JPH04304896A - Production of composition containing angiotensin converting enzyme inhibitor - Google Patents

Abstract

PURPOSE:To produce a composition containing angiotensin converting enzyme inhibitor useful as a hypotensor or a hypotensive food from a natural material. CONSTITUTION:Protein is hydrolyzed with at least one kind of enzyme selected from neutral protease produced by Bacillus subtilis, acid protease produced by Aspergillus niger and acid protease produced by Rhizopus delemar.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention can be prepared from natural products,
The present invention relates to a method for producing an angiotensin-converting enzyme inhibitor-containing composition that is particularly useful as a blood pressure-lowering agent or a blood pressure-lowering food.

0002

[Prior Art] Angiotensin-converting enzyme is mainly present in the lungs, vascular endothelial cells, and renal proximal tubules.
The dipeptide (His-Pro-Phe-His-Leu) is released from the C-terminus of angiotensin I.
-Leu) to produce angiotensin II, which has a strong pressor effect.

[0003] This enzyme also has the effect of decomposing and inactivating bradykinin, which is a hypotensive substance in the body, and is strongly involved in the pressor system. It has been conventionally believed that inhibiting the activity of angiotensin converting enzyme lowers blood pressure and is clinically effective in preventing and treating hypertension.

Recently, captopril, a proline derivative, was synthesized and its antihypertensive activity was confirmed. Since then, research has been active in the synthesis of various angiotensin-converting enzyme inhibitors, and efforts are also being made to obtain them from natural products. . This is because angiotensin-converting enzyme inhibitors derived from natural products can be obtained from foods or food materials, and are therefore expected to be low-toxic and highly safe antihypertensive agents.

[0005]

[Problems to be Solved by the Invention] However, strong angiotensin converting enzyme inhibitors found in natural products are extremely rare, and only teprotide (nonapeptide, SQ20881) obtained from Brazilian and Japanese snake venoms is found in natural products.
), and the metabolite IS83 of actinomycetes belonging to the genus Streptomyces (Japanese Unexamined Patent Publication No. 177920/1983) are only known. In addition, as angiotensin-converting enzyme inhibitors obtained by enzymatically treating natural products, peptides obtained by decomposing milk casein with tryptocin are known (Japanese Patent Laid-Open Nos. 58-109425, 5
No. 9-44323, No. 59-44324, No. 61-3
No. 6226, No. 61-36227) The development of new inhibitors is desired.

[0006]

[Means for Solving the Problems] In order to solve the problems, the present inventors have diligently searched for angiotensin-converting enzyme inhibitors that are natural substances and have few side effects.As a result, the present inventors have found that proteins, especially muscle proteins, bonito or bonito-derived substances, Fish, pork, beef or chicken meat is treated with Bacillus subtilis.
Neutral protease produced by Aspergillus subtilis, Aspergillus niger
Acidic protease produced by Rhizopus niger and Rhizopus delemer
The present inventors have completed the present invention by discovering that peptides having angiotensin converting enzyme inhibitory activity are present in a composition hydrolyzed by at least one enzyme selected from acidic proteases produced by E. mar).

Compositions having the activity of the present invention can be obtained particularly effectively when using enzymes produced by the above-mentioned specific microorganisms, and even if other known enzymes are used to decompose proteins, they will not be as potent as those of the present invention. No active composition is obtained. Commercially available enzymes that can be used in the present invention include the following. Bacillus subtilis producing enzyme; aloase AP-1
0 (manufactured by Yakult)
Orientase 90N (manufactured by Ueda Chemical)
Protin PC10F (manufactured by Daiwa Kasei) Aspergillus niger production enzyme; protease Y
P-SS (manufactured by Yakult)
Protin FA (
Made by Daiwa Kasei)
Morsin (manufactured by Seishin Pharmaceutical)

Denapsin (manufactured by Nagase Sangyo) Rhizopus deremer-producing enzyme; XP-415 (manufactured by Nagase Sangyo)

000
8] Proteins derived from animals or microorganisms can be arbitrarily used, but particularly useful ones are muscle protein, bonito or bonito-derived substances, fish meat such as sardines, pork, beef, and chicken.

[0009] In order to hydrolyze a protein with the enzyme of the present invention, the treatment method differs depending on the properties of the protein, but if the protein is poorly soluble, the protein is mixed with hot water and homogenized with strong stirring, and then the enzyme is added to the protein. 0.05-10% by weight based on the solution
Preferably 0.1 to 2% by weight is added and the temperature is 10 to 60°C.
Preferably, under the reaction conditions of 20 to 40°C and reaction time of 10 minutes to 3 days, the peptide bond of the hydrophobic amino acid has a decomposition rate of 5.
% or more, the reaction is continued under standing or stirring until the desired product is obtained. The pH at this time is 4-9, preferably 6-8 when using neutral protease, and 0.1-4, preferably 0.5-3 when using acidic protease.

[0010] The decomposition rate is the percentage of amiso nitrogen to total nitrogen.
Expressed as However, Journal of Agriculture
tural and Food Chemist
ry 24 No. 6 1090-1093 (19
76). The angiotensin-converting enzyme inhibitor-containing composition thus obtained is a mixture of various peptides, and may be used as is or after post-treatment. The administration route for the peptides obtained in the present invention may be oral administration, parenteral administration, or intrarectal administration, but oral administration is preferred. The dosage of the peptides of the present invention varies depending on the type of compound, administration method, patient's symptoms, age, etc., but is usually 0.001 to 100 mg/dose.
0mg, preferably 0.01-10mg once per day
~3 times.

The peptides of the present invention are usually administered in the form of a preparation prepared by mixing it with a pharmaceutical carrier. As a pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the peptides of the present invention is used. Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, 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 silicic anhydride, magnesium stearate, talc, tragacanth, bentonite, vegum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, Examples include fatty acid glycerin ester, purified lanolin, 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, injections, and the like. These formulations are prepared according to conventional methods. In the case of a liquid preparation, it may be dissolved or suspended in water or other suitable medium before use. Furthermore, tablets and granules may be coated by a well-known method. In the case of injections, the peptides of the present invention are prepared by dissolving them in water, but if necessary, they may be dissolved in physiological saline or glucose solution, and buffers and preservatives may be added. Good too.

[0012] These preparations contain 0 of the peptides of the present invention.
．． It can be contained in a proportion of 0.01% or more, preferably 0.5 to 70%. These formulations may also contain other ingredients of therapeutic value.

[0013]

[Operation] The present invention can produce an angiotensin-converting enzyme inhibitor-containing composition that can be prepared from natural products and is particularly useful as a hypotensive agent or a hypotensive food.

[0014]

[Examples] The present invention will be explained in more detail below with reference to Examples. Examples 1 to 8 Add 40 ml of water to 5 g of bonito flakes and homogenize thoroughly.
After acting with the protease shown in Table 1, hydrolysis was performed and the reaction solution was centrifuged, and the supernatant liquid was concentrated, and the angiotensin-converting enzyme inhibitory activity was measured for the composition.

(Protease action conditions) When neutral protease is used, the reaction solution is purified with sodium hydroxide.
The mixture was reacted at 37° C. for 13 hours at H7.0, and then boiled for 10 minutes. When using acidic protease, use hydrochloric acid to
As H2.5, the reaction was carried out at a reaction temperature of 37° C. for 3 hours, and then boiled for 10 minutes. The enzyme amount is all 1 for bonito flakes
/100 parts by weight was added.

(Measurement of angiotensin converting enzyme inhibitory activity) The measurement of angiotensin converting enzyme inhibitory activity is carried out by Cheung and C.
ushman's method [Biochemical Ph
aramacology 20, 1637 (1971
)] using the following method. Enzyme substrate; Bz (benzyl)-Gly-His-L
eu (a solution of 86 mg dissolved in 8 ml of water and 8 ml of phosphate buffer) Enzyme; Rabbit lung acetone powder (manufactured by Sigma)
(Supernatant after pulverizing 1g in 10ml of 50mM phosphate buffer and centrifuging) 100μl of the above enzyme substrate and 12μl of the enzyme solution.
and the peptide of the present invention at a predetermined concentration were mixed, the total volume was made up to 250 μl with water, and 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 was added to the reaction completed solution, stirred with Vortex for 15 seconds, and centrifuged. Take 1.0 ml from the ethyl acetate layer, distill off the ethyl acetate, add 1 ml of distilled water to it, and dissolve the residue.
The value of ultraviolet absorption of extracted hippuric acid at 228 nm (OD22
8) was measured.

[0018] The inhibition rate is the OD when reacting without an inhibitor.
OD22 when 228 is 100% and reaction time is 0 minutes
8 as 0%, and the activity was expressed as the concentration IC50 (mg/ml) of the inhibitor (peptide composition of the present invention) when the inhibition rate was 50%. The results are shown in Table 1.

[0019]

[Table 1]

[0020]

According to the present invention, an angiotensin-converting enzyme inhibitor-containing composition useful as a hypotensive agent or a hypotensive food can be produced by decomposing proteins using an enzyme produced by a specific microorganism.

Claims (4)

[Claims] Claim 1: The protein is at least one selected from neutral protease produced by Bacillus subtilis, acidic protease produced by Aspergillus niger, and acidic protease produced by Rhizopus deremer.
A method for producing an angiotensin-converting enzyme inhibitor-containing composition, which is hydrolyzed with a seed enzyme. [Claim 2] The production method according to claim 1, wherein bonito or a bonito-derived substance is used as the protein. [Claim 3] Claim 1 in which fish meat is used as the protein.
Manufacturing method described [Claim 4] The production method according to Claim 1, wherein pork, beef, or chicken is used as the protein.