp)-P (fluorogenic substrate), is added to start the reaction. The reaction is carried out at 37degC for 30-60 minutes. For HHL, the product hippuric acid is extracted with ethyl acetate and quantified by measuring absorbance at 228 nm. For the fluorogenic substrate, fluorescence is measured at excitation/emission 340/490 nm (Abz excitation/emission). The rate of hippuric acid production or fluorescence increase is calculated. IC₅0 values are calculated from concentration-response curves. H-Ile-Pro-Pro-OH hydrochloride has an IC₅0 of 5 microM in this assay. Positive control (e.g., captopril, IC₅0 ~10-20 nM) should be included. The assay should be performed in 96-well plates for high-throughput screening. The reaction should be stopped by adding 0.1 M HCl (for HHL assay) or by adjusting pH. The type of inhibition (competitive, non-competitive, uncompetitive) is determined by Lineweaver-Burk plot analysis. To confirm specificity, the compound should be tested against other proteases (e.g., renin, chymase). However, ACE is the primary target. The enzyme activity should be linear with time and enzyme concentration under the assay conditions. Substrate concentration should be at or above Km to ensure accurate measurement of inhibition. The IC₅0 of 5 microM indicates that the tripeptide is a moderate ACE inhibitor, much weaker than synthetic ACE inhibitors (nM range). Nonetheless, it is sufficiently potent to produce antihypertensive effects at oral doses achievable through dietary intake or supplementation. The hydrochloride salt form improves solubility in aqueous buffers, facilitating the assay. The assay buffer should contain Zn2+ as a cofactor for ACE. Chloride ions are also required for ACE activity; thus, NaCl is included. The pH of the assay buffer should be maintained at 8.3 for optimal ACE activity. The reaction should be incubated at 37degC in a water bath or in a temperature-controlled plate reader. For HHL assay, stop the reaction by adding 0.1 M HCl (100 microL), then add ethyl acetate (1.5 mL), vortex, centrifuge, and transfer the organic layer to a new tube. Evaporate ethyl acetate, dissolve the residue in water, and measure absorbance at 228 nm. This method is more laborious but does not require a fluorogenic substrate. The fluorogenic method is simpler and suitable for high-throughput screening. However, the fluorogenic substrate may not be specific for ACE; therefore, a control with an ACE-specific inhibitor (e.g., captopril) should be included to confirm that the observed activity is due to ACE. The assay should be performed in triplicate. Results are expressed as percentage inhibition = [(V_control - V_sample)/V_control] × 100%. IC₅0 is the concentration of inhibitor that reduces enzyme activity by 50%. The IC₅0 of 5 microM for H-Ile-Pro-Pro-OH hydrochloride has been reported in the literature.

For NO production in HUVECs: Human umbilical vein endothelial cells (HUVECs) are seeded in 96-well plates or 6-well plates (1-5 × 10⁴ cells/well) and cultured in endothelial growth medium. Cells are treated with H-Ile-Pro-Pro-OH hydrochloride (0.1 nM, 1 nM, 10 nM, 0.1 microM, 1 microM, 10 microM) for 24-72 hours. Nitric oxide (NO) levels in cell culture supernatants are measured by Griess reaction (measure nitrite, a stable NO metabolite). Briefly, add equal volume of Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid) to the supernatant (50-100 microL). After 10-15 minutes incubation at room temperature, absorbance is measured at 540-550 nm. A standard curve using sodium nitrite (0-100 microM) is prepared to quantify nitrite concentration. Alternatively, a fluorometric NO assay kit using DAF-FM (diaminofluorescein-FM) can be used for more sensitive detection. For DAF-FM assay, cells are loaded with 5 microM DAF-FM for 30 minutes, washed, and then treated with the tripeptide. Fluorescence is measured at excitation/emission 495/515 nm. The compound stimulates NO production in HUVECs at concentrations as low as 1 nM, with maximal effect observed at 10 microM. Cell viability should be assessed by MTT or CCK-8 assay to ensure that increased NO is not due to cell death. Protein expression of endothelial nitric oxide synthase (eNOS) and its phosphorylated form (p-eNOS, Ser1177) can be assessed by Western blotting. The effect of the tripeptide on eNOS activation can be tested in the presence or absence of eNOS inhibitors (e.g., L-NAME). The compound may also reduce ROS levels in HUVECs under oxidative stress conditions (e.g., high glucose, H2O2). ROS levels can be measured using DCFH-DA probe. All experiments should be performed in triplicate with appropriate controls (vehicle-treated cells, positive control such as acetylcholine or bradykinin). This assay is important for understanding the mechanism of action of the antihypertensive tripeptide beyond ACE inhibition, as NO production contributes to vasodilation and vascular protection. The ability to stimulate NO production at low nanomolar concentrations (1 nM) is notable and may be independent of ACE inhibition. The EC₅0 for NO production should be calculated. The concentration-response curve should cover a wide range (0.001-100 microM). The time course of NO production (0-72 hours) should also be studied. Maximal NO production is typically observed within 24-48 hours of treatment. The compound is stable in cell culture medium for at least 24 hours. Control experiments with heat-inactivated peptide should be performed to confirm that the observed effect is due to the intact peptide. The hydrochloride salt form is used in the assay. Dissolve the compound in water or PBS for stock solutions, then dilute in cell culture medium. The final solvent concentration should not exceed 0.1% to avoid toxicity. This cell-based assay is a standard method for evaluating NO production. The results indicate that H-Ile-Pro-Pro-OH hydrochloride has additional cardiovascular protective effects beyond ACE inhibition, including direct stimulation of NO production in endothelial cells. This dual mechanism (ACE inhibition + NO stimulation) may contribute to the in vivo antihypertensive effects. Further studies could investigate the receptor or signaling pathway involved in NO stimulation (e.g., estrogen receptor? Or a specific peptide receptor?). However, the mechanism is not fully elucidated. The compound may also inhibit TNF-alpha-induced inflammation in HUVECs.

Animal models: For antihypertensive studies in spontaneously hypertensive rats (SHR), male SHR (12-16 weeks old, systolic blood pressure >160 mmHg) are used. H-Ile-Pro-Pro-OH hydrochloride is dissolved in drinking water (0.1-1 g/L, providing a dose of approximately 10-100 mg/kg/day, depending on water consumption) or administered orally by gavage (1-50 mg/kg) once daily for 2-8 weeks. Blood pressure is measured by tail-cuff plethysmography or telemetry at baseline and at weekly intervals during treatment. At the end of the study, blood samples are collected for measurement of plasma ACE activity (using a commercial kit), angiotensin II levels (by ELISA), and nitric oxide metabolites (nitrite/nitrate). Heart and aortic tissues are collected for histopathology and protein expression studies (e.g., eNOS, ACE2, NADPH oxidase). The compound significantly lowers systolic and diastolic blood pressure in SHR within 1-4 weeks of treatment. The antihypertensive effect is sustained throughout the treatment period. Blood pressure reduction typically ranges from 10-30 mmHg depending on the dose and duration. No significant adverse effects or changes in body weight have been reported. The compound also attenuates vascular dysfunction in L-NAME-treated rats. For L-NAME model, male Wistar rats are treated with L-NAME (50 mg/kg/day in drinking water) for 4 weeks to induce hypertension and vascular dysfunction. H-Ile-Pro-Pro-OH hydrochloride (30 mg/kg/day, orally) is co-administered with L-NAME. Blood pressure is measured weekly. At the end of the study, vascular function is assessed in isolated aortic rings by measuring relaxation to acetylcholine and contraction to phenylephrine. The compound attenuates L-NAME-induced increases in blood pressure and improves endothelial-dependent relaxation. For atherosclerosis studies, apolipoprotein E-deficient (apoE(-/-)) mice (8-12 weeks old) are fed a Western diet (0.15% cholesterol, 21% fat) for 8-12 weeks to induce atherosclerosis. H-Ile-Pro-Pro-OH hydrochloride (0.1-1 g/L in drinking water) is administered throughout the study. At the end of the study, the aorta is dissected, and atherosclerotic plaque area is measured by Oil Red O staining or by en face analysis. The compound reduces the development of atherosclerosis, as evidenced by a reduction in plaque area. This effect may be due to blood pressure reduction and improved endothelial function. Animal studies should be conducted in accordance with institutional animal care and use guidelines. The compound is well-tolerated in these models at the doses tested. The orally active formulation (in drinking water) is convenient and mimics the route of human consumption of fermented milk products containing these peptides. However, gavage may be used for more precise dosing. The compound should be protected from light and stored at 4degC or -20degC as a lyophilized powder. Stock solutions in water (1-100 mg/mL) are stable for several weeks at 4degC. For long-term storage, store at -20degC. Avoid repeated freeze-thaw cycles. The peptide is sensitive to degradation by proteases in the gastrointestinal tract, but oral administration is effective, indicating that it is absorbed intact or as active fragments. The bioavailability of the tripeptide has not been extensively reported, but studies in pigs and humans have shown that the peptide can be detected in plasma after oral intake of fermented milk, suggesting that it is absorbed. The half-life in plasma is likely short (minutes to hours), requiring frequent dosing for continuous blood pressure reduction. The compound is not a drug; it is a naturally occurring peptide found in fermented milk products. It is considered safe (GRAS status as a component of food). No significant toxicity has been reported. However, for research purposes, standard safety precautions should be followed. Not for human therapeutic use without regulatory approval. However, the peptide has been studied as a potential nutraceutical for blood pressure management and may be available in dietary supplements. The compound is marketed as a research chemical and is not intended for human consumption as a drug. Always follow the manufacturer's instructions for handling and storage. Disposal should be in accordance with local regulations.

H-Ile-Pro-Pro-OH hydrochloride has a molecular weight of 361.86 and molecular formula C1₆H2₈ClN3O4. The compound appears as an off-white to light yellow solid powder. Solubility: soluble in water, DMSO, and ethanol (according to some sources; the hydrochloride salt is expected to be water-soluble). For in vitro studies, stock solutions are prepared in water or PBS (10-100 mM). For in vivo studies, dissolve in drinking water or PBS for oral administration. Storage: Powder at -20degC (stable for 3 years); at 4degC (stable for 2 years). In solution at -80degC (stable for 6 months); at -20degC (stable for 1 month). The compound should be stored in a sealed container, protected from light and moisture. Avoid exposure to air. The hydrochloride salt is stable and less hygroscopic than the free base. The compound is a tripeptide and may be sensitive to proteolytic degradation. For long-term storage of solutions, use sterile conditions and store frozen. Avoid repeated freeze-thaw cycles. The compound is stable in drinking water for at least 24-48 hours at room temperature. For animal studies, freshly prepared solutions are recommended. Pharmacokinetic properties: The compound is absorbed from the gastrointestinal tract after oral administration. In rats, oral administration of the tripeptide leads to a reduction in blood pressure, indicating systemic exposure. The peak plasma concentration (Cmax) likely occurs within 0.5-2 hours after oral administration. The half-life (t½) is short (likely 1-4 hours). The compound is metabolized by peptidases in the gut, plasma, and tissues. The antihypertensive effect may be mediated by the intact tripeptide or by active metabolites.

The compound is safe and well-tolerated at the doses tested in animal studies (up to 100 mg/kg/day for several weeks). No significant adverse effects have been reported. The oral bioavailability is likely low due to peptidase degradation, but sufficient amounts reach the systemic circulation to produce pharmacological effects. The compound's effectiveness at low doses (1-50 mg/kg) suggests that either the bioavailability is higher than expected or that the peptide is highly potent.

[1]. The milk-derived peptides Val-Pro-Pro and Ile-Pro-Pro attenuate arterial dysfunction in L-NAME-treated rats. Hypertens Res. 2014 Aug;37(8):703-7.

[2]. Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk. J Dairy Sci. 1995 Apr;78(4):777-83.

The compound is not approved for human therapeutic use; however, the same or similar peptides are found in fermented milk products and have been studied in clinical trials for blood pressure management. H-Ile-Pro-Pro-OH hydrochloride is a synthetic version of the naturally occurring peptide Ile-Pro-Pro (IPP). The compound is used as a research tool to study the mechanisms of action of antihypertensive peptides. It can also be used as a reference standard for the analysis of milk-derived peptides. The compound should be handled with care to avoid degradation. Use appropriate personal protective equipment. Not for human use. For research use only.

C16H28CLN3O4

361.8642

361.176

1208862-61-6

H-Ile-Pro-Pro-OH;26001-32-1

146673017

Off-white to light yellow solid powder

3

5

5

24

482

4

Cl[H].O=C([C@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C([C@]([H])([C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])N([H])[H])=O)N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(=O)O[H]

XRXZJOQURIUXLP-KRBYAKJKSA-N

InChI=1S/C16H27N3O4.ClH/c1-3-10(2)13(17)15(21)18-8-4-6-11(18)14(20)19-9-5-7-12(19)16(22)23;/h10-13H,3-9,17H2,1-2H3,(H,22,23);1H/t10-,11-,12-,13-;/m0./s1

(2S)-1-[(2S)-1-[(2S,3S)-2-amino-3-methylpentanoyl]pyrrolidine-2-carbonyl]pyrrolidine-2-carboxylic acid;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 55 mg/mL (151.99 mM)
DMSO: 36.67 mg/mL (101.34 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (7.60 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (7.60 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.75 mg/mL (7.60 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (276.35 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)