JPH06179695A - Angiotensin i converting enzyme inhibitory tripeptide, its production and food containing the same tripeptide - Google Patents

Abstract

PURPOSE:To provide angiotensin I converting enzyme inhibitory peptides reduced in side-effect, their suitable production method and their use. CONSTITUTION:Angiotensin I converting enzyme inhibitory peptides respectively having a structure of Leu-Lys-Phe, Ile-Arg-Trp or Phe-Lys-Pro. These peptides can be applicable to various kinds of foods or medicines. Since Leu-Lys-Phe, Ile-Arg-Trp and Phe-Lys-Pro exhibit an angiotensin I converting enzyme inhibitory effect, the blood pressure can be reduced.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an angiotensin I converting enzyme inhibitory peptide and a method for producing and using the same.

[0002]

2. Description of the Related Art Hypertension has a maximum blood pressure of 160 mmHg.
The above is the minimum blood pressure of 95 mmHg or higher, or both of them. In Japan, the number of patients is said to be about 20 million, which is a highly prevalent disease. Hypertension is known to cause various complications over a wide range of organs such as cerebral hemorrhage, cerebral infarction, subarachnoid hemorrhage, angina, myocardial infarction, renal sclerosis, renal failure, and retinal vein occlusion, Effective therapeutic agents are desired. As one of the mechanisms that regulates blood pressure in vivo, the pressor system renin-angiotensin system and the antihypertensive system kallikrein-
There is a kinin system. In the renin-angiotensin system, the enzyme renin is produced in renal juxtaglomerular cells (JG cells) and acts on angiotensinogen, which is a renin substrate in blood vessels, to produce angiotensin I. The enzyme that converts angiotensin I into angiotensin II is an angiotensin I converting enzyme, and the produced angiotensin I acts on arterioles to cause contraction.
Angiotensin II also acts on the adrenal cortex, promotes synthesis and secretion of aldosterone, promotes reabsorption of sodium in the kidney, and also has a function of maintaining body fluid volume. Thus, angiotensin II raises blood pressure.

On the other hand, in the kallikrein-quinine system, kallikrein, which is a proteolytic enzyme, acts on kininogen, which is a substrate, to produce quinine. Kinin has the function of dilating blood vessels and lowering blood pressure, but kininase I
Subject to decomposition by I. Kininase II is known to be the same substance as angiotensin I converting enzyme. From the above, it is considered possible to treat hypertension by inhibiting angiotensin I converting enzyme. Based on this idea, synthetic drugs such as captopril, enalapril, and alacepril are currently being developed.
It is also known that naturally derived peptides derived from casein, gelatin, fish meat, livestock blood and the like also have an action of inhibiting angiotensin I converting enzyme. (JP-A-59-44324, JP-A-64-5497, JP-A-01-313498)

[0004]

[Problems to be solved by the invention] Captopril is
It shows a strong antihypertensive effect when taken orally. However, improper usage results in renal dysfunction and hypotension. Further, it is said that the SH group existing in the molecule causes rash and taste abnormality. Peptides derived from casein, gelatin, fish meat, which are angiotensin I-converting enzyme inhibitors made from inexpensive natural products, are difficult to separate and purify, and the yields are poor. In the present invention, the present invention was completed by finding a peptide that inhibits angiotensin I converting enzyme, establishing a method for producing the peptide, and developing a food product to which the peptide is applied.

[0005]

The present invention relates to angiotensin I converting enzyme inhibitory peptides. This peptide is
Fmoc (9-fluorenylmethyloxycarbonyl)
-Amino acid and t-Boc (t-butoxycarbonyl)-
All known chemical synthesis methods such as solid-phase synthesis method using amino acids, peptide synthesis method using protease [Biochemistry Experiment Course 1, Protein Chemistry IV Part II Peptide Synthesis (P.205), edited by the Japanese Biochemical Society Tokyo Chemistry Doujin, Sequel Biochemistry Experiment Course 2 Under protein chemistry, Chapter 20, Peptide Synthesis (P.641), edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin]. In addition, a protein containing the peptide sequence is abundant in nature, and can be efficiently obtained by hydrolysis by a suitable method such as using a proteolytic enzyme.

[0006]

EXAMPLES The present invention will be described in detail below with reference to examples. (Example 1) Using a peptide synthesizer Peptide Synthesizer 430A manufactured by Applied Biosystems,
The peptides represented by Sequence Listing 1 to 3 were synthesized according to the operation manual of the same company. Peptide is extracted and treated with anion exchange resin column, then reverse phase HPL using ODS column
The peptide of the present invention was purified by C. As a result of measuring the amino acid composition, it was in good agreement with the value expected from the peptide sequence. [Measurement of Angiotensin I-Converting Enzyme Inhibitory Activity] The measurement of angiotensin I-converting enzyme inhibition is performed by the method of Kashman et al. (Biochemical Pharmacology 20 Vol. 1
According to the method of Maruyama et al. (Agricultural Biological Chemistry, Vol. 46, No. 5 (1393 to 1394 (1982)), which is a modification of pp. 637 to 1648 (1971)).

To the test tube, 30 μl of the peptide aqueous solution and 250 μl of a borate buffer of pH 8.3 containing L-Hipryl histidyl leucine (manufactured by Sigma) and NaCl as an enzyme substrate were added, and the mixture was pretreated at 37 ° C. for 10 minutes. Incubated. Then, 100 μl of angiotensin I converting enzyme-containing solution was added to start the enzymatic reaction. At this time, the concentration of borate buffer was 0.1 M, the concentration of L-hypril histidyl leucine was 5 mM, and the concentration of NaCl 300 was 300.
The enzyme activity is 8m in the case of mM and no inhibition.
U.

After the reaction was carried out while incubating at 37 ° C. and pH 8.3 for 30 minutes, 250 μl of 1N HCl was added to stop the reaction. In addition, as a blind test, 1N HCl was added before adding the angiotensin I converting enzyme-containing solution, and the same treatment was performed. 1.5 ml of ethyl acetate was added, and the mixture was shaken for 15 seconds to extract hippuric acid generated by the enzymatic reaction.
Centrifuge at 000 rpm for 10 minutes to obtain ethyl acetate layer 1.
0 ml was collected in a test tube. The ethyl acetate was heated in a hot dry bath at 120 ° C. for 30 minutes to completely remove it, and then left at room temperature for 5 minutes. Then, 1.0 ml of water was added, and the amount of hippuric acid produced was determined by measuring the absorbance at 228 nm. The angiotensin I-converting enzyme-containing solution used for the enzymatic reaction was 1 g of rabbit rung acetone powder (manufactured by Sigma) in 0.1 M borate buffer (pH 8.
3) Dissolve in 10 ml and stir well, then 4 ° C, 4000
Centrifuge at 0 g for 40 minutes. The supernatant was diluted with 0.1 M borate buffer (pH 8.3). The angiotensin I converting enzyme inhibitory activity was determined using the following formula 1.

[0009]

[Equation 1] A: Absorbance upon addition of distilled water (228 nm) B: Absorbance upon addition of inhibitor (228 nm) a, b: Blinded absorbance for each (228n)
m) In this way, the IC ₅₀ of the above peptides was 47.9 respectively.
μM (Sequence Listing 1), 1.6 μM (Sequence Listing 2), 27.5
μM (Sequence Listing 3).

(Example 2) Examples of food products of the present invention are shown below, but the use of the present peptide is not limited to the following examples. A peptide-containing hamburger was prepared as follows. 50 mg of one or more kinds of peptides were added per 60 g of hamburger having a composition specific gravity of beef 30, chicken 30, onions 25, bread crumbs 10, eggs 4, and starch 1 and mixed well. afterwards,
It was baked in an oven to prepare a peptide-containing hamburger.
The peptide content of this hamburger can be increased or decreased depending on the subject who eats the hamburger.

[0011]

As described above, according to the present invention,
A novel angiotensin I converting enzyme inhibitory peptide was synthesized and could be applied to food.

[0012]

[Sequence list]

SEQ ID NO: 1 Sequence length: 3 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 2 Sequence length: 3 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 3 Sequence length: 3 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

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Claims (3)

[Claims] 1. An angiotensin I converting enzyme inhibitory peptide having the following structure. Leu-Lys-Phe Ile-Arg-Trp Phe-Lys-Pro 2. A method for producing an angiotensin I-converting enzyme inhibitory peptide having the structure according to claim 1, wherein the peptide is synthesized by a chemical synthesis method. 3. A food containing at least one angiotensin I converting enzyme inhibitor peptide having the structure according to claim 1.