WO2005118619A1 - Dipeptide exhibiting antihypertensive action - Google Patents

Abstract

A novel substance having an inhibitory activity on angiotensin I converting enzyme and exhibiting an antihypertensive action; a process for producing the novel substance; and a food or beverage, or functional food, or pharmaceutical composition comprising the novel substance. There are provided alanylphenylalanine (Ala-Phe), glycylphenylalanine (Gly-Phe), and salts thereof. Further, there is provided an inhibitory composition for angiotensin I converting enzyme comprising the same, and provided, comprising them, a material for food or beverage, food or beverage, or pharmaceutical composition.

Description

 Specification

 Dipeptide with hypotensive action

 Technical field

 The present invention relates to a dipeptide having a blood pressure lowering effect, a composition containing the dipeptide, a method for extracting and producing the composition from brewer's yeast, a food and beverage, a functional food, and a food or beverage containing the dipeptide or the composition. About pharmaceuticals.

 Background art

 [0002] At present, one in four people is said to have high blood pressure. Hypertension is called a silent killer and has no major symptoms. If left unchecked, it can cause a variety of illnesses, including cerebrovascular disorders, which are the leading causes of death, heart disease, and even arteriosclerosis. Various treatments are currently used to treat hypertension, but there are concerns about the side effects of some of them.

 [0003] Angiotensin II (Asp-Arg-Val-Try-Ile-His-Pro-Phe), a potent vasoconstrictor known as a causative agent of hypertension, is an inactive peptide, angiotensin. From I (Asp-Arg-Val_Try-Ile-His-Pro-Phe-His-Leu), the C-terminal His-Leu is cleaved by angiotensin converting enzyme (ACE) while it circulates through the lung and kidney. It is known that it is generated by performing In recent years, the renin-angiotensin system (RAS) in the kidneys associated with the conversion of angiotensin I to angiotensin II has been closely linked to the development of essential hypertension, which accounts for about 90% of hypertension. Have been reported. On the other hand, angiotensin converting enzyme (ACE) also catalyzes the reaction that inactivates the degradation of the antihypertensive peptide bradykinin in the Kun-kallikrein system. If these ACE activities are inhibited, it is expected that an increase in blood pressure will be suppressed, leading to suppression of hypertension.

[0004] Various peptide compounds that inhibit ACE activity have been synthesized so far (Patent Documents 1 and 2). In addition, peptides derived from milk proteins, seafood and plant origin have been found to act as a substrate that antagonizes the substrate for angiotensin I converting enzyme, and as a result, maintain a low blood pressure elevation level. For a long time as those foods It has been reported that ingestion of barrel is effective for prevention of hypertension (Non-Patent Documents 1 to 3). More recently, it has been shown to specifically inhibit ACE and, similarly to angiotensin I, act as a substrate for ACE to produce relatively low levels of ACE activity, thereby suppressing blood pressure elevation and increasing hypertension. There are active attempts to create, search for, and isolate components that improve and prevent food from various food-derived proteins (Patent Documents 3 to 6, Non-Patent Documents 4 to 9). In addition, it has been found that the protein contained in royal jelly (RJ), which is one of the functional food materials, significantly enhances the ACE inhibitory activity by proteolytic enzyme treatment (Non-Patent Document 10). It has also been reported that oral administration of peptides released at that time lowers the blood pressure of spontaneously hypertensive rats (HSHR) (Non-Patent Document 11).

Patent Document 1: JP-A-57-53447

Patent Document 2: JP-A-2-282395

Patent Document 3: Japanese Patent Application Laid-Open No. 5-958

Patent Document 4: JP-A-11 29594

Patent Document 5: Japanese Unexamined Patent Publication No. 2002-29995

Patent Document 6: JP-A-2002-291452

Non-patent Document 1: Seki Eiji et al., "Hypertensive effect of sardine protein-derived peptide and Valy tyrosine", Journal of Japan Nutrition 'Food Association, (1999) 52, p.271-277

Non-Patent Document 2: Ohtsuru Katsu et al., "Effects of Maitake Administration on Blood Pressure and Body Weight of Spontaneously Hypertensive Rats", Journal of the Japan Food Industry Association, (1999) 46, p.806-814.

Non-Patent Document 3: Shunro Kawagishi, Ed., "Biochemical Chemistry Experimental Method 38: Research Methods for Biological Function Modulating Substances in Foods", Gakkai Shuppan Center, (1996), p.116-129

Sato, M. et al., Angiotensin I_ onverting Enzyme Inhibitory Peptides Derived from Wakame (Undaria pinnatifida) and Their Antihypertensive Effect in Spontaneously Hypertensive Rats., J. Agric. Food. Chem., ( 2002) 50, p. 6245-6252 Non-Patent Document 5: Nakamura, Y. et al., Purification and characterization of Angiotensin-converting enzyme inhibitors from sour milk., J. Dairy Sci., (1995) 78, p. 777 -783 Non-Patent Document 6: Hiroyuki Ueda et al., "Preparation of ΑΝΏ-converting enzyme-inhibiting peptide from sardine protein hydrolyzate and its separation", Journal of the Japanese Society of Agricultural Chemistry, (1991) 65, ρ.1223-1228 Patent Document 7: Matsui, T. et al., Preparation and Characterization of Novel Bioactive Peptides Responsible for Angiotensin I-Converting Enzyme Inhibition from Wheat Germ., J. Peptide ScL, (1999) 5, p.289-297

 Non-Patent Document 8: Hiroyuki Ueda et al., "Angiotensin I-converting enzyme inhibitory peptide present in pepsin hydrolyzate of heated sardine muscle", Journal of the Japanese Society of Agricultural Chemistry, (1992) Vol.66, N 0.1, pp.25-29

 Non-Patent Aiden 9: Saiga, A. et al., Angiotensin onverting Enzyme Inhibitory Peptides in a Hydrolyzed Cmcken Breast Muscle Extract., J. Agric. Food Chem. (2003) 51, pp.1741-1745

 Non-Patent Document 10: Kazumichi Suzuki et al., “Angiotensin-converting enzyme inhibitory activity of royal jelly hydrolyzate by protease”, Journal of Japan Society for Food Science and Technology, (2003) 50, p.286-288 Non-patent Document 11: Kazumichi Suzuki Et al., "Blood pressure regulating effect of proteolytic enzyme-treated oral jelly on spontaneously developing blood pressure in rats", Journal of Japan Society for Food Science and Technology, (2003) 50, p.47-462.

 Problems to be solved by the invention

[0005] The present invention provides a substance having an inhibitory activity on angiotensin I converting enzyme and exhibiting an antihypertensive effect, a method for producing the substance, and foods, beverages, functional foods and pharmaceuticals containing the substance. The purpose is to:

 Means for solving the problem

[0006] The present inventors conducted intensive studies to solve the above-mentioned problems, and when a peptide fraction derived from a hydrolyzate of dried brewer's yeast was administered to spontaneously hypertensive rats (SHR), the blood pressure of rats Found to descend. Thus, the present inventors searched for a peptide that inhibits angiotensin I converting enzyme (ACE) activity from the hydrolyzate of dried brewer's yeast, and found that two types of peptides, Ala_Phe and Gly_Phe, which have a hypotensive effect. Was found. The present invention has been completed based on these findings, that is, as follows:

[1] A dipeptide or a salt thereof which is araelfeniralanine (Ala-Phe).

[2] A dipeptide which is dalysylphenylalanine (Gly-Phe) or a salt thereof. [3] A composition for inhibiting angiotensin-converting enzyme, comprising at least one selected from the group consisting of arayurpheniralanine (Ala-Phe), glycylfuranlaranine (Gly_Phe), and a salt thereof. .

 [4] A method for producing a composition for inhibiting angiotensin converting enzyme, comprising the following steps a) to c):

[0011] a) a step of hydrolyzing brewer's yeast,

 b) a step of fractionating the hydrolyzate by a chromatographic method, and c) a step of fractionating a fraction containing alanylpheniralanine (Ala-Phe) and / or glycylphenylalananine (Gly-Phe).

 [5] The above-mentioned [4], wherein the fraction containing araelpheniralanine (Ala-Phe) and / or glycylphenylalanine (Gly_Phe) is a fraction containing a peptide having a molecular weight of 150 to 2,000. The described method.

 [6] A composition for inhibiting angiotensin I converting enzyme produced by the method according to the above [4] or [5].

 [7] A material for food or drink, comprising the dipeptide or the salt thereof according to the above [1] or [2].

 [8] A food or drink comprising the dipeptide or the salt thereof according to the above [1] or [2].

 [9] A food or drink, to which the dipeptide or the salt thereof according to the above [1] or [2] is added and increased.

 [10] A food or drink material comprising the composition according to the above [3] or [6].

 [11] A food or drink to which the material for food or drink according to the above [10] is added.

 [12] The food or beverage according to the above [8], [9] or [11], which is a beverage.

 [13] The food or drink according to the above [8], [9], [11] or [12] for lowering blood pressure.

 [14] A pharmaceutical composition comprising the dipeptide according to the above [1] and [2] or a salt thereof.

 [15] A pharmaceutical composition comprising the composition according to the above [3] or [6].

 [16] The pharmaceutical composition according to the above [14] or [15], which is a blood pressure lowering agent.

 [17] A method for producing a composition containing arayurpheniralanine (Ala-Phe) and Z or dalysylphenylalananine (Gly-Phe), comprising the following steps a) to c):

[0024] a) a step of hydrolyzing brewer's yeast,

b) passing the hydrolyzate through a column filled with a hydrophobic adsorbent, and c) A step of eluting the adsorbed substance from the hydrophobic adsorbent using an aqueous ethanol solution having a concentration of 50 to 100%.

 The invention's effect

 [0025] The peptides Ala_Phe, Gly-Phe and salts thereof according to the present invention, and compositions containing them have angiotensin I converting enzyme (ACE) inhibitory activity. The peptide of the present invention, a salt thereof, and a composition containing the same exhibit an effect of suppressing an increase in blood pressure. The material for food and drink, food and drink, and the pharmaceutical composition containing the peptide of the present invention, a salt thereof, or a composition containing the same can suppress an increase in blood pressure, for example, by oral administration.

 [0026] This description includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application No. 2004-166223, which is a priority document of the present application.

 Brief Description of Drawings

 FIG. 1 is a schematic diagram showing a procedure for preparing a dried beer yeast hydrolyzate.

 FIG. 2 is a schematic diagram showing the procedure for measuring the ACE inhibitory activity of a hydrolyzate of dried brewer's yeast.

 FIG. 3 is a diagram showing a feed feeding scheme for each experimental group.

 FIG. 4 is a graph showing changes in blood pressure in each experimental group.

 FIG. 5 is a diagram showing a blood pressure change amount in each experimental group.

 FIG. 6 is a schematic diagram showing a procedure for measuring angiotensin I converting enzyme activity in serum.

 [Fig. 7] Fig. 7 is a schematic diagram showing a procedure for preparing a sampnole for measuring angiotensin I converting enzyme activity of renal power.

 FIG. 8 is a schematic diagram showing an experimental procedure used to identify a 50% EtOH-eluting fractional ACE inhibitor.

 FIG. 9 is a diagram showing an elution pattern of a yeast alcalase hydrolyzate by S-marked hadex_G-25 column chromatography and an ACE inhibitory activity of the eluted fraction.

 FIG. 10 is a view showing a reverse phase preparative HPLC chromatogram of fraction 18. Peak fractions AC are shown.

FIG. 11 is a view showing a reverse phase preparative HPLC chromatogram of fraction 18. Peak fraction D to F are shown.

 FIG. 12 is a view showing an HPLC chromatogram obtained by subjecting a mixture of fraction B and fraction E to gel filtration HPLC.

 FIG. 13 shows the mass spectrum of fraction G and the chemical structural formula of phenylalanine.

 FIG. 14 shows the mass spectrum of fraction H and the chemical structural formula of arayurfeniralanin.

 [Fig. 15] Fig. 15 shows the mass spectrum of fraction I and the chemical structural formula of dalysylphenylalanine.

 FIG. 16 is a graph showing changes in systolic blood pressure following a single oral administration of AF or GF.

 FIG. 17 is a graph showing changes in the maximum systolic blood pressure change associated with a single oral administration of AF or GF.

 BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

 [0029] 1) Peptides Ala-Phe, Gly-Phe and salts thereof having angiotensin I converting enzyme (ACE) inhibitory activity, and a composition for inhibiting angiotensin I converting enzyme containing the same according to the present invention The peptides, arayurpheniralanine (Ala-Phe), dalysylphenyliralanine (Gly-Phe), and their salts each have angiotensin converting enzyme (ACE) inhibitory activity.

 [0030] In the present invention, arayurpheniralanine (Ala_Phe) means a dipeptide containing an N-terminal alanine residue and a C-terminal feniralanine residue. Examples of the salt of arauelfeniralanine in the present invention include a sodium salt and a potassium salt.

 [0031] In the present invention, dalysylphenylalanine (Gly-Phe) means a dipeptide containing an N-terminal glycine residue and a C-terminal fenylalanine residue. Examples of the salt of glycinolephenylalanine in the present invention include a sodium salt and a potassium salt.

[0032] The peptide Ala_Phe, Gly-Phe or a salt thereof of the present invention can be prepared by a peptide synthesis method known to those skilled in the art, for example, but not limited to, a chemical or enzymatic cleavage method, a chemical synthesis method (solution Phase method, solid phase method, column method, batch method, etc.), chromatographic purification It can be manufactured by utilizing a method such as a method. For details of the peptide synthesis method, see, for example, "The Peptides: Analysis, Synthesis, Biology, Vol. 1-5, edited by E. ross, J. Meienhofer; Vol. 6-9, S. Udenfriend, J. Meienhofer, Academic Press. And New York (1979-1987), “Basics and Experiments of Peptide Synthesis” (Nobuo Izumiya et al., Maruzen Co., Ltd. (1985)) and the like.

[0033] Further, a composition containing at least one selected from the group consisting of the peptides Ala-Phe and Gly-Phe of the present invention and a salt thereof also has angiotensin converting enzyme inhibitory activity. Therefore, such a composition of the present invention can be used as a composition for inhibiting angiotensin I converting enzyme (ACE). The composition containing at least one selected from the group consisting of the peptides Ala_Phe, Gly-Phe and salts thereof according to the present invention comprises a peptide fraction obtained from brewer's yeast hydrolyzate (hereinafter referred to as AF / GF brewer's yeast). (Referred to as the derived fraction).

 [0034] The fraction derived from AF / GF brewer's yeast according to the present invention can be prepared using any method known to those skilled in the art by the following steps a) to:

 a) hydrolyzing brewer's yeast,

 b) a step of fractionating the hydrolyzate by a chromatographic method, and c) a step of fractionating a fraction containing alanylpheniralanine (Ala-Phe) and / or glycylphenylalananine (Gly-Phe). Can be produced. The molecular weight of the peptide contained in the beer yeast-derived fraction is preferably in the range of 150 to 2,000, but is not limited thereto.

 [0035] The brewer's yeast used for the production of the fraction derived from the AF / GF brewer's yeast may be any yeast used for brewing beer, but may be Saccharomyces cerevisiae, Saccharomyces cerevisiae (Saccharomyces pastorianus), Saccharomyces bayanus and the like are preferred. The brewer's yeast according to the present invention may be in a dry state (for example, in a powder form or a granular form) or may be in a culture. As used herein, the term “culture” includes a culture solution containing cells (cells) in culture and a medium, and culture cells separated from the culture solution.

[0036] Hydrolysis methods applied to brewer's yeast typically include enzymatic or chemical hydrolysis methods. Enzymatic hydrolysis methods include, for example, alkaline protease, neutral Examples include a method of treating with a protease containing a protease and an acidic protease. As the protease used in the present invention, an alkaline protease (Alcalase 2.4L FG; manufactured by novozymes) and the like are preferable, but not limited thereto. Examples of the chemical hydrolysis method include a method of treating with a strong acid or strong alkali. As the hydrolysis conditions, any conditions used in known hydrolysis methods may be used. The amount of enzyme added in the enzymatic method is usually 0.001% or more, preferably 0.1 to 10%, per 1 g of protein. The reaction pH and reaction temperature are preferably set near the optimum pH and the optimum temperature of the enzyme used. The reaction time can be appropriately set by those skilled in the art according to the type of enzyme, the amount added, the reaction temperature, and the reaction pH, but is usually in the range of 30 minutes to 40 hours. The hydrolysis reaction can be stopped according to a known method including heating of the reaction mixture, inactivation of the enzyme by a change in pH, etc., filtration of the enzyme by ultrafiltration, and the like.

As a technique for fractionating the fraction derived from AF / GF brewer's yeast, various techniques used for analysis of organic substances can be used. Examples of such a fractionation technique include membrane filtration such as ultrafiltration and microfiltration, size exclusion chromatography such as gel permeation chromatography and gel filtration chromatography (eg, high performance liquid chromatography), and partition chromatography. , Adsorption chromatography, ion exchange chromatography and the like. Specifically, for example, it is preferable to fractionate the hydrolyzate of brewer's yeast by chromatography, identify a fraction containing Ala-Phe and Z or Gly-Phe, and fractionate it. The beer yeast hydrolyzate may be sequentially subjected to different chromatography to obtain a more purified fraction. In this case, the beer yeast hydrolyzate is first passed through a chromatography packed with an adsorbent (for example, Amberlite ™ XAD-2 ™ resin) to which a substance having hydrophobicity is adsorbed, and the organic solvent (for example, ethanol (50 to 100% The fraction containing the hydrophobic substance is collected by eluting the adsorbed fraction with (EtOH) or methanol), and the fraction is further fractionated by a chromatographic method such as HPLC. The ACE inhibitory activity may be measured, a fraction having high ACE inhibitory activity may be collected, and the obtained fraction may be further fractionated by HPLC, etc. An adsorbent that adsorbs a substance having hydrophobicity Amberlite ( ^R ) XAD-2 ™ resin (manufactured by Rohm &Haas; polystyrene dibutylbenzene copolymer, average pore diameter 90A, specific surface area 300m ² / g , A pore volume of 42% a true density of ^{1.02g / ml), Amberlite 1 M} XAD-4 1 M resins. The beer yeast hydrolyzate is preferably subjected to centrifugation or filtration to remove solid components before being subjected to chromatography.

[0038] Examples of a specific method for producing a fraction derived from AF / GF brewer's yeast are described in Examples 1 and 4.

 [0039] The fraction derived from AF / GF brewer's yeast produced in this manner contains 8 1 & 1½ (molecular weight 236.12), Gly-Phe (molecular weight 222.10) and / or a salt thereof. If fractionation is performed to a fraction containing only a single peak and amino acid analysis or mass spectrometry is performed, Ala-Phe, Gly-Phe or a salt thereof contained in that fraction can be more clearly confirmed. it can. Amino acid analysis and mass analysis may be performed according to methods known to those skilled in the art. For example, amino acid analysis can be performed using an amino acid analyzer (ATO MLC-703) according to the manufacturer's instructions. Mass spectrometry can be performed using a liquid chromatograph / tandem mass spectrometer (LC / MS / MS; L-Q Advantage ion trap mass spectrometer (Thermo Finmgan)) or the like.

 The above-mentioned fraction derived from AF / GF brewer's yeast can be used as it is as a solution for inhibiting angiotensin converting enzyme in a solution state. Alternatively, the AF / GF brewer's yeast-derived fraction that has been subjected to further treatment such as concentration and / or removal of ethanol may be used as the composition for inhibiting angiotensin converting enzyme. Any method may be used for the concentration, and examples thereof include a heat concentration method, a heat concentration method under reduced pressure, a concentration method using ethanol precipitation, and a concentration method using activated carbon or an ion exchange resin. AF / GF beer yeast-derived fractions can be dried and used as a composition for inhibiting angiotensin converting enzyme. Examples of the method for such drying include an arbitrary method such as an air drying method, a freeze drying method, a spray drying method, a reduced pressure drying method, and a heating drying method. The composition for inhibiting angiotensin M converting enzyme (ACE) of the present invention may contain, in addition to Ala-Phe, Gly_Phe and / or a salt thereof, optionally any excipient conventionally added to a drug. (Water, stabilizers, buffers, preservatives, antioxidants, etc.).

[0041] The present invention relates to alanylpheniralanine (Ala-Phe) and / or glycylph, such as the above-mentioned AF / GF brewer's yeast-derived fraction or a single peak fraction further fractionated from the fraction. The present invention also relates to a method for producing a composition containing enylalanine (Gly-Phe). The process for the preparation of such a composition according to the invention, which has already been described herein above, comprises at least the following steps a) to c):

 a) hydrolyzing brewer's yeast,

 b) passing the hydrolyzate through a column packed with a hydrophobic adsorbent, and c) hydrophobic adsorption using an aqueous ethanol solution having a concentration of 100 to 100% (preferably 50 to 100%). Eluting the adsorbed substance from the agent,

 including.

 [0042] In this production method, the hydrolysis method applied to brewer's yeast in step a) is as described above. The hydrophobic adsorbent used in step b) includes, but is not limited to, Amberlite XAD resin series adsorbents such as Amberlite XAD_2, Amberlite XAD_4, Amberlite XAD_7, and Amberlite XAD-10 (Rohm & Haas, USA). Amberlite XAD-2 is particularly preferred as the hydrophobic adsorbent used in step b).

 Further, in step c) of this production method, an aqueous ethanol solution having an arbitrary concentration within a range of 0 to 100% (excluding 0%), preferably 50 to 100%, may be appropriately used. it can. As the elution method in this step c), various column elution procedures such as stepwise elution and gradient elution can be used.

 [0044] The adsorbed substance eluted in the above step c) may include both arayurpheniralanine (Ala-Phe) and dalysylfeniralanine (Gly_Phe). In this case, the adsorbed substance eluted in step c) is further separated by a known fractionation method such as chromatography, thereby containing arayurpheniralanine (Ala_Phe) or glycylfuranalanine (Gly-Phe) alone. Isolate the fractions.

 [0045] 2) Ala_Phe, Glv_Phe, and salts thereof, and angiotensin I-converting enzyme containing them. Angiotensin I-converting enzyme of harmful composition.

 The angiotensin I converting enzyme (ACE) inhibitory activity of Ala_Phe, Gly_Phe, their salts, and compositions containing them can be measured by the method described in Non-Patent Document 3. An example of the measurement method will be described below.

[0046] The method for measuring ACE inhibitory activity in the present invention is based on the in vitro extraction method of ethyl acetate. Angiotensin I converting enzyme (ACE) inhibition rate is calculated, and the ACE inhibition activity is expressed using the calculated rate as an index (see Non-Patent Document 3, FIG. 2). Specifically, first, a test sample to be tested for ACE inhibitory activity is prepared using distilled water to a concentration of 10 mg / ml. The angiotensin converting enzyme was prepared by dissolving the tripeptide Hip-His-Leu and NaCl in a borate buffer (pH 8.3) so that the final concentrations in the reaction solution were 5 mM and 400 mM, respectively. Prepared as a substrate solution. Next, add 125 µl of the prepared substrate solution to 15 µl of the test sample solution, and incubate at 37 ° C for 5 minutes. Then, add 50 μl of angiotensin converting enzyme (ACE) solution (prepared with borate buffer (pH 8.3) to 60 mU / ml), and incubate at 37 ° C for 30 minutes. Next, add 125 μl of IN HC1 to stop the reaction, add 0.75 ml of ethyl acetate, mix well, and centrifuge (3,000 rpm, 15 ° C, 10 minutes). The upper ethyl acetate layer is collected and dried under reduced pressure and dissolved in 1.0 ml of distilled water. Finally, the absorbance at 228 nm is measured.

 [0047] As a control sample, add 125 µl of IN HC1 to 50 µl of the test sample, incubate at 37 ° C for 5 minutes, and then add 125 µl of the substrate solution and 50 / il of the ACE solution, and add After incubating for 30 minutes, add 0.75 ml of ethyl acetate in the same manner as above, and perform a series of subsequent operations to prepare a sample blank. Alternatively, add 50 μl of borate buffer instead of the test sample solution, incubate with 125 μl of the substrate solution at 37 ° C for 5 minutes, and then calcine 125 μl of IN HC1 and then add 50 μl of the ACE solution. In addition, after incubating at 37 ° C for 30 minutes, add 0.75 ml of ethyl acetate in the same manner as above, and perform a series of subsequent operations to prepare a blank. Furthermore, a control is prepared by performing a series of operations similar to the above except that distilled water is added instead of the test sample solution. The absorbance of the sample blank, blank and control is measured in the same manner as described above.

 [0048] Based on the measured absorbance values thus obtained, the ACE inhibition rate (%) is calculated by the following equation.

 [0049] Inhibition rate (%) = {(Ec-Eb)-Es} / (Ec-Eb) x 100

 Ec: Absorbance of controller

 Eb: Absorbance of blank

Es: Absorbance of test sample Absorbance of sampnole blank In the present invention, this inhibition rate (%) indicates ACE inhibitory activity. The higher the inhibition rate (%), the higher the ACE inhibitory activity. The lower the inhibition rate, the lower the ACE inhibitory activity.

 [0050] 3) Ala_Phe, Glv_Phe, or a salt thereof, or the angiotensin-converting enzyme (ACE) of the present invention II. Food and drink and food and drink material containing the harmful composition

 The Ala-Phe, Gly_Phe, or a salt thereof, or the composition for inhibiting angiotensin I converting enzyme (ACE) containing the same of the present invention can be used as a material for food or drink. By adding an appropriate amount of this food / drink material, functionality such as, for example, a blood pressure lowering effect can be imparted to the food / drink. The food and drink material may be, but is not limited to, liquid, powder, granule, or solid. Examples of the food and drink material of the present invention include yeast extract and the like. Preferably, the food or drink material contains a high content of Ala-Phe, Gly-Phe, or a salt thereof, or a composition for inhibiting angiotensin converting enzyme (ACE) containing the same.

 [0051] The present invention also relates to foods and beverages to which Ala-Phe, Gly_Phe, or a salt thereof, or a composition for inhibiting angiotensin I converting enzyme (ACE) containing the same is added. As used herein, “food and drink” includes, but is not limited to, beverages, foods, and functional foods.

 [0052] The amount of Ala-Phe, Gly_Phe, or a salt thereof, or a composition for inhibiting ACE containing the same, in a food or drink is not particularly limited. For example, Ala-Phe, Gly-Phe and salts thereof Examples of the compounding amount that gives a total power of 0.001 to 100% by weight can be exemplified. However, the actual blending amount can be appropriately determined by those skilled in the art in consideration of the type of food or drink, the desired taste or texture.

 [0053] A further preferred embodiment of the present invention provides a food or drink product in which Ala-Phe, Gly_Phe, or a salt thereof, or an ACE-inhibiting composition containing the same has been added to increase the amount thereof (additionally increased amount). It is. Such a food or drink according to the present invention may contain Ala-Phe, Gly-Phe, or a salt thereof, or an ACE-inhibiting composition containing them, in any effective amount of 4 or a combination thereof in an effective amount. It is particularly preferred to contain.

[0054] Ala-Phe, Gly_Phe, or a salt thereof, or a composition for inhibiting angiotensin converting enzyme (ACE) containing the same is contained in a food or drink by any appropriate method available to those skilled in the art. I'll do it. For example, Ala-Phe, Gly_Phe, or their salts, or The ACE composition containing them may be incorporated into foods after being prepared in liquid, solid or granular form. Alternatively, they may be directly mixed or dissolved in food or drink, or may be carried in food or drink. Ala-Phe, Gly_Phe, or a salt thereof, or a composition for inhibiting ACE containing the same may be applied, coated, penetrated, or sprayed on food. Ala-Phe, Gly_Phe, or a salt thereof, or an ACE-inhibiting composition containing them may be uniformly distributed or unevenly distributed in food or drink. Alternatively, Ala-Phe, Gly_Phe, or a salt thereof, or a composition for inhibiting ACE containing the same may be unevenly distributed at a specific site in a food. When Ala_Phe, Gly_Phe, or a salt thereof, or an ACE-inhibiting composition containing them is dissolved in a beverage in a freeze-dried state, for example, after dissolving in water and uniformly mixing by stirring, It is preferably added to beverages, water and the like. When Ala_Phe, Gly_Phe, or a salt thereof, or an ACE-inhibiting composition containing them is mixed with a solid food, for example, it is preferably added to a food material, mixed uniformly by stirring, and then processed. . In addition, foods and beverages containing Ala_Phe, Gly_Phe, or a salt thereof, or a composition for inhibiting ACE containing them, can be further processed. Such processed products are also included in the scope of the present invention. Alternatively, Ala_Phe, Gly_Phe, or a salt thereof, or an ACE-inhibiting composition itself containing the same, which is solid-formed or formulated in the form of a capsule, a sugar-coated tablet, or the like, is also included in the food and drink of the present invention. Included.

In the production of the food and drink of the present invention, various additives commonly used in food and drink may be used. Additives include, but are not limited to, color formers (such as sodium nitrite), coloring agents (such as gardenia pigment, red 102, etc.), flavors (such as orange flavors), and sweeteners (such as stevia, astel palm). , Preservatives (sodium acetate, sorbic acid, etc.), emulsifiers (sodium chondroitin sulfate, propylene glycol fatty acid ester, etc.), antioxidants (disodium EDTA, vitamin C, etc.), pH adjusters (taenoic acid, etc.), chemical seasoning Additives (sodium inosinate, etc.), thickeners (xanthan gum, etc.), swelling agents (calcium carbonate, etc.), defoamers (calcium phosphate, etc.), binders (sodium polyphosphate, etc.), nutrient enhancers ( Calcium enhancer, vitamin A, etc.). In addition, functional materials such as Panax ginseng extract, Ezodokogi extract, Eucalyptus and Tochu tea extract may be added. [0056] The type of beverage of the present invention is not particularly limited. The beverage of the present invention includes, for example, tea beverages (unfermented tea such as brown rice tea and green tea, fermented tea such as black tea, semi-fermented tea such as oolong tea and jasmine tea, Tochu tea, persimmon leaf tea, Kumasa tea, Beverages including gear baron tea, corn tea, hub tea, chrysanthemum tea, etc.), fruit and vegetable beverages (orange, apple, grape, peach, strawberry, banana, lemon, etc., tomato, carrot, cabbage, celery) Beverages containing vegetable juices, etc.), alcoholic beverages (beverages, low-malt beer, whiskey, wine, liqueurs, etc.), carbonated beverages, lactic acid beverages, dairy beverages (coffee milk, fruit milk, functional milk, etc.) ), Soft drinks, low-calorie drinks, and the like. With respect to the production method of various beverages, etc., it is possible to refer to existing reference books, for example, “Latest 'Soft Drinks'” (2003) (Korin Co., Ltd.).

 [0057] The type of food of the present invention is not particularly limited. The food of the present invention may be a fresh food or a processed food. For example, baked sweets such as cookies, breads, cakes, rice crackers, etc., Japanese sweets such as yokan, cold desserts such as pudding, jelly and ice cream, sweets such as chewing gum and candy, snacks such as crackers and chips, nosta, udon , Buckwheat, etc., fish paste products such as rikamaboko, ham, fish sausage, miso, soy sauce, dressing, mayonnaise, seasonings such as sweeteners, tofu, konnyaku, other tsukudani, dumplings, coco Specific examples include various dishes such as cookie, salad, soup, stew, bread, cut vegetables, fish fillets, processed meat, and the like.

 [0058] The food or drink containing the above-mentioned composition for inhibiting Ala-Phe, Gly-Phe, or a salt thereof, or an angiotensin I converting enzyme (ACE) containing the same is preferably a functional food. The “functional food” of the present invention means a food having a certain function, for example, a health functional food including a special health food and a nutritional food, a special use food (food for a sick person, a pregnant woman and a nursing woman). Foods, infant formulas, foods for the elderly, etc.) as well as general health foods such as dietary supplements, health supplements, supplements, and beauty foods (eg, diet foods). The functional food of the present invention also includes a health food to which a health claim based on a food standard of Codex (FAO / WHO Joint Food Standards Committee) is applied.

[0059] The functional food of the present invention includes solid preparations such as tablets, granules, powders, pills, and capsules; It may be in the form of liquid preparations such as liquid preparations, suspensions and syrups, or preparations such as jewel preparations, or in the form of ordinary foods and drinks (for example, beverages, powdered tea leaves, confectionery, etc.). Good

[0060] Ala-Phe, Gly-Phe, a salt thereof, or a composition for inhibiting angiotensin I converting enzyme (ACE) containing them in a functional food, a compounding method, a compounding method, and a functional food The possible additives are the same as those described for the food and drink above.

 [0061] The functional food of the present invention is not limited, but is preferably used for lowering blood pressure. Specifically, the functional food is intended for those who want to lower blood pressure. It is preferable that the power is good. In the present invention, "a person whose blood pressure is desired to be lowered" generally means a person whose blood pressure is high, and although the blood pressure is significantly higher than the average value of people of the same age of the same age, the blood pressure is still high. Hypertension refers to individuals who have not been diagnosed with hypertension, but also includes those who subjectively perceive that their blood pressure is routinely high and that they need to lower their blood pressure. `` Targeting a person whose blood pressure is desired to be lowered '' indicates or indicates that the food or drink is suitable for the intake of a person whose blood pressure is desired to be reduced, and the blood pressure elevation level of the person who took it is indicated. This means that the effects of suppressing blood pressure and lowering blood pressure are expected. The statement or labeling that the food / drink is suitable for ingestion by persons for whom hypotension is desired has been approved in accordance with the statutory provisions for functional health foods such as special health foods and nutritional foods. It may comply with the function labeling (nutrition ingredient function labeling or health use labeling).

 [0062] 4) Ala-Phe. Glv_Phe, or a salt thereof, or angiotensin-converting enzyme (ACE) containing them II. Pharmaceutical composition containing harmful composition

 The present invention also relates to a pharmaceutical composition containing, as an active ingredient, Ala-Phe, Gly-Phe, or a salt thereof, or a composition for inhibiting angiotensin I converting enzyme (ACE) containing the same.

[0063] The pharmaceutical composition of the present invention may contain a carrier or additive acceptable for pharmaceutical preparations. Examples of such carriers and additives are water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, carboxymethyl. Starch sodium, penchi , Xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum anolebumin, mannitol, sorbitol, ratatose, acceptable as a pharmaceutical additive And artificial cell structures such as ribosomes. The additives to be used are selected appropriately or in combination according to the dosage form of the preparation. The pharmaceutical composition of the present invention may further contain other pharmacological components.

[0064] The pharmaceutical composition of the present invention can be administered orally or parenterally, but is preferably administered orally. The pharmaceutical composition of the present invention to be orally administered includes solid preparations such as tablets, granules, powders, pills, and capsules, and jewels, and liquid preparations such as liquids, suspensions, and syrups. It can be in shape. When used as a liquid preparation, the pharmaceutical composition of the present invention may be supplied as a dry product intended to be redissolved when used.

 [0065] Of the above dosage forms, the oral solid preparation may contain additives generally used in pharmacy, such as a binder, an excipient, a lubricant, a disintegrant, and a wetting agent. Further, the liquid preparation for oral use may contain additives generally used in medicine, such as stabilizers, buffers, flavoring agents, preservatives, fragrances and coloring agents.

 [0066] Since the pharmaceutical composition of the present invention has a blood pressure lowering effect, it can be used as a blood pressure lowering agent. The antihypertensive agent of the present invention can significantly suppress the blood pressure elevation level or significantly lower the blood pressure. The antihypertensive agent of the present invention can reduce, for example, systolic blood pressure to a level of 90 to 60% of the blood pressure before administration by 8 hours after a single administration.

 [0067] The dose of the pharmaceutical composition of the present invention varies depending on the age and weight of the subject to be administered, the route of administration, and the number of administrations, and can be widely varied according to the discretion of those skilled in the art. For example, in the case of oral administration, the composition of the present invention for inhibiting Ala-Phe, Gly_Phe, or a salt thereof, or angiotensin I converting enzyme (ACE) inhibitor containing the same, for one day, Nikkei body weight, preferably between lmg and lg per kg. The pharmaceutical composition of the present invention may be administered once, or may be administered repeatedly at intervals of 6 to 8 hours.

[0068] Subjects to which the pharmaceutical composition of the present invention is administered include humans, domestic animals, pet animals, and experimental (test) animals. And other mammals. In particular, mammals whose blood pressure (systolic blood pressure) is significantly higher than the average of healthy individuals on a daily basis, and mammals whose blood pressure (systolic blood pressure) tends to be higher than the average of healthy individuals. Alternatively, mammals having a predisposition (genetic or environmental predisposition) to hypertension are preferred as subjects to which the pharmaceutical composition of the present invention is administered. The pharmaceutical composition of the present invention can be very usefully used for continuous use, since there is little concern about side effects.

[0069] 5) Ala-Phe. Glv_Phe and salts thereof, and angiotensin-converting yeast containing them

 Ala-Phe, Gly-Phe and their salts, and the composition for inhibiting angiotensin I converting enzyme (ACE) of the present invention have a blood pressure lowering effect. This blood pressure lowering effect can be confirmed by a method known to those skilled in the art. In the present invention, for example, the following can be confirmed.

 [0070] First, spontaneously hypertensive rats (SHR) (male, which can be purchased from Japan SLC) which had been fasted for 12 hours were treated with Ala_Phe, Gly-Phe or a salt thereof of the present invention prepared as 16 mg / ml H0.

 2

 The composition for inhibiting angiotensin converting enzyme (ACE) containing them is orally administered by 1 ml per animal. Immediately before oral administration (0 hour) and 2, 4, 6 and 8 hours after oral administration, blood pressure is measured by the tail cuff method using a non-invasive automatic blood pressure device BP-98A manufactured by Softron Co., Ltd. I do. The blood pressure measurement is performed by pre-incubating the rats at 37 ° C for about 10 to 15 minutes, and immediately thereafter. The blood pressure measurement is preferably performed a plurality of times (2 to 3 times) continuously.

 The blood pressure is expressed as an average of a plurality of measured values of the measured maximum systolic blood pressure value.

 It is preferable to statistically process the data by analysis of variance (ANOVA), Duncan's multiple comparison test, Student's t_test, etc. to examine the significant differences and significance between the experimental groups.

[0072] The blood pressure lowering effect in the present invention can be evaluated based on the blood pressure values thus measured and calculated. In the present invention, the systolic blood pressure after administration (systolic blood pressure) is 95-60 as compared to the systolic blood pressure immediately before administration. Up to the level of / ₀ , preferably 90. / _0-60. When the blood pressure drops to the level of / ₀ , it is assumed that there is a blood pressure lowering effect.

[0073] All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety. Example

 Hereinafter, the present invention will be described more specifically with reference to Examples. However, the technical scope of the present invention is not limited to these embodiments.

Example 1 Preparation of Fraction Derived from Dry Beer Yeast Hydrolyzate

 FIG. 1 shows an outline of the procedure for preparing the fraction derived from the hydrolyzate of dried beer yeast used in this example.

 First, 50 g of dried beer yeast powder (Saccharomyces cerevisiae; trade name: Kirin dried beer yeast A) manufactured by Kirin Brewery Co., Ltd., 0.05 N NaOH 500 ml, and alcalase (Alcalase 2.4L FG; novozymes) 10 ml) and hydrolyzed while stirring at 50 ° C. for 12 hours. Thereafter, the mixture was centrifuged at 10,000 X g for 20 minutes at 4 ° C, and the supernatant was filtered using Toyo No. 2 filter paper. The filtrate was passed through an XAD-2 column (manufactured by Rohm & Haas) to obtain a permeate containing a non-adsorbed fraction. On the other hand, the adsorbed fraction on the XAD-2 column was eluted using 50% EtOH and 100% EtOH in sequence. Each of the obtained fractions was concentrated under reduced pressure, and further freeze-dried. The freeze-dried product (powder) of each fraction thus obtained was used in the following Examples.

 Example 2 In Vitro Angiotensin I Converting Enzyme (ACE) Inhibitory Activity of Fraction Derived from Dried Beer Yeast Hydrolyzate

 The angiotensin I converting enzyme (ACE) inhibitory activity was calculated by calculating the angiotensin I converting enzyme (ACE) inhibition rate according to the `` ethyl acetate extraction method '' described in Non-Patent Document 3, and expressed as an index. . The ethyl acetate extraction method is a method that utilizes the cleavage of the peptide His-Leu from Hip (hippuric acid) -His-Leu using ACE, and the extraction of Hip released thereby with ethyl acetate. . FIG. 2 shows an outline of the procedure for measuring the ACE inhibitory activity used in this example.

 [0078] Table 1 shows the calculated inhibition rates (%) as indexes of the ACE inhibitory activity.

1] ACE inhibition (%) of fractions derived from dried peel yeast hydrolyzate

 Test sample inhibition rate (%)

 XAD-2 column non-adsorption surface 5 6

 50% EtOH elution fraction from XAD-2 column 8 1

 100% P, t, 0H elution surface from XAD-2 column 9 5

[0079] As shown in Table 1, the 50% EtOH-eluted fraction and the 100% EtOH-eluted fraction both showed high inhibition rates, indicating that they had high ACE inhibitory activity.

 [0080] The ACE inhibitory activity of the non-adsorbed fraction of the XAD-2 column was low, indicating that the ACE inhibitor contained in the hydrolyzate of dried brewer's yeast had hydrophobicity.

 Example 3 In Vivo Blood Pressure Inhibitory Effect of Fraction Derived from Dried Beer Yeast Hydrolyzate and Dried Beer Yeast

 (1) Feeding the fraction derived from the hydrolyzate of dried beer yeast to experimental animals

 Spontaneously hypertensive rats (SHR) (male, 10 weeks old, initial weight average 300 g) purchased from Japan SLC were used.

 [0082] Breeding was carried out in individual wire gauges, at room temperature of 22 ° C ± 2 ° C, humidity of 40 to 60%, and light / dark cycle of 12 hours (7:00 to 19:00). The breeding period was 22 days. Food was given from 10 pm to 11 am the following day, and water was available ad libitum.

 [0083] The feed given to the rats is a basic feed (here, referred to as "Con (-)"), a feed obtained by adding 1% of NaCl to the basic feed (here, referred to as "Con (+)"), Con (+ ) To 50% EtOH-eluting fraction (“Alka”) 0.4% added to the feed (herein referred to as “(+) + Alka”), and Con (+) to the dried beer yeast powder (rkouboj). A feed supplemented with 3% (here, referred to as “(+) + Koubo”) was used. Table 2 shows the feed composition.

[Table 2] Feed composition

 Ingredients Con (-) Con (+) C ÷) + Alka (+) + Koubo casein 20 20 20 20 h-cornstarch-sucrose 6.5 64.1 611.5 5s = 2: 1

 Senonloose Udaichi 5 5 5 5 Corn oil 5 5 5

 3.5 3.5 3.5 3.5 Mixing vitamins (MN-ys-VX) 1 1 1 1

 NaCl ― T I 1

 Alka one-0.4. One

 Yeast powder 1-3

 B'p 100 100 100 100 100

[0084] The experimental group was composed of a control group (Con (-) group) fed Con (-) as an experimental diet, a group fed Con (+) as an experimental diet (Con (+) group), and an experimental group. In the first half, (+) + Alka was fed as an experimental meal ((+) + Alka group), and in the second half of the experiment, (+) + Koubo was fed as an experimental meal instead of (+) + Alka ((+) + Koubo) Group), and each group had 5 rats.

 [0085] Feeding of feed to rats in each experimental group was performed as shown in FIG. In the present example, the date when feeding of the feed (Con (-)) to each experimental group was started was set to the 0th day. For the first two days, all experimental groups were given Con (-), and for the next four days, each experimental group was given an experimental diet (Con (-), Con (+), (+) + Alka, respectively). For the next 4 days, Con (+) was given to the Con (+) group and (+) + Alka group, and Con (-) was given to the Con (-) group. After that, the experimental food of each experimental group was given again until the end of the experiment, except that (+) + Alka group was given (+) + Koubo instead of (+) + Alka, and (+) + Koubo group Called.

 [0086] With respect to the rats in each experimental group, the food intake and body weight were measured every day from 9:00 to 11:00. 2

 Table 3 shows body weight on day, cumulative feed intake for 6 days, and body weight gain. Each value represents the mean soil standard error (SEM) of 5 rats in each experimental group.

[Table 3] Feed intake and body weight increase of rats between 6 B

 —Experiment! ~

 Con (-) Con (+) (+) + Alka

Day 2 (g) 301.06 + 4 235. SS ± 7 30 ₀ 742 ± 5 ~

 6 Cumulative feed intake per hour (g) 112.84 ± 丄. 3 120.26 Sat 2.40 1.44 ± 2.8

 6 days rest increase (g) 5.24 ± 1.12 10.98 ± 2.72 6.76 Sat 1.75

[0087] Table 4 shows the body weight on the sixth day, the cumulative feed intake and body weight gain for the 16 days up to the 22nd day, and the liver weight at the time of dissection. Each value represents the mean soil standard error (SEM) for the 5 rats included in each experimental group.

 [Table 4]

Feed intake and weight gain of rats from day 6

 Male group

 Con (-) Con (-i-) (+) + Koubo

 Day 6 weight (g) 311.94 ± 5 3307.3 Sat 5.9

 Cumulative feed intake for 16 days (g) 278. 7- 3 319. 1 person L6. 0 27B. 7 Sat. G, 5

 Weight gain between 16 (g) 22. 32 ± 1.7 7 29.34 ± 4.3 22.9 ± 2.6 Liver weight at autopsy (% of body weight) 3, 96 above 0.03, 86 ± 0.13.73 ± 0.06

[0088] As shown in Table 3, no significant difference was observed between the groups in any of the cumulative feed intake and body weight gain up to the sixth day. From the 6th day to the 22nd day, as shown in Table 4, the (+) Koubo group showed a significant increase in both cumulative feed intake and weight gain compared to the Con (+) group, as shown in Table 4. Showed a low level or a low tendency. There was no significant difference in liver weight (weight ratio) at the time of dissection among the Con (-) group, Con (+) group, and (+) Koubo group.

 [0089] (2) Blood pressure measurement

 In this example, the blood pressure of rats was measured by the tail cuff method using a non-invasive automatic blood pressure device BP-98A manufactured by SOFTRON CORPORATION. Rats were pre-incubated at 37 ° C for approximately 10-15 minutes before measurement, and then three consecutive blood pressure measurements were performed. The measured value was expressed as the average of the three measurements. The change in blood pressure based on the obtained blood pressure measurement values is shown in Fig. 4, and the change in blood pressure based on the blood pressure change amount when the blood pressure measurement value on day 2 was set to 0 in each rat is shown in Fig. 5.

[0090] Regarding the data shown in Fig. 5, among the three groups of the Con (-) group, the Con (+) group and the (+) + Alka group, the Con (-) group, the Con (+) group, and the ( Analysis of variance (ANOVA) was performed for the three groups of +) + Koubo to examine the significance. In addition, those significant differences were examined by Duncan's multiple comparison test method. did. In addition, the significant difference between the two groups was also tested by Student's t-test.

[0091] As a result, blood pressure measurement after feeding for 3 days after changing to the experimental diet (Con (-), Con (+), (+) + Alka) showed that the (+) + Alka group The blood pressure elevation level was significantly lower than that of the (+) group (Fig. 5). Then, after feeding Con (_), Con (+), and (+) + Alka diets four times, Con (+) diet and Con (+) diet for Con (+) group and (+) + Alka group Blood pressure measurement for 4 days in which the group was fed the Con (-) diet without any change showed no significant difference in blood pressure elevation between the experimental groups (Figure 5). The Con (-) group was fed a Con (-) diet, the Con (+) group was fed a Con (+) diet, and the (+) + Alka group was fed a (+) + Koubo diet. On the other hand, in the blood pressure measurement on the 12th to 20th days, the (+) + Koubo group was fed to the (+) + Alka group (here, referred to as the (+) + Koubo group) compared to the Con (+) group. Showed a significantly lower blood pressure elevation level (Figure 5).

 [0092] From the above results, it was shown that the (+) + Alka diet and the (+) + Koubo diet had an effect of suppressing an increase in blood pressure. As shown in (4) and (5) below, there is no significant difference in ACE activity in serum between the Con (+) group and the (+) + Koubo group, The activity was lower in the (+) + Koubo group than in the Con (-) group and the Con (+) group, and the level was lower than that in the Con (-) group. It was inferred, at least in part, that ACE activity in the kidney was kept low.

 [0093] (3) Preparation of serum

 On the last day of breeding (day 22), the rats in each experimental group were laparotomized under Nembutal anesthesia (0.1 ml / 100 g body weight), and blood was directly collected from the heart using a syringe. The collected blood was placed in a test tube, left at room temperature for 1 hour, and then centrifuged (3,000 rpm, 15 ° C., 15 min) to separate serum.

 [0094] (4) Measurement of angiotensin I converting enzyme activity in serum

Serum angiotensin I converting enzyme (ACE) activity in serum was measured by Lieberman et al. (Jack Lieberman, MD: Elevation of Serum Angiotensin—Convertmg—Enzyme (A and E) Level in S arcoidosis. The American Journal of Medicine, 59, pp. 365-3 7 (1975)), and Kasaha ra et al. (Kasahara, Y. and Ashihara, Y .: Colorimetry of angiotensin-I converting enzyme activity in serum.Clin. Chem., Pp. 1922-1925 (1981)) according to the method of ethyl acetate extraction. Ethyl acetate extraction method uses ACE to extract dipeptides from Hip-His-Leu. This method is based on the cleavage of His-Leu and extraction of hippuric acid (Hip) released with it with ethyl acetate. FIG. 6 shows an outline of the procedure for measuring ACE activity in serum used in this example.

 [0095] Table 5 shows the ACE activity in the serum. With respect to the data shown in Table 5, analysis of variance (ANOVA) was performed between the three groups of the Con (-) group, the Con (+) group, and the (+) + Koubo group, and the significance was examined. These significant differences were examined by Duncan's multiple comparison test. Tests using Student's t_test were also performed for significant differences between the two groups. As a result, the ACE activity in the serum showed no significant difference among the three groups of the Con (-) group, the Con (+) group and the (+) Koubo group.

 [Table 5] ACR activity in tftl 淸

 Experiment group

 Con (-) C. n (+) (+) + Koubo

 ACE (U / ff 淸 1ml) 10.9 ± 0.6 10.8 ± 0 6 12.0 ± 0.1

[0096] (5) Measurement of ACE enzyme activity in kidney

 Fig. 7 shows the outline of the procedure for preparing a sample for measuring ACE activity from kidney. About 0.7 g of frozen kidney tissue is placed in a Teflon homogenizer, and 10 volumes of a buffer (50 mM

 Two

Mix B O and 200 mM HBO, adjust to pH 8.3), homogenize, and

4 7 3 3

 Centrifugation (10,000 卬 m, 20 minutes). The supernatant collected with a Pasteur pipette was used as a measurement sample.

 [0097] The ACE activity was measured in the same manner as in the case of the serum described in (4) above, by quantifying hippuric acid using the ethyl acetate extraction method, and calculating based on the measured value.

 [0098] Furthermore, in order to calculate ACE activity per mg of protein contained in kidney tissue, a Lowry method (Lowry, OH., Rosenbrough, NJ., Farr, AJ. And Randall RJ .: Protein measurement with According to the Folin phenol reagent. J. Biol. Chem., 193, pp. 265-275 (1951)), the amount of protein contained in about 0.7 g of kidney tissue used was quantified.

[0099] From the obtained measured values, the ACE activity per mg of the protein contained in the kidney tissue was calculated. Table 6 shows the ACE activity in the kidney. Regarding the data in Table 6, analysis of variance (ANOVA) was conducted between the Con (-) group, Con (+) group and (+) + Koubo group to examine the significance. did. Those significant differences were examined by Duncan's multiple comparison test. Tests using Student's t_test were also performed for significant differences between the two groups. ACE activity in the kidney was significantly lower and higher in the (+) Koubo group than in the Con (-) and Con (+) groups (p <0.01).

[Table 6]

 ACEffi-sexuality in the kidney

 Experimental group

 Con (-) Con (+) (+) + Koubo

 ACE (U) 60.27 ± 4.8 8 55.8I ± 3.4 32.70 ± 11,99

 ACE (U / protein lg) 44.62 ± 8, 14 41.31 ± 1.63 14, 58 ± 3.97 *

 *: Significant difference compared to control group (p <0.01) ""

[Example 4] Search and structural analysis of angiotensin I converting enzyme inhibitor

 (1) Fractionation by column chromatography

 An angiotensin I-converting enzyme inhibitor was identified from the 50% EtOH-eluted fraction showing high ACE inhibitory activity in Example 2.

 [0101] First, in the same manner as in Example 1, dried brewer's yeast was hydrolyzed with alcalase, centrifuged, and filtered to obtain a supernatant. The supernatant was passed through an XAD-2 column, and the column was washed with water. The peptides were eluted using% EtOH. Further, the fraction eluted with 50% EtOH was concentrated on a rotary evaporator to remove ethanol, and then lyophilized to prepare a lyophilized product of the fraction eluted with 50% EtOH. FIG. 8 shows the procedure used in this example to identify an ACE inhibitor from this freeze-dried product.

 After dissolving the lyophilized product in distilled water, the soluble fraction was subjected to Sephadex-G-25 column chromatography (developing solvent: distilled water) to fractionate into 18 fractions. FIG. 9 shows the obtained elution pattern by S-marked hadex-G-25 column chromatography. The absorbance at 280 nm was measured for each fraction. The ACE inhibitory activity of the fraction having a high absorbance was measured in the same manner as in Example 2. Fractions 17 to 19, which exhibited high absorbance at 280 nm, also had high ACE inhibitory activities (inhibition rates) of 61%, 62%, and 64%, respectively. The ACE inhibitory activities of the eluted fractions 17, 18, 19, 20 and 24 are shown in Table 7 (Table 7). Fractions 17 to 19 showing high ACE inhibitory activity were concentrated under reduced pressure using a rotary evaporator, and then lyophilized.

[Table 7] ACE inhibitory activity of the fraction derived from the yeast allylase hydrolyzate Fraction number% inhibition

 Fraction 1 7 6 1

 Fraction 1 8 6 2

 Fraction 1 9 6 4

 Fraction 2 0 4 7

 Fraction 2 1 2 1

[0103] (2) Fractionation and purification by reversed-phase preparative HPLC

 The lyophilized product of the fraction showing high ACE inhibitory activity prepared in the above (1) was dissolved in distilled water and then subjected to reverse phase preparative HPLC. In reverse phase preparative HPLC, Develosil C30-UG-5 (25 mm x 250 mm) was used as a column, and 1) 5% MeCN and 2) 20% MeCN were used as developing solvents. Was developed with a linear gradient (linear gradient) of 100%. The flow rate was 2.0 ml / min and detection was at 215 nm.

 FIGS. 10 and 11 show HPLC chromatograms obtained by subjecting fraction 18 prepared in (1) above to reverse phase preparative HPLC (Develosil C30-UG-5 (25 mm × 250 mm)). Show me. Major peaks were observed around 30, 50 and 70 minutes (elution time). The main peaks are shown as A to C in FIG. 10 and D to F in FIG. The ACE inhibitory activity of the main peak was measured in the same manner as in Example 2. The ACE inhibitory activity (inhibition rate) of the fraction containing peak A (fraction A) and the fraction containing peak B (fraction B) were 18% and 23%. Similarly, the ACE inhibitory activities (inhibition rates) of the fractions containing peaks E and F (fractions E and F) were 18% and 14%, respectively.

 [0105] Next, Fraction B and Fraction E showing high ACE inhibitory activity were mixed, and subjected to gel filtration HPLC. In gel filtration HPLC, Develosil 300 Diol-5 (10 mm × 250 mm) was used as a column, and distilled water was used as a developing solvent. Detection was performed at 215 nm, and the ACE inhibitory activity was measured for the major peak fraction in the same manner as described above. The gel filtration HPLC chromatograph obtained is shown in FIG. Three major peaks G to I were obtained, and the fractions containing those peaks (fractions G to I) also showed ACE inhibitory activity (Table 8).

[Table 8] Fraction inhibition rate (%)

 Fraction G 8 7

 Fraction H 2 0

 Fraction I 4 5

[0106] The peak fractions G to I were analyzed again by reverse-phase preparative HPLC analysis using Develosil C30-UG-5 (4.6 mm x 250 mm) to confirm the uniformity of the peaks. .

 [0107] (3) Amino acid analysis and mass analysis (LC / MS * MS)

 An amino acid analysis was performed for each of the peak fractions G to I including the single peak obtained in (2) above. 0.5 ml of the peak fraction was taken, 0.5 ml of 12N HC1 was added thereto, and a drop of α-mercaptoacetic acid was further added, and the air in the test tube was replaced with a gas. Then aluminum foil

 2

 Hydrolysis was performed at 110 ° C for 24 hours using a reaction heater in a state where the light was shielded from light. After cooling, suction filtration was performed using a G-4 glass filter. After concentrating the filtrate to dryness, 2 ml of 0.2 N sodium citrate buffer (pH 2.2) was added to dissolve the amino acids, and the mixture was subjected to finolator filtration (hydrophilicity: 0.2 zm) to obtain a sample for amino acid analysis. And For the analysis, an amino acid analyzer (ATO MLC-703 type) was used.

 As a result of amino acid analysis, only Phe was detected as an amino acid in fraction G. On the other hand, in fraction H, alanine (Ala): phenylalanine (Phe) = 1: 1, and in fraction I, glycine (Gly): phenylalanine (Phe) = 2: 1 to 1: 1 amino acid molar ratio was detected. Was done. Thus, all of fractions GI contained fenilalanine.

 [0109] Subsequently, the fractions G to I were subjected to level analysis using a liquid chromatograph Z tandem mass spectrometer (LC / MS / Ms; LCQ Advantage ion trap mass spectrometer (Thermo Pinmgan)).

[0110] As a result, Fraction G was identified as phenylalanine (Phe or F) because it gave m / z 166 [M + H] ⁺ . FIG. 13 shows the mass spectrum obtained for Fraction G and the chemical structural formula of phenylalanine. Fraction H gave m / z 237 [M + H] ⁺ and m / z 166 [M + H] ⁺ as a secondary ion for MS / MS analysis, indicating that alanylpheniralanine (Ala- Phe or AF). Figure 14 shows the mass spectrum and Arani obtained for fraction H. 1 shows a chemical structural formula of rufiniralanine. Fraction I gave m / z 223 [M + H] ⁺ and further gave m / z 166 [M + H] ⁺ as a secondary ion, and thus was identified as glycylphenylalanine (Gly_Phe or GF). Was done. FIG. 15 shows the mass spectrum obtained for Fraction I and the chemical structural formula of glycylpheniralanine.

[Example 11] Blood pressure lowering effect of synthetic AF and GF

 Synthetic alayurfenite obtained by outsourcing the synthesis to BACHEM (Bachem AG, Hauptstrasse 144, CH-4416, Budendorf) t

 1+ luananine (AF) or glycylpanolananine (GF) was given to SHR rats (male, 21 weeks old, 366g-396g; purchased from Japan SLC) fasted for 12 hours at 16mg / ml H0 as lml / animal. Was orally administered. Immediately before oral administration (0 hour) and 2, 4, 6 and 8 hours after oral administration, blood pressure is measured by the tail cuff method using a non-invasive automatic blood pressure measurement device BP-98A manufactured by Softron Co., Ltd. Was performed twice. The experimental group was an AF administration group (2 animals), a GF administration group (2 animals), and a control group (1 ml oral administration of distilled water instead of AF or GF; 1 animal).

 [0112] The results are shown in Table 9. The data of the AF administration group and the GF administration group in the table are the mean values of the standard errors of the blood (SEM) of the blood pressure measurement values of the rats in each group.

 [Table 9] Experimental group

 AF group (mHg) GF group (mmHg) Control group (mm （)

2 hours on lti9 7.5 179.75 ± 6,0 160 m 173.75 Sat 4.9 169.75 ± 4.1

 S time 161.5 ± 8.3 151.5 ± 7, 2 170

3 hours 159 ± 4.9 9 152.5 ±: 8 201.5

[0113] Fig. 16 shows a graph showing the transition of systolic blood pressure based on this data. In addition, a blood pressure change value when the blood pressure at 0 hour in each rat is set to 0 is calculated, and a change in the blood pressure change amount based on the calculated value is shown in a graph in FIG.

[0114] As shown in Fig. 16, both AF and GF showed a blood pressure lowering effect in spontaneously hypertensive (SHR) rats. At 2, 4 and 6 hours after administration, the blood pressure drop in the AF group and the GF group was not clearly shown due to the large fluctuations in the blood pressure of the control rats. AF group and GF group showed clearly lower blood pressure than control group [0115] As shown in Fig. 17, in the AF administration group and the GF administration group, the blood pressure was maintained at a low level until at least 8 hours after the administration.

 Industrial applicability

[0116] The peptides and compositions according to the present invention can effectively inhibit the activity of angiotensin I converting enzyme. In addition, by incorporating the peptide and the composition according to the present invention into foods and drinks, functional foods and pharmaceuticals, foods and drinks, functional foods and pharmaceuticals having a hypotensive effect can be produced.

Claims

The scope of the claims

 [I] A dipeptide which is araelfeniralanine (Ala-Phe) or a salt thereof.

[2] A dipeptide which is dalysylphenylalanine (Gly-Phe) or a salt thereof.

 [3] Angiotensin-converting enzyme inhibitor containing at least one selected from the group consisting of araelpheniralanine (Ala-Phe), glycylphenylalanan (Gly-Phe), and salts thereof. Composition.

 [4] A method for producing a composition for inhibiting angiotensin I converting enzyme, comprising the following steps a) to c).

 a) hydrolyzing brewer's yeast,

 b) a step of fractionating the hydrolyzate by a chromatographic method, and c) a step of fractionating a fraction containing alanylpheniralanine (Ala-Phe) and / or glycylphenylalananine (Gly-Phe).

 [5] Arayurpheniralanine (Ala-Phe) and / or glycylfuranylalanine (Gly_Phe)

5. The method according to claim 4, wherein the fraction containing the peptide having a molecular weight of 150 to 2,000.

 [6] A composition for inhibiting angiotensin I converting enzyme produced by the method according to claim 4 or 5.

 [7] A food and drink material containing the dipeptide according to claim 1 or 2 or a salt thereof.

[8] A food or drink comprising the dipeptide according to claim 1 or 2 or a salt thereof.

[9] A food or drink to which the dipeptide according to claim 1 or 2 or a salt thereof is added to increase the amount.

[10] A material for food or drink, comprising the composition according to claim 3 or 6.

 [II] A food or drink to which the food or drink material according to claim 10 is added.

[12] The food or drink according to claim 8, 9 or 11, which is a drink.

 [13] The food or drink according to claim 8, 9, 11 or 12 for lowering blood pressure.

[14] A pharmaceutical composition comprising the dipeptide according to claim 1 or 2 or a salt thereof.

[15] A pharmaceutical composition comprising the composition according to claim 3 or 6.

[16] The pharmaceutical composition according to claim 14 or 15, which is a hypotensive agent.

[17] A method for producing a composition containing alanylpheniralanine (Ala-Phe) and / or glycylpheniralanine (Gly-Phe), comprising the following steps a) to c). a) hydrolyzing brewer's yeast,

 b) passing the hydrolyzate through a column packed with a hydrophobic adsorbent, and c) eluting the adsorbed substance from the hydrophobic adsorbent using a 50-100% ethanol aqueous solution