An ACE-inhibiting peptide and its application in the preparation of antihypertensive drugs
By synthesizing black bean hydrolyzed peptides KDFPPR, VVPPGHPF, and DTFPYPR, the problem of side effects from synthesized ACE inhibitory drugs has been solved, providing a highly effective and safe blood pressure lowering solution suitable for the health food and pharmaceutical fields.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI AGRICULTURAL UNIVERSITY
- Filing Date
- 2023-04-18
- Publication Date
- 2026-07-03
AI Technical Summary
Existing synthetic ACE inhibitors have side effects with long-term use, and there is a lack of green, natural, and safe treatment options for hypertension.
By studying the hydrolyzed peptides of black soybeans, three ACE-inhibiting peptides were synthesized: KDFPPR, VVPPGHPF, and DTFPYPR. These peptides were then used to prepare antihypertensive drugs, utilizing their ACE-inhibiting activity to lower blood pressure.
It provides low-cost, easily isolated and purified ACE inhibitory peptides with high ACE inhibitory activity, significantly reducing hypertension, suitable for industrial production, and long-term consumption can prevent and alleviate hypertension with few side effects.
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Figure CN122325549A_ABST
Abstract
Description
[0001] This invention is a divisional application of application number 202310429836.2, filed on April 18, 2023, entitled "Three Stable and Efficient ACE Inhibitory Peptides and Their Application in Antihypertensive Drugs". Technical Field
[0002] This invention relates to the field of ACE inhibitory peptide technology, and in particular to an ACE inhibitory peptide and its application in antihypertensive drugs. Background Technology
[0003] Hypertension is a highly prevalent chronic disease and a major risk factor for cardiovascular and kidney diseases. As a complex disease caused by both genetic and environmental factors, hypertension has multiple causes. Among these, dysregulation of the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) in the kidneys is the most common pathway leading to hypertension. The blood pressure-raising pathway controlled by the RAS is as follows: angiotensinogen in the plasma (synthesized by the liver and released into the plasma) continuously generates angiotensin I (Ang I), which has a low vasoconstrictive effect. Subsequently, angiotensin I-converting enzyme (ACE) in the blood and lung tissue catalyzes the conversion of angiotensin I (Ang I) to angiotensin II (Ang II), thereby exerting a strong blood pressure-raising effect.
[0004] ACE is a Zn-containing 2+ Carboxypeptidase, with a relative molecular mass of 120,000–150,000, is widely present in human tissues and plasma, primarily within vascular endothelial cells. Studies have shown that the maintenance of normal blood pressure is closely related to the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS), with angiotensin-converting enzyme (ACE) playing a crucial role in the dynamic balance of both systems. In normal physiological activities, renin hydrolyzes angiotensinogen in plasma to release Ang I, a decapeptide. ACE cleaves a dipeptide from the C-terminus of inactive Ang I, forming the octapeptide ang II, a vasoconstrictor. Simultaneously, ACE inactivates excess bradykinin, a nonapeptide-like vasodilator, thus maintaining the dynamic balance of blood pressure. However, in hypertensive patients, excessive ACE catalyzes the production of excessive Ang II, disrupting this balance and leading to elevated blood pressure.
[0005] In current clinical treatment of hypertension, many drugs such as lisinopril and captopril are synthetic ACE inhibitors. While they can effectively improve blood pressure in hypertensive patients, long-term use of these drugs leads to side effects such as coughing, loss of taste, and rashes, potentially causing organ damage. Therefore, developing other greener, more natural, safer, and side-effect-free antihypertensive biological products to prevent hypertension and to replace or partially replace synthetic drugs has become a major focus in the prevention and treatment of hypertension.
[0006] ACE inhibitory peptides are a class of polypeptides with ACE-inhibiting activity, which lower blood pressure by inhibiting ACE activity. They have a greater affinity for the active ACE region than Ang I and bradykinin for ACE, and once bound to the active ACE region, they are difficult to release. This hinders the two biochemical reactions catalyzed by ACE to hydrolyze Ang I to Ang II and to hydrolyze bradykinin to inactive fragments, thus lowering blood pressure. Summary of the Invention
[0007] This invention studies the effective blood pressure-lowering components in hydrolyzed black bean peptides and artificially synthesizes three ACE-inhibiting peptides, which possess strong blood pressure-lowering functions and stability. The amino acid sequences of the ACE-inhibiting peptides are: KDFPPR (Lys-Asp-Phe-Pro-Pro-Arg), VVPPGHPF (Val-Val-Pro-Pro-Gly-His-His-Pro-Phe), and DTFPYPR (Asp-Thr-Phe-Pro-Tyr-Pro-Arg).
[0008] A stable and highly effective ACE inhibitory peptide, wherein the ACE inhibitory peptide is formed by the sequential tail-end condensation of the following amino acids Lys-Asp-Phe-Pro-Pro-Arg, with the amino acid sequence: KDFPPR; chemical structure as shown. Figure 1 As shown.
[0009] A stable and highly effective ACE inhibitory peptide, wherein the ACE inhibitory peptide is formed by the sequential condensation of the following amino acids Val-Val-Pro-Pro-Gly-His-His-Pro-Phe, with the amino acid sequence: VVPPGHPF; the chemical structural formula is as follows. Figure 2 As shown.
[0010] A stable and highly effective ACE inhibitory peptide, wherein the ACE inhibitory peptide is formed by the sequential tail-end condensation of the following amino acids Asp-Thr-Phe-Pro-Tyr-Pro-Arg, with the amino acid sequence: DTFPYPR, and the chemical structural formula is as follows. Figure 3 As shown.
[0011] A stable and efficient mixture of ACE inhibitory peptides, the ACE inhibitory peptide composition comprising a mixture of two or three of the following: an ACE inhibitory peptide with the amino acid sequence KDFPPR, an ACE inhibitory peptide with the amino acid sequence VVPPGHPF, and an ACE inhibitory peptide with the amino acid sequence DTFPYPR.
[0012] The content of the ACE inhibitory peptide with the amino acid sequence KDFPPR is 30-45%, the content of the ACE inhibitory peptide with the amino acid sequence VVPPGHPF is 30-55%, and the content of the ACE inhibitory peptide with the amino acid sequence DTFPYPR is 0-40%.
[0013] The ACE inhibitory peptide mixture was used for dietary intervention and compared with antihypertensive drugs. After feeding to hypertensive model mice, it significantly reduced the blood pressure of the hypertensive model mice.
[0014] Beneficial effects
[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0016] (1) The active peptides provided by this invention have small molecular weight, low cost, simple preparation process, easy separation and purification, mild reaction conditions, safe and reliable, and the hydrolysis products have light color and good sensory acceptance, making them suitable for industrial production.
[0017] (2) The multiple small molecule peptides provided by this invention all possess high ACE inhibitory activity. In vitro experiments show that the optimal combination achieves an ACE inhibition rate of 87.48±2.90%. Furthermore, the invented ACE inhibitory peptides can effectively inhibit ACE activity under low-dose conditions, inhibiting its catalytic conversion of angiotensin I (Ang I) to angiotensin II (Ang II), thereby significantly inhibiting the rise in blood pressure. The small molecule peptides provided by this invention are extracted from black bean protein hydrolysate and belong to green natural products. They can be used for long-term consumption to prevent, alleviate, and assist in the treatment of hypertension. They can be used to develop related health foods or drugs for the treatment of hypertension and have broad market prospects in many fields such as health foods and medicines. Attached Figure Description
[0018] Figure 1 The chemical formula of the peptide KDFPPR in this invention.
[0019] Figure 2 The chemical formula of the peptide VVPPGHPF in this invention.
[0020] Figure 3 The chemical formula of the peptide DTFPYPR in this invention.
[0021] Figure 4The ACE inhibitory activity assay of the three polypeptides synthesized in this invention and their equal-proportion mixture is shown in the figure.
[0022] Figure 5 The liquid chromatogram of the synthesized high-purity peptide KDFPPR in this invention.
[0023] Figure 6 The mass spectrum of the high-purity peptide KDFPPR synthesized in this invention.
[0024] Figure 7 The liquid chromatogram of the synthesized high-purity peptide VVPPGHPF in this invention.
[0025] Figure 8 The mass spectrum of the high-purity peptide VVPPGHPF synthesized in this invention.
[0026] Figure 9 The liquid chromatogram of the high-purity peptide DTFPYPR synthesized in this invention.
[0027] Figure 10 The mass spectrum of the high-purity peptide DTFPYPR synthesized in this invention.
[0028] Figure 11 The structural diagram of the binding of peptide KDFPPR to ACE in this invention.
[0029] Figure 12 The structural diagram of the binding of peptide VVPPGHPF to ACE in this invention.
[0030] Figure 13 The structural diagram of the binding of peptide DTFPYPR to ACE in this invention.
[0031] Figure 14 The secondary mass spectra of the three polypeptides synthesized in this invention.
[0032] Figure 15 The blood pressure-lowering effect of the inhibitory peptide mixture of this invention in mouse experiments is shown in the figure. Detailed Implementation
[0033] The present invention will be further described in detail below with reference to the embodiments.
[0034] Example 1
[0035] This embodiment synthesizes a stable and highly effective ACE inhibitory peptide, which is formed by the sequential tail-condensation of the following amino acids: Lys-Asp-Phe-Pro-Pro-Arg. The amino acid sequence is: KDFPPR; the chemical structure is shown in [reference needed]. Figure 1 .
[0036] Example 2
[0037] This embodiment synthesizes a stable and highly effective ACE inhibitory peptide, which is formed by the sequential tail-end condensation of the following amino acids: Val-Val-Pro-Pro-Gly-His-His-Pro-Phe. The amino acid sequence is: VVPPGHPF; the chemical structure is shown in [reference needed]. Figure 2 .
[0038] Example 3
[0039] This embodiment synthesizes a stable and highly efficient ACE inhibitory peptide, which is formed by the sequential tail-condensation of the following amino acids Asp-Thr-Phe-Pro-Tyr-Pro-Arg, with the amino acid sequence DTFPYPR. Its chemical structure is shown in [reference needed]. Figure 3 .
[0040] Example 4
[0041] This embodiment synthesizes a stable and efficient ACE inhibitory peptide mixture, which is composed of KDFPPR from Example 1 and VVPPGHPF from Example 2, wherein KDFPPR accounts for 50% and VVPPGHPF accounts for 50%.
[0042] Example 5
[0043] This embodiment synthesizes a stable and efficient mixture of ACE inhibitory peptides, which are composed of KDFPPR from Example 1, VVPPGHPF from Example 2, and DTFPYPR from Example 3, wherein KDFPPR accounts for 33.3%, VVPPGHPF accounts for 33.3%, and DTFPYPR accounts for 33.3%.
[0044] Example 6
[0045] The synthesis methods of the ACE inhibitory peptides in Examples 1 to 3 are as follows:
[0046] 1. Based on the first amino acid at the C-terminus of the polypeptide sequence, select 0.5 mmol of the corresponding Fmoc protected amino acid - WangResin, add it to the solid-phase reactor, add DCM swelling resin for 30 min, dry it under vacuum, wash it three times with DMF, add 20% hexahydropyridine DMF solution, react for 5 min, add 20% hexahydropyridine DMF solution again and react for 10 min, wash with DMF once in between, dry it under vacuum after the reaction is finished, and wash with DMF three times.
[0047] 2. Following the sequence of the peptide from C-terminus to N-terminus, condensation and Fmoc removal reactions were carried out alternately in 1.5 mmol reaction amounts to condense all remaining amino acids of the peptide onto the resin. After the last amino acid was condensed, a 20% (v / v) hexahydropyridine DMF solution was added, and the reaction was carried out for 5 min. Then, another 20% (v / v) hexahydropyridine DMF solution was added, and the reaction was carried out for 10 min. DMF was used for washing once in between. After the reaction was completed, the solution was dried under vacuum, and DMF was used for washing three times.
[0048] 3. Add 1 mmol of FITC and N-methylmorpholine to the reactor and react for 5-10 minutes. Check whether the reaction is complete with ninhydrin. After the reaction is complete, wash three times alternately with DMF and DMC, and then wash with methanol to shrink and obtain dried polypeptide-resin.
[0049] 4. Place the peptide-resin in a round-bottom flask and slowly add the prepared lysis buffer (the reagent formula for the lysis buffer has a volume ratio of TFA: benzyl sulfide: phenol: triisopropylsilane: water = 82.5: 7.5: 5: 3: 2) at 0°C. Stir slowly and react at low temperature for 0.5 hours, then at room temperature for 2 hours. Filter to obtain the lysis buffer. Slowly add the lysis buffer to anhydrous ice-cold ether and stir. Filter to separate the crude peptide. Wash the crude peptide three times with ice-cold ether to obtain the crude peptide.
[0050] 5. Use mass spectrometry to check if the molecular weight of the crude product is correct. If it is correct, purify and separate it using high performance liquid chromatography, freeze-dry it, and then obtain the pure peptide.
[0051] The three ACE inhibitory peptide samples from Examples 1-3 were identified by mass spectrometry using a Nano-HPLC system. The active peptide sequences identified included:
[0052] KDFPPR (Lys-Asp-Phe-Pro-Pro-Arg), VVPPGHPF (Val-Val-Pro-Pro-Gly-His-His-Pro-Phe), DTFPYPR (Asp-Thr-Phe-Pro-Tyr-Pro-Arg). (See secondary mass spectra for...) Figure 14 )
[0053] ACE inhibitory activity assay
[0054] The assay for ACE inhibitory activity was performed according to the following method:
[0055] Using furanylacryl tripeptide (FAPGG) as a substrate, the ACE inhibitory activity of black soybean peptide was determined using a microplate reader. 0.606 g of tris(hydroxymethyl)aminomethane was dissolved in 70 mL of pure water, the pH was adjusted to 7.5 with 0.1 mol / L HCl, 1.753 g of NaCl was added, and the volume was brought to 100 mL with pure water to obtain a Tris-HCl buffer solution. 3.994 mg of FAPGG was added to a 10 mL volumetric flask and brought to volume with the above buffer solution. 10 mg of the sample was added to a 1 mL volumetric flask and brought to volume with the buffer solution. 10 μL of the ACE aqueous solution (0.25 U / mL) and 10 μL of the sample solution were added to a microplate, 150 μL of FAPGG was added, and the mixture was stirred at room temperature. The absorbance (A) was measured at 340 nm. 1， The reaction was then carried out at 37°C for 30 min, and the absorbance (A2) was measured again at 340 nm. In the blank group, buffer solution was used instead of the sample. The ACE inhibition rate was calculated by the following formula:
[0056]
[0057] ΔA=A1-A2
[0058] The ACE inhibitory activity of the peptides was tested using ACE inhibitory activity as an indicator. The inhibitory activity of the black bean ACE inhibitory peptides extracted from black bean protein in Examples 1 and 2 was tested and analyzed. The specific results are shown in Table 1 below.
[0059] Table 1 Results of ACE inhibitory activity in Examples 1-5
[0061] As shown in Table 1, the polypeptides synthesized in Examples 1 and 2 of this invention all possess strong ACE inhibitory activity. Long-term consumption can achieve the purpose of preventing, alleviating, controlling, and adjuvantly treating hypertension, and can be used to develop health foods or drugs for the treatment of hypertension. In summary, the ACE inhibitory peptides of this invention can not only be obtained through alkaline protease heating hydrolysis and separation purification, but also through chemical solid-phase synthesis. This invention, through ACE inhibitory activity determination, found that the sequences KDFPPR, VVPPGHPF, and DTFPYPR have strong ACE inhibitory activity, with an inhibition rate reaching 35-70%. The ACE inhibition rate can be further improved at the same concentration when KDFPPR and VVPPGHPF are mixed, or when KDFPPR, VVPPGHPF, and DTFPYPR are mixed. Long-term consumption can achieve the purpose of preventing, alleviating, and adjuvantly treating hypertension, and has broad application prospects in health foods, medicines, and other fields. The above description is only a preferred embodiment of this invention, but the scope of protection of this invention is not limited thereto. All equivalent changes and modifications made within the scope of the claims of this invention should be included within the scope of protection of this invention.
[0062] Example 7
[0063] This embodiment provides the application of a stable and efficient ACE inhibitory peptide mixture in antihypertensive drugs. The mixture of KDFPPR, VVPPGHPF, and DTFPYPR from Example 5 is mixed with maltodextrin and compressed into tablets to prepare a drug for lowering blood pressure. The content of the ACE inhibitory peptide mixture is 50%, and the content of maltodextrin is 50%.
[0064] Seventy-four male C57BL6j mice, 7 weeks old, weighing 18-20g, SPF grade, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. (Nanjing). The mice were housed at a temperature of 18-22℃ and humidity of 50-60%, fed a normal diet. Fifty mice were used to establish a hypertension model, while the remainder served as a blank control group, a blank + high-dose group, and a hypertension prevention group (n=8 each). After model establishment, successfully modeled hypertensive mice were randomly assigned to three groups: a hypertension model group, a lisinopril drug group, and high, medium, and low dose inhibitory peptide groups (calculated at inhibitory peptide equivalents of 100mg / kg, 50mg / kg, and 25mg / kg, respectively). The experiment lasted for 9 weeks, with the first week being an adaptation feeding period. Results are as follows: Figure 15 As shown.
[0065] The results showed that although the blood pressure-lowering effect was weaker than that of the lisinopril drug group, the blood pressure of mice in the ACE inhibitory peptide feeding group was significantly lower than that of the hypertension model group. Therefore, the ACE inhibitory peptide composition provided in Example 5 has a blood pressure-lowering effect on hypertensive model mice and can be used in the preparation of health foods and antihypertensive drugs.
[0066] Similarly, those skilled in the art will know that maltodextrin is merely a mixed excipient. Through experiments, it has been found that a mixture of ACE inhibitory peptides with a content of 30-70% and a maltodextrin content of 30-70% can both achieve the blood pressure lowering function of Example 7.
Claims
1. An ACE-inhibiting peptide, characterized in that, The ACE inhibitory peptide is formed by the sequential tail-condensation of the following amino acids Asp-Thr-Phe-Pro-Tyr-Pro-Arg, with the amino acid sequence DTFPYPR, and its chemical structure is as follows: 。 2. The application of the ACE inhibitory peptide according to claim 1, characterized in that: The ACE inhibitory peptide mixture is used to prepare antihypertensive drugs.
3. A blood pressure-lowering drug, characterized in that: The antihypertensive drug includes an ACE inhibitory peptide with the amino acid sequence DTFPYPR.
4. The antihypertensive drug according to claim 3, characterized in that: The blood pressure-lowering drugs also include maltodextrin.
5. The blood pressure lowering drug according to claim 3, characterized in that: The ACE inhibitory peptide mixture also includes an ACE inhibitory peptide with the amino acid sequence KDFPPR and an ACE inhibitory peptide with the amino acid sequence VVPPGHPF.
6. The blood pressure lowering drug according to claim 3, characterized in that: By mass ratio, the content of the ACE inhibitory peptide with the amino acid sequence VVPPGHPF is 30-55%, the content of the ACE inhibitory peptide with the amino acid sequence KDFPPR is 30-45%, and the content of the ACE inhibitory peptide with the amino acid sequence DTFPYPR is 0-40%.
7. The blood pressure lowering drug according to claim 4, wherein the content of the ACE inhibitory peptide is 30-70% and the content of maltodextrin is 30-70%.