JP2010024165A - Krill protein-derived angiotensin converting enzyme inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a component having blood pressure reduction which has safety with no side effect and has hypotensive effects, and can be taken in as a drug and a food. <P>SOLUTION: An angiotensin converting enzyme inhibitor has at least any one kind of Ile-Thr-Arg-Tyr, Val-Phe-Glu-Arg, Ile-Trp-Ala-Lys, Val-Asp-Tyr, Ala-Leu-Pro-His, Phe-Glu-Gln, Ile-Thr-Ala, Leu-Gly-Asp-Tyr-Asn, Phe-Asn-Pro, Val-Asp-Pro, Val-Tyr-Glu-Gly, Phe-Arg-Ala-Gly, and Ile-Ile-Gly-Glu-Tyr as an active ingredient. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a composition comprising a peptide having angiotensin converting enzyme inhibitory activity and having a blood pressure lowering action, an angiotensin converting enzyme inhibitor comprising the composition as an active ingredient, and a blood pressure reducing agent comprising the composition as an active ingredient. And a functional food that can be used for prevention and improvement of hypertension.

It is known that renin-angiotensin-aldosterone (RAA) system, kallikrein-kinin system, prostaglandin synthesis system and the like are complementarily involved in blood pressure regulation in vivo. Above all, the RAA system is said to play a central role in regulating pressure in body fluid systems. That is, angiotensinogen produced in the liver is broken down into 10 amino acid residues of angiotensin-I in the blood by the kidney-derived enzyme renin, and then the action of angiotensin-II converting enzyme (ACE) mainly present in the lung Converts to 8 amino acid residues of angiotensin-II. Angiotensin-II is the most potent vasopressor in the body, not only has a direct vasopressor action to contract vascular smooth muscle, but also stimulates aldosterone secretion in the adrenal gland and increases sodium and water storage. Involved in indirect blood pressure increase. Therefore, it is clinically known that the inhibition of RAA system enhancement is effective in suppressing blood pressure elevation, and the use of ACE inhibitors such as captopril or angiotensin-II receptor antagonists such as losartan is effective for pharmacotherapy. It is.
In recent years, research on ACE-inhibiting substances targeting food ingredients has progressed, and it is known that various protein-derived peptides exhibit effective ACE-inhibiting activity and further blood pressure lowering action (Patent Documents 1-5, etc.) .

Japanese Patent Laid-Open No. 3-81291 Japanese Patent Laid-Open No. 5-271297 JP-A-2005-145827 JP 2008-37766 A JP 2001-112470 A

  An object of the present invention is to provide a blood pressure lowering component that is safe without side effects, has a blood pressure lowering action, and can be taken as a pharmaceutical or food.

  It is known that peptides derived from various proteins have angiotensin converting enzyme inhibitory activity. The present applicant has previously found that a tripeptide (Leu-Lys-Tyr) obtained from krill has an angiotensin I converting enzyme (ACE) inhibitory activity (Patent Document 1). In order to find a more effective ACE inhibitor in the present invention, krill was decomposed with various degrading enzymes, and peptides contained therein were studied to complete the present invention.

The gist of the present invention is the angiotensin converting enzyme inhibitor (1) to (3), the antihypertensive agent (4) to (6), and a functional food.
(1) An angiotensin converting enzyme inhibitor comprising as an active ingredient any one or more of peptides having the amino acid sequences of (i) to (xiii) below or salts thereof:
(I) Ile-Thr-Arg-Tyr
(Ii) Val-Phe-Glu-Arg
(Iii) Ile-Trp-Ala-Lys
(Iv) Val-Asp-Tyr
(V) Ala-Leu-Pro-His
(Vi) Phe-Glu-Gln
(Vii) Ile-Thr-Ala
(Viii) Leu-Gly-Asp-Tyr-Asn
(Ix) Phe-Asn-Pro
(X) Val-Asp-Pro
(Xi) Val-Tyr-Glu-Gly
(Xii) Phe-Arg-Ala-Gly
(Xiii) Ile-Ile-Gly-Glu-Tyr

(2) The angiotensin converting enzyme inhibitor according to (1), comprising a degradation product of a krill protein by a proteolytic enzyme.
(3) The angiotensin converting enzyme inhibitor according to (2), wherein the proteolytic enzyme is thermolysin.

(4) An antihypertensive agent comprising the angiotensin converting enzyme inhibitor according to any one of (1) to (3) as an active ingredient.
(5) A functional food containing any one of the angiotensin converting enzyme inhibitors (1) to (3).
(6) The functional food according to (5), which is a food indicating that it is effective in preventing and improving hypertension.

  Since the peptide of the present invention has an angiotensin converting enzyme inhibitory activity and has a blood pressure lowering effect by oral administration, it is effective for prevention and treatment of hypertension by oral administration.

In the present invention, krill is preferably Antarctic krill (Euphausia pacifica), which has abundant resources, but other krill can be used as well. The active ingredient of the present invention is a peptide obtained by degrading these krill proteins with a proteolytic enzyme.
The enzyme degradation product of the krill protein of the present invention is pretreated so that the krill is suitable for enzyme treatment, and then proteolytic enzyme treatment is performed to produce a peptide, which is separated, filtered, concentrated and sterilized as necessary. Can be obtained.

  The composition of the present invention is produced from krill as a raw material, but it may be either with the head and shell attached or with the head and shell removed. Easy to handle is stripped skin with the head and shell removed. If it is stripped, it can be directly subjected to enzyme treatment. Even with a head or shell, the enzyme treatment can be performed as it is or after cutting. Preferably, the enzyme treatment can be efficiently performed by cutting with a homogenizer or the like. After the enzyme treatment, the shells that have not been decomposed are removed by filtration or the like.

  Enzyme treatment is carried out by adding 1/2 to 20 times, preferably 10 to 10 times the amount of the raw material, and then adding an alkaline agent such as aqueous ammonia or sodium hydroxide (potassium) solution, Adjust the pH to the appropriate value for the proteolytic enzyme to be used, warm the temperature to the appropriate enzyme temperature (depending on the enzyme used, 20-65 ° C, reacts well even at room temperature), add the proteolytic enzyme, and add 30 minutes Treat for ~ 30 hours (preferably 5-20 hours).

As the proteolytic enzyme, all enzymes can be used alone or in combination as long as they are capable of degrading proteins under neutral or alkaline conditions. Its origin can be found in microorganisms in addition to animals and plants, and in addition to pepsin, renin, trypsin, chymotrypsin, papain, bromelain, bacterial protease, filamentous fungal protease, actinomycete protease, etc. can be widely used. These enzymes are usually commercially available. However, unpurified enzymes, enzyme-containing culture fluids, and solid or liquid enzyme-containing materials such as sputum should also be used as required. Can do. The amount of enzyme added may be about 0.1% to 5.0%.
Preferred proteases include Aspergillus oryzae genus protease, Bacillus subtilis genus protease, Aspergillus oryzae genus protease, Aspergillus melleus genus protease, Bacillus stearothermophilus genus protease, Bacillus subtilis genus protease, Rhizopus Examples include peptidases derived from oryzae strains, peptidases derived from Aspergillus oryzae strains, and the like. Particularly preferred is thermolysin, which is a protease derived from the genus Bacillus stearothermophilus.

  After the enzyme treatment, neutralization treatment is performed if necessary, and then kept at a temperature of 70 ° C. (preferably 80 ° C.) for 2 to 60 minutes (preferably 5 to 30 minutes) to deactivate the enzyme. Ensure good separation later. After heat deactivation treatment, it is roughly separated by filtration with a hybro screen or the like, and if necessary, subjected to a jet treatment and then centrifuged to remove suspended matters and precipitates. Since it has angiotensin converting enzyme inhibitory activity as it is, it may be used as it is, but since it has a unique taste and smell derived from the raw material, it is further deodorized, decolorized and purified by filtration, activated carbon treatment, etc. depending on the purpose of use. Sterilization, spray drying, freeze drying, etc. are performed as necessary.

Since the angiotensin converting enzyme inhibitor comprising the protein enzyme degradation product of the krill protein of the present invention as an active ingredient was confirmed to have excellent ACE inhibitory activity as shown in the Examples, it was confirmed that the ACE inhibitor or blood pressure lowering was achieved. It can also be used as the intended medicine, health food, especially food for specified health use. When used as a food, the enzyme degradation product can be added as it is, or used in combination with other foods or food ingredients as appropriate according to conventional methods. According to a conventional method, tablets, granules, powders, capsules and powders can be prepared.
The standard for using the krill protein enzyme degradation product, which is an angiotensin converting enzyme inhibitor of the present invention, is about 0.1 to 6000 mg / day, and is preferably orally administered once or twice a day. If necessary, it can be used in combination with other drugs. The enzyme-decomposed product of krill protein of the present invention is an enzyme-decomposed product of krill that has a long dietary experience as food, and is highly safe and can be used with confidence.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

Manufacture of Krill Protein-Derived Peptide Powder After adding an equal amount of water to 1 kg of krill flakes ( manufactured by Nihon Suisan Co., Ltd.) and heating to 70 ° C, Samoaase PC is 0.15% by weight with respect to the krill flakes -10F (manufactured by Amano Enzyme) was added, and the enzyme reaction was performed at 70 ° C. for 20 hours with stirring. After the enzyme reaction, the enzyme was inactivated by incubating at 100 ° C. for 30 minutes. After removal of the residue by centrifugation and filtration, concentration using an evaporator was performed, and a peptide dry powder was obtained using spray drying (75 g) (product of the present invention). The peptide powder was stored at -20 ° C until use.

Gel electrophoresis analysis In order to examine the molecular weight distribution of the peptide derived from krill protein, a Superdex Peptide PC 3.2 / 30 gel filtration column (GE) was used on the SMART system (GE healthcare bioscience).
gel filtration chromatography using healthcare bioscience). As a solvent, 50 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and 6 M guanidine hydrochloride is used. Used.
As a result of gel filtration chromatography, it was confirmed that the krill protein-derived peptide was distributed with a molecular weight of 10,000 or less (FIG. 1). This is a state where the protein is sufficiently degraded.

Measurement of angiotensin converting enzyme (ACE) inhibitory activity Rabbit lung ACE, ACE substrate N- [3- (Furyl) acryloyl] -Phe-Gly-Gly (FAPGG), and valyltyrosine used as a positive control is Sigma The product made by company was used. The ACE inhibitory activity was measured by modifying the method of Peterson et al. (Anal. Biochem. 125; 420-426 (1982)). That is, 18 μL of 250 mM HEPES pH 8.3, 1.5 M NaCl, 10 μL of ACE prepared at 90 μg / mL, and an appropriately diluted test substance were mixed to make 90 μL and preincubated at room temperature for 15 minutes at room temperature. It was. Next, 10 μL of 50 mM HEPES pH 8.3, 300 mM NaCl, and 1.5 mM FAPGG were added to the reaction solution, followed by incubation at room temperature for 2-3 hours. The inhibitory activity was measured by measuring the absorbance at 320 nm, and the inhibitory activity was expressed with IC ₅₀ as the amount of the test substance that inhibits ACE by 50%.
The ACE inhibitory activity of the peptide derived from the krill protein of the present invention produced in Example 1 was about 1.9 mg / mL with an IC ₅₀ .

Antihypertensive effect of krill-derived peptide after single oral administration In this study, 7-week-old female rats (SHR / Izm, SPF) (Japan SLC) were used. Rats were given solid feed (Lab MR Stock (Nippon Agricultural Industries)) under conditions of room temperature 22 ± 3 ° C, relative humidity 50 ± 20%, lighting time 12 hours / day, ventilation rate 13-17 times / hour. Breeding and drinking water was inoculated freely.
Seven-week-old rats were preliminarily bred for 1 week in the above environment and acclimated to perform grouping so that the systolic blood pressure was almost the same (6 mice in each group). Rats fasted overnight after grouping were forcibly administered a single oral dose of test substance, and 2, 4, 6, 8 and 24 hours after administration, small animal non-warming type non-invasive blood pressure monitor MK-2000 (Muromachi Kikai) Blood pressure and heart rate were measured. As a test substance, the krill protein-derived peptide powder produced in Example 1 was used as an aqueous solution in 1 mg / kg, 10 mg / kg and 100 mg / kg administration groups. The same amount of water was administered to the control group. )

  In the administration group of 1, 10, or 100 mg / kg krill protein-derived peptide, the decrease in systolic blood pressure 2 hours after administration was 17 ± 1.59 mmHg, 28 ± 4.97 mmHg, and 24 ± 4.15 mmHg, respectively. There was a significant decrease (Figure 2). Thereafter, systolic blood pressure almost returned to its original value after 8 hours. Moreover, in the krill protein-derived peptide administration group and the control group, there was no significant difference in the heart rate after administration of the krill protein-derived peptide.

Isolation and structural analysis of ACE inhibitory peptide Ion exchange chromatography using SP10 / 16XL cation exchange column (GE healthcare bioscience) on AKTAexplorer 100 system (GE healthcare bioscience) after dissolving peptide powder in 26.5 mM formic acid It was used for. Elution was performed with a linear gradient of 0-1 M NaCl, and the chromatography flow rate was 2 mL / min. The eluted peptide was monitored by measuring the absorbance at 214 nm. Fractions with high ACE inhibitory activity were lyophilized and subjected to gel filtration chromatography. Gel filtration chromatography was performed using a Superdex Peptide 10/300 GL gel filtration column (GE healthcare bioscience) on an AKTAexplorer 100 system after dissolving the lyophilized fraction in distilled water. Elution was performed using distilled water, and the flow rate was 0.9 mL / min. The eluted peptide was monitored by measuring the absorbance at 214 nm. The fraction with the highest ACE inhibitory activity was lyophilized, dissolved in 26.5 mM formic acid, and subjected to reverse phase chromatography using an ODS-80 ™ column (Tosoh corp.) Column on the SMART system. Elution was performed with a linear gradient of 0-40% acetonitrile and a flow rate of 0.5 mL / min. The eluted peptide was monitored by measuring the absorbance at 214 nm. The fraction with high ACE inhibitory activity was lyophilized and then dissolved in 0.1% trifluoroacetic acid, and the peptide was isolated using a μRPC C2 / C18 SC 2.1 / 10 column (GE healthcare bioscience). The isolated peptide was sequenced using a Procise 494 HT sequencer (Applied biosystems).

Results Cation exchange chromatography of the krill protein-derived peptide and the ACE inhibitory activity of each fraction are shown in FIG. Fractions 24-25 with high ACE inhibitory activity were then subjected to gel filtration chromatography. The gel filtration chromatography of fractions 24-25 and the ACE inhibitory activity of each fraction are shown in FIG. Fraction 19 with high ACE inhibitory activity was then subjected to reverse phase chromatography. The ACE inhibitory activity of the eluted fraction is shown in FIG. The peptide contained in the obtained ACE inhibitory activity peaks I-VI was isolated and subjected to structural analysis using an amino acid sequencer. The obtained peptide sequences were Ile-Thr-Arg-Tyr, Val-Trp, Val-Phe-Glu-Arg, Ile-Trp-Ala-Lys, Leu-Lys-Tyr, Val-Asp-Tyr, Ala-Leu. -Pro-His, Phe-Glu-Gln, Ile-Thr-Ala, Leu-Gly-Asp-Tyr-Asn, Phe-Asn-Pro, Val-Asp-Pro, Val-Tyr-Glu-Gly, Phe-Arg -Ala-Gly, Ile-Ile-Gly-Glu-Tyr.
Among them, Val-Trp and Leu-Lys-Tyr contained in Peaks II and III showed high ACE inhibitory activity (IC ₅₀ = 2.75 μg / mL and 4.02 μg / mL, respectively), and these were peptides derived from krill protein. It was thought that this greatly contributed to the blood pressure lowering action of the blood.

  Since the enzyme degradation product of krill protein of the present invention has an angiotensin converting enzyme inhibitor and has a blood pressure lowering effect, it can provide a therapeutic / preventive agent for hypertension.

It is the photograph which showed the result of the gel filtration chromatography performed in order to see the molecular weight distribution of the enzyme degradation product of krill protein. It is a figure which shows the effect with respect to the blood pressure of the rat of the single oral administration of the enzyme degradation product of krill protein. (*: P <0.05, **: p <0.01, Dunnet test) It is the figure which fractionated the krill protein enzyme degradation product using cation exchange chromatography, and showed ACE inhibitory activity of each fraction. It is the figure which fractionated fractions 24-25 of Drawing 3 by gel filtration chromatography, and showed ACE inhibitory activity of each fraction. FIG. 5 is a diagram showing the ACE inhibitory activity of each fraction by further fractionating fraction 19 in FIG. 4 by reverse phase chromatography.

Claims (6)

An angiotensin converting enzyme inhibitor comprising as an active ingredient any one or more of peptides having the amino acid sequences of (i) to (xiii) below or salts thereof:
(I) Ile-Thr-Arg-Tyr
(Ii) Val-Phe-Glu-Arg
(Iii) Ile-Trp-Ala-Lys
(Iv) Val-Asp-Tyr
(V) Ala-Leu-Pro-His
(Vi) Phe-Glu-Gln
(Vii) Ile-Thr-Ala
(Viii) Leu-Gly-Asp-Tyr-Asn
(Ix) Phe-Asn-Pro
(X) Val-Asp-Pro
(Xi) Val-Tyr-Glu-Gly
(Xii) Phe-Arg-Ala-Gly
(Xiii) Ile-Ile-Gly-Glu-Tyr   The angiotensin converting enzyme inhibitor according to claim 1, which comprises a degradation product of krill protein by a proteolytic enzyme.   The angiotensin converting enzyme inhibitor according to claim 2, wherein the proteolytic enzyme is thermolysin.   An antihypertensive agent comprising the angiotensin converting enzyme inhibitor according to any one of claims 1 to 3 as an active ingredient.   The functional food used for the prevention and / or treatment of hypertension containing the angiotensin converting enzyme inhibitor in any one of Claims 1 thru | or 3.   6. The functional food according to claim 5, wherein the functional food is effective for preventing and / or improving hypertension.

Images