JP3726106B2 - Angiotensin converting enzyme inhibitor and antihypertensive agent - Google Patents

Description

Ａ：阻害剤を含まない場合の 228nm吸収値
Ｂ：阻害剤添加の場合の 228nm吸収値
また阻害率50％のときの本オリゴペプチドの濃度をIC₅₀値とした。
結果を表１に示す。
【００５８】

【００５９】

【００６０】
実施例５ 血圧降下作用
動物は１０週令高血圧自然発症ラット( SHR )(日本ラット（株) 、♂、体重240 〜280g、１群４〜６匹）を用いた。試料を生理食塩水に溶解し、有効成分オリゴペプチド（フリー）として100mg/kgを腹腔内に投与した。対照として生理食塩水を投与した。血圧はラット・マウス用非観血血圧計TK-350( ユニコム社製)を用い、投与前及び投与後経時的にTail-Cuff 法で測定した。
結果を( 投与前最高血圧値−投与後最高血圧値）±S.E.で表２に示した。
【００６１】

【００６２】
実施例６ 静脈注射剤
本オリゴペプチドを20〜 100倍( 容積／重量）の滅菌生理食塩水に溶解し、無菌的にフィルター（孔径0.45μm)で濾過した濾液を注射剤とする。
【００６３】
実施例７ 錠剤
本オリゴペプチド 7 部
ヒドロキシプロピルセルロース 1 部
ラクトース 10.9部
ポテトスターチ 1 部
ステアリン酸マグネシウム 0.1部
ヒドロキシプロピルセルロース１部を含む60％エタノール水溶液20部を調製し、本オリゴペプチド７部およびラクトース10.9部を加えて十分に混練した後、減圧下で乾燥し、得られた乾燥物にポテトスターチ１部及びステアリン酸マグネシウム0.1 部を加えて混和して、打錠機により製錠する。
【００６４】
【発明の効果】
トウモロコシタンパク質α−ゼイン由来のＡＣＥ阻害作用を有する新規オリゴペプチド、及び特定オリゴペプチドを含有するＡＣＥ阻害剤及び血圧降下剤が提供される。[0001]
[Industrial application fields]
The present invention relates to a novel oligopeptide derived from corn protein and an angiotensin converting enzyme inhibitor and an antihypertensive agent containing a specific oligopeptide derived from corn protein as active ingredients. This drug is expected to be useful for the treatment or prevention of hypertension.
[0002]
[Prior art]
It is well known that the renin-angiotensin system is deeply involved in the development of hypertension, and this renin-angiotensin system contains an angiotensin-converting enzyme (EC3.4.15.1, hereinafter also referred to as ACE). Plays an important role. In this case, ACE is used for angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu), which is an angiotensinogen secreted in the liver, which is degraded by the enzyme renin produced in the kidney. This is converted into angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). This angiotensin II has actions such as contracting the vascular wall smooth muscles to increase blood pressure and further acting on the adrenal cortex to promote aldosterone secretion. In addition, the enzyme kallikrein present in plasma degrades a protein called kininogen to produce bradykinin that dilates blood vessels and lowers the blood pressure, but this bradykinin is degraded and inactivated by the action of ACE. Thus, ACE, on the one hand, produces vasopressor peptide (angiotensin II) and, on the other hand, degrades antihypertensive peptide (bradykinin), resulting in an increase in blood pressure. Therefore, it is possible to prevent an increase in blood pressure (hypertensive pressure) by suppressing this enzyme activity.
[0003]
A number of synthetic substances such as captopril (D-2-methyl-3-mercaptopropanoyl-L-proline) are known as ACE activity inhibitors, including several peptide inhibitors obtained from snake venom. Of these, captopril has already been put into practical use as an oral antihypertensive agent.
[0004]
In recent years, ACE inhibitors have been found in microorganisms and various foods, and their practical application as antihypertensive agents has been studied (Kunio Sueami, Fermentation and Industry).46(No.3), 179-182 (1988)). Furthermore, Susumu Maruyama, Bioscience and Industry on ACE inhibitors derived from food proteins, especially casein and corn seed protein47 (No.11), 38-42 (1989) and Susumu Maruyama, Chemistry and Biologytwenty two (No.8), 485-487 (1984). As for ACE inhibitors derived from corn seed protein, Susumu Maruyama et al., 23rd Annual Meeting of the Fermentation Engineering Society of Japan in 1988 (1988), Susumu Maruyama et al., 8th Annual Meeting of the Japanese Society of Agricultural Chemistry, 8th page (1989) ), Shinsuke Miyoshi et al., Japanese Society of Nutrition and Food Science, Abstracts, 113 pages (1989), Shinsuke Miyoshi et al., Journal of Japanese Society of Agricultural Chemistry64(3), also reported on page 555 of the abstract of the 1990 conference.
Further, the following applications have been filed by the present applicant for ACE inhibitors and antihypertensive agents derived from corn seed protein: Japanese Patent Application Nos. 63-185467 and 63-185468, Japanese Patent Application Nos. 1-59549 and 1-59550.
[0005]
[Problems to be solved by the invention]
New and useful antihypertensive agents, and thus angiotensin converting enzyme inhibitors, are constantly being sought and will lead to an enrichment of technology.
[0006]
It is an object of the present invention to provide a novel oligopeptide having an excellent angiotensin converting enzyme inhibitory action, and an angiotensin converting enzyme inhibitor and a blood pressure lowering agent having extremely high safety using such oligopeptide as an active ingredient.
[0007]
[Means for Solving the Problems]
The above object of the present invention is achieved by the following invention relating to an angiotensin converting enzyme inhibitor comprising a novel oligopeptide derived from α-zein, which is a kind of corn protein, and a specific oligopeptide derived from α-zein as an active ingredient. It was done.
[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-Leu-L-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-Val-L-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 salt, L-Leu-L-Arg-L-Pro and its acid addition salt, and L-Ile-L-Arg-L-Ala-L- Gln-L-Gln and its acid addition salts.
[0009]
2. L-Tyr-L-Val, L-Leu-L-Tyr, L-Leu-L-Phe, L-Phe-L-Tyr, L-Tyr-L-Arg or pharmacologically acceptable acid addition Salt, 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-Leu-L-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-Val-L-Ala-L-Tyr, L-Leu-L-Ala-L-Tyr, L-Leu-L-Ser-L-His or pharmacologically acceptable L-Ile-L-Arg-L-Ala or a pharmacologically acceptable acid addition salt thereof, L-Leu-L-Arg-L-Pro or a pharmacologically acceptable acid addition salt thereof, And an angiotensin converting enzyme inhibitor containing as an active ingredient at least one selected from the group consisting of L-Ile-L-Arg-L-Ala-L-Gln-L-Gln or a pharmacologically acceptable acid addition salt thereof .
[0010]
3. L-Tyr-L-Val, L-Leu-L-Tyr, L-Leu-L-Phe, L-Phe-L-Tyr, L-Tyr-L-Arg or a pharmacologically acceptable acid addition salt thereof, 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- Leu-L-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-Val-L-Ala-L-Tyr, L-Leu-L-Ala-L-Tyr, L-Leu-L-Ser-L-His or its pharmacologically acceptable Acid addition salts, L-Ile-L-Arg-L-Ala or pharmacologically acceptable acid addition salts thereof, L-Leu-L-Arg-L-Pro or pharmacologically acceptable acid addition salts thereof, and L -Ile-L-Arg-L-Ala-L-Gln-L-Gln or an antihypertensive agent containing as an active ingredient at least one selected from the group consisting of pharmacologically acceptable acid addition salts thereof
[0011]
In the above, acid addition salts are pharmaceutically acceptable acid (inorganic and organic acid) addition salts such as hydrochloride, hydrobromide, sulfate, nitrate, acetate, benzoate, maleate, fumarate. Acid salts, succinates, tartrate, citrate, oxalate, methanesulfonate, toluenesulfonate, aspartate, glutamate and the like.
[0012]
The oligopeptide used in the present invention can be obtained by enzymatic hydrolysis of corn protein, a method of introducing amino acids stepwise by an organic chemical synthesis method, a peptide synthesis method utilizing reverse reaction of hydrolase, a genetic engineering method, etc. Can be manufactured.
[0013]
The enzymatic hydrolysis method of corn protein will be described. Although gluten meal may be used as the raw material corn protein, the case where α-zein obtained by further purifying it will be described. As α-zein, a commercially available product may be used, or it may be separated from zein separated from gluten meal by a known method.
[0014]
As the enzyme, endo-type proteases such as papain and thermolysin can be used, and thermolysin is particularly preferable. Thermolysin may be of high purity or of industrial grade (for example, Samoaise (Daiwa Kasei)).
[0015]
Hereinafter, α-zein thermolysin treatment will be described. This treatment is usually carried out simply in water or in a buffer (eg Tris-HCl buffer, phosphate buffer). The substrate concentration may be any as long as it is within a range where stirring and mixing can be performed during the reaction, but it is preferably performed within a protein concentration range of 2 to 20% (w / v) that facilitates stirring. The addition amount of the enzyme thermolancin varies depending on the titer, but is usually 0.01% by weight or more, preferably 0.1 to 10% by weight per protein. A part of thermolysin may be added during the reaction. The pH and temperature of the reaction may be in the vicinity of the optimal pH of thermolysin. The pH is preferably 6 to 10, preferably 7 to 8, and the temperature is 30 to 80 ° C, preferably 60 to 70 ° C. The pH during the reaction is adjusted with an aqueous sodium hydroxide solution, hydrochloric acid or the like as necessary.
[0016]
The reaction time is not constant because it varies depending on the amount of enzyme added, reaction temperature, and reaction pH, but it is usually about 1 to 50 hours.
[0017]
The hydrolysis reaction is stopped by heating the hydrolyzate or by changing the pH 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 carried out according to a known method such as inactivation of the enzyme or filtration of the enzyme with an ultrafiltration membrane or the like. Next, the hydrolyzed liquid is subjected to solid-liquid separation (for example, centrifugation, filtration, etc.), and the separated liquid is fractionated by ultrafiltration, gel filtration, or the like to obtain a liquid containing a fraction having a molecular weight of 10,000 or less. This solution contains the target oligopeptide of the present invention, and this solution or its concentrate (for example, lyophilized product) is further fractionated to obtain individual target oligopeptides.
[0018]
The lyophilized product is referred to herein as an α-zein hydrolyzed lyophilized product, and the case of using this will be further described.
[0019]
The α-zein hydrolyzed lyophilizate is, as a method, first subjected to an anion exchange resin, for example, a weakly basic anion exchange resin (eg DEAE-Toyopearl 650M, manufactured by Tosoh Corporation). Or a cation exchange resin such as a weakly acidic cation exchange resin (for example, SP-Toyopearl 650M (manufactured by Tosoh Corporation)).
[0020]
In the first fractionation method through an anion exchange resin, each oligopeptide held in the resin is eluted with a linear concentration gradient (for example, when using DEAE-Toyopearl 650M, elution with NaCl in Tris buffer → Tris buffer at an appropriate concentration) Sort by. Fractionate the eluate into several fractions, gel filtration (e.g. using Sephadex LH-20), cation exchange resin treatment (e.g. SP-Toyopearl 650M), reverse phase HPLC (e.g. CAPCELL PAK C)₁₈ (Shiseido) (trade name of octadecylsilane), Senshu PAK 1251-Y (Senshu Kagaku) (trade name of octadecylsilane, etc.), etc.] and so on, and so on.
[0021]
In addition, there are two types of organic chemical synthesis methods: liquid phase method and solid phase method, both of which are conventional methods, for example, Nobuo Izumiya, Tetsuo Kato, Toshihiko Aoyagi, and Noriaki Wakimichi, “Basics and Experiments of Peptide Synthesis” , Maruzen Co., Ltd., 1985. In the liquid phase method, for example, an amino acid that should be located at the C-terminus of the oligopeptide and whose carboxyl group is protected with a benzyl group (Bzl), t-butyl group (t-Bu), etc., and the C-terminus An amino acid that should be located next to an amino acid and whose α-amino group is protected with a t-butyloxycarbonyl group (Boc), benzyloxycarbonyl group (Z), etc. is converted into dimethylformamide (DMF), dimethylacetamide, etc. Dissolve and react them in the presence of dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBT), usually at room temperature overnight. Next, the dipeptide derivative after removal of the amino protecting group of the product by a conventional method is reacted in the same manner as the third amino acid with the amino group protected if necessary, the amino protecting group is removed, and the same procedure is performed as necessary. This oligopeptide derivative is obtained repeatedly. If the amino acid to be reacted has a hydroxyl group, a guanidino group or an imidazolyl group, these groups should generally be protected prior to the reaction. Protecting groups for alcoholic hydroxyl groups include Bzl, t-Bu, etc., protecting groups for phenolic hydroxyl groups include Bzl, etc., protecting groups for guanidino groups include tosyl groups (Tos), and protecting groups for imidazolyl groups include Tos etc. . After completion of the final reaction, all protecting groups are removed to obtain the oligopeptide. Introduction and removal of these protecting groups can be carried out by conventional methods.
[0022]
On the other hand, as a 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, phenylacetamidomethyl (PAM) resin L-Xaa-O-CH to which an amino acid located at the C-terminal of this oligopeptide is bonded.₂-PAM (where Xaa is an amino acid residue) (available from Applied Biosystems) from the N-side, the α-amino acid (Boc-amino acid) whose amino group is protected with Boc is repeated by repeating peptide bonds and removal of Boc Extended. Boc-L-Arg (Mts) (Mts is mesitylene-2-sulfonyl) and Boc-L-Gln are subjected to extension reactions via their 1-hydroxybenzotriazole (HOBT) esters as intermediates, and other Boc-L The amino acid is subjected to an extension reaction via its symmetrical anhydride as an intermediate by use of DCC. Boc-amino acid or L-Xaa-O-CH₂In -PAM, if there are reactive functional groups that should not participate in the reaction, they should generally be protected by a suitable protecting group. In the context of the present invention, protected Boc-amino acids and L-Xaa-O-CH₂-PAM as Boc-L-Arg (Mts), Boc-L-Ser (Bzl), Boc-L-Tyr (Br-Z) (Br-Z is 4-bromobenzyloxycarbonyl), L-Tyr (Br- Z) -O-CH₂-PAM L-His (DNP) -O-CH₂-PAM (DNP is 2,4-dinitrophenyl) and L-Arg (Mts) -O-CH₂-PAM is mentioned. All of these are available from Applied Biosystems. In the synthesis system using the 430A type peptide synthesizer, in addition to the amino acid raw material, the following reagents and solvent are used: N, N-diisopropylethylamine (TFA neutralizing agent), TFA (Boc cleavage), MeOH (dissolution of the generated 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 material and these reagents and solvent are charged in place, and their use is automatically performed by the peptide synthesizer, and the reaction temperature and time are adjusted automatically, but the reaction temperature is usually room temperature. Oligopeptide-O-CH in which reactive groups in oligopeptide are protected by the above procedure₂-PAM is obtained. The actual operation of the above solid phase peptide synthesis is by Applied Biosystems.
430A type peptide synthesizer user's manual.
[0023]
The resulting reactive functional group protected oligopeptide-O-CH₂-PAM is used as a scavenger to capture cations generated by conventional methods, for example, “Peptide Synthesis Fundamentals and Experiments” or the 430A Peptide Synthesizer User's Manual, for example, cleavage of a protecting group, and / or ethane. Treatment with trifluoromethanesulfonic acid (TFMSA) with TFA in the presence of dithiol (TFA is a diluent of TFMSA) cleaves the resin and protecting groups, thereby obtaining the desired oligopeptide. In the above TFMSA method, protected oligopeptide-0-CH₂When -PAM has an L-His (DNP) residue, it is subjected to the above treatment after removal of DNP with thiophenol.
[0024]
The acid addition salt of this oligopeptide can be manufactured by a conventional method. For example, it can be obtained by reacting the present oligopeptide (containing a basic amino acid residue) with 1 equivalent of an appropriate acid in water and freeze-drying.
[0025]
This oligopeptide and its acid addition salt have an ACE inhibitory action and thus a blood pressure lowering action, and are expected to be effective in the treatment and prevention of hypertension in mammals including humans.
[0026]
The oligopeptides and acid addition salts thereof are used as such or usually in at least one pharmaceutical adjuvant and pharmaceutical composition.
[0027]
The oligopeptide and its acid addition salt can be administered parenterally (that is, intravenous injection, rectal administration, etc.) or orally, and can be formulated into a form suitable for each administration method.
[0028]
The preparation form as an injection usually includes a sterile aqueous solution. Formulations in the above form are also buffer pH adjusters (sodium hydrogen phosphate, citric acid, etc.), isotonic agents (sodium chloride, glucose, etc.), preservatives (methyl paraoxybenzoate, propyl P-hydroxybenzoate, etc.) And other pharmaceutical adjuvants other than water. The preparation can be sterilized by filtration through a bacteria-retaining filter, mixing of a bactericide into the composition, irradiation of the composition or heating. The preparation can also be produced as a sterilized solid composition and dissolved in sterilized water before use.
[0029]
Orally administered drugs are formulated in a form suitable for absorption by the gastrointestinal tract. Tablets, capsules, granules, fine granules, powders are conventional pharmaceutical adjuvants such as binders (syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone, hydroxypropylcellulose, etc.), excipients (lactose , Sugar, corn starch, calcium phosphate, sorbit, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch, carboxymethyl cellulose, etc.), wetting agents (sodium lauryl sulfate, etc.) Can be included. Tablets can be coated by conventional methods. The oral solution can be made into an aqueous solution or a dry product. Such oral solutions may contain conventional additives such as preservatives (methyl or propyl p-hydroxybenzoate, sorbic acid, etc.).
[0030]
The amount of the oligopeptide or the acid addition salt thereof in the ACE inhibitor and thus the antihypertensive agent can vary, but it is usually 5 to 100% (w / w), particularly 10 to 60% (w / w). Is appropriate. The dose of the present ACE inhibitor and thus the antihypertensive agent is suitably 10 to 200 mg / kg / day as an active ingredient when administered to humans. The acute toxicity of this peptide is LD₅₀(ICR mice, oral administration)> 3 g / kg.
[0031]
In addition, since this oligopeptide has the advantage of not adversely affecting the living body even if it is ingested in a large amount, it can be used as it is, with various nutrients added, or contained in foods and drinks to lower blood pressure and prevent hypertension. It may be eaten as a functional food or health food with a function. That is, for example, by adding nutrients such as various vitamins and minerals, for example, liquid foods such as energy drinks, soy milk and soup, solid foods of various shapes, and powders as they are or added to various foods. You can also. The content and intake of the active ingredient in the ACE inhibitor as a functional food and health food, and thus the antihypertensive agent, may be the same as the content and dosage in the above-mentioned pharmaceuticals, respectively.
[0032]
【Example】
Next, the present invention will be described with reference to examples.
Example 1
(1) Preparation of α-zein hydrolyzed freeze-dried product
100 g of α-zein (Wako Pure Chemical Industries, Ltd.) was added to 2000 ml of distilled water, 1 g of thermolysin (Daiwa Kasei) was added, the temperature was raised from room temperature to 65 ° C. over 1 hour with stirring, and the mixture was reacted at 65 ° C. for 17 hours. During this reaction, 5NNaOH was appropriately added dropwise to keep the pH at 8.0. Subsequently, 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. To inactivate the enzyme, it was kept at 105 ° C. for 5 minutes in an autoclave.
The resulting hydrolyzate is centrifuged at 5000 rpm for 10 minutes, and the supernatant is treated with Amicon.
It was subjected to ultrafiltration using PM-10 (ultrafiltration membrane, molecular weight cut off 10,000, Amicon), and the filtrate was lyophilized to obtain 97.3 g of α-zein hydrolyzed lyophilizate.
[0033]
(2) 2 g of the α-zein hydrolyzed lyophilizate obtained above was dissolved in 2 l of distilled water, added to an anion exchange resin, DEAE-Toyopearl 650M column (2.6 x 70 cm), and 1500 ml of 5 mM Tris. A buffer solution (pH 8.3) was allowed to flow, and the non-adsorbed fraction was collected and designated as solution A. Thereafter, elution was carried out with a linear concentration gradient of 1000 ml of 5 mM Tris buffer (pH 8.3) and 1000 ml of 5 mM Tris buffer (pH 8.3) containing 0.3 M NaCl.
The elution fraction of 320 to 520 ml was designated as B solution and the elution fraction of 620 to 700 ml as C solution after elution by the linear concentration gradient was started.
[0034]
(3) Processing of fraction A
The solution A was freeze-dried, added to a gel filtration resin Sephatex LH-20 column (1.6 × 100 cm) manufactured by Pharmacia, and desalted by elution with 30% methanol. The desalted solution A was further added to the cation exchange resin SP-Toyopearl 650M column (2.6 × 70 cm), and after flowing 500 ml of 5 mM sodium acetate buffer (pH 4.0), 1000 ml of 5 mM sodium acetate. Elution was performed with a linear concentration gradient using 1000 ml of 5 mM sodium acetate buffer (pH 4.0) containing buffer (pH 4.0) and 0.6 M NaCl. The elution fraction from 340 to 600 ml was designated as A1 solution and the elution fraction from 950 to 1050 ml was designated as A2 solution after elution by concentration gradient was started.
[0035]
The A1 solution was further fractionated by a semi-preparative high-speed liquid chromatograph manufactured by Nihon Millipore Limited (hereinafter the same). The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10-60% acetonitrile gradient in the presence of 0.1% trifluoroacetic acid (hereinafter abbreviated as TFA) (required time 30 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under these conditions, the elution fraction of 4.0 to 6.3 minutes was A1-1, the elution fraction of 6.3 to 7.7 minutes was A1-2, and the elution fraction of 9.1 to 9.3 minutes was A1-3.
[0036]
A1-1 was further fractionated with a semi-preparative high-speed liquid chromatograph. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 0 to 20% acetonitrile gradient in the presence of 0.1% TFA (required time 20 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under these conditions, the elution fraction from 14.3 to 14.5 minutes was designated as A1-1-1.
A1-2 was further fractionated with a semi-preparative high-speed liquid chromatograph. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 0 to 20% acetonitrile gradient in the presence of 0.1% TFA (required time 20 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under these conditions, the elution fraction from 14.9 to 15.1 minutes was designated as A1-2-1, and the elution fraction from 16.9 to 17.1 minutes was designated as A1-2-2.
[0037]
The A2 liquid was further fractionated by a semi-preparative high-speed liquid chromatograph. The elution conditions are as follows.
Column: Kasell Pack C18 (1.5 x 25cm)
Eluent: 0 to 20% acetonitrile gradient in the presence of 0.1% TFA (required time 20 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under this condition, the elution fraction of 14.9 to 15.1 minutes was designated as A2-1.
[0038]
(4) Processing of B fraction
The solution B was fractionated by a semi-preparative high-speed liquid chromatograph after lyophilization. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (required time 30 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under these conditions, the elution fraction of 6.8 to 7.8 minutes was B1, the elution fraction of 7.8 to 8.3 minutes was B2, the elution fraction of 8.3 to 9.1 was B3, and the elution fraction of 14.2 to 14.4 minutes was B4.
[0039]
B1 was further fractionated with a semi-preparative high-speed liquid chromatograph. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10% acetonitrile in the presence of 0.1% TFA
Flow rate: 8 ml / min
Detection: UV 210nm
Under this condition, the elution fraction from 6.9 to 7.1 minutes was defined as B1-1.
[0040]
B2 was further fractionated with a semi-preparative high-speed liquid chromatograph. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10% acetonitrile in the presence of 0.1% TFA
Flow rate: 8 ml / min
Detection: UV 210nm
Under this condition, the elution fraction from 11.2 to 11.4 minutes was defined as B2-1.
[0041]
B3 was further fractionated with a semi-preparative high-speed liquid chromatograph. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10% acetonitrile in the presence of 0.1% TFA
Flow rate: 8 ml / min
Detection: UV 210nm
Under this condition, the elution fraction of 15.0 to 16.0 minutes was defined as B3-1.
[0042]
(5) Processing of C fraction
The solution C was freeze-dried, added to a gel filtration resin Sephadex LH-20 column (1.6 × 100 cm), and desalted by elution with 30% methanol. The desalted C solution was further fractionated with a semi-preparative high-speed liquid chromatograph. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (required time 30 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under this condition, the elution fraction from 17.6 to 17.8 minutes was defined as C1.
[0043]
(6) Peptide identification
The elution fractions of A1-1-1, A1-2-1, A1-3, A2-1, B1-1, B2-1, B3-1, B4 and C1 are each single by each high-speed liquid chromatography. Showed a peak.
Next, these elution fractions were dried and dissolved in distilled water, and the following structural analysis was performed using these aqueous solutions.
Table 1 shows amino acid analysis values after hydrolysis with 6N hydrochloric acid at 110 ° C for 24 hours (using Hitachi L-8500 type amino acid analyzer), mass analysis values (using JEOL JMX-DX 303), amino acid sequence analysis data (Shimadzu) Protein sequencer PSQ-1 system).
[0044]
Example 2
(1) Cation exchange resin SP-Toyopearl was prepared by dissolving 2 g of α-zein hydrolyzed lyophilizate prepared in the same manner as in Example 1 (1) in 0.02 M acetic acid aqueous solution and adjusting the pH to 500 ml and pH 4.0. The mixture was added to a 650M column (2.6 × 76 cm), and 1500 ml of 0.02M ammonium acetate buffer (pH 4.0) was passed through to remove the non-adsorbed fraction. Then elute with 1000 ml of 0.02M ammonium acetate buffer (pH 4.0) and 1000 ml of 0.05M ammonium acetate buffer (pH 8.6), followed by 0.05M ammonium acetate buffer (pH 8.6). And then eluted.
After elution with a linear concentration gradient, the elution fraction of 850 to 1000 ml is liquid D, the elution fraction of 1000 to 1100 ml is liquid E, the elution fraction of 1100 to 1200 ml is liquid F, and the elution fraction of 1400 to 1500 ml Solution G, 1920-2120 ml elution fraction was designated H solution, and 2200-2270 ml elution fraction was designated solution I.
[0045]
(2) D fraction processing
The liquid D was fractionated by a semi-preparative high-speed liquid chromatograph after lyophilization. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (required time 30 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under this condition, the elution fraction of 10.5 to 11.0 minutes was defined as D1.
[0046]
(3) E fraction processing
The solution E was fractionated by lyophilization and semi-preparative high-speed liquid chromatography. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (required time 30 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under this condition, the elution fraction from 13.0 to 13.2 minutes was defined as E1.
[0047]
(4) F fraction processing
The liquid F was fractionated by a semi-preparative high-speed liquid chromatograph after lyophilization. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (required time 30 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under these conditions, the elution fraction from 10.3 to 10.7 minutes was F1, the elution fraction from 12.5 to 13.2 minutes was F2, and the elution fraction from 17.0 to 18.0 minutes was F3.
[0048]
(5) Processing of G fraction
The G solution was lyophilized and fractionated with a semi-preparative high-speed liquid chromatograph. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (required time 30 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under these conditions, the elution fraction of 5.0 to 5.2 minutes was designated G1, and the elution fraction of 13.8 to 15.0 minutes was designated G2.
[0049]
(6) H fraction processing
The liquid H was fractionated with a semi-preparative high-speed liquid chromatograph after lyophilization. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (required time 30 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under these conditions, the elution fraction from 7.7 to 8.7 minutes was designated as H1, and the elution fraction from 9.6 to 9.9 minutes was designated as H2.
[0050]
H2 was further fractionated with a semi-preparative high-speed liquid chromatograph. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10% acetonitrile in the presence of 0.1% TFA
Flow rate: 8 ml / min
Detection: UV 210nm
Under this condition, the elution fraction of 12.6 to 13.2 minutes was designated as H2-1.
[0051]
(7) Processing of I fraction
The solution I was fractionated with a semi-preparative high-speed liquid chromatograph after lyophilization. The elution conditions are as follows.
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 10 to 60% acetonitrile gradient in the presence of 0.1% TFA (required time 30 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
Under this condition, the elution fraction of 6.5 to 7.2 minutes was designated as I1.
[0052]
(8) Peptide identification
The structural analysis of each of the elution fractions was performed in the same manner as in Example 1 (6).
The results are shown in Table 1.
[0053]
Example 3    Synthesis of L-Leu-L-Arg-L-Pro acetate
  0.5mmol of Boc-L-Pro-O-CH₂-PAM resin and 2 mmol each of Boc-L-Arg (Mts) and Boc-L-Leu-, N, N-diisopropylamine, TFA, MeOH, 0.5M HOBT / DMF, 0.5MDCC / DCM, DCM, DMF and A peptide synthesizer (430A type) (Applied Biosystems) filled with a neutralizing agent (70% ethanolamine + 29.5% methanol) is packed and subjected to the active ester method using HOBT. Boc-L-Leu-L -Arg (Mts) -L-Pro-O-CH₂-PAM was synthesized and then vacuum dried.
[0054]
1 g of dry peptide resin, 1 ml of thioanisole and 500 ml of ethanedithiol were placed in a 100 ml round bottom flask and the mixture was stirred for 10 minutes. While the flask was cooled with ice water, 10 ml of TFA was gradually added to the mixture and then stirred for 10 minutes. 1 ml of TFMSA was gradually added, the flask was removed from the ice water, and the reaction was continued for 30 minutes with stirring at room temperature. Cold diethyl ether was added until no new peptide precipitation occurred (about 50 ml) and then stirred for 1 minute. The entire contents of the flask were transferred onto a glass filter (medium hole) and washed with cold diethyl ether. A beaker containing about 200 ml of cold diethyl ether was placed under a glass filter and stirred. TFA was added to the glass filter to dissolve the peptide and transferred into ether (precipitation of the peptide occurred in ether). TFA was added until no new precipitation occurred. The peptide precipitate in ether was transferred onto a glass filter (medium pore), filtered and washed several times with diethyl ether. Next, 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-speed liquid chromatograph. The elution conditions were as follows:
Column: Capsule Pack C18 (1.5 x 25cm)
Eluent: 5 to 80% acetonitrile gradient in the presence of 0.1% TFA (time required 45 minutes)
Flow rate: 8 ml / min
Detection: UV 210nm
The eluate fraction containing the main peak by 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, 100 mg of L-Leu-L-Arg-L-Pro acetate was obtained as a powder.
[0055]
Example 4    ACE inhibitory activity
The ACE inhibitory activity of the oligopeptide obtained as described above was measured as follows. That is, firstly, 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.
[0056]
Add 0.03 ml of each aqueous solution of this oligopeptide to each test tube, add 0.25 ml of hippurylhistidylleucine (containing 5 mM final concentration and 300 mM NaCl) as a substrate, and then add the above angiotensin converting enzyme. 0.1 ml of the solution was added and reacted at 37 ° C. for 30 minutes. Thereafter, 0.25 ml of IN hydrochloric acid was added to stop the reaction, 1.5 ml of ethyl acetate was added, and the absorption value of hypuric acid extracted into ethyl acetate was measured at 228 nm, which was defined as the enzyme activity. . Under these conditions, the absorption value at 228 nm when this oligopeptide was not contained was 0.35.
[0057]
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 when inhibitor is added
The concentration of this oligopeptide when the inhibition rate is 50% is IC₅₀Value.
The results are shown in Table 1.
[0058]

[0059]

[0060]
Example 5    Blood pressure lowering effect
The animals used were 10-week-old spontaneously hypertensive rats (SHR) (Japan Rat Co., Ltd., rabbits, body weight 240-280 g, 4-6 animals per group). The sample was dissolved in physiological saline, and 100 mg / kg was administered intraperitoneally as an active ingredient oligopeptide (free). Saline was administered as a control. Blood pressure was measured by the Tail-Cuff method using a non-invasive blood pressure monitor TK-350 (manufactured by Unicom) for rats and mice before and after administration.
The results are shown in Table 2 as (maximum blood pressure value before administration−maximum blood pressure value after administration) ± S.E.
[0061]

[0062]
Example 6    Intravenous injection
This oligopeptide is dissolved in 20 to 100 times (volume / weight) of sterilized physiological saline, and the filtrate obtained by aseptically filtering with a filter (pore size 0.45 μm) is used as an injection.
[0063]
Example 7    tablet
7 parts of this oligopeptide
Hydroxypropyl cellulose 1 part
Lactose 10.9 parts
1 potato starch
Magnesium stearate 0.1 parts
Prepare 20 parts of 60% ethanol aqueous solution containing 1 part of hydroxypropylcellulose, add 7 parts of this oligopeptide and 10.9 parts of lactose and knead well, then dry under reduced pressure. And 0.1 parts of magnesium stearate are added and mixed, and tableted with a tableting machine.
[0064]
【The invention's effect】
A novel oligopeptide having an ACE inhibitory action derived from corn protein α-zein, and an ACE inhibitor and an antihypertensive agent containing the specific oligopeptide are provided.

Claims (2)

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 pharmacologically acceptable acid addition salt thereof, L-Ile-L-Arg-L-Ala or pharmacologically acceptable An angiotensin converting enzyme inhibitor comprising, as an active ingredient, an acid addition salt and one or more selected from the group consisting of L-Leu-L-Arg-L-Pro or a pharmacologically acceptable acid addition salt thereof. 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 pharmacologically acceptable acid addition salt thereof, L-Ile-L-Arg-L-Ala or pharmacologically acceptable An antihypertensive agent comprising, as an active ingredient, an acid addition salt and one or more selected from the group consisting of L-Leu-L-Arg-L-Pro or a pharmacologically acceptable acid addition salt thereof.