JPH0616568A - Angiotensin converting enzyme inhibitor - Google Patents

Abstract

PURPOSE:To provide a new excellent angiotensin converting enzyme inhibitor composed of a tripeptide having a regular amino acid sequence. CONSTITUTION:In case the C-terminal position of this inhibitor obtained by synthesis is an aromatic amino acid, especially Trp or Tyr, the angiotensin converting enzyme inhibitory activity is high. Further, in case the central amino acid is Ile or Val or in case the N-terminal amino acid is Ile or Cys, the activity is high. In addition, Ile-Ile-Tyr can be obtained also by enzymatic degradation of wheat protein.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an angiotensin converting enzyme (hereinafter referred to as ACE) inhibitor consisting of a tripeptide having a regular amino acid sequence.
The ACE inhibitor of the present invention can be used as a base for an antihypertensive drug or an antihypertensive drug.

[0002]

2. Description of the Related Art The usefulness of ACE inhibitors has been widely recognized as a type of therapeutic drug for hypertension and is used as a first-line drug. In addition, for the treatment or prevention of hypertension, ACE
It has been proposed to use peptides with inhibitory action.

As an example of a peptide having an ACE inhibitory action, a protein hydrolyzate of corn α-zein or gluten meal (Japanese Patent Laid-Open No. 240028) or an animal erythrocyte protein hydrolyzate (Japanese Patent Laid-Open No. 3-280028). 66626), and a peptide obtained from a hydrolyzate of krill protein (JP-A-3-81291).
Japanese patent publication) and peptides derived from sardines (Japanese Patent Laid-Open No. 3-6329).
No. 5), etc., and any of them can be used as an ACE inhibitor. In addition, there have been proposals for peptides having a short chain length obtained by a synthetic method, but there are few proposals, and the regularity of the amino acid sequence of the peptide is unknown.

[0004]

With regard to hypertension, new and useful antihypertensive agents are constantly being sought, and there are many peptide ACE inhibitors used for the purpose of preventing and suppressing hypertension through daily diet. Although proposed,
In addition, it is insufficient. Therefore, the present invention provides excellent ACE.
Proteins that have an inhibitory action and are commonly available and can be used for food, especially various peptides that are regularly correlated with a simple amino acid sequence having a structure that can be easily chemically synthesized from wheat-derived gluten The development of an ACE inhibitor consisting of

[0005]

The present invention relates to an ACE inhibitor comprising a tripeptide in which the C-terminal amino acid is an aromatic amino acid, particularly Trp or Tyr, and the ACE inhibitor according to the present invention is Since the amino acid sequence of the constituent tripeptides is selected from a specific amino acid group at each sequence position, it can be easily obtained by a peptide synthesis method, and Il
The tripeptide having an amino acid sequence represented by e-Ile-Tyr can also be extracted by enzymatically decomposing gluten of wheat protein.

The present invention will be described in detail below. The tripeptide in the present invention can be synthesized by a usual peptide synthesis method. In addition, a kind of tripeptide obtained by decomposing wheat protein with a proteolytic enzyme followed by a purification step is also included in this. The wheat protein used here may be any one, and may be commercially available gluten.

The proteolytic enzyme used for enzymatic degradation is
Pepsin as an acidic protease, PD enzyme (trade name:
Morishin Pharmaceutical Co., Ltd.), and chymotrypsin is preferable as the alkaline protease, and these proteases can be used alone or in combination, or a plurality of proteases can be sequentially acted to perform the treatment. Furthermore, even with other acidic proteases, alkaline proteases, thiol proteases, and metalloproteases, hydrolysis can be promoted by subjecting these to the same treatment as described above.

[0008] Next, the purification of the wheat protein-origin tripeptide of the present invention is carried out by gel filtration chromatography,
After removal of high molecular weight peptides by fractionation by molecular weight such as ultrafiltration or extraction with a water-soluble organic solvent such as ethanol, ion exchange chromatography, reverse phase chromatography, or hydrophobic chromatography is performed. This can be achieved by performing one or more of the above processes.

The tripeptide of the present invention can also be obtained by a peptide synthesis method using the constituent amino acids as raw materials. As the peptide synthesis method, either a liquid phase method or a solid phase method may be used, and a commercially available peptide synthesizer may also be used. Whatever method is used for peptide synthesis, the tripeptide can be purified and isolated by reverse-phase high performance liquid chromatography (hereinafter referred to as reverse-phase HPLC).

The amino acid sequence of the tripeptide of the present invention can be confirmed by using a gas phase protein sequencer (Model 477A (manufactured by Applied Biosystems)) for analysis of the amino acid sequence.

The method for measuring the ACE inhibitory activity of the tripeptide in the present invention is the method of Cheung et al. (Bioch).
m. Pharmacol. 20 , 1637 (197)
According to 1)), change a part and follow the procedure below. First,
125 mM borate buffer containing 1 M NaCl (pH 8.
50 μl of the ACE solution prepared in 3) having been extracted from rabbit rung acetone powder (manufactured by Sigma) and having an ACE activity of 5 to 10 mU was placed in a test tube, and 50 μl of a sample for assaying ACE inhibitory activity dissolved in distilled water ,
150 μl of a substrate solution in which 8.3 mM hypryl histidyl leucine (manufactured by Peptide Institute) was dissolved in a borate buffer solution having the same composition as described above was sequentially added, and the enzyme reaction was carried out at 37 ° C. for 30 minutes. Then, the enzymatic reaction is stopped by adding 250 μl of 1N HCl.

After the enzymatic reaction is stopped, in order to extract hippuric acid produced in the reaction system as a result of the reaction, 300 μl of ethyl acetate is added to the reaction solution, stirred and then centrifuged. 200 of the upper layer (ethyl acetate layer) of the separated two layers
After transferring μl to a test tube and evaporating to dryness, and further re-dissolving the dried product in distilled water, the hippoic acid extracted was measured for absorbance at 228 nm and quantified to calculate the ACE inhibition rate.

The control test is carried out by adding 50 μl of distilled water instead of the sample solution for assaying the ACE inhibitory activity. Further, the blank test was carried out by partially modifying the above procedure and first adding 1N HCl.
After deactivating CE, a substrate solution is added and the measurement is performed in the same manner. Further, the calculation of the ACE inhibition rate follows the following formula. Inhibition rate (%) = {1- (TB) / (CB)} × 10
0 Here, T is a value indicating the absorbance at 228 nm when a sample is added, B is a value indicating the absorbance at 228 nm in the blank test, and C is 228n in the control test.
It is a value representing the absorbance at m. Then, when the inhibitory activity is displayed, the inhibition rate is measured according to the concentration gradient of the ACE activity-inhibiting sample, and the 50% inhibitory concentration is calculated and expressed as an IC50 value.

[0014]

The ACE inhibitor according to the present invention is acidic from wheat protein gluten (hereinafter referred to as wheat gluten), as is apparent from Example 1, Example 2 and Examples 3 to 23 described later. It can be produced by hydrolysis with either a protease or an alkaline protease, and can also be produced by an ordinary peptide synthesis method using constituent amino acids. The IC50 value (μM) of the obtained ACE-inhibiting activity of the tripeptide of the present invention is 1.1 as shown in Table 1 below.
23, which is much higher than the IC50 value (μM) of 30 to 50, which is generally said to be effective as an ACE inhibitor.

In the amino acid sequences of the tripeptides listed in Table 1 below, all the C-terminal amino acids are aromatic amino acids represented by Trp or Tyr, and in this case, the central amino acid is Ile or Val.
When, the ACE inhibitory activity is increased. Furthermore, when the N-terminal amino acid is Ile or Cys, an extremely high ACE inhibitory activity value is shown. Also, Ile-Ly
s-Trp also showed a high activity value. Therefore, the ACE inhibitor according to the present invention prevents hypertension, which is currently a social issue,
It can be said that it can be sufficiently used for drugs to be suppressed or foods for specified insurance.

Hereinafter, the present invention will be described in more detail with reference to examples. Example 1. 6.7 g of wheat gluten with a water content of 70% is 0.0
Dissolve in 200 ml of 2N acetic acid and adjust the pH to 3.5 with hydrochloric acid.
Then, pepsin having a protein weight ratio of 1/250 was added and subjected to hydrolysis treatment at 37 ° C. for 24 hours. Then, the decomposition solution was heated at 100 ° C. for 10 minutes to inactivate pepsin, and then freeze-dried to obtain 2.0 g of a pepsin decomposition product powder.

The pepsin decomposition product powder was treated with 0.02N acetic acid 2
Redissolve in 0 ml, and Sephadex G-25 column (φ
Gel filtration chromatography with 2.6 x 95 cm) was performed. Of the four peaks having ACE inhibitory activity, the peak with the slowest elution was collected and freeze-dried in order to obtain a lower molecular weight fraction. 250 mg of the powder obtained here was redissolved in 12 ml of 0.02N acetic acid, and subsequently, Sephadex G-10 column (φ1.9 × 95c).
Gel filtration chromatography according to m) was performed. By using this Sephadex G-10 column
Three peaks having CE inhibitory activity were obtained, and again, 75 ml of a peak composed of a low-molecular substance with the slowest elution was collected and concentrated under reduced pressure to 0.5 ml.

The vacuum concentrate was subjected to reverse phase HPLC using an octadecylsilyl column (trade name, Cosmo Seal 5C18AR: manufactured by Nacalai Tesque) (φ0.46 × 15 cm).
Was fractionated by. Elution in the fractions was performed by a concentration gradient method using 0.05% trifluoroacetic acid as a basic solvent and increasing the concentration of acetonitrile. At that time, the most prominent ACE inhibitory activity was observed in the peak eluted at a retention time of 29 to 30 minutes, so the peak portions were collected, concentrated under reduced pressure, and then subjected to reverse phase HPLC with a gentle gradient of acetonitrile concentration again. Primary purification was performed.

As a result of the primary purification, the peak portion could be further separated into two main peaks and some secondary peaks. Of the two major peaks, only the first eluting peak showed strong ACE inhibitory activity. Therefore, the first eluting peak was subjected to reverse phase HPLC with a gentle concentration gradient of acetonitrile again to perform secondary purification. And the ingredients. 21 to detect peptides in the elution pattern
The UV absorption at 0 nm was read. Then, the single peak portion of the secondary purification was dried under reduced pressure to obtain 0.2 mg of a tripeptide standard product. As a result of analyzing the amino acid sequence of this preparation, it was confirmed to be Ile-Ile-Tyr. The IC50 value for ACE of the tripeptide preparation of the ACE inhibitor of the present invention obtained here is 3.7 (μM).
Met.

Example 2. Boc-L-Ile 1 mmol (equivalent to 231 mg), Boc-L-Tyr (Br-
Z) PAM resin 2 mmol (Boc-L-Tyr (Br
-Z) as a raw material) using a fully automatic peptide synthesizer, model 431A (manufactured by Applied Biosystems) as a raw material, and Boc-L-Ile-L-Ile-L-Tyr (B) by DCC-HOBt method.
The r-Z) PAM resin, a resin bound protected peptide, was synthesized. (Here, Boc is t-butyloxycarbonyl protecting group, Br-Z is 2-bromobenzyloxycarbonyl protecting group, PAM resin is phenylacetamide resin, DC.
C is dicyclohexylcarbodiimide, HOBt is 1-
Represents hydroxybenzotriazole. ) Subsequently, the resin-bound protected peptide was stirred with a mixture of thioanisole and ethanediol, trifluoroacetic acid (hereinafter referred to as TFA) was added, and the mixture was stirred at room temperature for 3 hours.
Ile-Ile-Tyr was cleaved from the deprotecting group and resin by treatment with trifluoromethanesulfonic acid for 0 minutes.
A crude peptide preparation of

The crude peptide preparation was washed with diethyl ether, dried, redissolved in distilled water and purified by reverse phase HPLC. At that time, the column is Nakarai Tesuku 5
5 using C18AR column in the presence of 0.05% TFA
A gradient elution of -80% acetonitrile was performed. The detection of the tripeptide was performed by measuring the ultraviolet absorption at 210 nm. The fraction corresponding to the main peak was dried under reduced pressure, redissolved in 2N acetic acid, and further lyophilized to give purified Ile-Ile-Tyr powder 83.
mg was obtained. The purified Ile-Ile-Tyr obtained here was analyzed using a protein sequencer, and it was confirmed that the amino acid sequence was correct. In addition, the ACE of the tripeptide obtained by the peptide synthesis method of this Example
The IC50 value for the compound was 4.0 (μM), and
Which is a tripeptide derived from wheat protein shown in
The value was extremely close to the ACE inhibitory activity of Ile-Tyr.

Examples 3 to 23. Fully automatic peptide synthesizer, model 431A, as in Example 2.
(Manufactured by Applied Biosystems) was used to synthesize various tripeptides related to the ACE inhibitor of the present invention. That is, Example 3 is Ile-Ile-Trp. Example 4 is C
ys-Ile-Trp. Example 5 is Ile-Val-T.
rp. Example 6 is Cys-Val-Trp. Example 7 is Tyr-Val-Trp. Example 8 is Pro-Val-
Trp. Example 9 is Ile-Lyr-Trp. Example 1
0 is Cys-Ile-Tyr. Example 11 is Lys-I
le-Tyr. Example 12 is Glu-Ile-Tyr.
Example 13 is Tyr-Ile-Tyr. Example 14 is T
rp-Ile-Tyr. Example 15 is Ala-Ile-
Tyr. Example 16 is Pro-Ile-Tyr. Example 17 is Met-Ile-Tyr. Example 18 is Ile-
Val-Tyr. Example 19 is Cys-Val-Ty.
r. Example 20 is Ile-Lys-Tyr. Example 21
Is Ile-Tyr-Tyr. Example 22 is Ile-Tr
p-Tyr. Example 23 is Ile-Ala-Tyr. Then, the IC50 value showing the ACE inhibitory activity of each tripeptide was determined according to a method in which a part of the method of Cheung et al. Was modified, and the results are shown in Table 1 together with the results of Example 1 and Example 2.

That is, the ACE inhibitory activities of the tripeptides of the examples listed in Table 1 all far exceed IC50 values of 30 to 50 (μM), which are generally considered to be effective for the activity,
It was an excellent ACE inhibitor. Then, in the amino acid sequence of the tripeptide, the C-terminal amino acid is an aromatic amino acid represented by Trp or Tyr, and in this case, the central amino acid is Ile or Va.
When it is 1, the ACE inhibitory activity is increased, and in addition, N
I when the terminal amino acid is Ile or Cys
The C50 value was 1.1 to 4.0 (μM), showing a stable and extremely high ACE inhibitory activity value. Ile-Lys-Trp also showed a high activity value.

[0024]

[Table 1]

[0025]

The ACE inhibitor of the present invention has excellent ACE.
Since it has an inhibitory activity, it is useful as a medicine or a food for specified health use for preventing and suppressing hypertension. Moreover,
Since the tripeptide relating to the ACE inhibitor of the present invention is a low molecular weight peptide having a regular amino acid sequence, not only can its activity be predicted relatively easily to carry out chemical synthesis, but a part thereof It can also be produced by a method of enzymatic decomposition using wheat gluten as an edible protein as a raw material.

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Claims (10)

[Claims] 1. An angiotensin converting enzyme inhibitor comprising a tripeptide in which the C-terminal amino acid is an aromatic amino acid. 2. The angiotensin converting enzyme inhibitor according to claim 1, which comprises a tripeptide whose aromatic amino acid is Trp. 3. The angiotensin converting enzyme inhibitor according to claim 2, which comprises a tripeptide in which the central amino acid is Ile. 4. The C-terminal amino acid is an aromatic amino acid T.
The angiotensin converting enzyme inhibitor according to claim 3, which comprises a tripeptide which is yr. 5. The angiotensin converting enzyme inhibitor according to claim 2, which comprises a tripeptide in which the central amino acid is Val. 6. The T-terminal amino acid having a C-terminal position is an aromatic amino acid.
The angiotensin converting enzyme inhibitor according to claim 5, which comprises a tripeptide which is yr. 7. The angiotensin converting enzyme inhibitor according to claim 3, claim 4, claim 5 or claim 6, which comprises a tripeptide in which the amino acid at the N-terminal position is Ile. 8. A tripeptide comprising an N-terminal amino acid as Ile and a central amino acid as Lys.
The angiotensin converting enzyme inhibitor described. 9. The angiotensin converting enzyme inhibitor according to claim 3, claim 4, claim 5, or claim 6, which comprises a tripeptide whose N-terminal amino acid is Cys. 10. N obtained by enzymatically degrading wheat protein
An angiotensin-converting enzyme inhibitor comprising a tripeptide in which the terminal amino acid is Ile, the central amino acid is Ile, and the C-terminal amino acid is Tyr.