Angiotensin-converting enzyme inhibitor peptides

Abstract

To provide a novel peptide with higher angiotensin-converting enzyme inhibitory activity than conventional peptides by searching for peptides that strongly bind to the substrate recognition site of angiotensin-converting enzyme using structural biological methods.SOLUTION: The present invention provides a peptide consisting of the following amino acid sequence: Trp-Ile-Asp. The present invention also provides an angiotensin-converting enzyme inhibitor containing the peptide as an active ingredient. The present invention also provides food and drink for inhibiting angiotensin-converting enzyme and pharmaceuticals for inhibiting angiotensin-converting enzyme, which contain the peptide as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a novel peptide. The present invention also relates to an angiotensin-converting enzyme inhibitor and a food or drink for angiotensin-converting enzyme inhibition, each containing the novel peptide as an active ingredient. [Background technology]

[0002] Angiotensin-converting enzyme (ACE) is an enzyme that acts on the renin-angiotensin system to regulate blood pressure. Angiotensinogen, with a molecular weight of approximately 57,000, is converted to angiotensin I by renin, which migrates from the kidney into the bloodstream. While angiotensin I is an inactive form that exhibits little vasoconstriction, ACE generates angiotensin II, which has a strong hypertensive effect, and also inactivates bradykinin, which has a hypotensive effect. Because of these effects, angiotensin-converting enzyme inhibitors are used as over-the-counter drugs to treat hypertension. Angiotensin-converting enzyme inhibitors are also used to treat many conditions, including heart disease, kidney disease, cerebrovascular disease, arteriosclerotic disease, diabetes, metabolic syndrome, and elderly hypertension.

[0003] Peptides with angiotensin converting enzyme inhibitory activity have been found in natural products (Patent Documents 1 to 3). These documents disclose angiotensin converting enzyme inhibitors that contain peptides with specific structures obtained from bovine casein as active ingredients. The IC50 (concentration required to inhibit 50% of angiotensin converting enzyme activity) of the peptide in Patent Document 1 is 7.7 × 10- 5 M, and the IC50 of the peptide in Patent Document 2 is disclosed to be 15.5 μM. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Unexamined Patent Publication No. 58-109425 [Patent Document 2] Japanese Patent Application Publication No. 6-277090 [Patent Document 3] Patent No. 3567012 Summary of the Invention [Problem to be solved by the invention]

[0005] As described above, peptides derived from milk casein are known to have angiotensin-converting enzyme inhibitory activity, but the angiotensin-converting enzyme inhibitory activity of these peptides is still insufficient. Therefore, there is a need to obtain natural peptides with even higher angiotensin-converting enzyme inhibitory activity and to apply them to foods, medicines, etc. The present invention aims to provide a novel peptide having high angiotensin-converting enzyme inhibitory activity, and to provide an angiotensin-converting enzyme inhibitor and a food or drink for angiotensin-converting enzyme inhibition that contain the peptide as an active ingredient. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above-mentioned problems. Specifically, they have used structural biology techniques to search for peptides that strongly bind to the substrate recognition site of angiotensin-converting enzyme. As a result, they have discovered novel peptides that have higher angiotensin-converting enzyme inhibitory activity than conventional peptides, and have completed the present invention.

[0007] That is, the present invention has the following configuration. (1) A peptide consisting of the following amino acid sequence: Trp-Ile-Asp (2) An angiotensin-converting enzyme inhibitor containing the following amino acid sequence as an active ingredient: Trp-Ile-Asp (3) A food or beverage for inhibiting angiotensin-converting enzyme, containing as an active ingredient a peptide having the following amino acid sequence: Trp-Ile-Asp [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a graph showing the angiotensin-converting enzyme inhibition rate at various concentrations of the peptide of the present invention. [Figure 2] 1 shows a chromatogram of the reaction solution in an angiotensin-converting enzyme inhibition test in which the peptide of the present invention was added at 400 μM. [Figure 3] 1 is a chromatogram of the reaction solution from an angiotensin-converting enzyme inhibition test when the peptide of the present invention is not added. DETAILED DESCRIPTION OF THE INVENTION

[0009] Preferred embodiments of the present invention will be described in detail below. However, the present invention is not limited to the following preferred embodiments and can be freely modified within the scope of the present invention. In this specification, percentages are expressed by mass unless otherwise specified.

[0010] (Peptide having the sequence represented by Trp-Ile-Asp) The peptides of the present invention have a sequence represented by Trp-Ile-Asp. In the present invention, Trp represents an L-tryptophan residue, Ile represents an L-isoleucine residue, and Asp represents an L-aspartic acid residue. The peptides of the present invention may also be in the form of pharmaceutically acceptable acid addition salts and base addition salts of the peptides. Examples of acid addition salts include salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid; and salts with organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, succinic acid, malic acid, tartaric acid, and citric acid. Examples of pharmaceutically acceptable base addition salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt; and salts with amines such as ammonium, ethanolamine, triethylamine, and dicyclohexylamine.

[0011] The peptides of the present invention can be produced by chemical synthesis. Chemical synthesis of the peptides of the present invention can be carried out by liquid-phase or solid-phase methods commonly used in oligopeptide synthesis. The synthesized peptides are deprotected as necessary, and unreacted reagents, by-products, etc. are removed. Such peptide synthesis can be carried out using a commercially available peptide synthesizer. The peptides of the present invention are preferably isolated and purified from the synthesized product. Peptide purification can be carried out by an appropriate combination of techniques similar to those commonly used in oligopeptide purification, such as various chromatographic methods such as ion exchange chromatography, adsorption chromatography, reverse-phase chromatography, partition chromatography, and gel filtration chromatography, as well as solvent precipitation, salting out, and partitioning between two liquid phases. During the purification of the peptides of the present invention, fractions containing the target substance can be determined using the angiotensin-converting enzyme inhibitory activity described below as an indicator, and the active components of these fractions can be identified by mass spectrometry and / or protein sequencing.

[0012] The peptides of the present invention can also be produced by expressing recombinant DNA encoding the peptides in suitable host cells. The vectors and hosts required for producing the recombinant DNA can be those commonly used for producing proteins and peptides. The peptides of the present invention may be extracted from natural products, and can be extracted from natural products using angiotensin-converting enzyme inhibitory activity as an indicator. The degree of purification of the extracted fraction may be determined depending on the required level of angiotensin-converting enzyme inhibitory activity. Therefore, not only highly purified peptides of the present invention but also crude fractions containing the peptides of the present invention are included in the scope of peptides as active ingredients of the present invention.

[0013] (angiotensin-converting enzyme inhibitors) The angiotensin-converting enzyme inhibitor of the present invention contains, as an active ingredient, a peptide consisting of the sequence represented by Trp-Ile-Asp. Examples of the angiotensin-converting enzyme inhibitor of the present invention include foods and beverages, feed, cosmetics, quasi-drugs, and pharmaceuticals. In particular, foods and beverages are sometimes referred to as angiotensin-converting enzyme-inhibiting foods and beverages, and pharmaceuticals are sometimes referred to as angiotensin-converting enzyme-inhibiting pharmaceuticals.

[0014] Angiotensin-converting enzyme inhibitors containing the peptides of the present invention as active ingredients may be administered orally or parenterally. Parenteral administration includes transdermal administration, intravenous injection, rectal administration, inhalation, etc. When administering the peptides of the present invention orally, the active ingredient Trp-Ile-Asp can be administered as is, or it can be added to food or drink, or formulated into powders, granules, tablets, capsules, drinks, etc., according to standard methods.

[0015] (Food and drink for inhibiting angiotensin-converting enzyme) The angiotensin-converting enzyme-inhibiting food and beverage of the present invention contains as an active ingredient a peptide consisting of the sequence represented by Trp-Ile-Asp. Such foods and beverages are sometimes referred to as foods for specified health uses, foods and beverages with functional claims, or dietary supplements and beverages. These foods and beverages may be in any form, such as liquid, paste, solid, or powder, and include tablets, liquid diets, dairy products such as cheese and fermented milk, confectioneries such as jelly, paste products such as sausages, noodles such as udon and soba, fruit juice drinks, and dairy drinks.

[0016] (angiotensin-converting enzyme inhibitors) The angiotensin-converting enzyme inhibitory pharmaceutical of the present invention contains, as an active ingredient, a peptide consisting of the sequence Trp-Ile-Asp. When formulating the pharmaceutical, additives such as carriers, excipients, binders, disintegrants, lubricants, colorants, stabilizers, diluents, and injectable solvents can be used. Specific examples of pharmaceutical formulations include tablets, powders, capsules, granules, pills, and lozenges. In the present invention, oral preparations such as powders, granules, tablets, and capsules can be formulated by conventional methods using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, cornstarch, inorganic salts, etc. In addition to the excipients, binders, disintegrants, surfactants, lubricants, flow enhancers, colorants, flavors, etc. may also be used appropriately in these types of preparations.

[0017] Examples of binders include starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, sodium carboxymethylcellulose, methylcellulose, crystalline cellulose, ethyl cellulose, and polyvinylpyrrolidone, and examples of disintegrants include starch, hydroxypropyl starch, carboxymethylcellulose, sodium carboxymethylcellulose, cross-linked sodium carboxymethylcellulose, and crystalline cellulose. Examples of surfactants include soybean lecithin and sucrose fatty acid esters. Examples of lubricants include talc, wax, sucrose fatty acid esters, and hydrogenated vegetable oils. Examples of flow enhancers include anhydrous silicic acid, dried aluminum hydroxide, and magnesium silicate.

[0018] (Other uses) The peptide of the present invention can also be incorporated as an active ingredient into feed (including feed for pets) to be processed into feed having angiotensin converting enzyme inhibitory activity. Furthermore, the peptide of the present invention can be incorporated as an active ingredient into cosmetics or quasi-drugs to produce cosmetics or quasi-drugs having angiotensin converting enzyme inhibitory activity.

[0019] In the angiotensin-converting enzyme inhibitor of the present invention (in the form of food, drink, pharmaceutical, or other use), the amount of the peptide of the present invention is preferably 0.001% by mass or more relative to the final composition of the angiotensin-converting enzyme inhibitor.

[0020] The dosage of the angiotensin-converting enzyme inhibitor of the present invention varies depending on age, symptoms, etc., but is usually 0.001 mg to 1000 mg of the peptide of the present invention, and may be administered once a day or in divided doses. Furthermore, when the angiotensin-converting enzyme inhibitor of the present invention is ingested or taken, the effect of the present invention is sufficiently exhibited regardless of whether it is taken before, between, or after meals. [Example]

[0021] The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

[0022] Example 1 [Angiotensin-converting enzyme inhibitory effect of peptides] (1) Test method (1-1) Measurement of angiotensin-converting enzyme inhibition Angiotensin-converting enzyme inhibition was measured according to the method of Cushman et al. [Biochemical Pharmacology, Vol. 20, pp. 1637-1648 (1971)]. The chemically synthesized peptide of the present invention, Trp-Ile-Asp (GL Biochem), was used as a sample.

[0023] (i) The sample was dissolved in 0.1 M borate buffer (containing 0.3 M NaCl, pH 8.3), and 0.08 ml of the solution was placed in a test tube. 0.2 ml of the enzyme substrate (hippurylhistidylleucine, Sigma) adjusted to 5 mM with 0.1 M borate buffer (containing 0.3 M NaCl, pH 8.3) was added, and the mixture was incubated at 37°C for 3 minutes. Next, 0.02 ml of rabbit lung angiotensin-converting enzyme (Sigma) adjusted to 0.1 U / ml with distilled water was added, and the mixture was incubated at 37°C for 30 minutes.

[0024] (ii) After that, 0.25 ml of 1N hydrochloric acid was added to terminate the reaction, 1.7 ml of ethyl acetate was added, and the mixture was stirred vigorously for 20 seconds. After centrifugation at 3000 rpm for 10 minutes, 1.4 ml of the ethyl acetate layer was collected. The resulting ethyl acetate layer was heated to remove the solvent, and 1.0 ml of distilled water was added. The absorbance of the extracted hippuric acid (absorbance at 228 nm) was measured and used as the enzyme activity.

[0025] (1-2)IC 50 How to decide The inhibitory activity is calculated from the following formula, and the inhibition rate is plotted on the vertical axis and the sample concentration on the horizontal axis to obtain the inhibition curve formula, and then the IC 50 The sample concentration (μM) required to inhibit the activity of angiotensin converting enzyme by 50% was determined.

[0026] Inhibition rate (%) = (AB) / (AC) × 100 A: Enzyme activity without sample (peptide) (absorbance at 228 nm) B: Enzyme activity when sample is added (absorbance at 228 nm) C: Enzyme activity without enzyme or sample (absorbance at 228 nm)

[0027] (2) Test results The test results are shown in Table 1, Figure 1, Figure 2, Figure 3 and Table 2. From Table 1 and Figure 1, the IC concentration of the sample (peptide of the present invention) at which the inhibition rate was 50% 50 was 0.6 μM (Table 2). Figure 2 shows a chromatogram of the reaction mixture (i) with 400 μM Trp-Ile-Asp added, and Figure 3 shows a chromatogram of the reaction mixture without Trp-Ile-Asp added. Figure 2 shows that the substrate hippurylhistidylleucine is not decomposed to hippuric acid, inhibiting the function of angiotensin converting enzyme. Figure 3 also shows that the substrate hippurylhistidylleucine is decomposed to hippuric acid by the action of angiotensin converting enzyme. Therefore, it was revealed that the peptide of the present invention consisting of the sequence Trp-Ile-Asp has potent angiotensin-converting enzyme inhibitory activity.

[0028] [Table 1]

[0029] [Table 2] [Industrial Applicability]

[0030] According to the present invention, a novel peptide having high angiotensin-converting enzyme inhibitory activity is provided. Angiotensin-converting enzyme inhibitors containing the peptide as an active ingredient can be used as pharmaceuticals. Furthermore, since the peptide is composed entirely of natural (L-form) amino acids, it is highly safe and can also be used as a food or beverage for angiotensin-converting enzyme inhibition.

Claims

1. A peptide consisting of the following amino acid sequence: Trp-Ile-Asp

2. An angiotensin-converting enzyme inhibitor containing the following amino acid sequence as an active ingredient: Trp-Ile-Asp

3. A food or drink for inhibiting angiotensin converting enzyme, containing as an active ingredient a peptide consisting of the following amino acid sequence: Trp-Ile-Asp

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