JP4187633B2 - Angiotensin I-converting enzyme inhibitors and their method of manufacture - Google Patents

Description

The present invention relates to a peptide compound having angiotensin I-converting enzyme inhibitory activity that is believed to be capable of regulating increases in blood pressure, and a composition containing the same, which can be widely used in pharmaceuticals, quasi-drugs, functional foods, etc. for preventing and treating hypertension.

The risk factors for coronary artery disease, such as myocardial infarction, which can cause sudden death, are hypertension, hyperlipidemia, impaired glucose tolerance, and obesity, also known as the deadly quartet. According to the Japanese Society of Hypertension, hypertension is one of these risk factors, and there are an estimated 33 million patients in Japan. If hypertension is left untreated, it often progresses without symptoms and often leads to death unless the condition is severe, so it is known as a silent killer. There is a strong demand to improve hypertension, and the development of various antihypertensive drugs and functional foods that regulate blood pressure is underway.

The mechanisms that regulate blood pressure in the body include the renin-angiotensin system, which is a hypertensive system, and the kallikrein-kinin system, which is a hypotensive system. In the former renin-angiotensin system, the enzyme renin is secreted from the juxtaglomerular cells of the kidney into the circulating blood and acts on the substrate angiotensinogen, which is biosynthesized in the liver and present in the blood, to generate angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu). The enzyme that converts this angiotensin I to angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) is angiotensin I converting enzyme, which is mainly present in vascular endothelial cells, lungs, and renal proximal tubules. The angiotensin II generated in this way has the effect of contracting vascular smooth muscle. In addition, angiotensin II acts on the adrenal cortex to promote the production and secretion of aldosterone, and also acts on the renal proximal tubules to increase the reabsorption of sodium filtered by the renal glomerulus. As a result, blood pressure rises.

Meanwhile, in the latter kallikrein-kinin system, the enzyme kallikrein acts on the substrate kininogen to produce kinin. This kinin has the effect of lowering blood pressure by dilating vascular smooth muscle, but angiotensin I converting enzyme is known to break down this kinin. In this way, angiotensin I converting enzyme has the effect of simultaneously activating the renin-angiotensin system, which is a blood pressure increasing system, and inactivating the kallikrein-kinin system, which is a blood pressure decreasing system, resulting in an increase in blood pressure. Therefore, substances that inhibit the activity of angiotensin I converting enzyme are expected to regulate increases in blood pressure, and pharmaceuticals and functional foods that focus on this are being developed in various fields.

As angiotensin I-converting enzyme inhibitors, synthetic compounds such as captopril (D-3-mercapto-2-methylpropanoyl-L-proline), which was published by Ondetti et al. in 1977, have been put to practical use as pharmaceuticals. In addition, many angiotensin I-converting enzyme inhibitory peptides have been found in various foods in recent years, and foods to which peptides derived from milk casein, fermented milk, and fish meat have been added have been put to practical use as foods for specified health uses (Patent Documents 1 to 6 ).
JP 58-109425 A (JP 60-23085 B) Title of invention: "Angiotensin I converting enzyme inhibitor" (obtained from bovine casein) JP 59-44323 A (JP 59-44324 B) Title of invention: "Angiotensin I converting enzyme inhibitor" (obtained from bovine casein) JP 59-44324 A (JP 61-44324 B) Title of invention: "Angiotensin-converting enzyme inhibitor" (obtained from bovine casein) JP 61-36226 A (JP 61-51562 B) Title of invention: "Angiotensin-converting enzyme inhibitor" (obtained from bovine casein) JP 61-36227 A (JP 61-51564 B) Title of invention: "Angiotensin-converting enzyme inhibitor" (obtained from cow-derived casein) JP 5-271297 A, title of invention: "Novel peptide α-1000" Novel peptide α-1000 derived from fish meat, angiotensin-converting enzyme inhibitors, antihypertensive agents, and foods for specified health uses each of which contains novel peptide α-1000 as an active ingredient, and methods for producing the same.

As mentioned above, many angiotensin-converting enzyme inhibitors have already been reported, but as pharmaceuticals, they are expensive because they are produced synthetically. Furthermore, although these synthetic angiotensin-converting enzyme inhibitors have a strong antihypertensive effect, inappropriate dosages can cause renal dysfunction and low blood pressure, so they must be used carefully under a doctor's supervision.

On the other hand, although many food-derived angiotensin I-converting enzyme inhibitor peptides are known, as mentioned above, only a few of these have been put to practical use. The reasons for this include that they often have weak effects when taken orally, or each has its own unique taste, smell, color, etc., making them unsuitable for practical use.

The inventors conducted extensive research to find food-derived angiotensin I converting enzyme inhibitor peptides that can regulate blood pressure rise, and discovered that a protease-treated product made by treating shark cartilage with a protease strongly inhibits angiotensin I converting enzyme. They then found seven types of powerful angiotensin I converting enzyme inhibitor peptides in the protease-treated product.

They were peptide compounds having angiotensin I-converting enzyme inhibitory activity and represented by the amino acid sequences (1) to (7) below.
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
He-Val-Gly (7).

Of the peptide compounds represented by the above seven types of amino acid sequences, Leu-Ile-Gly (5) is a novel peptide compound that has not been described in the literature. The other six types of peptide compounds have been reported to be obtained by proteolytic enzyme treatment, peptide synthesis, genetic engineering, etc., but with the exception of Phe-Tyr (2) and Phe-Ala (3), there have been no reports that they have angiotensin I-converting enzyme inhibitory activity.

The inventors have measured the angiotensin I converting enzyme inhibitory activity of these seven types of shark cartilage-derived peptide compounds represented by amino acid sequences, and have found that each of them has a strong angiotensin I converting enzyme inhibitory activity with high intravascular absorption, stability, and safety. The inventors therefore intend to develop and provide angiotensin I converting enzyme inhibitors, medicines (including pharmaceutical raw materials), quasi-drugs, and foods for improving and treating hypertension, using the newly discovered three types of shark cartilage-derived angiotensin I converting enzyme inhibitory peptide compounds, excluding Phe-Tyr (2), Phe-Ala (3) , Leu-Gly-Val (6), and Ile-Val-Gly (7) , out of the seven types of shark cartilage-derived peptide compounds represented by amino acid sequences (1) to (7).

In order to solve the above-mentioned problems, the present inventors have discovered seven powerful angiotensin I-converting enzyme inhibitory peptide compounds present in proteolytic enzyme-treated shark cartilage, and have utilized three of these angiotensin I-converting enzyme inhibitory peptide compounds to complete the invention of angiotensin I-converting enzyme inhibitors and a method for producing the same .

The present invention relates to an angiotensin I-converting enzyme inhibitor characterized by containing, as an active ingredient, at least one of a peptide compound represented by the amino acid sequence Tyr-Phe (1), a peptide compound represented by the amino acid sequence Leu-Val-Gly (4), a peptide compound represented by the amino acid sequence Leu-Ile -Gly (5), and a pharma- ceutically acceptable salt thereof , and a method for producing the same.

In this specification, Tyr means tylosine, Phe means phenylalanine, Ala means alanine, Leu means leucine, Val means valine, Gly means glycine, and Ile means isoleucine. The symbols representing other amino acid residues are also based on the conventional notation method in amino acid chemistry. Furthermore, all of these amino acids are in the L-form unless otherwise specified.

That is, the first invention to be patented is an angiotensin I-converting enzyme inhibitor containing as an active ingredient at least one of the peptide compounds having angiotensin I-converting enzyme inhibitory activity represented by (1), (4), and (5 ) below.
Tyr-Phe (1),
Leu-Val-Gly (4),
Leu-Ile-Gly (5).

The peptide compounds represented by the three types of amino acid sequences each have a strong angiotensin I-converting enzyme inhibitory activity with high intravascular absorbability, stability, and safety. Therefore, the angiotensin I-converting enzyme inhibitor according to the first invention may be one type of peptide compound having angiotensin I-converting enzyme inhibitory activity, or may be a suitable combination of two or more types.

In addition, the peptide compounds represented by the three types of amino acid sequences can be obtained from a protein having the same amino acid sequence as the compound by the same proteolytic enzyme treatment as described above. However, these peptides can also be obtained by general organic chemical liquid-phase or solid-phase peptide synthesis in which amino acids are introduced stepwise, genetic engineering techniques, or the like.

Examples of proteolytic enzymes used in the present invention include enzymes produced by the genus Bacillus (e.g., Bacillus subtilis, Bacillus thermoproteolyticus, Bacillus licheniformis, etc.), enzymes produced by the genus Aspergillus (e.g., Aspergillus oryzae, Aspergillus niger, Aspergillus mellens, etc.), enzymes produced by the genus Rhizopus (e.g., Rhizopus niveus, Rhizopus delemar, etc.), pepsin, pancreatin, papain, etc. These enzymes may be used alone or in combination of two or more kinds.

The second invention for which a patent is being sought is an angiotensin I converting enzyme inhibitor containing as an active ingredient at least one selected from the group consisting of a peptide compound represented by the amino acid sequence Tyr-Phe, a peptide compound represented by the amino acid sequence Leu-Val-Gly, and a peptide compound represented by the amino acid sequence Leu-Ile -Gly in the form of a salt formed with an inorganic acid, an organic acid, an inorganic base, or an organic base.

The angiotensin I converting enzyme inhibitor described in the second invention contains, as an active ingredient, at least one selected from the group of peptide compounds having angiotensin I converting enzyme inhibitory activity described in the first invention and a pharma- ceutical acceptable salt formed from an inorganic acid, an organic acid, or an inorganic base, or an organic base.
The active ingredient described in the second invention is a peptide compound having angiotensin I-converting enzyme inhibitory activity and a pharma- ceutical acceptable salt thereof, obtained regardless of origin (including those obtained by protease treatment, peptide synthesis, and genetic engineering techniques). It was newly found that the peptide compound has angiotensin I-converting enzyme inhibitory activity, and this invention was completed based on this finding.

The peptide compound obtained by the present invention can be formed into a salt with an inorganic or organic acid or an inorganic or organic base as necessary, but a pharma- ceutically acceptable salt is preferred. Examples of acid addition salts include hydrochlorides, nitrates, sulfates, methanesulfonates, p-toluenesulfonates, and salts with dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, and salts with monocarboxylic acids such as acetic acid, propionic acid, and butyric acid. Inorganic bases suitable for forming salts of the peptide compound obtained by the present invention include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, calcium, magnesium, and aluminum. Examples of salts with organic bases include mono-, di-, and tri-alkylamine salts such as methylamine, dimethylamine, and triethylamine, mono-, di-, and tri-hydroxyalkylamine salts, guanidine salts, and N-methylglucosamine salts.

The third invention for which a patent is being sought is an angiotensin I converting enzyme inhibitor comprising as an active ingredient at least one selected from the group consisting of a peptide compound having an amino acid sequence represented by Tyr-Phe, a peptide compound having an amino acid sequence represented by Leu-Val-Gly, and a peptide compound having an amino acid sequence represented by Leu-Ile -Gly, which is obtained by decomposing shark cartilage or a processed product thereof with a protease, and the resulting decomposition solution.

The third invention is an angiotensin I converting enzyme inhibitor comprising as an active ingredient at least one selected from a group of peptide compounds having angiotensin I converting enzyme inhibitory activity represented by three types of amino acid sequences obtained by decomposing shark cartilage or a processed product thereof with a proteolytic enzyme and extracting the decomposition liquid.

The three peptide compounds derived from shark cartilage, which are the active ingredients of the angiotensin I converting enzyme inhibitor of the third invention, each have strong angiotensin I converting enzyme inhibitory activity, so it goes without saying that one of them may be used, or two or more of them may be used in appropriate combination.

The type of shark from which the peptide compound is obtained is not particularly limited, but the following are preferably used: blue shark (Prionace glauca), porcupine shark (Lamna ditropis), cat shark (Heterodontus japonicus), mako shark (Isurus oxyrinchus), scalloped hammerhead shark (Sphyrna lewini), reef shark (Carcharhinus plumbeus), starry hound shark (Mustelus manazo), thresher shark (Alopias valpinus), flathead shark (Rhizoprionodon acutus), Fuji whale (Etmopterus lucifer), etc.

The protease in the third invention is the same as that described in the first invention, and it goes without saying that these enzymes may be used alone or in combination of two or more kinds.

The pharmaceutical can be prepared by using at least one selected from the group consisting of a peptide compound having an amino acid sequence represented by Tyr-Phe, a peptide compound having an amino acid sequence represented by Leu-Val-Gly, and a peptide compound having an amino acid sequence represented by Leu-Ile-Gly and having angiotensin I-converting enzyme inhibitory activity, or a salt thereof, as an active ingredient.

The three types of peptide compounds having angiotensin I converting enzyme inhibitory activity and their pharma- ceutical acceptable salts have strong antihypertensive effects and bradykinin inactivation inhibitory effects, and therefore can be used as medicines for improving or treating hypertension.

The peptide compounds used in the present invention can be mixed with additives such as excipients as necessary and formulated into formulations such as injections, oral liquids, tablets, granules, powders, capsules, suppositories, ointments, nasal drops, patches, etc.

Additives used in the above-mentioned various preparations include, for example, magnesium stearate, talc, lactose, dextrin, starches, methylcellulose, fatty acid glycerides, water, propylene glycol, macrogols, alcohol, crystalline cellulose, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, carmellose, povidone, polyvinyl alcohol, calcium stearate, etc. In this case, colorants, stabilizers, antioxidants, preservatives, pH regulators, isotonicity agents, solubilizing agents and/or pain-relieving agents can be added as necessary. Granules, tablets, or capsules can be coated with a coating base, such as hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, etc. These preparations can contain 0.01% or more, preferably 0.1 to 70%, of the peptide compound obtained by the present invention.

When preparing the formulation, odorants such as menthol, citric acid and its salts, and fragrances can be used as necessary. Furthermore, the angiotensin I converting enzyme inhibitor obtained by the present invention can contain or be used in combination with other therapeutically useful ingredients, such as known antihypertensive drugs such as captopril and enalapril.

Pharmaceuticals containing at least one of the peptide compounds used in the present invention as an active ingredient are administered orally or parenterally (e.g., transdermally, intravenously, intraperitoneally, etc.) to mammals, including humans. The dosage varies depending on the animal species, the race, sex, symptoms, weight, age, level of blood pressure, administration method, etc. of the target patient, and cannot be generalized, but when administered orally to a typical adult human, the dosage is usually 0.1 to 200 mg, preferably 1 to 150 mg, per kg of body weight per day, and this is usually administered once a day or in divided doses 2 to 3 times a day. However, the dosage can be selected appropriately depending on the severity of the symptoms.

The quasi-drug can be prepared by containing at least one selected from the group consisting of a peptide compound having an amino acid sequence represented by Tyr-Phe, a peptide compound having an amino acid sequence represented by Leu-Val-Gly, and a peptide compound having an amino acid sequence represented by Leu-Ile-Gly and having angiotensin I-converting enzyme inhibitory activity, or a salt thereof .

At least one selected from the group consisting of a peptide compound represented by an amino acid sequence represented by Tyr-Phe, a peptide compound represented by an amino acid sequence represented by Leu-Val-Gly, and a peptide compound having an amino acid sequence represented by Leu-Ile-Gly and having angiotensin I-converting enzyme inhibitory activity, or a salt thereof, can be used in food.

The three peptide compounds used in the present invention have excellent angiotensin I converting enzyme inhibitory activity, and exhibit antihypertensive effects and bradykinin inactivation suppression effects. Moreover , the three peptide compounds obtained in the present invention have no particular unpleasant smell, taste, or color, and are therefore easy to take orally. Therefore, the peptide compounds obtained in the present invention or their salts can be used not only as medicines, but also as ingredients or additives in foods such as jellies, candies, granulated confectioneries, tablets, beverages, soups, noodles, rice crackers, Japanese confectioneries, Western confectioneries, frozen confectioneries, and baked confectioneries, and can also be used as cosmetics or quasi-drugs.

The fourth invention to be patented is a method for producing an angiotensin I converting enzyme inhibitor containing at least one active ingredient selected from the group consisting of a peptide compound represented by the amino acid sequence Tyr-Phe, a peptide compound represented by the amino acid sequence Leu-Val-Gly, and a peptide compound having an amino acid sequence represented by Leu-Ile -Gly, which is characterized by reacting shark cartilage or a processed product thereof with a protease to decompose it, and separating the active ingredient from the decomposition liquid .

The method for producing the peptide compound used in the present invention from shark cartilage or a processed product thereof will be specifically described. The method for decomposing shark cartilage or a processed product thereof with a protease is carried out according to a conventional method. For example, after crushing the shark cartilage as desired, purified water is added, and the pH and temperature are adjusted to optimal values as necessary, and an appropriate protease is added and incubated. Next, after neutralization as necessary, the enzyme is inactivated to obtain an enzyme decomposition solution. The decomposition solution is filtered, for example, using filter paper and/or a filter aid, to remove insoluble matter, and the obtained filtrate is treated with ultrafiltration or the like to obtain a crude peptide fraction having a molecular weight of 50,000 to 10,000 or less. The obtained crude peptide fraction is treated with activated carbon as necessary, filtered, and then concentrated to obtain a crude peptide mixture. The crude peptide mixture is fractionated by column chromatography via liquid-liquid partitioning as desired to obtain a peptide compound having excellent angiotensin I converting enzyme inhibitory activity. The present invention relates to a method for producing angiotensin I converting enzyme inhibitors, which contain as an active ingredient one or an appropriate combination of two or more of the thus obtained peptide compounds represented by the amino acid sequence Tyr-Phe, the peptide compounds represented by the amino acid sequence Leu-Val-Gly, and the peptide compounds having an amino acid sequence Leu-Ile-Gly, or salts thereof.

The peptide compound used in the present invention has strong angiotensin I converting enzyme inhibitory activity, and exhibits strong blood pressure lowering effects and bradykinin inactivation suppression effects. Therefore, the present invention is useful as a preventive or therapeutic agent for hypertension such as essential hypertension, renal hypertension, and adrenal hypertension, a diagnostic agent for these diseases, an antihypertensive agent used in various pathological conditions, an agent for reducing myocardial infarction, and an agent for improving the pathological condition of congestive heart failure.

The peptide compound used in the present invention is easy to take orally because it has no particular unpleasant smell, taste, or color. Therefore, the peptide compound obtained in the present invention, or various preparations containing the compound, can be blended or added to foods such as jellies, candies, granulated confectioneries, tablets, beverages, soups, noodles, rice crackers, Japanese sweets, Western sweets, frozen desserts, and baked goods. It can also be used as health foods, foods for specified health uses, and functional foods that have the above-mentioned useful effects. It can also be provided as cosmetics or quasi-drugs.

Examples of the present invention will be described in detail below, but it goes without saying that the present invention is not limited to these examples.

Example 1
<Production Example 1> Production of angiotensin I converting enzyme inhibitory peptide compound 3 kg of cartilage from the blue shark (Prionace glauca) was crushed, 9 kg of purified water was added, and the mixture was heated to 55°C, after which 7.5 g of papain (Carica Papaya) was added and stirred for 14 hours to carry out an enzymatic decomposition reaction. The reaction solution was heated to 95°C to inactivate the enzyme activity, and after cooling, it was filtered using Celite with a median pore size of 7 microns. The filtrate was passed through an ultrafiltration membrane (ROMICON HF1.0-43-PM50) to remove fractions with a molecular weight of 50,000 or more, and then spray-dried to obtain 260 g of angiotensin I converting enzyme inhibitory peptide mixture.

The crude mixture of angiotensin I converting enzyme inhibitory peptides obtained by the above method was subjected to HPLC analysis under the following conditions, and the chromatogram shown in FIG. 7 was obtained.
<HPLC analysis conditions>
Column Waters
μBONDASPHERE C18 3.9×150mm
Column temperature: 40°C
Mobile phase A H ₂ O (contains 0.1TFA)
Mobile phase B: Acetonitrile (containing 0.1 TFA)
Gradient: Linear gradient from 5% solution A to 40% solution B over 0-60 min, 40% solution B over 60-75 min
Analysis time 75 minutes Flow rate 0.4ml/min
Injection volume: 15 μL
Detection UV 220nm

As a result, as shown in Figure 7, it was confirmed that the crude mixture of angiotensin I-converting enzyme inhibitory peptides obtained from the protease-treated product of shark cartilage was found to contain seven types of potent angiotensin I-converting enzyme inhibitory peptides.
Tyr-Phe (1),
Phe-Tyr (2),
Phe-Ala (3),
Leu-Val-Gly (4),
Leu-Ile-Gly (5),
Leu-Gly-Val (6),
He-Val-Gly (7).

Therefore, we decided to separate each of the seven types of potent angiotensin I-converting enzyme inhibitor peptides contained in the crude peptide mixture.

First, 60.0 g of the peptide mixture was dissolved in 1200 mL of water and the pH was adjusted to 2.0 with 1 M hydrochloric acid. This aqueous solution was extracted with 1200 mL of n-butanol, and the aqueous layer was further extracted twice with 600 mL of n-butanol. The n-butanol layer was concentrated under reduced pressure to obtain 10.3 g of extract.

5.78 g of this extract was subjected to silica gel column chromatography (Fuji Silysia Chemical, BW-820MH, developing solvent: n-butanol-pyridine-water-acetic acid 10:1:2:1),
Silica gel column chromatography (BW-820MH, developing solvent: ethanol-water 10:1),
Ion exchange resin column chromatography (Aldrich, Amberlite (trademark) CG-50, developing solvent: methanol),
Silica gel column chromatography (Kanto Chemical, silica gel 60N for flash chromatography, developing solvent: n-butanol-ethanol-chloroform-5% ammonia water 6:4:2:1)
As a result, 45 mg of fraction A and 30 mg of fraction B, each of which exhibited high angiotensin I converting enzyme inhibitory activity, were obtained.

Fraction A: 29 mg was dissolved in 0.87 mL of 5% hydrogen chloride-methanol, allowed to stand at room temperature for 20 hours, and then concentrated to dryness under reduced pressure to obtain 32 mg of residue.

This was separated by silica gel column chromatography (BW-820MH, developing solvent: n-butanol-ethanol-chloroform-water 8:1:2:1) to obtain fraction A2: 6.6 mg. Fraction A2: 4.3 mg was dissolved in 0.22 mL of water, and then 4.5 mg of sodium bicarbonate was added. 43 μL of a tetrahydrofuran solution containing 3.8 μL of benzyloxycarbonyl (hereinafter abbreviated as Z) chloride was added dropwise to this, and the mixture was stirred at room temperature for 6 hours, after which the tetrahydrofuran was distilled off. Extraction was performed twice with 0.2 mL of ethyl acetate, and the combined organic layer was concentrated to dryness under reduced pressure to obtain 6.5 mg of residue.

This was separated by silica gel column chromatography (BW-820MH, developing solvent: toluene-ethyl acetate 2:1 and 5:1) to obtain fraction A3: 0.8 mg. As a result of comparing ^{the 1} H-NMR with the synthetic compound described in Preparation Example 4, the structure of fraction A3 was confirmed to be Z-Tyr(Z)-Phe-methyl ester (hereinafter abbreviated as OMe). Tyr-Phe can be obtained from fraction A3 by general organic chemistry techniques. This Tyr-Phe was found to be identical to the peptide compound described in Biochimica et Biophysica Acta (1980), 625(2), 266-273, but its angiotensin I converting enzyme inhibitory activity has not been reported.

Example 2
Next, 23 mg of fraction B was dissolved in 0.70 mL of 5% hydrogen chloride-methanol, and the mixture was left at room temperature for 16 hours, and then concentrated to dryness under reduced pressure to obtain 28 mg of residue. This was separated by silica gel column chromatography (BW-820MH, developing solvent: n-butanol-ethanol-chloroform-water 8:1:2:1) to obtain fractions B2: 4.6 mg and B3: 6.1 mg. 3.5 mg of fraction B2 was dissolved in 0.25 mL of water, and then 7.3 mg of sodium bicarbonate was added. 31 μL of a tetrahydrofuran solution containing 6.2 μL of Z chloride was added dropwise to this, and the mixture was stirred at room temperature for 5 hours, after which tetrahydrofuran was distilled off. Then, the mixture was extracted twice with 0.3 mL of ethyl acetate, and the combined organic layer was concentrated to dryness under reduced pressure to obtain 5.8 mg of residue. This was separated by silica gel column chromatography (BW-820MH, developing solvent: toluene-ethyl acetate 1:1) to obtain fraction B21: 0.4 mg. As a result of comparing the structure of this synthetic compound with ¹ H-NMR, the structure of fraction B21 was confirmed to be Z-Leu-Ile-Gly-OMe. Leu-Ile-Gly can be obtained from fraction B21 by general organic chemistry techniques. Leu-Ile-Gly is a novel peptide compound that has not been described in the literature.

(Reference Example 1)
Next, 5.1 mg of fraction B3 was dissolved in 0.25 mL of water, and 5.4 mg of sodium bicarbonate was added. 25 μL of a tetrahydrofuran solution containing 4.6 μL of Z chloride was added dropwise to the mixture, and the mixture was stirred at room temperature for 4 hours, after which tetrahydrofuran was distilled off. Then, the mixture was extracted twice with 0.2 mL of ethyl acetate, and the combined organic layer was concentrated to dryness under reduced pressure to obtain 7.0 mg of residue. This was separated by silica gel column chromatography (BW-820MH, developing solvent: toluene-ethyl acetate 1:1 → toluene-ethyl acetate 1:3), and fraction B31: 0.7 mg and fraction B32: 1.0 mg were obtained. As a result of comparing ^{the 1} H -NMR with the synthetic compound described in Preparation Example 5, the structure of fraction B31 was confirmed to be Z-Phe-Ala-OMe. Phe-Ala can be obtained from fraction B31 by general organic chemistry techniques. It was found that the Phe-Ala was identical to the peptide compound described in Science, (1981) 212(4499), 1153-1155, but its angiotensin I converting enzyme inhibitory activity was not reported.

Example 3
The structure of fraction B32 obtained by the above method was determined to be Z-Leu-Val-Gly-OMe. Leu-Val-Gly can be obtained from fraction B32 by general organic chemistry techniques. This Leu-Val-Gly was found to be identical to the peptide compound described in Journal of Experimental Medicine (1985), 161(4), 805-815, but its angiotensin I converting enzyme inhibitory activity has not been reported.

Example 4
A mixture of fraction A and fraction B (0.12 g) was obtained by the same method as above. This was dissolved in 3.6 mL of 5% hydrogen chloride-methanol, left at room temperature for 24 hours, and then concentrated to dryness under reduced pressure to obtain 0.13 g of residue. This was dissolved in 4.0 mL of 33% dioxane water, and 0.11 g of sodium bicarbonate and 92 μL of Z chloride were added and stirred at room temperature for 16 hours. The solvent was removed to obtain 95 mg of residue. This was separated by silica gel column chromatography (Kanto Chemical, silica gel 60N for flash chromatography, developing solvent: toluene-ethyl acetate 2:1 → toluene-ethyl acetate 1:3 → ethyl acetate → methanol) to obtain 10 mg of a mixture of Z-Phe-Tyr(Z)-OMe and Z-Tyr(Z)-Phe-OMe, and 2.3 mg of a mixture of Z-Phe-Tyr-OMe and Z-Tyr-Phe-OMe. These structures were confirmed by comparing ^{the 1} H-NMR with the synthetic compounds described in Preparation Examples 3 and 4. From these, Phe-Tyr and Tyr-Phe can be obtained by general organic chemistry techniques. Phe-Tyr was found to be identical to the compound reported in Journal of Nutritional Biochemistry (1998), 9(7), 415-419 as an angiotensin I converting enzyme inhibitor peptide derived from garlic.

(Reference Example 2)
The crude mixture of angiotensin I converting enzyme inhibitory peptides was subjected to HPLC analysis under the following conditions, and the chromatogram shown in Fig. 7 was obtained. Leu-Gly-Val was then separated under the HPLC conditions, and the structure was determined by comparing it with the sample synthesized by Production Method 8. However, no angiotensin I converting enzyme inhibitory activity has been reported for this Leu-Gly-Val.

(Reference Example 3)
<Preparation Example 2> Synthesis of Phe-Tyr 30 mg of Z-Phe-Tyr was dissolved in 1.2 mL of dioxane, and 0.6 mL of water was added. After adding 6 mg of 10% palladium-carbon, the mixture was stirred for 4 hours under a hydrogen atmosphere. After removing the catalyst by cotton plug filtration, the filtrate was concentrated to dryness under reduced pressure to obtain 24 mg of residue. This was purified by G-25 column chromatography (1.2 g, developing solvent: water-acetic acid 20:1, 1 fraction 0.3 mL) to obtain 17 mg of Phe-Tyr (yield 81%).

(Reference Example 4)
<Production Example 3> Synthesis of Z-Phe-Tyr(Z)-OMe 10 mg of Z-Phe-Tyr was dissolved in 0.50 mL of 5% hydrogen chloride methanol solution, and the solution was left at room temperature for 2 hours, and then concentrated to dryness under reduced pressure to obtain 10 mg of Z-Phe-Tyr-OMe (quantitative yield). 10 mg of Z-Phe-Tyr-OMe was dissolved in 0.50 mL of tetrahydrofuran, and 7 μL of triethylamine and 6 μL of Z chloride were added at 0° C. and stirred at room temperature for 30 minutes. After concentrating under reduced pressure to remove tetrahydrofuran, 2 mL of ethyl acetate, 0.5 mL of water, and 0.2 mL of saturated aqueous potassium hydrogen sulfate solution were added to separate the organic layer. The aqueous layer was extracted once with 1 mL of ethyl acetate, and the combined organic layer was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (BW-820MH 1.0 g, toluene-ethyl acetate 4:1, 1 fraction 0.5 mL) to obtain 6.2 mg of Z-Phe-Tyr(Z)-OMe (yield 48%).

(Example 5)
<Production Example 4> Synthesis of Z-Tyr(Z)-Phe-OMe 20 mg of Tyr-Phe was dissolved in 0.60 mL of 5% hydrogen chloride methanol solution, and the solution was left to stand at room temperature for 12 hours, and then concentrated to dryness under reduced pressure to obtain 23 mg of Tyr-Phe-OMe.HCl (quantitative yield). 23 mg of the Tyr-Phe-OMe.HCl was dissolved in 0.60 mL of dichloromethane, and 23 μL of triethylamine and 21 μL of Z chloride were added at 0° C. and stirred at room temperature for 3.5 hours. 23 μL of triethylamine was added and stirred at room temperature for 30 minutes, and the mixture was concentrated under reduced pressure to remove dichloromethane, and then 2 mL of ethyl acetate and 0.5 mL of a saturated aqueous potassium hydrogen sulfate solution were added to separate the organic layer. The aqueous layer was extracted once with 1 mL of ethyl acetate, and the combined organic layer was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (BW-820MH 1.9 g, chloroform-ethyl acetate 10:1, 1 mL per fraction) to give 11 mg of Z-Tyr(Z)-Phe-OMe (yield 30%).

(Reference Example 5)
Preparation Example 5: Synthesis of Z-Phe-Ala-OMe and Phe-Ala 0.31 g of Phe was suspended in 3.1 mL of water, and 0.32 g of sodium bicarbonate and 0.27 mL of Z chloride were added at 0° C. The mixture was stirred at 0° C. for 1 hour and at room temperature for 18 hours. The mixture was washed once with 4 mL of diethyl ether, and 3 mL of 2M hydrochloric acid was added, and the mixture was extracted once with 8 mL of ethyl acetate and concentrated to dryness under reduced pressure to obtain 0.49 g of Z-Phe (yield 88%). 0.49 g of Z-Phe was dissolved in 15 mL of tetrahydrofuran, and 0.22 g of commercially available Ala-OMe.HCl was added. 0.31 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter abbreviated as EDCI.HCl) and 0.22 mL of triethylamine were added at 0° C., and the mixture was stirred for 2 hours at 0° C. and for 63 hours at room temperature. After adding 2 mL of water, the mixture was concentrated under reduced pressure to remove tetrahydrofuran, and then 20 mL of ethyl acetate, 1 mL of water, and 0.5 mL of a saturated aqueous solution of potassium hydrogen sulfate were added, and the organic layer was separated, washed once with 4 mL of a saturated aqueous solution of sodium hydrogen carbonate and once with 4 mL of a saturated aqueous solution of sodium chloride, and concentrated to dryness under reduced pressure.

The residue was purified by silica gel column chromatography (BW-820MH 15 g, toluene-ethyl acetate 3:2, 1 fraction 4 mL) to obtain 0.34 g of Z-Phe-Ala-OMe (yield 53%). 0.15 g of Z-Phe-Ala-OMe was dissolved in 6 mL of tetrahydrofuran, and then 1.5 mL of water was added. 9 mg of lithium hydroxide was added and stirred at room temperature for 2.5 hours, then 1 mg of lithium hydroxide was added and stirred at room temperature for 6 hours. 1 mg of lithium hydroxide was added and stirred at room temperature for another 10 hours, then concentrated to dryness under reduced pressure. 5 mL of ethyl acetate, 1 mL of water, and 0.5 mL of saturated aqueous potassium hydrogen sulfate solution were added to the residue, and the organic layer was separated. The aqueous layer was extracted once with 2 mL of ethyl acetate, and the combined organic layer was concentrated to dryness under reduced pressure to obtain 0.14 g of Z-Phe-Ala (yield quantitative).

50 mg of Z-Phe-Ala was dissolved in 2 mL of dioxane, and 1 mL of water was added. 10 mg of 10% palladium-carbon was added, and the mixture was stirred under a hydrogen atmosphere for 3 hours. After removing the catalyst by filtration through a cotton plug, the filtrate was concentrated to dryness under reduced pressure to obtain 38 mg of residue. This was purified by G-25 column chromatography (1.2 g, water only, 1 fraction 0.4 mL) to obtain 32 mg of Phe-Ala (quantitative yield).

Example 6
Preparation Example 6: Synthesis of Z-Leu-Val-Gly-OMe and Leu-Val-Gly 0.25 g of Val was suspended in 2.5 mL of water, and 0.35 g of sodium bicarbonate and 0.30 mL of Z chloride were added at 0° C. The mixture was stirred at 0° C. for 1 hour and at room temperature for 18 hours. The mixture was washed once with 4 mL of diethyl ether, and 3 mL of 2M hydrochloric acid was added. The mixture was extracted once with 8 mL of ethyl acetate and concentrated to dryness under reduced pressure to obtain 0.54 g of Z-Val (quantitative yield).

0.54 g of Z-Val was dissolved in 16 mL of tetrahydrofuran, and 0.30 g of commercially available Gly-OMe.HCl was added. 0.49 g of EDCI.HCl and 0.36 mL of triethylamine were added at 0°C, and the mixture was stirred at 0°C for 2 hours and at room temperature for 17 hours. 2 mL of water was added, and the mixture was concentrated under reduced pressure to remove tetrahydrofuran. 15 mL of ethyl acetate, 2 mL of water, and 0.5 mL of saturated aqueous potassium hydrogen sulfate solution were added, and the organic layer was separated. The organic layer was washed once with 3 mL of saturated aqueous sodium hydrogen carbonate solution and once with 3 mL of saturated aqueous sodium chloride solution, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (13 g of BW-820MH, toluene-ethyl acetate 3:2, 1 fraction 4 mL) to obtain 0.32 g of Z-Val-Gly-OMe (yield 46%). 73 mg of Z-Val-Gly-OMe was dissolved in 0.51 mL of 33% hydrogen bromide-acetic acid solution, allowed to stand at room temperature for 2.5 hours, and then concentrated under reduced pressure.

2 mL of diethyl ether was added to the residue, and the mixture was thoroughly shaken and allowed to stand. The supernatant was removed and the mixture was dissolved in 1 mL of methanol. The pH of this solution was 4. This was concentrated to dryness under reduced pressure to obtain 69 mg of crude Val-Gly-OMe.HBr (crude yield 113%). 2 mL of a tetrahydrofuran solution of 67 mg of Z-Leu was added to 68 mg of Val-Gly-OMe.HBr. 58 mg of EDCI.HCl and 42 μL of triethylamine were added at 0°C, and the mixture was stirred at 0°C for 2 hours and at room temperature for 24 hours. 1 mL of water was added, and the mixture was concentrated under reduced pressure to remove tetrahydrofuran. 4 mL of ethyl acetate, 1 mL of water, and 0.2 mL of saturated aqueous potassium hydrogen sulfate solution were added, and the organic layer was separated, and then concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (BW-820MH 4.3 g, toluene-ethyl acetate 1:1, 1 fraction 1.5 mL) to obtain 39 mg of Z-Leu-Val-Gly-OMe (yield 35%).

30 mg of Z-Leu-Val-Gly-OMe was dissolved in 1.2 mL of tetrahydrofuran, and then 0.3 mL of water was added. 2 mg of lithium hydroxide was added, and the mixture was stirred at room temperature for 1.5 hours, and then concentrated to dryness under reduced pressure. 1 mL of ethyl acetate, 0.3 mL of water, and 0.1 mL of saturated aqueous potassium hydrogen sulfate solution were added to the residue, and the organic layer was separated. The aqueous layer was extracted once with 1 mL of ethyl acetate, and the combined organic layers were concentrated to dryness under reduced pressure to obtain 29 mg of Z-Leu-Val-Gly (quantitative yield).

(Example 7)
28 mg of Z-Leu-Val-Gly was dissolved in 1.2 mL of dioxane, and 0.6 mL of water was added. After adding 6 mg of 10% palladium-carbon, the mixture was stirred under a hydrogen atmosphere for 4 hours. After removing the catalyst by cotton plug filtration, the filtrate was concentrated to dryness under reduced pressure to obtain 23 mg of residue. This was purified by silica gel column chromatography (BW-820MH 1.2 g, n-butanol-ethanol-chloroform-water 6:4:2:1, 1 fraction 0.6 mL) to obtain 10 mg of Leu-Val-Gly (yield 52%).

(Reference Example 6)
Preparation Example 7: Synthesis of Z-Leu-Gly-Val-OMe and Leu-Gly-Val 0.88 g of tert-butoxycarbonyloxy (hereinafter abbreviated as Boc)-Gly was dissolved in 26 mL of tetrahydrofuran, and 0.84 g of commercially available Val-OMe.HCl was added. 0.96 g of EDCI.HCl and 0.69 mL of triethylamine were added at 0°C, and the mixture was stirred at 0°C for 2 hours and at room temperature for 20 hours. 5 mL of water was added, and the mixture was concentrated under reduced pressure to remove tetrahydrofuran. 30 mL of ethyl acetate and 1 mL of saturated aqueous potassium hydrogen sulfate solution were added, and the organic layer was separated, washed once with 5 mL of saturated aqueous sodium hydrogen carbonate solution, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (BW-820MH 14 g, toluene-ethyl acetate 1:1, 1 fraction 4 mL) to obtain 1.2 g of Boc-Gly-Val-OMe (yield 86%).

0.50 g of Boc-Gly-Val- OMe was dissolved in 5.0 mL of trifluoroacetic acid, and the mixture was left at room temperature for 20 minutes, then concentrated to dryness under reduced pressure to obtain 0.64 g of Gly-Val-OMe.TFA (crude yield 123%). 0.43 g of Z-Leu in 13 mL of tetrahydrofuran solution was added to 0.64 g of Gly-Val-OMe.TFA. 0.40 g of EDCI.HCl and 0.29 mL of triethylamine were added at 0°C, and the mixture was stirred at 0°C for 2 hours and at room temperature for 14 hours. 2 mL of water was added, and the mixture was concentrated under reduced pressure to remove tetrahydrofuran. 15 mL of ethyl acetate, 2 mL of water, and 0.5 mL of saturated aqueous potassium hydrogen sulfate solution were added, and the organic layer was separated, washed once with 3 mL of saturated aqueous sodium hydrogen carbonate solution, and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (BW-820MH 23 g, toluene-ethyl acetate 1:2, 1 fraction 8 mL) to obtain 0.59 g of Z-Leu-Gly-Val-OMe (yield 84%).

100 mg of Z-Leu-Gly-Val-OMe was dissolved in 4 mL of tetrahydrofuran, and then 1 mL of water was added. 6 mg of lithium hydroxide was added and stirred at room temperature for 16 hours, after which 3 mg of lithium hydroxide was added and stirred at room temperature for 6 hours. 2 mg of lithium hydroxide was added to the mixture, and the mixture was further stirred at room temperature for 22 hours, and then concentrated to dryness under reduced pressure. 3 mL of ethyl acetate, 1 mL of water, and 0.5 mL of saturated aqueous potassium hydrogen sulfate solution were added to the residue, and the organic layer was separated. The aqueous layer was extracted once with 2 mL of ethyl acetate, and the combined organic layer was concentrated to dryness under reduced pressure. The resulting residue was purified by silica gel column chromatography (5.0 g of BW-820MH, ethyl acetate only, 1 mL per fraction) to obtain 31 mg of Z-Leu-Gly-Val with a purity of about 70% (yield 23%).

(Reference Example 7)
Furthermore, 29 mg of Z-Leu-Gly-Val with a purity of about 70% was dissolved in 1.2 mL of dioxane, and 0.6 mL of water was added. After adding 6 mg of 10% palladium-carbon, the mixture was stirred for 4 hours under a hydrogen atmosphere. After removing the catalyst by cotton plug filtration, the filtrate was concentrated to dryness under reduced pressure to obtain 22 mg of residue. This was purified by silica gel column chromatography (BW-820MH 1.1 g, n-butanol-ethanol-chloroform-water 6:4:2:1, 1 fraction 0.4 mL) to obtain 8 mg of pure Leu-Gly-Val (yield 36%). Leu-Gly-Val was found to be the same as the peptide compound described in JP-T-2003-504062, but its angiotensin I converting enzyme inhibitory activity has not been reported.

Test Example 1: Measurement of inhibitory activity of angiotensin I-converting enzyme inhibitors The inhibitory activity of peptide compounds having angiotensin I-converting enzyme inhibitory activity was measured by a partially modified method of Cushmann et al. (Biochemical Pharmacology, 20, 1637-1648 (1971)).

That is, 250 μL of 0.2 M borate-potassium phosphate buffer (containing 0.4 M NaCl, pH 8.3) of 5 mM Benzoyl-Glycyl-L-Histidyl-L-Leucine (manufactured by Peptide Institute) and 30 μL of aqueous solution of test compound prepared to a predetermined concentration were added to a 1.5 mL Eppendorf tube, and pre-incubated at 37°C for 5 minutes. Angiotensin I converting enzyme (derived from rabbit lung, manufactured by Sigma, enzyme number EC3.4.15.1) solution (60 mU/mL 0.2 M borate-potassium phosphate buffer) was added to this solution to start the enzyme reaction. After the reaction was carried out at 100 rpm in a shaker bath at 37°C for 60 minutes, 250 μL of 1 N hydrochloric acid was added to stop the reaction. 580 μL of ethyl acetate was added to this, and after shaking for 45 seconds, it was centrifuged at 3000 rpm for 10 minutes, and 500 μL of the supernatant ethyl acetate layer was collected. 520 μL of ethyl acetate was further added to the aqueous layer, and after shaking for 20 seconds, it was centrifuged at 3000 rpm for 10 minutes, and 500 μL of the supernatant ethyl acetate layer was collected. This operation was repeated again, and the resulting ethyl acetate layer (1.5 mL in total) was dried in a centrifugal evaporator at 3000 rpm for 30 minutes under reduced pressure conditions to completely remove the ethyl acetate. The dried product was dissolved in 3 mL of high-performance liquid chromatography buffer (20% acetonitrile/0.1% trifluoroacetic acid aqueous solution).

The hippuric acid produced was analyzed by reversed-phase high performance liquid chromatography (HPLC), and the peak area of hippuric acid produced by the enzyme reaction was determined by measuring the absorbance at 228 nm. A blank was also prepared by adding 1N hydrochloric acid in advance during the enzyme reaction and carrying out the same procedure. The HPLC column used was μBONDASPHERE C18 Φ3.9×150 mm (Waters), and the mobile phase was 20% acetonitrile/0.1% trifluoroacetic acid aqueous solution.

The inhibition rate was calculated using the following formula.
Angiotensin I-converting enzyme inhibition rate (%) = {1-B-C) / A} x 100
A: Peak area (228 nm) when distilled water is added
B: Peak area (228 nm) when inhibitor was added
C: Blank peak area (228 nm) when inhibitor was added

Table 1 shows the angiotensin I converting enzyme inhibition rate (%) for each sample described in Example 1 and Example 2 at 60 μg/mL.

<Test Example 2> Antihypertensive effect by tail vein administration in spontaneously hypertensive rats For the test, male spontaneously hypertensive rats (Wister-Okamoto derived Spontaneously Hypertensive Rats, hereinafter referred to as SHRs) with a body weight of 250±50 g, a systolic blood pressure of 200±20 mmHg, and a heart rate of 400±50 beats/min were used. Before the start of the test, six SHRs were kept in a 45×23×21 cm cage for 1 week for acclimation in an environment with a 12-hour light/dark cycle (lights on from 9:00 a.m. to 9:00 p.m.), room temperature of 21-23° C., humidity of 50-70%, and free access to food (Lab Diet manufactured by PMI Nutrition International) and drinking water (self-pumped water meeting tap water quality standards). The n-butanol extract concentrate of the peptide mixture obtained in Preparation Example 1 was dissolved in 5 mL of 0.9% saline per kg of SHR body weight at a dose of 30 mg per kg of SHR body weight, and administered once to the tail vein of SHRs (3 animals per group) and kept in an environment of 32±1° C. As a control group, SHRs (3 animals per group) were administered the same dose of 0.9% saline alone via the tail vein. Blood pressure was measured using a non-invasive blood pressure measuring device (Model 59, IITC, CA, USA) immediately before administration of the sample and 30, 60, and 240 minutes after administration. Table 2 shows the change in systolic blood pressure over time.

<Test Example 3> Antihypertensive effect by forced oral administration to SHRs SHRs were prepared in the same manner as in Test Example 1, and the n-butanol extract concentrate of the peptide mixture obtained in Preparation Example 1 was dissolved in 10 mL of 0.9% saline per kg of SHR body weight at a dose of 300 mg per kg of SHR body weight, and orally administered once to SHRs (3 animals per group) and kept in an environment of 32±1° C. As a control group, SHRs (3 animals per group) were orally administered only 0.9% saline at the same dose. Blood pressure was measured immediately before administration of the sample and 30, 60, and 240 minutes after administration using a non-invasive blood pressure measuring device (Model 59, IITC, CA, USA). Table 2 shows the change in systolic blood pressure over time.

The shark cartilage-derived peptide compound used in the present invention has strong angiotensin I converting enzyme inhibitory activity and exhibits a strong antihypertensive effect, and is therefore useful as a pharmaceutical agent such as a preventive or therapeutic agent for hypertension such as essential hypertension, renal hypertension, adrenal hypertension, etc., a diagnostic agent for these diseases, an antihypertensive agent used in various pathological conditions, an agent for reducing myocardial infarction, and an agent for improving the pathological condition of congestive heart failure. In addition, since the peptide compound of the present invention can be taken orally, it can be used as a health food, food for specified health uses, or functional food having the above-mentioned useful effects, and can also be used as a cosmetic or quasi-medical product.

1 shows the ¹ H-NMR spectrum of the peptide compound Phe-Tyr. 1 shows the ¹ H-NMR spectrum of peptide compound Phe-Ala. 1 shows the ¹ H-NMR spectrum of the peptide compound Leu-Val-Gly. 1 shows the ¹ H-NMR spectrum of the peptide compound Leu-Ile-Gly. 1 shows the ¹ H-NMR spectrum of the peptide compound Leu-Gly-Val. 1 shows the ¹ H-NMR spectrum of the peptide compound Ile-Val-Gly. 2 shows a chromatogram obtained by HPLC analysis of a crude peptide compound. Peptide compound Tyr-Phe derivative Z-Tyr(Z)-Phe-OMe ¹ The H-NMR spectrum is shown.

Claims (4)

An angiotensin I converting enzyme inhibitor comprising as an active ingredient at least one selected from the group consisting of a peptide compound represented by an amino acid sequence Tyr-Phe, a peptide compound represented by an amino acid sequence Leu-Val-Gly, and a peptide compound represented by an amino acid sequence Leu-Ile -Gly. An angiotensin I converting enzyme inhibitor comprising , as an active ingredient, at least one selected from the group consisting of a peptide compound represented by an amino acid sequence represented by Tyr-Phe, a peptide compound represented by an amino acid sequence represented by Leu-Val - Gly, and a peptide compound represented by an amino acid sequence represented by Leu-Ile-Gly, in the form of a salt formed with an inorganic acid, an organic acid, an inorganic base, or an organic base. An angiotensin I converting enzyme inhibitor comprising as an active ingredient at least one selected from the group consisting of a peptide compound having an amino acid sequence represented by Tyr-Phe, a peptide compound having an amino acid sequence represented by Leu-Val-Gly, and a peptide compound having an amino acid sequence represented by Leu-Ile -Gly, which is obtained by hydrolyzing shark cartilage or a processed product thereof with a proteolytic enzyme and extracting the hydrolyzed liquid. A method for producing an angiotensin I converting enzyme inhibitor containing at least one active ingredient selected from the group consisting of a peptide compound represented by the amino acid sequence Tyr-Phe, a peptide compound represented by the amino acid sequence Leu-Val-Gly, and a peptide compound having an amino acid sequence represented by Leu-Ile -Gly, characterized by reacting shark cartilage or a processed product thereof with a proteolytic enzyme to decompose it, and separating it from the decomposition liquid.

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