JPH11343297A - Production of peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a specific peptide being a natural substance having slight adverse action, useful as an inhibitor for angiotensin converting enzyme, by hydrolyzing a protein with thermolysin. SOLUTION: (A) A protein (preferably fish meat or dried bonito) is hydrolyzed with (B) thermolysin to give the objective compound of the formula: Leu-Lys-Pro (Leu is leucine; Lys is lysine; Pro is proline; all L-isomer). The method is carried out, for example, by mixing hot water with the component A, homogenizing the mixture by strong stirring, adding the component B to the homogenous mixture and hydrolyzing the mixture at about 0-85 deg.C for 0.1-48 hours. In the case of purification, the hydrolyzecl solution is filtered by centrifugal separation, etc., and purified by a means such as concentration, high-performance liquid chromatography, etc., preferably the obtained compound is orally administered and 0.01-10 mg/time is applied once to three times daily as a dosage.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a peptide having the following structure. Leu-Lys-Pro

[0002]

2. Description of the Related Art Angiotensin converting enzyme is mainly present in lungs, vascular endothelial cells and renal proximal tubules, and is angiotensin I (Asp-Arg-Val-Tyr-Ile-).
Acts on His-Pro-Phe-His-Leu) to dipeptide (Hi) from the C-terminus of angiotensin I
s ⁹ -Leu ¹⁰ ), which is an enzyme that releases angiotensin II having a strong pressor action. In addition, this enzyme also has the action of destroying and inactivating pradikinin, which is a hypotensive substance in the living body, and is strongly involved in the pressor system. Hitherto, it has been considered that inhibiting the activity of an angiotensin converting enzyme acts on blood pressure lowering, and is clinically effective for preventing and treating hypertension.

[0003] Recently, since captopril, a proline derivative, was synthesized and its antihypertensive activity was confirmed, studies on the synthesis of various angiotensin converting enzyme inhibitors have been actively conducted, and attempts are being made to obtain them from natural products. . This is because a natural product-derived angiotensin converting enzyme inhibitor is expected to be a low-toxicity and highly safe antihypertensive agent because it is obtained from foods or food raw materials.

[0004]

However, angiotensin converting enzyme inhibitors found in natural products are extremely rare, such as teprotide (nonapeptide, SQ20881) obtained from Brazilian or Japanese snake venom, and Streptomyces. Metabolite IS of actinomycetes belonging to the genus
No. 83 (JP-A-58-177920) is only known. Further, as an angiotensin converting enzyme inhibitor obtained by enzymatic treatment of a natural product, peptides and the like obtained by decomposing milk casein with trypsin are known (JP-A-58-109425, JP-A-58-109425). -4
No. 4323, No. 59-44324, No. 61-3622
No. 6, No. 61-36227) The development of new inhibitors is being demanded.

[0005]

Means for Solving the Problems The present inventors have intensively searched for an angiotensin converting enzyme inhibitor which is a natural substance and has few side effects in order to solve such a problem. Then, it was found that a substance having angiotensin converting enzyme inhibitory activity was produced, and the substance was identified as Leu-Lys-
The inventors have found that the peptide is Pro and completed the present invention.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The method of the present invention will be described below in detail. In the present invention, the protein is hydrolyzed with thermolysin. The treatment method differs depending on the properties of the protein, and when the protein is hardly soluble, the protein is mixed with hot water and homogenized with vigorous stirring to obtain a predetermined amount of the protein. -Add molysin and hydrolyze at a temperature of about 10 to 85 ° C for 0.1 to 48 hours. As such proteins, those derived from animals or microorganisms are arbitrarily used, and particularly useful ones are fish such as skipjack and sardines. Thermolysin is an endopeptidase that cleaves peptide bonds containing amino acid residues having large hydrophobic side chains such as leucine, isoleucine, valine, phenylalanine, and methionine.
The production temperature (enzymatic decomposition temperature) can be raised, and it has advantages such as being not affected by microbial contamination.

[0007] Leu-Lys-Pro is contained in the hydrolysis solution.
And other peptides other than the peptide exist. However, in practical use, the composition (mixture) may be used as it is, or may be used after purification (isolation) if necessary. You. Leu mentioned above means leucine, Lys means lysine, and Pro means proline, and all such amino acids are in L-form. In the case of purification (isolation), the hydrolyzate is filtered by a known operation such as centrifugation. Extraction, concentration, after applying dryness or the like, or without, as it is, the difference in adsorption affinity for various adsorbents, the difference in solubility or solubility in various solvents,
Purification of the target product by applying means utilizing the difference in distribution between two immiscible liquid phases, the difference in elution rate based on the size of the molecule, the difference in the precipitability from the solution or the difference in the deposition rate Is preferred. These methods may be used alone as needed, or combined in any order and applied repeatedly.

[0008] The administration route of the peptide produced by the method of the present invention may be any of oral administration, parenteral administration and rectal administration, but oral administration is preferred. The dosage of such a peptide varies depending on the type of the compound, the administration method, the symptoms and age of the patient, etc., but is usually 0.001 to 1000 m / time.
g, preferably 0.01 to 10 mg, 1 to 3 per day
Times. Such a peptide is usually administered in the form of a preparation prepared by mixing with a preparation carrier. As the pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the peptide is used. Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium metasilicate aluminate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose calcium, ion exchange resin, methylcellulose , Gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol,
Light silicic anhydride, magnesium stearate, talc,
Tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin,
Examples include glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water and the like. Dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments,
Creams, gels, patches, inhalants, injections and the like can be mentioned. These preparations are prepared according to a conventional method. still,
When used, the liquid preparation may be in the form of a solution or suspension in water or another suitable medium. Tablets and granules may be coated by a known method. In the case of an injection, the peptide is prepared by dissolving the peptide in water. If necessary, the peptide may be dissolved in a physiological saline or glucose solution, or a buffer or a preservative may be added.

These preparations contain the peptide produced by the method of the present invention in an amount of 0.01% by weight or more, preferably 0.5 to 70%.
% By weight. These formulations may also contain other therapeutically valuable components.

[0010]

Now, the present invention will be described more specifically below with reference to working examples. Example 1 (A) 40 ml of water was added to 5 g of skipjack and homogenized sufficiently, boiled at 100 ° C. for 10 minutes, and allowed to stand. 20 mg of thermolysin was added, and a hydrolysis reaction was carried out at 37 ° C. and pH 7 for 3 hours. After cooling, the solution was centrifuged, concentrated, and purified by high performance liquid chromatography (ODS-, ph- and CN-column) to obtain a peptide. The amino acid sequence of this product was analyzed by an automatic Edman degradation method using a gas-phase protein sequencer (type 477A, manufactured by Abride Biosystems) to obtain the following structure.

H-Leu-Lys-Pro-OH The physical properties of the peptide are as follows. TLC [n-butanol: acetic acid: pyridine: water = 15:
3:10:12] (silica gel plate, ninhydrin coloring) Rf: 0.31 m. p: 101.2 ° C Specific rotation [α] _D (c = 0.5, 24 ° C., water); −6
3.4

The angiotensin converting enzyme inhibitory activity was measured using the obtained peptide in the following manner. The measurement was performed according to the method of Cheung and Cushman [Bioc.
chemicalPharmacology 20,1
637 (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) Enzyme: Rabbit lung acetone powder (Sigma)
(Supernatant obtained by crushing 1 g in 10 ml of 50 mM phosphate buffer and centrifuging the mixture) 100 μl of the above enzyme substrate, 12 μl of the enzyme solution and a peptide of a predetermined concentration of the present invention were mixed, and the whole was mixed with water. 25
After adjusting the volume to 0 μl, the reaction was carried out at 37 ° C. for 30 minutes.

The reaction was terminated using 250 μl of 1N HCl. 1.5 ml of ethyl acetate is added to the reaction completion solution,
Stirred at ortex for 15 seconds and centrifuged. 1.0 ml was taken out from the ethyl acetate layer, ethyl acetate was distilled off, 1 ml of distilled water was added to dissolve the residue, and the ultraviolet absorption value of the extracted hippuric acid at ₂₂₈ nm (OD ₂₂₈ )
Was measured. Inhibition rate is OD when reacted without inhibitor
₂₂₈ as 100%, and OD ₂₂₈ at 0 minute
The activity was indicated by the concentration IC ₅₀ (μM) of the inhibitor (peptide of the present invention) at an inhibition rate of 50%. Table 1 shows the results. In addition, as a reference example, an inhibitor other than the present invention was measured, and is shown in Table 1.

[0014]

[Table 1] Inhibitor inhibitory activity IC ₅₀ (μM) Example 1 Reference for Leu-Lys-Pro 1.7 Reference Asn-Met 850

[0015]

According to the production method of the present invention, a peptide useful as an angiotensin converting enzyme inhibitor can be obtained.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 9/99 A61K 37/64

Claims (3)

[Claims] 1. A method for producing a peptide, comprising hydrolyzing a protein with thermolysin to produce a peptide of Leu-Lys-Pro. 2. The method for producing a peptide according to claim 1, wherein fish meat is used as the protein. 3. The method for producing a peptide according to claim 1, wherein katsuobushi is used as the protein.