JP3118236B2 - Method for producing angiotensin I converting enzyme inhibitor - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD TO WHICH THEINVENTION BELONGS TO: The present invention relates to a novel peptide, an angiotensin I converting enzyme inhibitor containing the peptide as an active ingredient, and a method for producing the same, which will play an extremely important role not only in the technical fields of biochemistry and protein chemistry, but also in the technical fields of antihypertensive agents and other medical treatments.

[0002]

2. Description of the Related Art Angiotensin I-converting enzyme (hereinafter referred to as AC
ACE (abbreviated as ACE) catalyzes the reaction of converting angiotensin I to angiotensin II, a strong hypertensive peptide, and inactivating bradykinin, a hypotensive peptide. Therefore, substances that specifically inhibit ACE have the effect of suppressing blood pressure rise, and from this perspective, development of antihypertensive agents is underway. On the other hand, in the food industry,
Research and development is currently being conducted to find peptides with ACE inhibitory activity in proteins from various biological resources, and to utilize these as food to help prevent hypertension.
There have already been many reports of novel peptides isolated and extracted from hydrolysates of soybean proteins, hydrolysates of casein, hydrolysates of fish proteins, etc., but the reality is that none of these have been fully put to practical use.

[0003]

[Problems to be Solved by the Invention] In recent years, research has been conducted into functional substances, focusing on the effects of components of various biological resources on the physiological functions of the human body. While there are still many unknown aspects, the discovery of new peptides in the technical field of the present invention, and the development of ACE inhibitors and foods that use these as active ingredients, etc., have attracted attention. In particular, in the fields of medicine and food, there has been a recent trend toward avoiding chemically synthesized products due to safety concerns and consumer sentiment, and the use of active ingredients from natural resources, which have long been a part of our dietary experience, has become an important issue. The object of the present invention is to provide a method for producing an ACE inhibitor that uses a novel peptide derived from food as an active ingredient.

[0004]

Summary of the Invention The present invention relates to a method for producing an ACE inhibitor, comprising the steps of:
The actin protein is hydrolyzed to produce a peptide having an amino acid sequence represented by the following formula (Chemical formula 1): Leu-Lys-Leu (Chemical formula 1).
The present invention is characterized by obtaining an ACE inhibitor having

[0005] With regard to ACE inhibitor peptides derived from various biological resources, there was a need for research into substances and extraction methods that were more suited to practical application, as well as food development. The present invention was completed as a result of extensive research conducted in response to the problems faced by the prior art.

[0006]

[Mode for Carrying Out the Invention] The present invention will now be described in detail. The novel peptides in the present invention were discovered by using protease enzymes with sardines as the raw material, and there is no problem with the safety of the novel peptides. For this reason, peptides consisting of this amino acid sequence can be safely administered to the human body, even if they are obtained from other biological resources or are produced by synthetic techniques. That is, in the present invention, sardine paste is used as the biological resource, and this is converted into peptides using commercially available protease enzymes. The AC of these peptides
The E inhibitory activity was determined according to the method of Cushman and Chung (
By studying the peptide fractions according to the method of An & Cheung, we were able to obtain peptide fractions with excellent ACE inhibitory activity. By further using various chromatographic techniques, we were able to find novel peptides that exhibit ACE inhibitory activity. In addition, by similarly hydrolyzing actin-based proteins, which are widely and abundantly present in nature, we were able to find novel peptides that contain at least one of the above three types of peptides that exhibit ACE inhibitory activity.

[0007] This novel peptide having excellent ACE inhibitory activity can be used in medicine as an antihypertensive and/or vasodilator, and can also be used in a variety of foods, such as health foods and functional foods, aimed at preventing hypertension.

Next, the present invention will be specifically explained with reference to experimental examples. (ACE inhibitory activity measurement) 50 μl of a sample was placed in a test tube.
100 μl of ACE (Sigma, 2.5 mM) solution was added to the mixture, and the mixture was incubated at 37° C. for 5 minutes.
0 μl of Bz-Gly-L-His-L-Leu (Peptide Institute, final concentration 5 mM, containing 400 mM NaCl) was added and reacted at 37° C. for 60 minutes. Then, 0.25 ml of 0.5N hydrochloric acid was added to stop the reaction, and 1.5 ml of ethyl acetate was added and vigorously stirred for 15 seconds. Then, the mixture was centrifuged at 3000 rpm for 10 minutes, and the ethyl acetate layer was heated at 140° C. for 20 minutes to remove the solvent. After removing the solvent, the mixture was dissolved in 3 ml of 1M NaCl aqueous solution, and the absorption (absorbance at 228 nm) of the extracted hippuric acid was measured, which was taken as the enzyme activity. Inhibition rate = [(A-B)/A] x 100 (%) A: Absorbance at 228 nm when no inhibitor was added B: Absorbance at 228 nm when inhibitor was added The sample concentration at which the inhibition rate was 50% was taken as _IC50 .

(Experimental Example) After adding water to sardine paste and crushing it if necessary, it is hydrolyzed using a protease. It would be even better if some method could be used to improve the mixing of the raw material and the enzyme. After hydrolysis under suitable conditions, the mixture is heated to inactivate the enzyme activity, and separated into a hydrolyzed liquid and a residue by centrifugation or other filtration methods. This hydrolyzed liquid is passed through a column packed with ODS resin. Next, the peptide fractions are fractionated in sequence using water and ethanol solutions of different concentrations, and the peptide portion with high ACE inhibitory activity is obtained by separating them.

The method for obtaining this peptide portion is characterized by the fact that the raw materials used are sardine paste, proteolytic enzymes, and ethanol, all of which are substances that have long been used as food, and by using ODS resin as the decomposition method, fractionation is mainly based on peptide chain length, rather than the collection of special fragments using a special solvent. In other words, the peptide portion can be given to humans as it is, without any problems, as food or as a food ingredient.

From this peptide portion, a peptide fragment is purified by a conventional method such as ion exchange chromatography, gel filtration chromatography, or reverse phase partition chromatography.
After obtaining the peptide fragments, their structures are determined using an amino acid sequencer and confirmed by an amino acid analyzer.

The peptide fragment thus obtained has a completely novel structure, and there is no evidence that it was previously known. In addition, it exhibits excellent ACE inhibitory activity, and can be used as a distinctive raw material in the above-mentioned application fields.

[0013]

EXAMPLES The present invention will now be described in more detail with reference to the following examples, but the present invention is not limited to these examples in any way.

Example 1 500 ml of water was added to 50 g of sardine paste, and the mixture was homogenized using a homogenizer/Polytron (Kinematica, Switzerland).
The homogenate was then homogenized for 2 minutes in 100% ethanol (Ica-mbH). The homogenate was incubated at 98°C for 15 minutes and the pH was adjusted to 3. 500 mg of pepsin (Boehringer Mannheim) was added and homogenized further with a Polytron for 1 minute. After hydrolysis for 5 hours at 40°C with shaking, the pH was adjusted to 7 and the enzyme was inactivated by heating at 98°C for 15 minutes to terminate the hydrolysis. The hydrolyzate was then diluted with 100% ethanol (Ica-mbH).
_The mixture was centrifuged at 1,000 rpm for 15 minutes, and the supernatant was filtered through a filter paper to obtain 500 ml of an enzymatic hydrolysis solution.
0) was passed through a column (3.5 cm x 14 cm),
500 ml of water (F-1) and 10% ethanol solution (F-
2), 25% ethanol solution (F-3), 50% ethanol solution (F-4), 99.5% ethanol solution (F-
5) Each fraction was collected using 500 ml of the solution. As shown in Table 1, each fraction had high ACE inhibitory activity.
The titer is significantly higher than before fractionation.

[0015]

Table 1

The novel peptides discovered by the present invention are
The 25% ethanol (F-3) elution fraction was concentrated and then purified by SP-MS.
-Sephadex C-25 (H ⁺ type) column (1.5
The fractions were then passed through a Toyopearl HW-40 column (1.5 cm x 10 cm) and eluted with a stepwise gradient of ammonium formate, and four major fractions with ACE inhibitory activity were collected. Each fraction was then passed through a Toyopearl HW-40 column (1.5 cm x 10 cm) and eluted with 40% methanol.
The active fraction was desalted by chromatography (O
The peptide fragments were further separated by DS reverse phase liquid chromatography. The ACE inhibitory activity of each separated peptide fragment was confirmed, and the structure was analyzed using an amino acid sequencer (ABI 477A type) to determine the amino acid sequence.
The novel peptide according to the present invention was obtained from fraction B of SP-Sephadex C-25 column chromatography (Figure 1). The ACE inhibition rate of each fraction is shown in Figure 1. The O2 concentration of fraction B after desalting is also shown in Figure 1.
The results of DS reverse phase liquid chromatography and the ACE inhibition rate are shown in Figures 2 and 3, respectively. In Figure 1, the horizontal axis indicates the fraction number (11 ml each) and the vertical axis indicates the fraction number (280 ml).
2, the horizontal axis indicates time (min), and the vertical axis indicates absorbance at ₂₂₀ nm _{( A220} , solid line) _{and CH3CN} (%, dotted line).
% indicates the change in concentration of acetonitrile used as the developing solvent. In FIG. 3, the horizontal axis indicates time (min), and the vertical axis indicates ACE
The inhibition rate (%) is shown.

The B-1, B-2, and B-3 fractions were purified again by ODS column chromatography, and the primary structures of each were determined.
A-1 is sequence number 1 in the sequence listing, B-2 is sequence number 2 in the sequence listing,
It was confirmed that B-3 has a structure represented by the formula (Chemical Formula 1). These peptides are novel peptides. The amino acid composition and ACE inhibitory activity of each peptide are shown in Table 2 below.

[0018]

Table 2

From Table 2, it is clear that the novel peptide obtained in the present invention exhibits excellent ACE inhibitory activity.
The peptide represented by the formula (Chemical Formula 1) of B-3 in Table 2 was prepared by the inventors of the present invention in a patent application filed in Japanese Patent Application Laid-Open No. 3-1109.
One of the peptides No. 7 is represented by the following formula (Chemical Formula 2): Asp-Lys-Gly-His-Leu-Lys-L
The peptide represented by formula (2) contains the amino acid sequence eu-Phe, and the _IC50 of the peptide represented by formula (2) is 109.
(μM), and it was revealed that the peptide represented by formula (Chemical Formula 1) had a higher ACE inhibitory activity.

Example 2 (Reference Example) Commercially available actin (Sigma, derived from chicken muscle) 100
To each of the 100 mg of peptide, 1 ml of water was added, and hydrolysis and fractionation were carried out in the same manner as in Example 1. The primary structure of the novel peptide thus obtained is the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, and its IC
₅₀ showed excellent ACE inhibitory activity at 83 μM.

[0021]

Effect of the Invention The novel peptide obtained by the present invention has excellent ACE inhibitory activity and is a highly safe substance obtained from sardines .
The invention relates to a method for producing the ACE inhibitor and the resulting ACE inhibitor.
The antihypertensive agent can be used as a blood pressure lowering agent and/or a vasodilator in various foods and medicines.

[0022]

[Sequence List]

SEQ ID NO: 1 Length of sequence: 5 Type of sequence: amino acids Number of chains: single chain Topology: linear Type of sequence: peptide

SEQ ID NO: 2 Length of sequence: 6 Type of sequence: amino acids Number of chains: single chain Topology: linear Type of sequence: peptide

[Brief description of the drawings]

FIG. 1 shows the enzymatic hydrolysis product of sardine surimi according to the present invention.
This is a graph showing the chromatography results when the F-3 fraction fractionated by resin treatment was further fractionated on an SP-Sephadex C-25 (H ⁺ type) column, and the ACE inhibition rates thereof.

FIG. 2 is a graph showing the results of ODS reversed-phase liquid chromatography after desalting of fraction B when fraction F-3 was refractionated, and the change in acetonitrile concentration.

FIG. 3 is a graph showing the ACE inhibition rate of fractions after desalting of fraction B.

───────────────────────────────────────────────────────── Continued from the front page (51) Int.Cl. ⁷ identification symbol FI C07K 5/08 C12N 9/99 C12N 9/99 A61K 37/64 // C07K 123:00 (56) References JP 3-81291 (JP, A) (58) Field surveyed (Int.Cl. ⁷ , DB name) C12P 21/02 - 21/06 C07K 5/08 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] Claim 1: By hydrolyzing actin-based proteins,
The peptide has an amino acid sequence represented by the following formula (Chemical Formula 1): Leu-Lys-Leu (Chemical Formula 1)
The present invention is directed to obtaining an angiotensin I converting enzyme inhibitor.
Method for producing angiotensin I converting enzyme inhibitors
Law.