JP2001122802A - Inhibitor of angiotensin converting enzyme and hypotensive agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a useful angiotensin converting enzyme(ACE) inhibitor and further a hypotensive agent. SOLUTION: This ACE inhibitor or a hypotensive agent comprises one or more kinds selected from the group consisting of L-Tyr-L-Val, L-Phe-L-Tyr, L-Leu-L-Ser-L-Pro, L-Val-L-Ser-L-Pro, L-Leu-L-Asn-L-Pro, L-Val-L-Ala-L-Tyr, L-Leu-L-Ala-L-Tyr, Leu-L-Gln-L-Pro, L-Leu-L-Ala-L-Ala, L-Val-L-Ala-L-Ala, L-Leu-L-Ser-L-His or its pharmacologically acceptable acid addition salt, L-Ile-L- Arg-L-Ala or its pharmacologically acceptable acid addition salt and L-Leu-L- Arg-L-Pro or its pharmacologically acceptable acid addition salt as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel oligopeptide derived from corn protein and an angiotensin converting enzyme inhibitor and a hypotensive agent containing a specific oligopeptide derived from corn protein as an active ingredient. This drug is expected to be useful for treating or preventing hypertension.

[0002]

2. Description of the Related Art It is well known that the onset of hypertension is closely related to the renin-angiotensin system, and the renin-angiotensin system includes an angiotensin converting enzyme (EC3.4.15.1, hereinafter referred to as EC3.4.15.1). ACE) plays an important role. In this case, ACE is angiotensin I (Asp-Arg-V) in which angiotensinogen secreted in the liver is degraded by the enzyme renin produced in the kidney.
al-Tyr-Ile-His-Pro-Phe-His-Leu) and convert this to angiotensin II (Asp-Arg-Val-Tyr-Ile-His
-Pro-Phe). And this angiotensin II has a function of contracting the vascular wall smooth muscle, increasing blood pressure, and further acting on the adrenal cortex to promote secretion of aldosterone. In addition, the enzyme kallikrein present in plasma degrades a protein called kininogen to produce bradykinin, which dilates blood vessels and lowers blood pressure. This bradykinin is degraded and inactivated by the action of ACE. Thus, ACE, on the other hand, has
Angiotensin II) and, on the other hand, degrade the antihypertensive peptide (bradykinin), resulting in
Increase blood pressure. Therefore, by suppressing this enzyme activity, it is possible to prevent an increase in blood pressure (lower blood pressure).

[0003] As ACE activity inhibitors, there are a number of synthetic inhibitors such as captopril (D-2-methyl-3-mercaptopropanoyl-L-proline), including several peptide inhibitors obtained from snake venom. It is known, of which captopril is already practically used as an oral antihypertensive agent.

[0004] In recent years, ACE inhibitors have also been found in microorganisms and various foods, and their practical use as antihypertensive agents has been studied (Kunio Sueami, Fermentation and Industry 46 (No. 3),
179-182 (1988)). Susumu Maruyama, Bioscience and Industry for ACE inhibitors derived from food proteins, especially casein and corn seed protein
47 (No.11), 38-42 (1989) and Susumu Maruyama, Chemistry and Biology 22
(No. 8), 485-487 (1984). For ACE inhibitor derived from corn seed protein, Susumu Maruyama et al., Abstracts of 1988 Annual Meeting of the Japan Fermentation Engineering Society
23 (1988), Susumu Maruyama et al., Abstracts of the Japan Society of Agricultural Chemistry, 8 pages (1989), Shinsuke Miyoshi et al., Abstracts of the Japanese Society of Nutrition and Food, 113 pages (1989), Shinsuke Miyoshi et al. A report has also been made in the Journal of the Japanese Society of Agricultural Chemistry 64 (3), and in the Abstracts of the 1990 Annual Meeting, p.555. In addition, the present applicant has filed the following applications for ACE inhibitors and antihypertensives derived from corn seed protein: Japanese Patent Application Nos. 63-185467 and 63-185468.
Japanese Patent Application Nos. 1-59549 and 1-59550.

[0005]

There is a constant need for new and useful antihypertensive agents and, thus, angiotensin converting enzyme inhibitors, which will enrich the technology.

An object of the present invention is to provide a novel oligopeptide having an excellent angiotensin converting enzyme inhibitory activity, and an extremely safe angiotensin converting enzyme inhibitor and a blood pressure lowering agent comprising such an oligopeptide as an active ingredient. .

[0007]

DISCLOSURE OF THE INVENTION The object of the present invention is to provide a novel oligopeptide derived from α-zein, a kind of corn protein, and an angiotensin converting enzyme comprising a specific oligopeptide derived from α-zein as an active ingredient. The invention has been achieved by the following inventions related to inhibitors.

[0008] 1. L-Leu-L-Ser-L-Pro, L-Leu-L-Gln-L-
Pro, L-Val-L-Ser-L-Pro, L-Leu-L-Leu-L-Pro, L-Le
uL-Asn-L-Pro, L-Phe-L-Leu-L-Pro, L-Leu-L-Ala-L-
Ala, L-Val-L-Ala-L-Ala, L-Leu-L-Gln-L-Gln, L-Va
lL-Ala-L-Tyr, L-Leu-L-Ala-L-Tyr, L-Leu-L-Ser-L-
His and its acid addition salts, L-Ile-L-Arg-L-Ala and its acid addition salts, L-Leu-L-Arg-L-Pro and its acid addition salts, and L-
Ile-L-Arg-L-Ala-L-Gln-L-Gln and acid addition salts thereof.

2. L-Tyr-L-Val, L-Leu-L-Tyr, L-Leu-
L-Phe, L-Phe-L-Tyr, L-Tyr-L-Arg or a pharmaceutically acceptable acid addition salt thereof, L-Leu-L-Ser-L-Pro, L-Leu-L-Gl
nL-Pro, L-Val-L-Ser-L-Pro, L-Leu-L-Leu-L-Pro,
L-Leu-L-Asn-L-Pro, L-Phe-L-Leu-L-Pro, L-Leu-L-Al
aL-Ala, L-Val-L-Ala-L-Ala, L-Leu-L-Gln-L-Gln, L
-Val-L-Ala-L-Tyr, L-Leu-L-Ala-L-Tyr, L-Leu-L-Ser
-L-His or its pharmacologically acceptable acid addition salt, L-Ile-L
-Arg-L-Ala or its pharmacologically acceptable acid addition salt, LL
eu-L-Arg-L-Pro or a pharmacologically acceptable acid addition salt thereof, and L-Ile-L-Arg-L-Ala-L-Gln-L-Gln or a pharmacologically acceptable acid addition salt thereof An angiotensin converting enzyme inhibitor comprising, as an active ingredient, at least one member selected from the group consisting of:

[0010] 3. L-Tyr-L-Val, L-Leu-L-Tyr, L-Leu-
L-Phe, L-Phe-L-Tyr, L-Tyr-L-Arg or a pharmaceutically acceptable acid addition salt thereof, L-Leu-L-Ser-L-Pro, L-Leu-L-Gl
nL-Pro, L-Val-L-Ser-L-Pro, L-Leu-L-Leu-L-Pro,
L-Leu-L-Asn-L-Pro, L-Phe-L-Leu-L-Pro, L-Leu-L-Al
aL-Ala, L-Val-L-Ala-L-Ala, L-Leu-L-Gln-L-Gln, L
-Val-L-Ala-L-Tyr, L-Leu-L-Ala-L-Tyr, L-Leu-L-Ser
-L-His or its pharmacologically acceptable acid addition salt, L-Ile-L
-Arg-L-Ala or its pharmacologically acceptable acid addition salt, LL
eu-L-Arg-L-Pro or a pharmacologically acceptable acid addition salt thereof, and L-Ile-L-Arg-L-Ala-L-Gln-L-Gln or a pharmacologically acceptable acid addition salt thereof An antihypertensive containing at least one selected from the group consisting of

The above-mentioned acid addition salts include pharmaceutically acceptable acid (inorganic acids and organic acids) addition salts such as hydrochloride, hydrobromide, sulfate, nitrate, acetate, benzoate, and maleic acid. Salts, fumarate, succinate, tartrate, citrate, oxalate, methanesulfonate, toluenesulfonate, aspartate, glutamate and the like are included.

The oligopeptide used in the present invention may be a method of enzymatic hydrolysis of corn protein, a method of introducing amino acids stepwise by an organic chemical synthesis method, a peptide synthesis method using a reverse reaction of a hydrolase, a genetic engineering method. It can be manufactured by a method or the like.

The enzymatic hydrolysis of corn protein will be described. Although gluten meal may be used as a raw material corn protein, a case where α-zein obtained by further purifying it will be described. α-Zein may be a commercially available one, or may be one separated from zein separated from gluten meal by a known method.

As the enzyme, an endo-type protease such as papain or thermolysin can be used, and thermolysin is particularly preferable. The thermolysin may be of high purity or of industrial grade (for example, samoase (Daiwa Kasei)).

Hereinafter, the thermolysin treatment of α-zein will be described. This treatment is usually performed simply in water or a buffer (eg, Tris-HCl buffer, phosphate buffer).
The substrate concentration may be any as long as the mixture can be stirred and mixed at the time of the reaction.
(w / v). The amount of the enzyme thermolansin varies depending on the titer, but is usually
A suitable amount is at least 0.01% by weight, preferably 0.1 to 10% by weight. Thermolysin may be partially added during the reaction.
The pH and temperature of the reaction may be around the optimum pH of thermolysin, and may be pH 6-10, preferably 7-8, and temperature 30-80.
80 ° C, preferably 60-70 ° C, is suitable. Adjustment of the pH during the reaction is carried out with an aqueous sodium hydroxide solution, hydrochloric acid or the like, if necessary.

The reaction time depends on the amount of enzyme added, the reaction temperature, and the reaction time.
Although it is not constant because it varies depending on the pH, it is usually about 1 to 50 hours.

The hydrolysis reaction is stopped by heating the hydrolyzate or by adding an organic acid such as citric acid or malic acid, an inorganic acid such as hydrochloric acid or phosphoric acid, or an alkali such as sodium hydroxide or potassium hydroxide. It can be performed according to a known method such as inactivation of the enzyme due to a change or the like, and filtration of the enzyme by an ultrafiltration membrane or the like. Then, the hydrolysis liquid is separated into solid and liquid.
(Eg, centrifugation, filtration, etc.), and the separated liquid is fractionated by ultrafiltration, gel filtration, or the like to obtain a liquid containing, for example, a fraction having a molecular weight of 10,000 or less. This liquid contains the target oligopeptide of the present invention, and the liquid or a concentrate thereof (for example, a lyophilized product) is further fractionated to obtain individual target oligopeptides.

The freeze-dried product is referred to as α-zein hydrolyzed freeze-dried product, and the use of the freeze-dried product will be further described.

The α-zein hydrolyzed lyophilizate is, in one method, firstly an anion exchange resin, for example a weakly basic anion exchange resin (eg DEAE-Toyopearl)
650M (Tosoh Corp.) or a cation exchange resin such as a weakly acidic cation exchange resin (eg, SP-Toyopearl 650M (Tosoh Corp.)).

First, in the fractionation method through an anion exchange resin, each oligopeptide retained in the resin is eluted with a linear gradient (for example, when DEAE-Toyopearl 650M is used, an appropriate concentration of Tris buffer → NaCl in Tris buffer)
Elution). The eluate was fractionated into several fractions, and each fraction was subjected to gel filtration (for example, Sephadex LH-2).
0), cation exchange resin treatment (for example, SP-Toyopearl 650M), reverse phase HPLC (for example, Capsule Pack (CAPCELL P
AK) _C18 (Shiseido) (brand name of octadecylsilane),
Senshu PAK 1251-Y (senshu science) (trade name of octadecylsilane), etc.] to separate into single oligopeptides.

As the organic chemical synthesis method, a liquid phase method,
There are two types of solid phase methods, both of which are common methods, for example, Nobuo Izumiya,
Tetsuo Kato, Higashihiko Aoyagi and Michinori Waki, "Basics and Experiments of Peptide Synthesis", Maruzen Co., Ltd., 1985. In the liquid phase method, for example, an amino acid to be located at the C-terminus of the present oligopeptide, whose carboxyl group is protected with a benzyl group (Bzl), t-butyl group (t-Bu) or the like; An amino acid to be located next to the amino acid, wherein the α-amino group is a t-butyloxycarbonyl group (Boc), a benzyloxycarbonyl group (Z)
Amino acid protected with dimethylformamide (DMF
), Dimethylacetamide or the like, and react them overnight at room temperature, usually in the presence of dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBT). Then, the dipeptide derivative after removing the amino protecting group of the product by a conventional method is reacted, if necessary, in the same manner as the third amino acid in which the amino group is protected to remove the amino protecting group. This oligopeptide derivative is obtained repeatedly. If the amino acid to be reacted has hydroxyl, guanidino or imidazolyl groups, these groups should generally be protected prior to the reaction. The protecting group for the alcoholic hydroxyl group is Bz
The protecting group for phenolic hydroxyl group such as l and t-Bu is B
Protecting groups for guanidino groups such as zl and the like include tosyl groups (Tos) and the like, and protecting groups for imidazolyl groups include Tos and the like. After completion of the final reaction, all the protecting groups are removed to obtain the present oligopeptide. Introduction and removal of these protecting groups can be carried out by a conventional method.

On the other hand, in the solid phase method, a method using a peptide synthesizer has been widely used in recent years. For example, the oligopeptide can be produced using a 430A type peptide synthesizer manufactured by Applied Biosystems. That is, basically, from the phenylacetamidomethyl that amino acids located at the C-terminus of the oligopeptide bound (PAM) resin _{L-Xaa-O-CH 2} -PAM (Xaa is an amino acid residue) (Applied Biosystems From the N side of (available), the α-amino acid (Boc-amino acid) whose amino group is protected with Boc is extended stepwise by repeated peptide bonding and removal of Boc. Boc-L-Arg (Mts
) (Mts is mesitylene-2-sulfonyl) and Boc-L-Gln
Is its 1-hydroxybenzotriazole (
HOBT) is subjected to an elongation reaction via an ester, and other Boc-
Amino acids undergo an elongation reaction via their symmetrical anhydride as an intermediate by use of DCC. In the Boc- amino acid or _{L-Xaa-O-CH 2} -PAM, it should be protected generally by a suitable protecting group if there is a reactive functional group which should not participate in the reaction. In accordance with the present invention, protected Boc-
Amino acids and L-Xaa-O-CH ₂ -PAM as Boc-L-Arg (Mts
), Boc-L-Ser (Bzl), Boc-L-Tyr (Br-Z) (Br-Z is 4-
Bromobenzyloxycarbonyl), L-Tyr (Br-Z) -OC
H ₂ -PAM L-His (DNP) -O-CH ₂ -PAM (DNP is 2,4-dinitrophenyl) and L-Arg (Mts) -O-CH ₂ -PAM.
All of these can be obtained from Applied Biosystems. In the synthesis system using the 430A type peptide synthesizer, the following reagents and solvents are used in addition to the amino acid materials: N, N-diisopropylethylamine (TFA neutralizer), TFA (Boc cleavage), MeOH (dissolution of the produced urea compound and Removal), HOBT (0.5M HOBT / DMF), DCC (0.5M
DCC / dichloromethane (DCM), DCM and DMF (solvent),
Neutralizer (70% ethanolamine, 29.5% methanol) (
Neutralization of waste liquid). The amino acid raw materials and these reagents and solvents are charged in predetermined locations. These uses are performed automatically by the peptide synthesizer. The reaction temperature and time are also adjusted automatically, but the reaction temperature is usually room temperature. Above procedure by oligopeptide -O-CH ₂ -PAM reactive group is protected in the oligopeptide is obtained. The actual operation of the solid-phase peptide synthesis is performed according to a 430A peptide synthesizer user's manual by Applied Biosystems.

The obtained oligopeptide-O-CH ₂ -PAM protected with a reactive functional group can be prepared by a conventional method, for example, a method described in the aforementioned “Basic and Experimental Peptide Synthesis” or the 430A Type Peptide Synthesizer User's Manual. For example,
Resin and protection by treatment with trifluoromethanesulfonic acid (TFMSA) (TFA is a diluent of TFMSA) with TFA in the presence of thioanisole and / or ethanedithiol as a scavenger to capture cations generated by cleavage of the protecting group Cleavage of the group, thereby obtaining the desired oligopeptide. In the above TFMSA method, when the protected oligopeptide-0-CH ₂ -PAM has an L-His (DNP) residue, it is subjected to the above treatment after removal of DNP with thiophenol.

The acid addition salt of the present oligopeptide can be produced by a conventional method. For example, it can be obtained by reacting the present oligopeptide (containing a basic amino acid residue) with one equivalent of an appropriate acid in water, followed by freeze-drying.

The oligopeptide and the acid addition salt thereof are AC
It has an E-inhibitory effect and thus a blood pressure lowering effect, and is expected to be effective in treating and preventing hypertension in mammals including humans.

The oligopeptide and the acid addition salt thereof are used as they are or usually in a pharmaceutical composition with at least one pharmaceutical auxiliary.

The present oligopeptide and its acid addition salt can be administered parenterally (ie, intravenous injection, rectal administration, etc.) or orally, and can be formulated into a form suitable for each administration method.

Preparations for injections usually include sterile aqueous solutions. Formulations of the above form are also buffer pH adjusters
(Sodium hydrogen phosphate, citric acid, etc.), isotonic agents (sodium chloride, glucose, etc.), preservatives (methyl parahydroxybenzoate, propyl P-hydroxybenzoate, etc.) and other pharmaceutical auxiliaries other than water. Can be contained. The preparation can be sterilized by filtration through a bacteria-retaining filter, by mixing a bactericide into the composition, or by irradiating or heating the composition. The preparation can also be produced as a sterilized solid composition and used after dissolving in sterilized water or the like at the time of use.

Oral formulations are formulated in a form suitable for absorption by the gastrointestinal tract. Tablets, capsules, granules, fine granules and powders are commonly used pharmaceutical auxiliaries such as binders (syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone, hydroxypropylcellulose, etc.), excipients (lactose) , Sugar, corn starch, calcium phosphate, sorbite, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch,
Carboxymethylcellulose) and wetting agents (such as sodium lauryl sulfate). Tablets can be coated by conventional methods. The oral solution can be made into an aqueous solution or the like or a dry product.
Such an oral solution is prepared by using a conventional additive such as a preservative (p-
Methyl or propyl hydroxybenzoate, sorbic acid, etc.).

The amount of the present oligopeptide or its acid addition salt in the present ACE inhibitor and thus the antihypertensive agent can vary, but is usually 5 to 100% (w / w), particularly 10 to 60% (w / w). w)
Is appropriate. The dose of the present ACE inhibitor and thus the antihypertensive agent is 10 to 20 as an active ingredient when administered to humans.
0 mg / kg / day is appropriate. The acute toxicity of the peptide is LD ₅₀ (ICR mouse, oral administration)> 3 g / kg.

The oligopeptide has an advantage that it does not adversely affect the living body even when ingested in large amounts. Therefore, the oligopeptide has an effect of lowering blood pressure as it is or by adding various nutrients or the like to food or drink. It may be eaten as a functional food or health food having a function of preventing hypertension. That is, for example, nutrients such as various vitamins and minerals are added and used, for example, liquid foods such as nutritional drinks, soy milk, soups and solid foods of various shapes, and further used as such in powder form or added to various foods. Can also.
The content and intake of the active ingredient in the present ACE inhibitor and thus the antihypertensive as such functional foods and health foods may be the same as those in the above pharmaceuticals.

[0032]

Next, the present invention will be described with reference to examples. Example 1 (1) Preparation of freeze-dried α-zein hydrolyzate 100 g of α-zein (Wako Pure Chemical) was added to 2000 ml of distilled water, 1 g of thermolysin (Daiwa Kasei) was added, and the mixture was stirred and stirred at room temperature.
The temperature was raised to 65 ° C. over 1 hour, and the reaction was carried out at 65 ° C. for 17 hours.
During this reaction, the pH was maintained at 8.0 by appropriately adding 5NNaOH dropwise.
Then, 1 g of thermolysin was further added, and the reaction was carried out at 65 ° C. for 24 hours while maintaining the pH at 8.0. The enzyme was kept at 105 ° C. for 5 minutes in an autoclave to inactivate the enzyme. The obtained hydrolyzate was centrifuged at 5000 rpm for 10 minutes, and the supernatant was
(Amicon) PM-10 (ultrafiltration membrane, molecular weight cut-off 10,000, Amicon) was subjected to ultrafiltration, and the filtrate was lyophilized to obtain 97.3 g of lyophilized α-zein hydrolyzate.

(2) 2 g of the lyophilized α-zein hydrolyzate obtained above was dissolved in 2 l of distilled water, and a column (2.6 × 70 cm) of anion exchange resin, DEAE-Toyopearl 650M was used.
And run 1500 ml of 5 mM Tris buffer (pH 8.3),
The non-adsorbed fraction was collected and used as solution A. Then 1000ml 5mM
5 mM with Tris buffer (pH 8.3) and 1000 ml of 0.3 M NaCl
Elution was performed with a linear concentration gradient using Tris buffer (pH 8.3). 320 to 520m from the start of elution by linear concentration gradient
The eluted fraction of l was designated as solution B, and the eluted fraction of 620-700 ml was designated as solution C.

(3) Treatment of Fraction A After freeze-drying, the solution A was added to a gel filtration resin Sephatex LH-20 column (1.6 × 100 cm) manufactured by Pharmacia, and the solution was added.
The mixture was desalted by elution with methanol. The solution A after desalting was further subjected to a cation exchange resin SP-Toyopearl 650M column (2.6
× 70 cm) and 500 ml of 5 mM sodium acetate buffer
(pH 4.0), and eluted with a linear concentration gradient of 1000 ml of 5 mM sodium acetate buffer (pH 4.0) and 1000 ml of 5 mM sodium acetate buffer (pH 4.0) containing 0.6 M NaCl. From the start of the elution by the concentration gradient, the eluted fraction of 340 to 600 ml was designated as solution A1, and the eluted fraction of 950 to 1,050 ml was designated as solution A2.

The A1 solution was further fractionated by a semi-preparative high-performance liquid chromatography device manufactured by Nippon Millipore Limited (the same applies hereinafter). The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 0.1% trifluoroacetic acid (hereinafter abbreviated as TFA)
Acetonitrile gradient of 10 to 60% in the presence (time required: 30 minutes) Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions, the eluted fractions of A1-1, 6.3 min
The eluted fraction of 7.77.7 minutes was designated as A1-2, and the eluted fraction of 9.1 to 9.3 minutes was designated as A1-3.

A1-1 was further fractionated by a semi-preparative high-performance liquid chromatograph. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 0 to 20% acetonitrile gradient (duration: 20 minutes) in the presence of 0.1% TFA Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions The eluted fraction at 14.5 minutes was designated as A1-1-1. A1-2 was further fractionated by a semi-preparative high-performance liquid chromatography device. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 0 to 20% acetonitrile gradient (duration: 20 minutes) in the presence of 0.1% TFA Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions The eluted fraction of 15.1 minutes was A1-2-1,
The eluted fraction from 16.9 to 17.1 minutes was designated as A1-2-2.

The solution A2 was further fractionated by a semi-preparative high-performance liquid chromatograph. The elution conditions are as follows. Column: Caselpack C18 (1.5 x 25 cm) Eluent: 0 to 20% acetonitrile gradient in the presence of 0.1% TFA (duration: 20 minutes) Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions 14.9-15.1 Fraction was designated as A2-1.

(4) Treatment of B fraction The B liquid was freeze-dried and fractionated by a semi-preparative high-performance liquid chromatograph. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (time required: 30 minutes) Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions The eluted fraction at 7.8 minutes was B1, 7.8 to 8.
The eluted fraction of 3 minutes is B2, the eluted fraction of 8.3 to 9.1 is B3,
The eluted fraction from 14.2 to 14.4 minutes was designated as B4.

B1 was further fractionated by a semi-preparative high-performance liquid chromatograph. The elution conditions are as follows. Column: Capsule pack C18 (1.5 × 25 cm) Eluent: 10% acetonitrile in the presence of 0.1% TFA Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions, the eluted fraction of 6.9 to 7.1 minutes is extracted from B1- It was set to 1.

B2 was further fractionated by a semi-preparative high-performance liquid chromatograph. The elution conditions are as follows. Column: Capsule Pack C18 (1.5 x 25 cm) Eluent: 10% acetonitrile in the presence of 0.1% TFA Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions, the eluted fraction from 11.2 to 11.4 minutes was B2- It was set to 1.

B3 was further fractionated by a semi-preparative high-performance liquid chromatograph. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 10% acetonitrile in the presence of 0.1% TFA Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions, the eluted fraction from 15.0 to 16.0 minutes is subjected to B3- It was set to 1.

(5) Treatment of the C fraction The solution C was freeze-dried, and then subjected to gel filtration resin Sephadex L.
It was added to an H-20 column (1.6 × 100 cm) and desalted by elution with 30% methanol. The solution C after desalting was further fractionated by a semi-preparative high-performance liquid chromatograph. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (time required: 30 minutes) Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions The eluted fraction at 17.8 minutes was designated as C1.

(6) Identification of peptide A1-1-1, A1-2-1, A1-3, A2-
The fractions eluted with 1, B1-1, B2-1, B3-1, B4 and C1 each showed a single peak by the high performance liquid chromatography. Next, these eluted fractions were dried and then dissolved in distilled water, and the following structural analysis was performed using these aqueous solutions. Table 1 shows amino acid analysis values (by Hitachi L-8500 type amino acid analyzer), mass spectrometry values (by JEOL JMX-DX303), amino acid sequence analysis data (Shimadzu) Protein sequencer PSQ-
1 depends on the system).

Example 2 (1) 2 g of the lyophilized α-zein hydrolyzate prepared in the same manner as in Example 1 (1) was dissolved in an aqueous 0.02 M acetic acid solution, and
l, adjusted to pH 4.0, added to a cation exchange resin SP-Toyopearl 650M column (2.6 x 76 cm), and
A non-adsorbed fraction was removed by flowing a 0.02 M ammonium acetate buffer (pH 4.0). After that, elution was carried out with a linear concentration gradient of 1000 ml of 0.02 M ammonium acetate buffer (pH 4.0) and 1000 ml of 0.05 M ammonium acetate buffer (pH 8.6).
Elution was continued with 0.05M ammonium acetate buffer (pH 8.6). 850-1000 after starting elution with linear gradient
ml of the eluted fraction, solution D of 1,000 to 1100 ml of the eluate,
The eluted fraction of 1100-1200 ml is solution F, the eluted fraction of 1400-1500 ml is solution G, the eluted fraction of 1920-2120 ml is solution H, 2200-2270
The eluted fraction of ml was used as solution I.

(2) Treatment of D fraction The D solution was freeze-dried and fractionated with a semi-preparative high-performance liquid chromatograph. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 10 to 60% acetonitrile gradient (time required: 30 minutes) in the presence of 0.1% TFA Flow rate: 8 ml / min Detection: UV 210 nm The elution fraction at 11.0 minutes was designated as D1.

(3) Treatment of E fraction The E solution was freeze-dried and fractionated by semi-preparative high-speed liquid chromatography. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 10 to 60% acetonitrile gradient (time required: 30 minutes) in the presence of 0.1% TFA Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions The eluted fraction at 13.2 minutes was designated as E1.

(4) Treatment of F fraction The above F liquid was freeze-dried and fractionated by a semi-preparative high-performance liquid chromatograph. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 10 to 60% acetonitrile gradient (time required: 30 minutes) in the presence of 0.1% TFA Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions The eluted fraction of 10.7 minutes was F1, 12.5-1.
The eluted fraction of 3.2 minutes is F2, and the eluted fraction of 17.0-18.0 minutes is F2.
It was set to 3.

(5) Treatment of the G fraction After the lyophilization, the G solution was fractionated by a semi-preparative high-performance liquid chromatograph. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (time required: 30 minutes) Flow rate: 8 ml / min Detection: UV 210 nm The elution fraction of 5.2 minutes was G1, 13.8-1
The eluted fraction at 5.0 minutes was designated as G2.

(6) Treatment of H fraction The H solution was freeze-dried and fractionated by a high-performance liquid chromatograph for semi-preparation. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (time required: 30 minutes) Flow rate: 8 ml / min Detection: UV 210 nm The eluted fraction at 8.7 minutes was designated as H1,
The eluted fraction of 9.99.9 minutes was designated as H2.

H2 was further fractionated by a high-performance semi-preparative liquid chromatography apparatus. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 10% acetonitrile in the presence of 0.1% TFA Flow rate: 8 ml / min Detection: UV 210 nm Under these conditions, the eluted fraction of 12.6- 13.2 minutes was H2- It was set to 1.

(7) Treatment of Fraction I The above Solution I was freeze-dried and fractionated by a semi-preparative high-performance liquid chromatograph. The elution conditions are as follows. Column: Capsule pack C18 (1.5 x 25 cm) Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (time required: 30 minutes) Flow rate: 8 ml / min Detection: UV 210 nm The elution fraction at 7.2 minutes was defined as I1.

(8) Identification of peptide Structural analysis of each eluted fraction was performed in the same manner as in Example 1 (6). Table 1 shows the results.

Example 3 Synthesis of L-Leu-L-Arg-L-Pro acetate 0.5 mmol Boc-L-Pro-O-CH ₂ -PAM resin and 2 mmol each of B
oc-L-Arg (Mts) and Boc-L-Leu- were converted to N, N-diisopropylamine, TFA, MeOH, 0.5M HOBT / DMF, 0.5MDCC / DCM
, DCM, DMF and neutralizer (70% ethanolamine + 29.5
% Methanol) and a peptide synthesizer (4
30A type) (manufactured by Applied Biosystems) and subjected to the active ester method using HOBT to obtain Boc-L-Leu-L
It was synthesized -Arg (Mts) -L-Pro- O-CH 2 -PAM, which was then vacuum dried.

1 g dry peptide resin, 1 thioanisole
ml and 500 ml of ethanedithiol were placed in a 100 ml round bottom flask and the mixture was stirred for 10 minutes. While cooling the flask with ice water, 10 ml of TFA was gradually added to the mixture, followed by stirring for 10 minutes. 1 ml of TFMSA was gradually added, the flask was taken out of ice water, and the reaction was continued at room temperature with stirring for 30 minutes. Cold diethyl ether was added until no further precipitation of peptide occurred (about 50 ml) and then stirred for 1 minute. The entire contents of the flask were transferred onto a glass filter (medium pore) and washed with cold diethyl ether. About 200m of cold diethyl ether
Place the beaker containing l under the glass filter,
Stirred. The peptide was dissolved by adding TFA to a glass filter and transferred into ether (precipitation of the peptide occurred in ether). Until no new precipitates form
TFA was added. The peptide precipitate in ether was transferred onto a glass filter (medium pore), filtered and washed several times with diethyl ether. Then, the precipitate on the glass filter was dissolved in 2N acetic acid and collected in an eggplant-shaped flask.
The solution was lyophilized. The lyophilized product was dissolved in distilled water and purified with a semi-preparative high-performance liquid chromatography device. The elution conditions were as follows: Column: Capsule Pack C18 (1.5 × 25 cm) Eluent: 5 to 80% acetonitrile gradient (time required: 45 minutes) in the presence of 0.1% TFA Flow rate: 8 ml / min detection Source: The eluate fraction containing the main peak by UV 210 nm detection was concentrated to dryness under reduced pressure, the residue was redissolved in 2N aqueous acetic acid and the solution was lyophilized. By this procedure, L-Leu-L-Arg
100 mg of -L-Pro acetate was obtained.

Example 4 ACE Inhibitory Activity The ACE inhibitory activity of the oligopeptide obtained as described above was measured as follows. First, 5 g of rabbit langacetone powder was dissolved in 50 ml of 0.1 M sodium borate buffer (pH 8.3), centrifuged at 40,000 G for 40 minutes, and the supernatant was further purified with hydroxyapatite. An angiotensin converting enzyme solution of 1 unit / mg protein was obtained.

Each of 0.03 ml of aqueous solutions of the oligopeptide of various concentrations was placed in a test tube, and 0.25
ml of hypryl histidyl leucine (final concentration 5 mM, NaCl
(Containing 300 mM), 0.1 ml of the above-mentioned angiotensin converting enzyme solution was added, and the mixture was reacted at 37 ° C. for 30 minutes. Thereafter, 0.25 ml of IN hydrochloric acid was added to stop the reaction, and then 1.
5 ml of ethyl acetate was added, and the absorption value of hyprilic acid extracted in ethyl acetate at 228 nm was measured, and this was defined as the enzyme activity. Under these conditions, the absorbance at 228 nm when the present oligopeptide was not contained was 0.35.

A plurality of such experiments were performed, and the inhibition rate was calculated from the following equation. A: Absorption value at 228 nm without inhibitor B: Absorption value at 228 nm with addition of inhibitor Further, the concentration of the present oligopeptide at 50% inhibition was determined as IC _50.
Value. Table 1 shows the results.

Table 1 Analytical values of corn-derived oligopeptide (No. 1) Elution fraction Amino acid sequence Amino acid analytical value Mass spectrometric value A1-1-1 L-Leu-L-Gln-L-Gln Leu (1.00) 388 (M + H) ⁺ Glu (1.92) A1-2-1 L-Val-L-Ala-L-Ala Val (1.00) 260 (M + H) ⁺ Ala (2.11) A1-2-2 L-Val-L- Ser-L-Pro Val (1.02) 302 (M + H) ⁺ Ser (1.06) Pro (1.00) A1-3 L-Leu-L-Asn-L-Pro Leu (1.02) 343 (M + H) ⁺ Asp (1.05) Pro (1.00) A2-1 L-Ile-L-Arg-L-Ala-Ile (1.00) 615 (M + H) ⁺ L-Gln-L-Gln Arg (0.93) Ala (1.06) Glu ( 2.04) B1-1 L-Leu-L-Ala-L-Ala Leu (1.00) 274 (M + H) ⁺ Ala (1.80) B2-1 L-Leu-L-Gln-L-Pro Leu (1.09) 357 (M + H) ⁺ Glu (0.93) Pro (1.00) B3-1 L-Leu-L-Ser-L-Pro Leu (1.06) 316 (M + H) ⁺ Ser (0.91) Pro (1.00) B4 L- Leu-L-Leu-L-Pro Leu (1.93) 342 (M + H) ⁺ Pro (1.00) C1 L-Phe-L-Leu-L-Pro Phe (1.00) 376 (M + H) ⁺ Leu (1.10 ) Pro (0.99) D1 L-Val-L-Ala-L-Tyr Ala (1.00) 352 (M + H) ⁺
Val (1.13) Tyr (0.94) E1 L-Leu-L-Ala-L-Tyr Ala (1.00) 366 (M + H) ⁺ Leu (1.07) Tyr (0.88) F1 L-Tyr-L-Val Val (1.03 ) 281 (M + H) ⁺ Tyr (1.00) F2 L-Leu-L-Tyr Tyr (1.00) 295 (M + H) ⁺ Leu (1.01) F3 L-Leu-L-Phe Leu (1.10) 279 (M + H) ⁺ Phe (1.00) G1 L-Leu-L-Ser-L-His Ser (0.86) 356 (M + H) ⁺ Leu (1.00) His (1.06) G2 L-Phe-L-Tyr Phe (1.03 ) 329 (M + H) ⁺ Tyr (1.00) H1 L-Ile-L-Arg-L-Ala Ala (1.04) 359 (M + H) ⁺ Ile (1.00) Arg (0.94) H2-1 L-Leu- L-Arg-L-Pro Leu (1.10) 385 (M + H) ⁺ Arg (1.00) Pro (1.00) I1 L-Tyr-L-Arg Tyr (1.00) 338 (M + H) ⁺ Arg (1.03)

[0059] Table 1 Analytical values of corn-derived oligopeptides (Part 2) gives eluted fraction IC ₅₀ also Percent inhibition and IC ₅₀ or inhibition constant concentration _{(μM) A1-1-1 IC 50 100 A1-2-1} IC _{50 13 A1-2-2 IC 50 10 A1-3 IC} 50 35 A2-1 IC 50 160 B1-1 IC 50 13 B2-1 IC 50 1.9 B3-1 IC 50 1.7 B4 IC 50 57 C1 IC 50 210 D1 IC ₅₀ 16 E1 IC ₅₀ 3.9 F1 40% 32 F2 58% 150 F3 48% 600 G1 51% 79 G2 IC ₅₀ 25 H1 IC ₅₀ 6.4 H2-1 IC ₅₀ 0.27 I1 58% 290

Example 5 Antihypertensive Action As the animal, a 10-week-old spontaneously hypertensive rat (SHR) (Japan Rat Co., Ltd., weight: 240 to 280 g, 4 to 6 animals per group) was used. The sample was dissolved in physiological saline, and 100 mg / kg of the active ingredient oligopeptide (free) was intraperitoneally administered. Saline was administered as a control. The blood pressure was measured by a Tail-Cuff method before and after administration using a non-invasive blood pressure monitor TK-350 (manufactured by Unicom) for rats and mice. The results are shown in Table 2 as (systolic blood pressure value before administration-systolic blood pressure value after administration) ± SE.

Table 2 Sample Blood pressure drop (mmHg) 5 hours later Physiological saline -1 ± 3 L-Leu-L-Arg-L-Pro acetate 14 ± 5 * * P <0.01, significant compared to control There is a difference

Example 6 Intravenous injection The present oligopeptide was dissolved in 20 to 100-fold (volume / weight) sterile physiological saline and aseptically filtered (pore size 0.45 μm).
The filtrate filtered in step is used as an injection.

Example 7 Tablets 7 parts of the present oligopeptide 1 part hydroxypropylcellulose 1 part lactose 10.9 parts potato starch 1 part magnesium stearate 0.1 part A 60% ethanol aqueous solution containing 1 part hydroxypropylcellulose 20 parts was prepared, and the oligopeptide 7 was prepared. And 10.9 parts of lactose, kneaded well, dried under reduced pressure, added 1 part of potato starch and 0.1 part of magnesium stearate to the obtained dried product, mixed, and made a tablet using a tableting machine. .

[0064]

Industrial Applicability The present invention provides a novel oligopeptide having an ACE inhibitory activity derived from corn protein α-zein, and an ACE inhibitor and a hypotensive agent containing a specific oligopeptide.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/99 C12P 21/06 // C12P 21/06 A61K 37/64 (72) Inventor Hideoki Tanaka Ibaraki 1-3-3 Higashi, Tsukuba City Institute of Industrial Science and Technology, Institute of Biotechnology and Industrial Technology (72) Inventor Hidekatsu Maeda 1-3-1 Higashi, Tsukuba City, Ibaraki Pref. Institute of Technology, Institute of Biotechnology and Industrial Technology (72) Inventor Shinsuke Miyoshi Chiba (72) Inventor Hiromi Ishikawa 2-20-2, Hinode, Funabashi City, Chiba Prefecture (72) Inventor Fumio Fukui 1-2-1, 117, Nakadai, Narita City, Chiba Prefecture

Claims (2)

[Claims] 1. L-Tyr-L-Val, L-Phe-L-Tyr, L-Leu-LS
er-L-Pro, L-Val-L-Ser-L-Pro, L-Leu-L-Asn-L-Pro
, L-Val-L-Ala-L-Tyr, L-Leu-L-Ala-L-Tyr, Leu-L-Gl
nL-Pro, L-Leu-L-Ala-L-Ala, L-Val-L-Ala-L-Ala,
L-Leu-L-Ser-L-His or a pharmaceutically acceptable acid addition salt thereof, L-Ile-L-Arg-L-Ala or a pharmaceutically acceptable acid addition salt thereof, and L-Leu-L An angiotensin converting enzyme inhibitor comprising, as an active ingredient, at least one selected from the group consisting of -Arg-L-Pro or a pharmacologically acceptable acid addition salt thereof. 2. L-Tyr-L-Val, L-Phe-L-Tyr, L-Leu-LS
er-L-Pro, L-Val-L-Ser-L-Pro, L-Leu-L-Asn-L-Pro
, L-Val-L-Ala-L-Tyr, L-Leu-L-Ala-L-Tyr, Leu-L-Gl
nL-Pro, L-Leu-L-Ala-L-Ala, L-Val-L-Ala-L-Ala,
L-Leu-L-Ser-L-His or a pharmaceutically acceptable acid addition salt thereof, L-Ile-L-Arg-L-Ala or a pharmaceutically acceptable acid addition salt thereof, and L-Leu-L An antihypertensive comprising as an active ingredient at least one selected from the group consisting of -Arg-L-Pro or a pharmacologically acceptable acid addition salt thereof.