JP4229933B2 - Salmon-derived angiotensin I converting enzyme-inhibiting peptide compound or peptide composition containing the same and method for producing them - Google Patents

Description

  TECHNICAL FIELD The present invention relates to a peptide compound having an angiotensin I converting enzyme inhibitory activity, which is said to be capable of regulating an increase in blood pressure, and a composition containing the same, and a pharmaceutical or quasi drug for preventing or treating hypertension It can be widely used for food additives, functional foods and the like.

  There are four risk factors for coronary artery disease such as myocardial infarction that can cause sudden death: hypertension, hyperlipidemia, impaired glucose tolerance, and obesity, and is also said to be a quartet of death. Hypertension, which is one of the risk factors, is reported to have 33 million patients in Japan according to the Japanese Society of Hypertension. If hypertension is left untreated, it is often called as a silent killer because it often progresses asymptomatic and results in death unless it is severe. Since there is a strong demand for improving such hypertension, various antihypertensive agents and functional foods that regulate blood pressure are being developed.

  One mechanism for regulating blood pressure in a living body is a renin-angiotensin system that is a pressor system and a kallikrein-kinin system that is a hypotensive system. In the former renin-angiotensin system, the enzyme renin is secreted from the glomerular cells of the kidney into the circulating blood, and is biosynthesized in the liver and acts on the substrate angiotensinogen present in the blood, thereby causing angiotensin I (Asp-Arg). -Val-Tyr-Ile-His-Pro-Phe-His-Leu). The enzyme that converts this angiotensin I into angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) is an angiotensin I-converting enzyme that exists mainly in vascular endothelial cells, lungs, and renal proximal tubules. . The angiotensin II produced in this way has the effect of contracting vascular smooth muscle. In addition, the angiotensin II acts on the adrenal cortex to promote the production and secretion of aldosterone, and acts on the renal proximal tubule to enhance the reabsorption of sodium filtered through the glomeruli. As a result, blood pressure increases.

  On the other hand, in the latter kallikrein-kinin system, the enzyme kallikrein acts on the substrate kininogen to produce kinin. The kinin works to dilate vascular smooth muscles and lower blood pressure, but angiotensin I converting enzyme is known to degrade the kinin. Thus, the angiotensin I converting enzyme has the action of simultaneously activating the renin-angiotensin system, which is a pressor system, and inactivating the kallikrein-kinin system, which is a hypotensive system, and as a result, increases blood pressure. There is an effect. Therefore, since substances that inhibit the activity of angiotensin I converting enzyme can be expected to regulate an increase in blood pressure, pharmaceuticals and functional foods focusing on this are being developed in various directions.

  As an angiotensin I converting enzyme inhibitory substance, a synthetic compound represented by captopril (D-3-mercapto-2-methylpropanoyl-L-proline) published by Ondetti et al. In addition, in recent years, many angiotensin I converting enzyme inhibitory peptides have been found in various foods, and among these, foods added with milk casein, fermented milk, and fish-derived peptides have been put to practical use as foods for specified health use. ing.

  Regarding peptides obtained from casein derived from cattle, JP-A-58-109425 (JP-B-60-2390) discloses Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly. Angiotensin I converting enzyme inhibitors having an amino acid sequence consisting of -Lys are described. JP-A-59-44323 discloses an angiotensin I converting enzyme inhibitor having an amino acid sequence consisting of Phe-Phe-Val-Ala-Phe, and JP-A-59-44324 discloses Val. An angiotensin I converting enzyme inhibitor having an amino acid sequence consisting of -Ala-Pro is described. Further, JP-A 61-36226 discloses an angiotensin I converting enzyme inhibitor having an amino acid sequence consisting of Ala-Val-Pro-Tyr-Pro-Gln-Arg, and JP-A 61-36227 discloses. Describes an angiotensin I converting enzyme inhibitor having an amino acid sequence consisting of Thr-Thr-Met-Pro-Leu-Trp. On the other hand, JP-A-5-271297 discloses an angiotensin converting enzyme inhibitor, antihypertensive agent, food for specified health use, and a method for producing these, containing peptide a-1000 having specific physical properties derived from fish meat as an active ingredient. Are listed.

Furthermore, the following reports are known for various di- or tripeptides.
Val-Leu: Fermentforschung, 11, 271-86 (1930);
Ile-Leu: Journal of Organic Chemistry, 20, 1169-72 (1955);
Val-Phe: Compt. Rend., 235, 180-2 (1952);
Ile-Phe: Journal of Organic Chemistry, 20, 1169-72 (1955);
Phe-Tyr: Annalen Der Chemie., 653, 76-80 (1962);
Tyr-Phe: Journal of the Chemical Society, Abstracts, 3658-69 (1960);
Leu-Phe: Biochemical Journal, 41, 596-602. (1947);
Leu-Ile: Journal of Biological Chemistry, 247 (4), 1208-10, (1972);
Ala-Phe-Leu: JOC (1971), 36 (1), 49-59;
Leu-Val-Leu: Zeitschrift fuer Lebensmittel-Untersuchung und -Forschung (1975), 159 (6), 329-36;
Ile-Val-Leu: Journal of Immunology (1998), 161 (5), 2465-2472;
Val-Ile-Leu: Biochemistry (1998), 37 (13), 4473-4481;
Val-Ile-Phe: Bulletin of the Chemical Society of Japan (1984), 57 (1), 103-7;
Tyr-Leu-Val: Quantative Structure-Activity Relationships (1989), 8 (3), 195-203;
Phe-Val-Leu: International Journal of Peptide & Protein Research (1996), 48 (2) 148-155;
Leu-Tyr: Fermentforschung, 11, 399-432 (1930);
Leu-Trp: Biochemical Journal, 39, 351-355 (1945);
Ile-Trp: Journal of Organic Chemistry, 32 (11), 3415-25 (1967);
Ile-Val-Trp: US Pat. No. 4,356,118 Oct. 26 (1982);
Japanese Laid-Open Patent Publication No. 58-109425 (Japanese Patent Publication No. 60-2390) JP 59-44323 A JP 59-44324 JP 61-36226 A JP 61-36227 A JP-A-5-271297 U.S. Pat.No. 4,356,118 Fermentforschung, 11, 271-86 (1930) Journal of Organic Chemistry, 20, 1169-72 (1955) Compt. Rend., 235, 180-2 (1952) Journal of Organic Chemistry, 20, 1169-72 (1955) Annalen Der Chemie., 653, 76-80 (1962) Journal of the Chemical Society, Abstracts, 3658-69 (1960) Biochemical Journal, 41, 596-602. (1947) Journal of Biological Chemistry, 247 (4), 1208-10, (1972) JOC (1971), 36 (1), 49-59 Zeitschrift fuer Lebensmittel-Untersuchung und -Forschung (1975), 159 (6), 329-36. Journal of Immunology (1998), 161 (5), 2465-2472. Biochemistry (1998), 37 (13), 4473-4481. Bulletin of the Chemical Society of Japan (1984), 57 (1), 103-7. Quantative Structure-Activity Relationships (1989), 8 (3), 195-203. International Journal of Peptide & Protein Research (1996), 48 (2), 148-155 Fermentforschung, 11, 399-432 (1930) Biochemical Journal, 39, 351-355 (1945); Journal of Organic Chemistry, 32 (11), 3415-25 (1967);

  As described above, many angiotensin I converting enzyme inhibitors have already been reported, but pharmaceuticals are expensive because they are made by synthetic methods. In addition, the angiotensin I converting enzyme inhibitor produced by the synthetic method has a strong antihypertensive effect, but if the dose is inappropriate, it causes renal dysfunction and hypotension. It is required to use.

  On the other hand, many food-derived angiotensin I converting enzyme inhibitory peptides are known, but only a few are practically used as described above. The reason for this is that the action and effects at the time of oral ingestion are weak, and the taste, smell, color, etc. are characteristic and are not suitable for practical use.

  An object of the present invention is to provide a peptide compound having a strong angiotensin I converting enzyme inhibitory activity and a pharmaceutically acceptable salt thereof, which are highly absorbable into the blood vessel, stable and safe. Another object of the present invention is to provide an angiotensin I converting enzyme inhibitor comprising as an active ingredient at least one of these peptide compounds and pharmaceutically acceptable salts thereof. Another object of the present invention is to provide a composition comprising these peptide compounds and pharmaceutically acceptable salts thereof, and a method for producing the same.

Examples of peptide compounds having angiotensin I converting enzyme inhibitory activity include peptide compounds represented by the amino acid sequences of the following formulas (i) to (xxi) .
Val-Leu (i),
Ile-Leu (ii),
Val-Phe (iii),
Ile-Phe (iv),
Phe-Tyr (v),
Tyr-Phe (vi),
Leu-Phe (vii),
Leu-Ile (viii),
Ala-Phe-Leu (ix),
Leu-Val-Leu (x),
Ile-Val-Leu (xi),
Val-Ile-Leu (xii),
Val-Ile-Phe (xiii),
Tyr-Leu-Val (xiv),
Phe-Val-Leu (xv),
Ile-Val-Phe (xvi),
Phe-Ile-Ala (xvii),
Leu-Tyr (xviii),
Leu-Trp (xix),
Ile-Trp (xx),
Ile-Val-Trp (xxi).

The peptide compounds, inorganic acids, organic acids, inorganic bases or organic bases, in may be formed pharmaceutically acceptable salts formed by.

The angiotensin I converting enzyme inhibitor according to the present invention includes a peptide compound represented by the amino acid sequence of Ile-Val-Phe (xvi), a peptide compound represented by the amino acid sequence of Phe-Ile-Ala (xvii), and these peptides An angiotensin I converting enzyme inhibitor comprising at least one selected from pharmaceutically acceptable salts of compounds as an active ingredient.

The angiotensin I converting enzyme inhibitor according to the present invention includes a peptide compound represented by the amino acid sequence of Tyr-Phe (vi) , a peptide compound represented by the amino acid sequence of Leu-Trp (xix), and an amino acid of Ile-Trp (xx) And further comprising at least one selected from peptide compounds represented by sequences and pharmaceutically acceptable salts of these peptide compounds, wherein all of the peptide compounds are obtained by reacting salmon or a processed product thereof with a proteolytic enzyme. It can be separated from the decomposition solution obtained by decomposition.

  The peptide compound obtained by the present invention has a strong angiotensin I converting enzyme inhibitory activity, and exhibits a strong blood pressure lowering action and a bradykinin inactivation inhibiting action. Therefore, the present invention is useful in the prevention and treatment of hypertension such as essential hypertension, renal hypertension, adrenal hypertension, diagnostic agents for these diseases, antihypertensive agents used in various pathologies, reduction of myocardial infarction, congestive heart failure. It is useful as a disease condition improving agent.

  Moreover, the peptide compound obtained by this invention is easy to ingest orally since the peculiar taste of smell, taste, and color is not recognized. Therefore, the peptide compound obtained in the present invention, or various preparations containing the compound, for example, jelly, rice cake, granule confectionery, tablet confectionery, beverage, supermarket, noodles, rice cracker, Japanese confectionery, Western confectionery, frozen confectionery, It can be formulated and added to foods such as baked goods and seasonings. It can be used as a health food having a useful action as described above, a food for specified health use, or a functional food. Furthermore, it can also be provided as a cosmetic or quasi-drug.

  The present inventors have conducted extensive research to find a food-derived angiotensin I-converting enzyme inhibitor peptide capable of regulating an increase in blood pressure. As a result, a processed proteolytic enzyme obtained by treating salmon muscle with a proteolytic enzyme is converted to angiotensin I. It was noticed that the enzyme was strongly inhibited, and 20 kinds of strong angiotensin I converting enzyme inhibitory peptides were found in the processed protease. Furthermore, it has been found that the processed product of proteolytic enzyme obtained by treating salmon liver with a proteolytic enzyme contains a component that strongly inhibits angiotensin I-converting enzyme. Seven types of strong angiotensin are included in the processed proteolytic enzyme. I-converting enzyme inhibitory peptides were found.

  These were di- or tripeptide compounds having angiotensin I converting enzyme inhibitory activity represented by any of the amino acid sequences (i) to (xxi) listed above. Among these, the formulas (i) to (vi) have been confirmed to exist in both salmon muscle and liver. Among the 21 types of peptide compounds having different amino acid sequences, (xvi) Ile-Val-Phe and (xvii) Phe-Ile-Ala are novel peptide compounds not described in any literature. Moreover, Val-Leu (i), Tyr-Phe (vi), Leu-Ile (viii), Ile-Val-Leu (xi), Val-Ile-Leu (xii), Val-Ile-Phe (xiii), Tyr-Leu-Val (xiv) and Phe-Val-Leu (xv) have been reported to be obtained by various general organic chemical methods or enzymatic degradation methods, but they have angiotensin I converting enzyme inhibitory activity. No report has been made. The other 11 types of peptide compounds have been reported separately as angiotensin I converting enzyme inhibitory activity, but they have not been reported as being derived from salmon or as a mixture containing them at a certain ratio.

  In addition, a mixture containing all seven types of angiotensin I-converting enzyme inhibitory peptides identified from the processed proteolytic enzyme derived from salmon liver in a specific ratio (the ratio included in the processed salmon liver enzyme) is compared to the original salmon. It was confirmed that the liver-derived enzyme degradation product had angiotensin I converting enzyme inhibitory activity of 50 to 100 times.

  Therefore, the present inventors measured the angiotensin I converting enzyme inhibitory activity of the peptide compound represented by any one of these 21 kinds of salmon-derived amino acid sequences. It was newly found to have a strong angiotensin I converting enzyme inhibitory activity with high safety. Therefore, the inventors have used a salmon-derived angiotensin I converting enzyme inhibitory peptide compound to improve or treat hypertension (including pharmaceutical raw materials), quasi drugs, food additives, We will develop and provide functional foods.

  Then, the present inventors have found 21 kinds of potent angiotensin I converting enzyme inhibitory peptides present in processed salmon muscle or liver proteolytic enzyme, and using these, angiotensin I converting enzyme inhibitory peptide compounds or The present invention has been completed for inventions of peptide compositions containing these and methods for producing them.

That is, the angiotensin I converting enzyme inhibitor according to the present invention includes a peptide compound represented by the amino acid sequence of Ile-Val-Phe (xvi), a peptide compound represented by the amino acid sequence of Phe-Ile-Ala (xvii), and these An angiotensin I converting enzyme inhibitor comprising, as an active ingredient, at least one selected from pharmaceutically acceptable salts of the peptide compounds.

  In this specification, Val or V is valine, Leu or L is leucine, Ile or I is isoleucine, Phe or F is phenylalanine, Tyr or Y is tylosin, Ala or A is alanine, Trp or W is tryptophan. This means that each symbol and notation representing other amino acid residues is also based on a conventional method in amino acid chemistry, and the amino acid sequence is N-terminal on the left side and C-terminal on the right side. These amino acids are all in the L form unless otherwise indicated.

First, the peptide compound having angiotensin I converting enzyme inhibitory activity used in the present invention is selected from (i) to (xxi) listed above, and is represented by the amino acid sequence of Ile-Val-Phe (xvi). At least one of the peptide compound and the peptide compound represented by the amino acid sequence of Phe-Ile-Ala (xvii) is used as an essential active ingredient of an angiotensin I converting enzyme inhibitor.

  The peptide compound according to the present invention can also be used in the form of a pharmaceutically acceptable salt. For the formation of this salt, an organic acid, an inorganic acid, an organic base, or an inorganic base can be used as desired. When a peptide compound is used as a pharmaceutically acceptable salt, one kind of salt obtained therefrom may be used for one kind of peptide compound, or the acid or base obtained from one kind of peptide compound is different. A combination of two or more salts may be used.

The above-mentioned 21 kinds of di- or tripeptide compounds having different amino acid sequences (i) to (xxi) each have a potent angiotensin I converting enzyme inhibitory activity with high absorbability, stability and safety in blood vessels. is doing.

  A peptide compound or peptide mixture comprising at least one of the above 21 amino acid sequences can also be obtained from a protein having the same amino acid sequence as the compound by the same proteolytic enzyme treatment as described above. Can also be obtained by peptide synthesis or genetic engineering techniques using general organic chemical liquid phase or solid phase methods in which amino acids are introduced stepwise.

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

  The peptide compound according to the present invention can also be used in the form of a pharmaceutically acceptable salt. This pharmaceutically acceptable salt is formed by one or more of the aforementioned angiotensin I converting enzyme inhibitory activity peptide compounds and an inorganic acid or organic acid, or an inorganic base or organic base. And a pharmaceutically acceptable salt.

  This pharmaceutically acceptable salt was also obtained regardless of its origin (including those obtained by proteolytic enzyme treatment, peptide synthesis, and genetic engineering techniques). Peptide compounds having angiotensin I converting enzyme inhibitory activity In contrast, it can be formed using acids or bases that can form pharmaceutically acceptable salts.

  In addition, the peptide compound obtained by this invention can form the salt with an inorganic acid or an organic acid, and the salt with an inorganic base or an organic base as needed. The acid or base can be selected according to the use of the salt, but the following pharmaceutically acceptable salts are preferable in consideration of the use for foods, cosmetics, pharmaceuticals and the like. Examples of the acid addition salt include hydrochloride, nitrate, sulfate, methanesulfonate, p-toluenesulfonate, and 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 or butyric acid. Inorganic bases suitable for forming a salt of the peptide compound obtained in the present invention are, for example, hydroxides such as ammonia, sodium, lithium, calcium, magnesium and aluminum, carbonates and bicarbonates. Examples of salts with organic bases include mono-, di- and tri-alkylamine salts such as methylamine, dimethylamine and triethylamine, mono-, di- and trihydroxyalkylamine salts, guanidine salts, N- Examples thereof include methyl glucosamine salt.

The peptide compound having angiotensin I converting enzyme inhibitory activity represented by the formulas (i) to (xxi) described above decomposes salmon muscle or salmon liver, or a processed product thereof with a proteolytic enzyme, Is obtained . This peptide compound can also be used in the form of a pharmaceutically acceptable salt.

  Here, the type of salmon from which the peptide compound is obtained is not particularly limited. ), Salmon trout (Oncorhynchus masou masou), charr (Salvelinus leucomaenis), brown trout (Salmo trutta), Atlantic salmon (Salmo salar salar), etc. are preferably used.

  The proteolytic enzyme used for decomposing this salmon or its processed product is the same proteolytic enzyme as described above, and these enzymes may be used alone or in combination of two or more. .

The angiotensin I converting enzyme inhibitor according to the present invention is selected from the group consisting of the above peptide compounds and pharmaceutically acceptable salts thereof, and is a peptide represented by the amino acid sequence of Ile-Val-Phe (xvi) A compound and a peptide compound represented by the amino acid sequence of Phe-Ile-Ala (xvii) and at least one selected from pharmaceutically acceptable salts of these peptide compounds are contained as essential active ingredients.

  Inhibition of angiotensin I converting enzyme by containing at least one peptide compound having an angiotensin I converting enzyme inhibitory activity and a pharmaceutically acceptable salt thereof according to the present invention in foods, cosmetics, pharmaceuticals or quasi drugs. Effects such as prevention, alleviation or treatment of hypertension derived from the activity can be obtained.

  The antihypertensive agent and / or prophylactic agent is prepared by mixing at least one of the above peptide compounds and pharmaceutically acceptable salts thereof with an additive such as an excipient as necessary, for example, an injection, It can be obtained by formulation in the form of oral liquids, tablets, granules, powders, capsules, suppositories, ointments, nasal drops, patches and the like.

  Examples of additives used in the above various preparations include magnesium stearate, talc, lactose, dextrin, starches, methyl cellulose, fatty acid glycerides, water, propylene glycol, macrogols, alcohol, Crystalline cellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, carmelloses, popidone, polyvinyl alcohol, calcium stearate and the like can be mentioned. In this case, a colorant, a stabilizer, an antioxidant, an antiseptic, a pH adjuster, a tonicity agent, a solubilizing agent and / or a soothing agent can be added as necessary. Granules, tablets, or capsules can be coated with a coating base such as hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, and the like. These preparations can contain the peptide compound obtained in the present invention in an amount of 0.01% by weight or more, preferably 0.1 to 70% by weight.

  In preparing the preparation, flavoring agents such as menthol, citric acid and salts thereof, and fragrance can be used as necessary. Furthermore, the angiotensin I converting enzyme inhibitor obtained in the present invention may contain other components useful for treatment, for example, known antihypertensive agents such as captopril and enalapril, or may be used in combination.

  An angiotensin I converting enzyme inhibitor characterized by comprising as an active ingredient at least one of the peptide compounds obtained in the present invention and pharmaceutically acceptable salts thereof is orally or non-humanly used in mammals including humans. It is administered orally (for example, transdermally, intravenously, intraperitoneally, etc.). The dose varies depending on the animal species, the race of the target patient, sex, symptoms, weight, age, blood pressure level, method of administration, etc., but it cannot be said unconditionally, but when administered orally to general human adults The dose is usually 0.1 to 200 mg / kg body weight per day, preferably 1 to 150 mg per day, and this is usually administered once a day or divided into 2 to 3 times a day. However, the dose can be appropriately selected according to the degree of symptoms.

  The peptide compound of the present invention has excellent angiotensin I converting enzyme inhibitory activity, and exhibits blood pressure lowering action and bradykinin inactivation inhibiting action. Moreover, the peptide compound obtained by the present invention is easy to be taken orally because no peculiar taste of smell, taste and color is observed. Therefore, the peptide compound obtained in the present invention or the composition described later is used not only as a pharmaceutical product, but also, for example, jelly, rice cake, granule confectionery, tablet confectionery, beverage, supermarket, noodle, rice cracker, Japanese confectionery, Western confectionery. It can be provided by adding to a food such as frozen confectionery, baked confectionery, seasoning, etc., and can also be provided as cosmetics or quasi-drugs. Quasi-drugs include, for example, hair restorers, hair nourishing agents, hair removal agents, hair dyes, depigmenting agents, permanent wave agents, bath preparations, medicated cosmetics / medicinal soaps, medicated dentifrices, refreshing agents, odor control agents, and tenka And powders.

  What is necessary is just to set the content of the peptide compound in these articles | goods according to the intended use and function of the articles | goods, for example, 0.01 weight% or more, Preferably it is selected from the range of 0.1 to 70 weight%. it can. In the case of cosmetics, in addition to various solid, semi-solid or liquid cosmetic base materials and active ingredients for obtaining the desired cosmetic effect, using peptide compounds or compositions, Various forms of cosmetics such as lotions, creams, powders, and emulsion gels can be constructed.

  When the compound having angiotensin I converting enzyme inhibitory activity according to the present invention is used in foods, cosmetics, pharmaceuticals, and quasi drugs, various compositions such as salmon extracts, crude peptide mixtures, and crude purified products ( It can also be used in the form of a preparation. This composition is obtained by extracting components containing the target peptide compound from salmon muscle or liver, or a degradation solution obtained by decomposing these processed products with a proteolytic enzyme, and purifying as necessary. As a prepared product. As a preferable example of this preparation (composition), the thing of the composition shown by the following Table 1-1 obtained by the process similar to the peptide mixture in the manufacture example 1 using a salmon muscle, and the salmon liver were used. The thing of the composition shown by the following Table 1-2 obtained by the process similar to the peptide mixture in manufacture example 3 can be mentioned.

  In addition, at least 1 sort of each isolated or synthesized peptide may be added to these peptide compositions, and the compounding quantity may be changed.

  Among the above peptide compositions, the angiotensin I converting enzyme inhibitory activity of a preparation (including components other than peptides) obtained by treating salmon liver or a processed product thereof with a proteolytic enzyme is the same as that of the peptide (i ) To (vi) and (xviii) were isolated by Test Example 8 to be described later to show a value of about 50 to 100 times that obtained by simply mixing the compositions shown in Table 1-2. Therefore, this preparation is one of the preferred embodiments for inhibiting angiotensin I converting enzyme.

  The preparation is preferably a step of reacting salmon muscle or liver or a processed product thereof with a proteolytic enzyme to obtain a degradation solution containing degradation products, and extracting a desired peptide compound from the degradation solution. It can obtain by the manufacturing method which has a process to do.

  A method for producing the peptide compound according to the present invention from salmon muscle or liver, or a processed product thereof will be specifically described. A method for decomposing salmon muscle or liver or a processed product thereof with a proteolytic enzyme is performed according to a conventional method. For example, if desired, salmon muscle or liver is crushed, purified water is added, pH and temperature are adjusted to optimum values as necessary, and an appropriate proteolytic enzyme is added and incubated. Next, after neutralization as necessary, the enzyme is deactivated to obtain an enzyme decomposition solution. The decomposition solution is filtered using, for example, filter paper and / or a filter aid to remove insoluble matter, and the resulting filtrate is treated by ultrafiltration to obtain a crude peptide fraction having a molecular weight of 50,000 to 10,000. obtain. The obtained crude peptide fraction is treated with activated carbon as necessary, filtered and concentrated to obtain a crude peptide mixture.

  This crude peptide mixture or a preparation obtained by removing impurities from this crude peptide mixture as necessary and purifying it can also be used as the composition described above. Furthermore, the crude peptide mixture can be fractionated by column chromatography through liquid-liquid partitioning or the like, if desired, to obtain each peptide compound having excellent angiotensin I converting enzyme inhibitory activity.

  As processed salmon muscle or liver, 0.1 to 10 equivalents of shredded salmon muscle or liver in an aqueous liquid medium, preferably ethanol, water, acetic acid, or a mixture of these in an appropriate ratio, Preferably, 1 to 5 equivalent volumes are added, and the mixture is stirred for 0.1 to 5 hours, preferably 0.5 to 3 hours under room temperature to 100 ° C., preferably 20 to 70 ° C. to remove impurities. Available to:

  As a specific example, 500 mL of ethanol was added to 200 g of shredded salmon muscle or liver and stirred for 2 hours. Ethanol was removed by suction filtration to obtain 185 g of a salmon-treated product, which was used in the production described later.

  Particularly preferred proteolytic enzymes and treatment conditions are as follows: salmon muscle or liver, or a treated product thereof, 0.5 to 10 equivalents, preferably 1 to 5 equivalents of water, and the liquid temperature is room temperature to 70 ° C., preferably Is kept at 30 to 60 ° C., and a suitable acid or alkali is used to adjust the pH to 1 to 10, preferably 3 to 8, preferably with addition of a proteolytic enzyme such as thermolysin or papain. The reaction is carried out for 12 hours, preferably 3 to 8 hours. As the acid and alkali to be used, hydrochloric acid, acetic acid, citric acid, sodium hydroxide, sodium hydrogen carbonate, aqueous ammonia and the like are selected.

  On the other hand, the liquid-liquid distribution can be performed using a suitable organic solvent by adjusting the pH of the salmon muscle or liver decomposition solution to 1 to 10, preferably 2 to 9, with acid or alkali as required. As the acid and alkali to be used, hydrochloric acid, acetic acid, citric acid, sodium hydroxide, sodium hydrogen carbonate, aqueous ammonia and the like are selected. The organic solvent to be used may be any one that is immiscible with water, and chloroform, ethyl acetate, butanol, octanol and the like are selected.

  Preferred separation liquids in the above column chromatography include chloroform, methanol, ethanol, butanol, isopropanol, ethyl acetate, toluene, acetonitrile, pyridine, diethyl ether, acetic acid, trifluoroacetic acid, formic acid, aqueous ammonia, water, and the like. Examples of the separation medium include normal phase silica gel, reverse phase silica gel, acidic ion exchange resin, and basic ion exchange resin.

Examples of the present invention will be described in detail below, but the present invention is not limited to these examples.
Example 1
<Production Example 1> Production of Angiotensin I Converting Enzyme Inhibiting Peptide Compound After cutting 3 kg of carrot trout (Oncorhynchus gorbuscha) muscle, adding 9 kg of purified water and warming to 55 ° C, adding 7.5 g of papain (Carica Papaya) and adding 8 The mixture was stirred for an hour to carry out an enzymatic decomposition reaction (pH 5-6). The reaction solution was heated to 95 ° C. to deactivate the enzyme activity, cooled, and filtered using Celite having an intermediate pore diameter of 7 μm. The filtrate was passed through an ultrafiltration membrane (Romicon HF1.0-43-PM50) to remove a fraction having a molecular weight of 50,000 or more and then spray-dried to obtain 360 g of an angiotensin I converting enzyme-inhibiting peptide mixture powder.

When the powder of the angiotensin I converting enzyme inhibitory peptide mixture obtained by the above method was subjected to HPLC analysis under the following conditions, a chromatogram as shown in FIG. 1 was obtained.
<HPLC analysis conditions>
Column: Waters μBONDASPHERE C18 3.9 × 150mm
Column temperature: 40 ° C
Mobile phase A: H ₂ O (containing 0.1% by volume TFA)
Mobile phase B: acetonitrile (containing 0.1 vol% TFA)
Gradient: A linear gradient from 5% by volume of B solution to 40% by volume of B solution from 0 to 60 minutes, and 40% by volume of B solution from 60 to 75 minutes.
Analysis time: 75 minutes Flow rate: 0.4ml / min
Injection volume: 15μL
Detection: UV220nm
(Example 2)
<Production Example 2>
Therefore, each of the strong angiotensin I converting enzyme inhibitory peptides contained in the peptide mixture crude was subjected to separation treatment.

  First, 300 g of the peptide mixture described in Production Example 1 was dissolved in 1200 mL of water, and the pH was adjusted to 8.0 with aqueous ammonia (28.0 wt%). This aqueous solution was extracted with 1,200 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 24 g of an extract.

  20 g of this extract was separated according to the method described in FIGS. A part of the isolated peptide was methyl esterified according to a general organic chemical method. A candidate peptide having a structure estimated by NMR measurement was synthesized with a peptide synthesizer and methyl esterified according to a general organic chemistry method as a standard substance. By comparing these with LC-MS, 20 types of peptide structures were determined.

The results of NMR and melting point (decomposition point) measurement of the free form and NMR of the methyl ester form are shown below for each of the separated Ile-Val-Phe and Phe-Ile-Ala.
About Ile-Val-Phe <Ecological data>
(1) ¹ H NMR data:
¹ H NMR (500 MHz, methanol-d ₄ ; TMS); d0.87 (3H, d, J = 7 Hz), 0.90 (3H, t, J = 7 Hz), 0.95 (3H, d, J = 7 Hz), 0.97 (3H, d, J = 7 Hz), 1.10 (1H, ddq, J = 14, 9 and 7 Hz), 1.47 (1H, ddq, J = 14, 4 and 7 Hz), 1.83 (1H , dddq, J = 9, 5, 4 and 7 Hz), 2.03 (1H, dqq, J = 7, 7 and 7 Hz), 2.97 (1H, dd, J = 14 and 9 Hz), 3.18 (1H, dd , J = 14 and 5 Hz), 3.72 (1H, d, J = 6 Hz), 4.22 (1H, d, J = 7 Hz), 4.62 (1H, dd, J = 9 and 5 Hz), 7.17 (1H , m) and 7.24 (4H, d, J = 4 Hz).
(2) Decomposition point 186 ℃
<Methyl ester data>
¹ H NMR data:
¹ H NMR (500 MHz, methanol-d ₄ ; TMS); d0.87 (3H, d, J = 7 Hz), 0.88 (3H, t, J = 7 Hz), 0.93 (3H, d, J = 7 Hz), 0.95 (3H, d, J = 7 Hz), 1.11 (1H, ddq, J = 14, 9 and 7 Hz), 1.46 (1H, ddq, J = 14, 4 and 7 Hz), 1.75 (1H , dddq, J = 9, 5, 4 and 7 Hz), 2.02 (1H, dqq, J = 7, 7 and 7 Hz), 2.97 (1H, dd, J = 14 and 9 Hz), 3.14 (1H, dd , J = 14 and 6 Hz), 3.41 (1H, d, J = 5 Hz), 3.67 (3H, s), 4.21 (1H, d, J = 7 Hz), 4.66 (1H, dd, J = 9 and 6 Hz), 7.17-7.22 (3H, m) and 7.24-7.27 (2H, m).
About Phe-Ile-Ala <Ecological data>
(1) ¹ H NMR data
¹ H NMR (500 MHz, methanol-d ₄ ; TMS); d0.92 (3H, t, J = 7 Hz), 0.99 (3H, d, J = 7 Hz), 1.19 (1H, ddq, J = 14 , 9 and 7 Hz), 1.41 (3H, d, J = 7 Hz), 1.60 (1H, ddq, J = 14, 4 and 7 Hz), 1.85 (1H, dddq, J = 9, 8, 4 and 7 Hz), 3.01 (1H, dd, J = 14 and 9 Hz), 3.25 (1H, dd, J = 14 and 5 Hz), 4.16 (1H, dd, J = 9 and 6 Hz), 4.27 (1H, d , J = 8 Hz), 4.33 (1H, q, J = 7 Hz) and 7.26-7.36 (5H, m).
(2) Decomposition point 188 ℃
<Methyl ester data>
¹ H NMR data:
¹ H NMR (500 MHz, methanol-d ₄ ; TMS); d0.89 (3H, t, J = 7 Hz), 0.93 (3H, d, J = 7 Hz), 1.12 (1H, ddq, J = 13 , 9 and 7 Hz), 1.39 (3H, d, J = 7 Hz), 1.46 (1H, ddq, J = 13, 4 and 7 Hz), 1.78 (1H, dddq, J = 9, 8, 4 and 7 Hz), 2.80 (1H, dd, J = 13 and 8 Hz), 3.03 (1H, dd, J = 13 and 6 Hz), 3.63 (1H, dd, J = 8 and 6 Hz), 3.70 (3H, s ), 4.22 (1H, d, J = 7 Hz), 4.37 (1H, q, J = 7 Hz), 7.19-7.22 (3H, m) and 7.25-7.29 (2H, m).
<Test Example 1> Measurement of inhibitory activity of angiotensin I converting enzyme inhibitory substance The inhibitory activity of a peptide compound having angiotensin I converting enzyme inhibitory activity was measured by the method of Cushmann et al. (Biochemical Pharmacology, 20, 1637-1648 (1971)). Partly modified.

  That is, 0.2 mM boric acid-potassium phosphate buffer (containing 0.4 M NaCl, pH 8.3) of 5 mM Benzoyl-Glycyl-L-Histidyl-L-Leucine (manufactured by Peptide Institute) in 1.5 mL Eppendorf tube Was added at 30 μL of a test compound aqueous solution prepared to a predetermined concentration, and preincubated at 37 ° C. for 5 minutes. To this solution, 100 μL of an angiotensin I converting enzyme (from rabbit lung, Sigma, enzyme number EC3.4.15.1) solution (60 mU / mL 0.2M boric acid-potassium phosphate buffer) was added, and the enzyme reaction was performed. Started. After reaction at 37 ° C. for 60 minutes in a shaker bath at 100 rpm, 250 μL of 1N hydrochloric acid was added to stop the reaction. To this was added 580 μL of ethyl acetate, and the mixture was shaken for 45 seconds, then centrifuged at 3000 rpm for 10 minutes, and 500 μL of the supernatant ethyl acetate layer was collected. An additional 520 μL of ethyl acetate was added to the aqueous layer, allowed to permeate for 20 seconds, and then centrifuged at 3000 rpm for 10 minutes, and 500 μL of the supernatant ethyl acetate layer was collected. This operation was repeated again, and a total of 1.5 mL of the obtained ethyl acetate layer was dried at 3000 rpm for 30 minutes under reduced pressure using a centrifugal evaporator to completely remove ethyl acetate. The dried product was dissolved in 3 mL of high-performance liquid chromatography buffer (20 vol% acetonitrile / 0.1 vol% trifluoroacetic acid aqueous solution).

  The generated hippuric acid was analyzed by reversed-phase high performance liquid chromatography (HPLC), and the peak area of hippuric acid generated by the enzymatic reaction was determined by measuring the absorbance at 228 nm. Further, a blank was prepared by performing the same operation by adding 1N hydrochloric acid in advance during the enzyme reaction. For HPLC, μBONDASPHERE C18 φ3.9 × 150 mm (Waters) was used for the column, and 20 volume% acetonitrile / 0.1 volume% trifluoroacetic acid aqueous solution was used for the mobile phase.

The inhibition rate was calculated using the following formula.
Angiotensin I converting enzyme inhibition rate (%) = {1 − ((B−C) ÷ A)} × 100
A: Peak area when adding distilled water (228nm)
B: Peak area when an inhibitor is added (228 nm)
C: Peak area of the blank when an inhibitor is added (228 nm)
On the other hand, when the peptide mixture containing the above 17 kinds of peptide compounds and each of the 17 kinds of isolated peptide compounds were inspected for odor, taste and color, no peculiar taste was found, and the taste of foods, etc. It was confirmed that it could be added without breaking.

  Table 2 shows the angiotensin I converting enzyme inhibition rate (%) in each sample of 197 μg / mL described in Example 1 and Example 2.

<Test Example 2> Antihypertensive effect of tail vein administration in spontaneously hypertensive rats Male Spontaneously hypertensive rats (Wister-Okamoto derived) with body weight 250 ± 50g, systolic blood pressure 200 ± 20mmHg, heart rate 400 ± 50 times / min Spontaneously Hypertensive Rat (hereinafter referred to as SHR) was used. Light / dark cycle 12 hours (lights on from 9:00 am to 9:00 pm) before the start of the test, room temperature 21-23 ° C, humidity 50-70%, feed (Lab Diet manufactured by PMI Nutrition International) and drinking water ) 6 SHRs were placed in a 45 x 23 x 21 cm cage in an environment of free consumption and acclimated for 1 week. The dried ultrafiltrate of the peptide mixture obtained in Production Example 1 was dissolved at a dose of 30 mg / kg SHR body weight in 0.9 ml of 0.9% by weight saline per 1 kg body weight of SHR. A single tail vein administration was carried out in an environment of 32 ± 1 ° C. As a control group, SHR (3 mice per group) was administered with 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 sample administration and 60, 120, and 240 minutes after administration. Table 3 shows changes over time in the systolic blood pressure.

<Test Example 3> Antihypertensive effect of SHR by oral gavage SHR was prepared in the same manner as Test Example 2, and the dried ultrafiltrate of the peptide mixture obtained in Production Example 1 was administered at a dose of 300 mg per kg SHR body weight. Thus, SHR was dissolved in 10 mL of 0.9% saline per 1 kg body weight, and was orally administered once to SHR (3 mice per group) and reared in an environment of 32 ± 1 ° C. As a control group, the same dose of 0.9% by weight saline was orally administered to SHR (3 mice per group). Blood pressure was measured using a non-invasive blood pressure measuring device (Model 59, IITC, CA, USA) immediately before sample administration and 60, 120, and 240 minutes after administration. Table 3 shows changes in systolic blood pressure over time.

(Example 3)
Production Example 3 Production of Angiotensin I Converting Enzyme Inhibitory Peptide Compound 21.2 kg of carrot trout (Oncorhynchus gorbuscha) liver was shredded and freeze-dried to obtain 4.66 kg of a dried product. About 5.66 volumes of ethanol (24 L) was added to 4.66 kg of this dried product, and the mixture was stirred at 70 ° C. for 1 hour, and ethanol-soluble components were removed by suction filtration. After the same operation was again performed on the obtained ethanol-insoluble matter, the solid content was dried under reduced pressure to obtain about 4.1 kg of salmon liver defatted product.

  After adding 80 kg of purified water to 4.1 kg of this salmon liver defatted material and heating to 55 ° C., 41 g of papain (Carica Papaya) was added and stirred for 8 hours to carry out an enzymatic decomposition reaction (pH 5-6). The reaction solution was heated to 95 ° C. to deactivate the enzyme activity, cooled, and filtered using Celite having an intermediate pore diameter of 7 μm. The filtrate was passed through an ultrafiltration membrane (Romicon HF1.0-43-PM50) to remove a fraction having a molecular weight of 50,000 or more, and then spray-dried to obtain 2.9 kg of an angiotensin I converting enzyme-inhibiting peptide mixture powder. .

When the powder of the angiotensin I converting enzyme inhibitory peptide mixture obtained by the above method was analyzed by HPLC under the following conditions, a chromatogram as shown in FIG. 4 was obtained.
<HPLC analysis conditions>
Column: Waters μBONDASPHERE C18 3.9 × 150mm
Column temperature: 40 ° C
Mobile phase A: H ₂ O (containing 0.1% by volume TFA)
Mobile phase B: acetonitrile (containing 0.1 vol% TFA)
Gradient: A linear gradient from 5% by volume of B solution to 60% by volume of B solution over 0 to 60 minutes, and 60% by volume of B solution maintained over 60 to 75 minutes.
Analysis time: 75 minutes Flow rate: 0.4ml / min
Injection volume: 15μL
Detection: UV 220 nm.

(Example 4)
<Production Example 4>
Therefore, each of the strong angiotensin I converting enzyme inhibitory peptides contained in the peptide mixture crude was subjected to separation treatment.

  First, 1000 g of the peptide mixture described in Production Example 3 was dissolved in 4000 mL of water, and the pH was adjusted to 8.0 with aqueous ammonia (28.0 wt%). This aqueous solution was extracted with 4000 mL of n-butanol, and the aqueous layer was further extracted twice with 2000 mL of n-butanol. The n-butanol layer was concentrated under reduced pressure to obtain 78 g of an extract.

20 g of this extract was separated according to the method described in FIG. A part of the isolated peptide was methyl esterified according to a general organic chemical method. A candidate peptide having a structure estimated by NMR measurement was synthesized with a peptide synthesizer and methyl esterified according to a general organic chemistry method as a standard substance. By comparing these with ¹ H-NMR and TOF-MS, seven types of peptide structures were determined.

<Test Example 5> Measurement of inhibitory activity of angiotensin I converting enzyme inhibitory substance The inhibitory activity of a peptide compound having angiotensin I converting enzyme inhibitory activity was measured by the method of Cushmann et al. (Biochemical Pharmacology, 20, 1637) as in Test Example 1. -1648 (1971) was partially modified.

  Table 4 shows the inhibition rate (%) of angiotensin I converting enzyme in each sample 197 μg / mL described in Examples 3 and 4.

  Table 5 shows the angiotensin I converting enzyme inhibition rate (%) in each sample 197 μg / mL described in Example 4.

<Test Example 6> Antihypertensive effect of tail vein administration on spontaneously hypertensive rats Male spontaneously hypertensive rats (Wister-Okamoto derived) with body weight 250 ± 50g, systolic blood pressure 200 ± 20mmHg, heart rate 400 ± 50 times / min Spontaneously Hypertensive Rat (hereinafter referred to as SHR) was used. Light / dark cycle 12 hours (lights on from 9:00 am to 9:00 pm) before the start of the test, room temperature 21-23 ° C, humidity 50-70%, feed (Lab Diet manufactured by PMI Nutrition International) and drinking water ) 6 SHRs were placed in a 45 x 23 x 21 cm cage in an environment of free consumption and acclimated for 1 week. The ultrafiltrate spray-dried product of the peptide mixture obtained in Production Example 4 was dissolved at a dose of 30 mg / kg SHR body weight in 0.9 ml of 0.9% by weight saline per 1 kg body weight of SHR, and dissolved in SHR (3 mice per group). On the other hand, the tail vein was single-administered and reared in an environment of 32 ± 1 ° C. As a control group, SHR (3 mice per group) was administered with 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 sample administration and 60, 120, and 240 minutes after administration. Table 6 shows changes over time in the systolic blood pressure.

<Test Example 7> Antihypertensive effect of SHR by oral gavage SHR was prepared in the same manner as in Test Example 6, and 300 mg / kg of SHR body weight was applied to the ultrafiltrate spray-dried product of the peptide mixture obtained in Production Example 3. The dose was dissolved in 10% 0.9% saline per kg of SHR body weight, and was orally administered to SHR (3 mice per group) in an environment of 32 ± 1 ° C. As a control group, the same dose of 0.9% by weight saline was orally administered to SHR (3 mice per group). Blood pressure was measured using a non-invasive blood pressure measuring device (Model 59, IITC, CA, USA) immediately before sample administration and 60, 120, and 240 minutes after administration. Table 6 shows changes over time in systolic blood pressure.

<Test Example 8>
Sample A prepared by preparing 59.1 μg / mL of the peptide mixture obtained in Production Example 3 (see Table 1-2 above for the composition), and the seven types of isolated peptides contained therein were shown in Table 1-2 above. When the ACE inhibition rate (ACEI activity) of sample B mixed with the amount corresponding to the ratio in the peptide mixture shown in Fig. 6 was measured in the same manner as in Test Example 1, A was about 50 to B relative to B as shown in Fig. 6. The inhibition rate was 100 times.

<LC-MS analysis conditions>
The LC-MS analysis for qualitative and quantitative determination of each peptide in the peptide mixture was performed under the following conditions.
<Enalyzing conditions for free form>
HPLC analysis conditions and equipment: Waters Alliance 2695 / PDA 2996 (Waters)
Column: Xterra (registered trademark) MS C ₁₈ 3.5μm, 4.6X100mm (Waters)
・ Solvent: Liquid A / acetonitrile (0.1% diacid), Liquid B / distilled water (0.1% formic acid)
・ Gradient: 0 minutes (A: B = 5: 95) → 30 minutes (A: B = 30: 70) → 35 minutes (A: B = 60: 40) → 40 minutes (A: B = 5: 95)
・ Flow rate: 0.2 mL / min
・ Analysis time: 40 minutes ・ Injection volume: 10 μL
・ Sample concentration (separated fraction 10μg / mL, synthetic peptide 1μg / mL)
MS detector conditions and equipment: JMS-LCmate JMS-BU30 (JEOL)
・ Ionization mode: ESI ⁺
・ Ion source: Needle KV / 2.5, Orifice 1/0, Ring lens / 30, Ion guide / 3
・ Detector: Multiplier / 450, Preamp gain / × 1, Filter / 1, Attenuator / 1
・ Inlet: Desolvating Plate / 230 ℃, Orifice 1/150 ℃
・ Mass selection: Mass Range / 1500, Accelerating volts / 2500, Scan range / 100-1000
・ Slit setting: Main slit / 750, Alpha slit / 4.0
<Methyl ester analysis conditions>
HPLC conditions and equipment: Waters Alliance 2695 / PDA 2996 (Waters)
Column: Xterra (registered trademark) MS C ₁₈ 3.5 μm, 4.6 × 100 mm (Waters)
・ Solvent: Liquid A / acetonitrile (0.1% diacid), Liquid B / distilled water (0.1% formic acid)
・ Elution gradient: 0 minutes (A: B = 18: 82) → 30 minutes (A: B = 35: 65) → 35 minutes (A: B = 60: 40) → 40 minutes (A: B = 18: 82) )
・ Flow rate: 0.2 mL / min
・ Analysis time: 40 minutes ・ Injection volume: 10 μL
・ Sample concentration (separated fraction 10μg / mL, synthetic peptide 1μg / mL)
Same as MS detector condition and educt

  The peptide compound obtained by the present invention has a strong angiotensin I converting enzyme inhibitory activity and exhibits a strong antihypertensive action. Therefore, a prophylactic or therapeutic agent for hypertension such as essential hypertension, renal hypertension, adrenal hypertension, In addition to being useful as a pharmaceutical for diagnostics of these diseases, antihypertensive agents used in various pathological conditions, reduction of myocardial infarction, pathological conditions in congestive heart failure, etc., the peptide compound of the present invention can be taken orally. Therefore, it can be used as a health food, a specific health food or a functional food having the above-mentioned useful action.

It is a figure which shows the chromatogram in the HPLC analysis of the angiotensin I converting enzyme inhibition peptide mixture obtained in manufacture example 1. FIG. It is a figure which shows the isolation | separation process (process to 4-1-9 fraction) of each mixture from the angiotensin I converting enzyme inhibition peptide mixture obtained by manufacture example 1. FIG. It is a figure which shows the isolation | separation process (process from a 4-1-9 fraction) of each mixture from the angiotensin I converting enzyme inhibition peptide mixture obtained in manufacture example 1. FIG. FIG. 4 is a diagram showing a chromatogram in HPLC analysis of an angiotensin I converting enzyme-inhibiting peptide mixture obtained in Production Example 3. It is a figure which shows the isolation | separation process of each mixture from the angiotensin I converting enzyme inhibition peptide mixture obtained in manufacture example 3. FIG. It is a figure which shows the comparison result of the angiotensin I converting enzyme inhibitory activity of the samples A and B in Test Example 8.

Claims (2)

A peptide compound represented by the amino acid sequence of Ile-Val-Phe (xvi), a peptide compound represented by the amino acid sequence of Phe-Ile-Ala (xvii) , and pharmaceutically acceptable salts of these peptide compounds. An angiotensin I converting enzyme inhibitor, comprising at least one active ingredient as an active ingredient . Peptide compound represented by the amino acid sequence of Tyr-Phe (vi) , peptide compound represented by the amino acid sequence of Leu-Trp (xix) , peptide compound represented by the amino acid sequence of Ile-Trp (xx), and these peptide compounds Further comprising at least one selected from pharmaceutically acceptable salts, wherein all of the peptide compounds are separated from a decomposition solution obtained by decomposing salmon or its processed product by reacting with a proteolytic enzyme The angiotensin I converting enzyme inhibitor according to claim 1 .

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