JPH05112465A - Preparation of composition containing angiotensin converting enzyme inhibitor - Google Patents

Abstract

PURPOSE:To obtain a composition containing angiotensin converting enzyme inhibitor useful as a hypotensor or hypotensive food from natural substance. CONSTITUTION:Protein is hydrolyzed by using at least one kind of enzyme selected from alkaline protease produced by Bacillus subtilis, a neutral or alkaline protease produced by Aspergillus melleus, a neutral protease produced by Aspergillus oryzae and a neutral protease originated from papaya.

Description

Detailed Description of the Invention

[0001]

This invention can be prepared from natural products,
In particular, the present invention relates to a method for producing a composition containing an angiotensin converting enzyme inhibitor which is useful as a blood pressure lowering agent or food for lowering blood pressure.

[0002]

BACKGROUND OF THE INVENTION Angiotensin converting enzyme is mainly present in the lung, vascular endothelial cells and renal proximal tubules, and is angiotensin I (Asp-Arg-Val-Tyr-Ile-).
It acts on His-Pro-Phe-His-Leu) to induce dipeptide (Hi) from the C-terminal of angiotensin I.
It is an enzyme that cleaves and releases s-Leu) to produce angiotensin II having a strong pressor action.

This enzyme also has the function of decomposing and inactivating bradykinin, which is an antihypertensive substance in vivo, and is strongly involved in the pressor system. It has been conventionally considered that if the activity of angiotensin converting enzyme is inhibited, it works to reduce blood pressure and is clinically effective for prevention and treatment of hypertension.

[0004] Recently, since the proline derivative captopril was synthesized and its antihypertensive activity was confirmed, various synthetic studies of angiotensin-converting enzyme inhibitory substances have been actively conducted, and their acquisition from natural products is being attempted. .. This is because the angiotensin converting enzyme inhibitor derived from a natural product is expected to be an antihypertensive agent with low toxicity and high safety since it is obtained from foods or food raw materials.

[0005]

However, the potent angiotensin-converting enzyme inhibitor found in natural products is extremely rare, and teprotide (nonapeptide, SQ2088) obtained from Brazilian and Japanese snake venoms was slightly found.
1) and the like and the metabolite IS83 of actinomycetes belonging to the genus Streptomyces (Japanese Patent Laid-Open No. 58-177920) are only known. As angiotensin-converting enzyme inhibitors obtained by enzymatically treating natural products, peptides obtained by decomposing milk casein with trypsin are known (JP-A-58-109425, JP-A-5-109425).
9-44323, 59-44324, 61-3
No. 6226, No. 61-36227) The development of new inhibitors is desired.

[0006]

Means for Solving the Problems As a result of diligent search for an angiotensin converting enzyme inhibitor which is a natural substance and has few side effects in order to solve such problems, the present inventors have found that proteins, particularly muscle proteins and skipjack or skipjack derived substances, Seafood meat,
Add pork, beef or chicken to Bacillus subtilis (Bati
Aspergillus mereus, an alkaline protease produced by L. subtilis.
Aspergillus oryzae (As), a neutral to alkaline protease produced by illusmelleus
It was found that peptides having angiotensin converting enzyme inhibitory activity are present in a composition hydrolyzed by at least one enzyme selected from a neutral protease produced by pergillus orzae) and a neutral protease derived from papaya. Has been completed.

The composition having the activity of the present invention can be obtained particularly effectively when the enzyme produced by the above-mentioned specific microorganism is used, and even if the protein is decomposed by other known enzymes, the composition having the strong activity of the present invention can be obtained. No working composition is obtained. The following are commercially available enzymes that can be used in the present invention. Bacillus subtilis-producing enzyme; orientase 10B
(Ueda Chemical Industry) Bioprase (Nagase Industry) Alcalase (Novo Industry Japan) Protease S (Amano Pharmaceutical) Pro Leather (Amano Pharmaceutical) Aspergillus meleus Producing Enzyme; Protease P
(Manufactured by Amano Pharmaceutical Co., Ltd.) Sumiteam MP (manufactured by Shin Nippon Chemical Co., Ltd.) Aspergillus oryzae-producing enzyme; Protease A
(Manufactured by Amano Pharmaceuticals) Denateam AP (manufactured by Nagase & Co., Ltd.) Punchase NP2 (manufactured by Yakult Honsha) Sumiteam LP (manufactured by Shin Nippon Chemical Co., Ltd.) Protease M (manufactured by Amano Pharmaceutical Co., Ltd.) Papaya-origin enzyme; papain (manufactured by Nagase & Co.) papain W40 (Amano) Pharmaceutical products)

Any protein derived from animals or microorganisms may be used as the protein. Particularly useful ones are muscle protein, skipjack or skipjack-derived substances, fish meat such as sardines,
Shellfish, pork, beef, chicken.

To hydrolyze a protein with the enzyme of the present invention, the treatment method varies depending on the properties of the protein. If the protein is poorly soluble, the protein is mixed with hot water and homogenized by vigorous stirring, and then the enzyme is added to the protein. 0.005 for dissolution or suspension
10 to 10% by weight, preferably 0.1 to 2% by weight is added, and the peptide bond of the hydrophobic amino acid is formed under the reaction conditions of temperature 5 to 90 ° C., preferably 20 to 70 ° C. and reaction time 1 minute to 3 days. The reaction is continued until the decomposition rate reaches 5% or more and the reaction is continued to obtain the desired product. The pH at this time is 4 to 9, preferably 6 to 8 when a neutral protease is used, and 7 to 12 when an alkaline protease is used, and preferably 8 to 8.
It is 10.

Decomposition rate is% of amino nitrogen to total nitrogen
It is represented by. However, Journal of Agricult
ural and Food Chemistry 2
4 No. 6 1090-1093 (1976). The angiotensin converting enzyme inhibitor-containing composition thus obtained is a mixture of various peptides,
It may be used as it is, or may be used after being post-treated. The administration routes of the peptides obtained in the present invention include:
It may be oral, parenteral or rectal, but oral administration is preferred.

The peptides of the present invention are usually administered in the form of a preparation prepared by mixing with a carrier for preparation. As the pharmaceutical carrier, substances that are commonly used in the pharmaceutical field and do not react with the peptides of the present invention are used. Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, saccharose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethyl cellulose, hydroxypropyl starch, calcium carboxymethyl cellulose, ion exchange resin, methyl cellulose. , Gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, bee gum,
Titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester,
Examples include purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water and the like. The dosage forms include tablets, capsules, granules, powders, syrups, suspensions,
Examples include injections. These preparations are prepared according to a conventional method. The liquid preparation may be dissolved or suspended in water or another appropriate medium before use. The tablets and granules may be coated by a known method.
In the case of an injectable preparation, it is prepared by dissolving the peptides of the present invention in water, but it may be dissolved in physiological saline or glucose solution, if necessary, and by adding a buffer or a preservative. Good.

These formulations can contain the peptides of the present invention in a proportion of 0.01% or more, preferably 0.5 to 70%. These formulations may also contain other therapeutically valuable ingredients.

[0013]

The present invention can prepare a composition containing an angiotensin-converting enzyme inhibitor which can be prepared from natural products and is particularly useful as a blood pressure lowering agent or food for lowering blood pressure.

[0014]

EXAMPLES The present invention will be described in more detail below with reference to examples. Examples 1 to 11 To 5 g of skipjack tuna, add 40 ml of water and homogenize thoroughly.
After the protease shown in Table 1 was allowed to act, the composition was hydrolyzed and the reaction solution was centrifuged to concentrate the supernatant, and the angiotensin converting enzyme inhibitory activity was measured.

(Protease Action Conditions) When a neutral protease is allowed to act, the reaction solution is treated with sodium hydroxide to p.
The reaction mixture was set to H7.0, reacted at 37 ° C for 13 hours, and then boiled for 10 minutes. When an alkaline protease was used, the pH was adjusted to 8.5 with sodium hydroxide, the reaction was carried out at a reaction temperature of 37 ° C. for 3 hours, and then boiling was performed for 10 minutes. The amount of enzyme was 1/100 parts by weight based on the skipjack sap.

(Measurement of Angiotensin-Converting Enzyme Inhibitory Activity) The angiotensin-converting enzyme inhibitory activity was measured by Ch
Eung and Cushman's method [Biochemic
al Pharmacology 20 , 1637
(1971)] according to the following method. Enzyme substrate; Bz (benzyl) -Gly-His-Leu (solution of 86 mg dissolved in 8 ml of water and 8 ml of phosphate buffer solution) Enzyme; Acetone powder of rabbit lung (Sigma) (1 g of 50 mM phosphoric acid Supernatant obtained by crushing in 10 ml of buffer solution and then centrifuging) 100 μl of the above enzyme substrate, 12 μl of the enzyme solution and a peptide having a predetermined concentration of the present invention were mixed, and the whole was mixed with water to 25
After making it 0 μl, the reaction was carried out at 37 ° C. for 30 minutes.

The reaction was terminated with 250 μl of 1N HCl. Ethyl acetate (1.5 ml) was added to the reaction-terminated liquid, and the mixture was stirred with Vortex for 15 seconds and then centrifuged. 1.0 ml was taken out from the ethyl acetate layer, ethyl acetate was distilled off, and 1 ml of distilled water was added to dissolve the residue.
Ultraviolet absorption of extracted hippuric acid value at 228 nm (O
D ₂₂₈ ) was measured.

The inhibition rate is the OD when the reaction was carried out without the inhibitor.
₂₂₈ is 100%, and OD ₂₂₈ when the reaction time is 0 minutes is 0
The activity was expressed by the concentration IC ₅₀ (μg / ml) of the inhibitor (peptide composition of the present invention) when the inhibition rate was 50%. The results are shown in Table 1.

[0019]

[0020]

INDUSTRIAL APPLICABILITY In the present invention, an angiotensin converting enzyme inhibitor-containing composition useful as a blood pressure lowering agent or a blood pressure lowering food can be produced by degrading a protein by using an enzyme produced by a specific microorganism and plant.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location // C12N 9/48 7823-4B C12P 21/06 8214-4B (C12N 9/48 C12R 1: 125 ) (C12N 9/48 C12R 1:66) (C12N 9/48 C12R 1:69)

Claims (4)

[Claims] 1. A protein comprising at least one protein selected from alkaline proteases produced by Bacillus subtilis, neutral to alkaline proteases produced by Aspergillus mereus, neutral proteases produced by Aspergillus oryzae, and neutral proteases derived from papaya. A method for producing an angiotensin converting enzyme inhibitor-containing composition, which comprises hydrolyzing with an enzyme 2. The method according to claim 1, wherein skipjack or a skipjack-derived substance is used as the protein. 3. The method according to claim 1, wherein seafood meat is used as the protein. 4. The method according to claim 1, wherein pork, beef or chicken is used as the protein.