JPH05294844A - Angiotensin converting enzyme inhibitor - Google Patents

Abstract

PURPOSE:To obtain an angiotensin converting enzyme inhibitor useful for the prevention and treatment of hypertension and having high safety by using a peptide originated from a natural source as an active component. CONSTITUTION:The objective angiotensin converting enzyme inhibitor comprises a peptide of (A) Arg-Phe or (B) Ile-Try-Pro-Arg-Try as an active component. The peptide exists in rice protein and is free from side action. The peptides A and B can be separated and purified from cereals, meat and other natural materials or their decomposition products, e.g. a substance produced by brewing refined rice wine from rice using a liquefaction liquid and decomposing the obtained sake lees with a proteinase or the peptides can be produced by a peptide synthesis process utilizing the reverse reaction of hydrolase or a peptide synthesis process using a genetic engineering technique or a technique of organic chemistry. Since the peptide contains exclusively natural-type amino acids, it has extremely high safety to enable continuous peroral administration. Accordingly, it can be used as an additive for conventional food to obtain a functional food or health food.

Description

Detailed Description of the Invention

[0001]

The present invention relates to Arg-Phe or I.
The present invention relates to an angiotensin converting enzyme inhibitor containing le, Try-Pro-Arg-Try as an active ingredient.

Arg-Phe or Ile used in the present invention
-Try-Pro-Arg-Try is a peptide derived from a natural product and is expected to contribute to the prevention and treatment of hypertension.

[0003]

2. Description of the Related Art Hypertension is said to be the largest disease of modern adult diseases, and its treatment or prevention has become an urgent and serious problem. Hypertension is classified into secondary hypertension (symptomatic hypertension) which is etiologically the cause of elevated blood pressure and primary hypertension of unknown origin (essential hypertension). Among the factors that are considered to be the causes of hypertension, endocrine hypertension and the latter essential hypertension.
The angiotensin system is considered to be an important factor. Angiotensin converting enzyme (EC3.4.5.1) plays a central role in this renin-angiotensin system. That is, angiotensin nogen secreted in the liver becomes angiotensin I due to renin existing in the kidney, and angiotensin II having a vascular wall smooth muscle contracting action is produced by angiotensin converting enzyme to increase blood pressure. Further, since angiotensin II has an action of promoting aldosterone production in the adrenal cortical globular layer, blood pressure is further increased. On the other hand, in the antihypertensive system, kininogen is decomposed by kallikrein present in blood to produce bradykinin having a vascular wall smooth muscle expanding action, which is decomposed and inactivated by angiotensin converting enzyme. Therefore, inhibition of this angiotensin converting enzyme suppresses the production of angiotensin II, which is a pressor peptide, and also suppresses the decomposition of prazikinin, which is a hypotensive peptide, to lower blood pressure.

Conventionally, a large number of synthetic substances such as captopril (D-2-methyl-3-mercaptopropanoyl-L-proline) have been put into practical use as pharmaceuticals as inhibitors of angiotensin converting enzyme. Further, various angiotensin inhibitors have been found in natural products, and their use as antihypertensive agents has been investigated.

However, a novel and safer angiotensin converting enzyme inhibitory peptide is required for the prevention and treatment of hypertension.

[0006]

Means for Solving the Problems As a result of diligent search for an angiotensin inhibitor having no side effects in natural substances, the present inventors have found that this inhibitor is present in rice proteins, and the substance is identified as Arg-Phe. Ile-Try-Pro-Arg-
The present invention was completed by finding out that the peptide has a structure of Try.

The two peptides used in the present invention are isolated and purified from natural products such as cereals, meats and the like, or degradation products thereof, and a peptide synthesis method utilizing the reverse reaction of hydrolase,
It can be produced by a peptide synthesis method using a genetic engineering method or an organic chemical method.

Separation and purification from natural products is carried out in the form or if necessary, decomposed with an acid, an alkali, or an enzyme, and if necessary, extracted and concentrated, and then the difference in adsorption affinity for various adsorbents. , Difference in solubility in various solvents,
A method usually used for separation and purification such as a difference in elution rate due to the molecular sieve effect, or a combination thereof may be appropriately used.

For example, in order to separate and purify the present peptide from rice protein, sake lees obtained when sake is brewed from rice by a liquefied liquid sake brewing method (Reference 1) is used as a protein source, and it is decomposed with a protease. Concentrated and using angiotensin inhibitory activity as an index, various chromatographic methods such as SP-
Sephadex C-25 (Pharmacia), SP-
Ion exchange chromatography with Toyopearl 650S (manufactured by Tosoh Corporation), Sephadex G-15 (manufactured by Pharmacia), gel filtration using Toyopearl HW-40 (manufactured by Tosoh Corporation), CAPCELL PACK18 (manufactured by Shiseido), Cosmosil C18 (manufactured by Nacalai Tesque) Made)
It can be carried out by high performance liquid chromatography such as.

The peptide thus obtained has an angiotensin converting enzyme inhibitory ability and is considered to be effective for the prevention and treatment of hypertension in mammals including humans.
For these purposes, the peptide may be administered parenterally or orally, and can be formulated into a form suitable for the administration method. In the form of injection, the peptide is a pharmaceutical adjuvant (pH regulator, isotonic agent, preservative, etc.)
It may be aseptically dissolved together. The orally-administered agent may be in the form of a drug (tablets, capsules, granules, etc.) together with pharmaceutical auxiliaries. Since this peptide contains only natural amino acids, it is extremely safe and can be continuously taken orally.Therefore, it can be included in existing foods to prevent hypertension,
Alternatively, it may be a functional food having a therapeutic function or a health food.

[References]

 1. Imanasu et al .: Farming. 63.971 (1989)

[0011]

【Example】

Example 1 Preparation of peptides and measurement of angiotensin converting enzyme inhibitory activity

[Arg-Phe and Ile-Try-Pro-Arg-
Isolation of Try] 100 g of sake lees (water content 36%) when brewing sake from rice by the brewing method using liquefied liquid
Suspended in water of Yamato Kasei Co., Ltd. proteolytic enzyme (Samoase)
After adding 2 g and reacting at 37 ° C. for 1 hour, the reaction was stopped by heating for 10 minutes in a boiling water bath. A lysate was obtained by centrifugation at 5,000 rpm for 10 minutes, and the lysate was lyophilized to decompose 5
I got g. 50 mM acetate buffer (pH 4.0) 1
1 and then equilibrated with the same buffer, added to SP-Sephadex C-25 (6 * 30 cm, flow rate 500 ml / hr) and eluted with a linear concentration gradient of O-0.3 M NaCl,
The angiotensin converting enzyme inhibitory fraction was collected. The collected active fraction was freeze-dried, and then CAPCELLPAKC18.
It fractionated by HPLC using (made by Shiseido). (HP
LC conditions: flow rate 5 ml / min, 5% acetonitrile in 10 mM (NH ₄ ) ₂ CO ₃ pH 9.0 to 35% acetonitrile in 10 mM (NH ₄ ) ₂ C
(O ₃ pH 9.0 linear concentration gradient) The recovered active fraction was lyophilized, and then each peptide was isolated by HPLC using Cosmosil C18 (manufactured by Nacalai Tesque). (HPLC conditions: flow rate 3 ml / min, 10%
A linear concentration gradient from acetonitrile in 0.1% TFA to 60% acetonitrile in 0.1% TFA)

Sequence analysis of the recovered peptide was carried out using a protein sequencer PSQ-1 manufactured by Shimadzu Corporation. Arg-Phe and I1e-Try-Pro-Arg were analyzed.
It was found that the sequence was -Try.

[0013]

[Method for measuring angiotensin converting enzyme inhibitory activity] 0.06 ml of each peptide solution was added to a test tube, and a hypryl histidyl leucine solution (final concentration 5 mM, N
aCl 385 mM, borate buffer pH 8.3 77 m
0.3 ml of M), and then 0.03 m of angiotensin converting enzyme solution (enzyme concentration 100 mU) manufactured by Sigma.
Then, the mixture was reacted at 37 ° C. for 30 minutes. After that, 0.5 ml of 1N hydrochloric acid was added to stop the reaction, 1.5 ml of ethyl acetate was added to extract the produced hypuric acid, and
The absorbance at 8 nm was measured and used as the enzyme activity. The inhibition rate was calculated by the following formula.

[0014]

[Formula 1]

The inhibitor concentration IC ₅₀ when the inhibition rate was 50% was as follows. IC ₅₀ (μM) Arg-Phe 90 Ile-Try-Pro-Arg-Try 9

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication C07K 7/06 ZNA Z 8318-4H C07K 99:00

Claims (2)

[Claims] 1. An angiotensin converting enzyme inhibitor containing Arg-Phe as an active ingredient. 2. Ile-Try-Pro-Arg-Tr
An angiotensin converting enzyme inhibitor containing y as an active ingredient.