Angiotensin-converting enzyme inhibitor

Abstract

To provide a composition that exhibits a superior angiotensin-converting enzyme inhibitory action and is effective against high blood pressure.SOLUTION: The present invention provides an angiotensin-converting enzyme inhibitor, a food composition for preventing or improving high blood pressure, or a pharmaceutical composition for preventing or improving high blood pressure, containing at least one selected from the group consisting of Pro-Leu, Pro-Ile, Leu-Val, Ile-Val, and salts thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an angiotensin-converting enzyme inhibitor, a food composition for preventing or ameliorating hypertension, and a pharmaceutical composition for preventing or treating hypertension, each of which contains a specific dipeptide or a salt thereof. [Background technology]

[0002] According to the 2019 National Health and Nutrition Survey Report (Ministry of Health, Labour and Welfare), the average systolic blood pressure is 134.3mmHg for men and 127.3mmHg for women, and the percentage of people with a systolic blood pressure of 140mmHg or higher is 34.6% for men and 24.8% for women, meaning that the proportion of people with high blood pressure is said to be very high in Japan.

[0003] Angiotensin-converting enzyme (ACE) is known as an enzyme that is closely involved in blood pressure elevation (Non-Patent Document 1). ACE is closely involved in blood pressure elevation by generating angiotensin II, which has a blood pressure increasing effect, from angiotensin I in the renin-angiotensin system, which is one of the blood pressure regulation mechanisms, and at the same time, by decomposing bradykinin, a hypotensive peptide (Non-Patent Documents 1 and 2).

[0004] In recent years, food ingredients with ACE inhibitory activity have been attracting attention, with many functional foods (functional food, food for specified health uses) aimed at preventing hypertension being sold. ACE inhibitor peptides isolated from foods and used in functional food include sardine peptide (Val-Tyr) isolated from sardines, lactotripeptide (Val-Pro-Pro, Ile-Pro-Pro) isolated from fermented milk, wakame peptide (Phe-Tyr, Val-Tyr, Ile-Tyr), bonito oligopeptide (Leu-Lys-Pro-Asn-Met), and sesame peptide (Leu-Val-Tyr) (Non-Patent Document 3). In addition, corn peptide (Leu-Pro-Pro) and seafood-derived dipeptides (Gly-Pro, Asn-Pro, Thr-Pro, Ala-Val, Gly-Val, Asp-Met, Asp-Leu) are also known to have ACE inhibitory activity (Patent Documents 1 and 2).

[0005] Black vinegar has a blood pressure lowering effect and is known to be effective against high blood pressure (Patent Document 3). It is also known that Ile-Tyr-Pro, Phe-Phe, Gln-Leu-Pro, and Asn-Pro, which are obtained by fractionating, isolating, and purifying black vinegar, have ACE inhibitory effects (Patent Document 4).

[0006] Kurozu has been produced in Fukuyama-cho, Kirishima City, Kagoshima Prefecture, using a unique method that has been in use for about 200 years. While rice vinegar is produced in about three months, kurozu is fermented and aged over a long period of one to three years. Kurozu is produced by a method in which all of these processes are carried out in one jar (Non-Patent Document 4). In this production method, the fermentation conditions vary slightly depending on the season, the year of production, and even each jar, so skilled craftsmen adjust the production process using color, taste, and other factors as indicators. Vinegar was also traditionally produced by craftsmen in this way, but nowadays, in order to maintain stable product quality, it is not fermented in a jar, but is produced in a factory by aeration fermentation by controlling the air and temperature in the tank, and kurozu can also be produced by aeration fermentation in the same way. However, most of the research on black vinegar to date has used black vinegar produced in a jar (both Patent Documents 3 and 4 refer to black vinegar produced by Sakamoto Jozo Co., Ltd.), and there has not been much research using black vinegar produced in a factory by aeration fermentation. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 10-212245 [Patent Document 2] JP 2003-24012 A [Patent Document 3] Japanese Unexamined Patent Publication No. 62-120320 [Patent Document 4] JP 2001-278893 A [Non-patent literature]

[0008] [Non-Patent Document 1] Toshiro Matsui, "Renin-angiotensin system and blood pressure regulation," Chemistry and Biology, Vol. 53, pp. 228-235, 2015. [Non-Patent Document 2] Yoichi Nagamura, "Blood Pressure and Healthy Foods," Journal of the Japan Food Safety Association, Vol. 13, pp. 219-222, 2018. [Non-Patent Document 3] Hirotaka Hayashi et al., "Foods labeled as 'Foods for Specified Health Uses' suitable for people with high blood pressure," Journal of the Japanese Society for Complementary and Alternative Medicine, Vol. 5, pp. 37-47, 2008 [Non-Patent Document 4] Kazuhiko Matsunaga, "GI-registered 'Kagoshima's pot-made black vinegar'", Journal of the Japanese Society for Food Science and Technology, Vol. 64, pp. 385-388, 2017 Summary of the Invention [Problem to be solved by the invention]

[0009] The inventors isolated peptides from black vinegar produced by aerobic fermentation in a factory and examined their activity, finding that four specific dipeptides or their salts have particularly high ACE inhibitory activity. [Means for solving the problem]

[0010] Therefore, the present invention provides an angiotensin-converting enzyme inhibitor, a food composition for preventing or ameliorating hypertension, and a pharmaceutical composition for preventing or treating hypertension, each of which contains at least one selected from four specific types of dipeptides and their salts. That is, the present invention provides the following. [1] An angiotensin-converting enzyme inhibitor comprising at least one selected from the group consisting of Pro-Leu, Pro-Ile, Leu-Val, Ile-Val, and salts thereof. [2] A food composition for preventing or ameliorating hypertension, comprising at least one selected from the group consisting of Pro-Leu, Pro-Ile, Leu-Val, Ile-Val, and salts thereof. [3] A pharmaceutical composition for preventing or treating hypertension, comprising at least one selected from the group consisting of Pro-Leu, Pro-Ile, Leu-Val, Ile-Val, and salts thereof. Effect of the Invention

[0011] Since the four specific types of dipeptides or salts thereof discovered by the present invention have high ACE inhibitory activity, the present invention can provide highly useful angiotensin-converting enzyme inhibitors, food compositions for preventing or ameliorating hypertension, and pharmaceutical compositions for preventing or treating hypertension. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a chromatogram showing the results of separation by liquid chromatography using a black vinegar sample (liquid sample) in the examples. [Diagram 2]FIG. 2 is a chromatogram showing the results of separation by liquid chromatography using a black vinegar sample (powder sample) in the examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] One embodiment of the present invention provides an angiotensin-converting enzyme inhibitor containing at least one selected from the group consisting of Pro-Leu, Pro-Ile, Leu-Val, Ile-Val, and salts thereof. Another embodiment of the present invention provides a food composition for preventing or ameliorating hypertension, comprising at least one selected from the group consisting of Pro-Leu, Pro-Ile, Leu-Val, Ile-Val, and salts thereof. Yet another embodiment of the present invention provides a pharmaceutical composition for preventing or treating hypertension, comprising at least one selected from the group consisting of Pro-Leu, Pro-Ile, Leu-Val, Ile-Val, and salts thereof.

[0014] Pro-Leu, Pro-Ile, Leu-Val, and Ile-Val (hereinafter collectively referred to as "the dipeptide of the present invention") contained in the above-mentioned angiotensin-converting enzyme inhibitor, food composition, and pharmaceutical composition can be isolated from black vinegar. The salt of the dipeptide of the present invention can be produced by substituting the C-terminal carboxyl group of the dipeptide to form a sodium salt, potassium salt, ammonium salt, etc., or by adding an acid such as hydrochloric acid or carboxylic acid to the N-terminal amino group, etc. to form an addition salt. These salts can be produced by a conventional salt formation reaction. The dipeptide of the present invention or a salt thereof may also be produced by a chemical synthesis method, and such a production method is known or well-known in the field of peptide synthesis.

[0015] The black vinegar used when isolating the dipeptide of the present invention from black vinegar may be black vinegar produced by a traditional outdoor production method using a jar, or may be black vinegar produced by aeration fermentation in a factory. Black vinegar produced by an outdoor production method using a jar is produced, for example, in the Satsuma region of Kagoshima Prefecture, by filling brewing jars lined up outdoors with rice, koji, etc., and aging them for a long time using solar energy. Black vinegar produced by aeration fermentation in a factory is produced, for example, by putting sake mash or seed vinegar made from rice, koji, etc. into a tank that can be fed with fine air by a high-speed agitator, fermenting it efficiently in a short period of time, and then aging it. However, as long as the dipeptide of the present invention can be produced, any black vinegar may be used as the raw material. Black vinegar may be in a liquid state, or may be powdered by drying and crushing, spray drying, or freeze drying the liquid black vinegar.

[0016] The method for obtaining peptides from black vinegar is not particularly limited as long as it can isolate the dipeptide of the present invention, and any known or publicly known method for isolating peptides can be used. For example, liquid black vinegar is dissolved in an appropriate solvent as necessary, or powdered black vinegar is dissolved in an appropriate solvent, and then the desired dipeptide is separated and purified by gel filtration chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), or the like. In particular, when isolating and producing the dipeptide of the present invention on a laboratory scale, it is preferable to use HPLC, and when isolating and producing it on a factory scale, it is preferable to use a method of removing polymeric substances using ultrafiltration, and then using a reverse osmosis membrane and a method of selectively recovering using an ion exchange resin. In addition, before subjecting to HPLC, it is preferable to perform ultrafiltration in order to extract peptides with a molecular weight of 3000 or less. HPLC is preferably performed using a reverse phase column (e.g., DeltaPak C18 Column), and a mixture of acetonitrile, trifluoroacetic acid, and water is preferably used as the mobile phase.

[0017] The amino acid sequence of the isolated and produced peptide can be analyzed using a publicly known or well-known amino acid sequence analysis method. For example, such an analysis method includes a method of analyzing the sequence of amino acids constituting the peptide from MS / MS spectrum using a liquid chromatography tandem mass spectrometer (LC / MS / MS). LC is preferably performed using a peptide column (e.g., Inertsil Peptides C18), and the mobile phase is preferably an aqueous formic acid solution or a formic acid acetonitrile solution.

[0018] The dipeptide of the present invention may be artificially produced by a chemical synthesis method known or well known in the field of peptide synthesis. For example, the dipeptide of the present invention can be produced by a solid-phase method or a liquid-phase method, and is preferably produced by a 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis method. When the peptide of the present invention is artificially produced, a peptide synthesizer can be used, which is convenient since a series of reaction operations can be performed automatically.

[0019] The dipeptide or a salt thereof of the present invention has an angiotensin converting enzyme (ACE) inhibitory effect and is therefore useful as an active ingredient of an ACE inhibitor. Since ACE inhibitors are known to be effective in treating hypertension, the dipeptide or a salt thereof of the present invention is useful as an active ingredient of a food composition for preventing or improving hypertension or a pharmaceutical composition for preventing or treating hypertension. In addition, the ACE inhibitor containing the dipeptide of the present invention or a salt thereof can also be used as an additive for imparting an ACE inhibitory effect or an antihypertensive effect to a food composition or pharmaceutical composition. The ACE inhibitory activity can be confirmed by a known or well-known method for measuring ACE inhibitory activity, or may be confirmed by using a commercially available kit for measuring ACE inhibitory activity, such as ACE kit-WST (manufactured by Dojin Chemical Laboratory).

[0020] The subject to which the ACE inhibitor, food composition, and pharmaceutical composition of the present invention are applied is not particularly limited, and can be applied to, for example, mammals such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, cows, horses, sheep, monkeys, humans, etc. The subject to which the ACE inhibitor, food composition, and pharmaceutical composition of the present invention are applied is preferably a human subject.

[0021] The method for applying the ACE inhibitor or pharmaceutical composition of the present invention to a subject is not particularly limited, and it can be applied orally or parenterally (for example, by injection, application, patch, etc.).

[0022] The ACE inhibitors, food compositions, and pharmaceutical compositions of the present invention may contain inactive ingredients that are publicly known or well-known in the manufacture of food compositions and pharmaceutical compositions, as necessary, and may also contain other active ingredients other than the dipeptide or salt thereof of the present invention in the hope of enhancing the effect or providing other effects.

[0023] Inactive ingredients that can be contained in the ACE inhibitors, food compositions, and pharmaceutical compositions of the present invention are not particularly limited as long as the desired action is achieved, and examples include carriers that are acceptable for use as foods or pharmaceuticals, such as excipients, binders, bulking agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, and diluents.

[0024] The specific dosage and application interval of the ACE inhibitor, food composition, and pharmaceutical composition of the present invention may vary according to the subject's body weight, sex, age, condition, application route, or other factors. The specific dosage and application interval can be appropriately determined by those skilled in the art. However, to give a non-limiting example, in the case of oral administration, the daily intake of the dipeptide or its salt of the present invention for one adult (body weight 60 kg) is, for example, 0.001 mg or more, preferably 0.005 mg or more, more preferably 0.01 mg or more, and more preferably 0.013 mg or more, and is preferably 500 mg or less, more preferably 300 mg or less, more preferably 280 mg, even more preferably 200 mg or less, even more preferably 100 mg or less, and particularly preferably 90 mg. In the present invention, it is preferable to apply such an amount once to several times a day.

[0025] The application period is not particularly limited, but repeated or continuous application is preferable, continuous application for 28 days or more is more preferable, continuous application for 8 weeks or more is even more preferable, and continuous application for 12 weeks or more is particularly preferable.

[0026] There are no particular limitations on the specific form of the ACE inhibitor, food composition, and pharmaceutical composition of the present invention, so long as the desired effect is achieved.

[0027] When the ACE inhibitor or pharmaceutical composition of the present invention is administered parenterally, it can be administered in the form of, for example, an external preparation for skin or an injection.

[0028] When the ACE inhibitor or pharmaceutical composition of the present invention is administered orally, it can be administered in the form of, for example, tablets, capsules, granules, powders, lozenges, liquids, syrups, suspensions, etc. Also, examples of the form of food compositions include soft drinks, tea drinks, coffee drinks, fruit juice drinks, carbonated drinks, milk drinks, jellies, wafers, biscuits, bread, noodles, sausages, etc. The ACE inhibitors, food compositions, and pharmaceutical compositions of the present invention are preferably in the form of solid preparations such as tablets, capsules, granules, powders, and lozenges, with tablets and capsules being more preferred due to their ease of ingestion, and tablets being particularly preferred.

[0029] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples. EXAMPLES

[0030] 1. Experimental Method (1) Sample The black vinegar concentrate (liquid sample) produced in the factory by aeration fermentation and the concentrate powdered by spray drying after concentration and sterilization (powder sample) were used. The liquid sample was used as it was for HPLC, and the powder sample was used after dissolving 2 g of the powder in 20 mL of pure water. In addition, powder of brown rice, the raw material of black vinegar, was used as a control.

[0031] (2) Method i) Separation of peptides by liquid chromatography (HPLC) To extract peptides with a molecular weight of 3000 or less, the HPLC sample prepared in (1) was ultrafiltered using Ultracel-3K (Amicon Co.), and the filtrate was then filtered through a 0.22 μm filter, and the resulting filtrate was subjected to HPLC. HPLC was performed under the following conditions. Liquid chromatograph: BioLogic HR (BioRad) Reverse phase column: DeltaPak C18 Column, 100Å, 5μm, 3.9mm×150mm (Waters) Sample injection volume: 200μL Mobile phase A: 0.05% acetonitrile, 0.1% trifluoroacetic acid, the remainder is water Mobile phase B: 80% acetonitrile, 0.1% trifluoroacetic acid, the remainder is water Elution was performed with a linear gradient at a flow rate of 0.5 mL per minute so that solution B reached 100% at the 50-minute point, and the solution of each peak was extracted with a fraction collector (BioRad) to prepare an analytical sample for each peak.

[0032] ii) Measurement of ACE inhibitory activity The ACE inhibitory activity of the analytical sample for each extracted peak was measured using ACE kit-WST (Dojindo Laboratories) according to the instruction manual.

[0033] iii) Amino acid sequence analysis The analytical samples with high ACE inhibitory activity were subjected to amino acid sequence analysis, which was outsourced to the Materials Science and Technology Foundation. The samples were analyzed by liquid chromatography tandem mass spectrometry (LC / MS / MS), and the sequences of amino acids constituting the peptides were analyzed from the MS / MS spectra. The analytical conditions for LC / MS / MS are as follows: <lc> Measurement equipment: Prominence UFLC (Shimadzu Corporation) Column: Inertsil Peptides C18 (1.0 mm x 150 mm, 4.0 μm) Column temperature: 40℃ Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile A:B=99.5:0.5(0min)→A:B=50:50(70min)→A:B=0:100(75min) ·Flow rate: 0.1mL / min Sample injection volume: 10μL Measurement equipment: Triple TOF 5600+ (AB SCIEX) Ionization method: ESI IonSpray Voltage Floating: 5.5kV (Positive mode) ·Mass range: m / z 50-1000

[0034] iv) Measurement of ACE inhibitory activity of the synthesized peptides To prove that a certain amino acid sequence exhibits a specific function, it is necessary to synthesize a peptide having that sequence and examine its reactivity. For this reason, we commissioned Cosmo Bio Co., Ltd. to synthesize the peptide for which the amino acid sequence analysis was performed in iii). The synthetic peptide used this time was synthesized using the 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis method. This synthetic peptide was dissolved in pure water and adjusted to a concentration of 10 mg / μL, and the ACE inhibitory activity of the synthetic peptide solution was measured using the ACE kit-WST (manufactured by Dojindo Laboratories) according to the instruction manual.

[0035] 2. Experimental Results i) HPLC analysis and ACE inhibitory activity of each peak By HPLC, five peaks were detected in the liquid sample and seven peaks in the powder sample (Figures 1 and 2). The ACE inhibitory activity values ​​of the five peaks in the liquid sample and the seven peaks in the powder sample were determined, and the results are shown in Tables 1 and 2 (the peak numbers in the tables correspond to the numbers assigned to the peaks in Figures 1 and 2).

[0036] [Table 1]

[0037] [Table 2]

[0038] For the liquid sample, peak No. 4 had the highest ACE inhibitory activity at 86.2%, while for the powder sample, peak No. 6 had the highest ACE inhibitory activity at 84.1%. ii) LC / MS / MS results LC / MS / MS analysis (described above) was performed on peaks No. 1 and 4 of the liquid sample, which had an ACE inhibitory activity of 80% or more, and peaks No. 4, 5, and 6 of the powder sample. When amino acid sequence analysis (described above) of the peptides contained in each peak was performed, it was estimated that peaks No. 1 and 4 of the liquid sample contained the peptides shown in Table 3, and peaks No. 4, 5, and 6 of the powder sample contained the peptides shown in Table 4. In addition, amino acid analysis was performed on the brown rice powder used as a control, and it was estimated that it contained the peptides shown in Table 5.

[0039] [Table 3]

[0040] [Table 4]

[0041] [Table 5]

[0042] iii) ACE inhibitory activity of synthetic peptide solution From Tables 3 to 5, peptides contained in black vinegar liquid sample, black vinegar powder sample, and brown rice were confirmed, and four types of peptides, Pro-Leu, Pro-Ile, Leu-Val, and Ile-Val, which are not contained in brown rice but are contained in black vinegar liquid sample and powder sample, were synthesized. The ACE inhibitory activity value of the synthesized peptide solution (10 mg / μL) was calculated using ACE kit-WST (manufactured by Dojindo Laboratories), and it was 90.8% for Pro-Leu, 91.3% for Pro-Ile, 99.6% for Leu-Val, and 98.2% for Ile-Val.

[0043] 3. Summary It was shown that black vinegar produced by aeration fermentation in a factory, rather than by using a jar, also contains peptides with ACE inhibitory activity. It was also shown that powdered black vinegar still contains peptides with ACE inhibitory activity. Eleven types of peptides were detected from the peaks with high ACE inhibitory activity obtained from the liquid sample of black vinegar, and seven types of peptides were detected from the peaks with high ACE inhibitory activity obtained from the powder sample of black vinegar. In addition, 11 types of peptides were detected from brown rice, the raw material for black vinegar. Therefore, we synthesized four types of peptides, Pro-Leu, Pro-Ile, Leu-Val, and Ile-Val, which are not contained in the raw material brown rice but are contained in both the liquid and powder samples, and measured their ACE inhibitory activity.All of them showed extremely high ACE inhibitory activity of over 90%. Therefore, these four peptides are considered to have extremely high antihypertensive effects. In addition, this study confirmed that peptides with ACE inhibitory activity remain not only in liquid black vinegar but also in powdered black vinegar. Because powdered black vinegar is easier to handle than liquid, powdered black vinegar is thought to be useful for developing new functional foods and supplements that are expected to have antihypertensive effects. < / lc>

Claims

1. An angiotensin-converting enzyme inhibitor comprising at least one selected from the group consisting of Pro-Leu, Pro-Ile, Leu-Val, Ile-Val, and salts thereof.

2. A food composition for preventing or improving hypertension, comprising at least one selected from the group consisting of Pro-Leu, Pro-Ile, Leu-Val, Ile-Val, and salts thereof.

3. A pharmaceutical composition for preventing or treating hypertension, comprising at least one member selected from the group consisting of Pro-Leu, Pro-Ile, Leu-Val, Ile-Val, and salts thereof.

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