JPH0466594A - New peptide and angiotensin transferase-inhibitor - Google Patents

Abstract

NEW MATERIAL:A peptide expressed by the formula Ile-Ala-Pro and salt of said peptide. USE:Used as an angiotensin transferase-inhibitor useful as a vasodepressing agent or a vasopression-suppressing agent. Said inhibitor exhibits effects with a very small administration dose and able to be very readily administrated with excellent absorptivity in a body. PREPARATION:For instance, wheat gluten is hydrolyzed by protease in a state of dispersed or dissolved in a liquid such as water and resultant water-soluble peptide mixture is separated and purified by a membrane fraction or ion- exchange fraction, etc., in a state of dissolved in water, etc., then treated with high-performance liquid chromatography, etc. Besides, as the protease, pepsin or aspartic proteinase derived from Aspergillus is preferable.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel peptide and an angiotensin-converting enzyme inhibitor containing the peptide or a salt thereof as an active ingredient.

[Conventional technology] Renin is a typical in-vivo factor that causes an increase in blood pressure.
The angiotensin system and the kallikrein-kinin system are known as typical in-vivo factors that act to lower blood pressure, and angiotensin converting enzyme (hereinafter referred to as rACEl) is heavily involved in both of these systems.

The mechanism will be briefly explained. First, in the renin-angiotensin system, the kidney enzyme renin secreted into the blood acts on angiotensinogen in the blood to generate the decapeptide angiotensin system. This angiotensin I does not exhibit an effect of increasing blood pressure, but when ACE acts on it, angiotensin ■, which is an octapeptide, is produced. This angiotensin■ constricts peripheral blood vessels and acts on the adrenal cortex to promote the production of aldosterone.Aldosterone acts on the kidneys, causing sodium reabsorption and an increase in body fluid volume, leading to an increase in cardiac output. Both of these significantly raise blood pressure.

On the other hand, in the kallikrein-kinin system, the blood enzyme kallikrein acts on the precursor protein kininogen in the blood to release kinin, which dilates peripheral blood vessels and activates phospholipase A to produce prosthesis. Promotes the synthesis of glassine and lowers blood pressure.

However, when ACE acts on this kallikrein-kinin system, it causes dilation of peripheral blood vessels and phospholipase A.
Since the above-mentioned kinin, which has the activating effect of No. 2, is decomposed and inactivated, no drop in blood pressure occurs.

Therefore, the presence of a substance (ACE inhibitor) that inhibits the above-mentioned functions of ACH suppresses the production of angiotensin, which is a substance that increases blood pressure, and prevents the decomposition of kinin, which acts as a substance that lowers blood pressure. It is possible to suppress the increase in blood pressure and lower blood pressure.

From this point of view, various research and development efforts have been made on ACE inhibitors in recent years, and it has been reported that specific peptides derived from natural proteins or synthesized have ACE inhibitory effects. A derived from natural proteins reported so far
Among the CE-inhibiting peptides, bradykinin potentiator B (Pyr-Gly-Leu-P
ro-Pr.

Arg-Pro-Lys-11e-Pro-Pro) and bradykinin potentuator C (Pyr-Gl
y-Leu-Pro-Pr.

-Gly-Pro-Pro-11e-Pro-Pro)
[Both H, 1 (at.

and T, 5uzuki, Biochemist
ry, 10. Phe-Ph, a peptide derived from milk casein]
e-Val-Ala-Pro-PhePro-Glu-
Vat-Phe-Gly-Lys (Tokuko Showa 60-23
No. 085), Phe-Phe-Vat-Ala-P
ro (Japanese Unexamined Patent Publication No. 59-44323), Thr-T
hr-Net-Pro-Leu-Trp
No. 263), Ala-Val-Pro-Tyr-P
ro-Gln-Arg.

Tyr-LysS, a peptide derived from fish protein
er-Phe-11e-Lys-Gly-Tyr-Pr
o-Val-Met, Pr.

1u-Glu-C+1u-Pro-His-Val-
Leu, a peptide derived from corn asein, Leu-Pro-Pro, Val
-Old 5-Leu-Pro-ProXVal-His-L
eu-Pro-Pr.

Most of them are peptides in which five or more amino acids are bonded.

[Contents of the Invention] Under the above-mentioned circumstances, the present inventors have also been conducting research on a substance that has an ACE inhibitory effect.

As a result, a novel tripeptide consisting of isoleucine-alanine-suthroline having an amino acid sequence different from that of the known ACE-inhibiting peptides lie -Ala
-Pro could be isolated from wheat gluten hydrolyzate, and it was found that this tripeptide has ACE inhibitory activity.

Therefore, the present invention provides the following formula %formula% These are the peptide represented by and its salt.

Furthermore, the present invention includes an ACE inhibitor containing a peptide represented by the above formula or a salt thereof as an active ingredient.

The above-mentioned peptide having ACE inhibitory activity of the present invention was first discovered as a product of gluten hydrolysis with protease, and in that case, the above three amino acids 1ieSAla and Pro are all L-amino acids. be. However, e: is not defined and any optical isomer may be used as long as the tripeptide has the above amino acid sequence, and all three types of amino acids are D.
- Tripeptides consisting of amino acids and tripeptides in which any one or two of the three amino acids are L-amino acids and the remainder are D-amino acids are also included, and they can be produced by chemical synthesis. .

Examples of the method for preparing the tripeptide of the present invention are as follows.

Method by Hydrolysis of Wheat Gluten Wheat gluten is hydrolyzed using protease to prepare a water-soluble gluten-derived peptide mixture. At this time, it is preferable to perform the hydrolysis while dispersing or dissolving gluten in a liquid such as water from the viewpoint of ease of operation, yield and purity of the target product.

Proteases include proteases that act in acidic conditions,
In particular, it is preferable to use aspartic acid/cuprotinase in which an aspartic acid residue and an aspartic acid carboxylic acid ion are involved in the active center of the enzyme. Examples of such proteases include pepsin, aspartic proteinase of Aspergillus origin, aspartic proteinase of Aspergillus origin, al.partic proteinase of Penicillium origin, and in particular pepsin, aspartic proteinase of Aspergillus origin. is preferable in that the target product can be obtained in high yield. Only one type of protease may be used, or multiple types may be used in combination as long as the proteases do not have an adverse effect on each other. When a plurality of proteases are used, the hydrolysis may be carried out by allowing the plurality of proteases to exist simultaneously, or by sequentially using one type at a time. Further, protease may be used in a free state or in an immobilized state. In each case, the amount of protease used is approximately 5,000-100.0 g of protease per 100 g of dried gluten.
It is better to use 00 units.

Here, all protease activities (units) in this specification are measured by the following method.

Measuring method for protease activity The substrate is Hammerstein casein 1 manufactured by Merck & Co., USA.
% solution, modified Anson-Hagiwara method [edited by Shibe Akahori, "Enzyme Research Methods", Vol. 2, p. 237 (January 10, 1960,
(Published by Breakfast Shoten)]. The reaction is 3 at 30@d.
The reaction was carried out for 0 minutes, and the amount of enzyme required to release an amount equivalent to 1 Mg of tyrosine in 1 minute was defined as 1 unit.

Protease treatment is performed using the optimal p
It is best to select conditions such as H, temperature, amount of protease, treatment speed, treatment time, etc. For example, when using the protease listed above, the pH is about 1.5 to 5.0, and the temperature is about 30 to 30. Hydrolysis may be carried out at 50° C. until the solubility in 0.75 M trichloroacetic acid is about 40-70%.

Once the desired hydrolysis state is achieved, the protease is inactivated by heating and/or pH adjustment to 8, and the inactivated enzyme and insoluble solids such as undegraded gluten are separated and removed by centrifugation or other appropriate means. Then, the peptide mixture contained in the residual liquid is recovered by drying or the like.

Next, this peptide mixture is dissolved in water or the like and separated and purified by membrane fractionation, ion exchange fractionation, gel filtration fractionation, etc., and further subjected to high performance liquid chromatography (for example, high performance liquid chromatography using a reversed phase column). chromatography, etc.) to isolate the tripeptide in pure form.

Separation and purification of the aqueous solution containing the peptide mixture and isolation of the tripeptide are performed as described above, for example, in the following (a) to (f).
).

(a) Adjust the pH of the aqueous solution containing the peptide mixture to about 3,
Q ~ 5.0 and subjected to ion exchange chromatography (for example, 5P-Toyo manufactured by Tosoh Corporation).
pearl, passed through a column packed with 55O3), and the components adsorbed in this chromatography were eluted with an aqueous NaCl solution with a linear concentration gradient from OM to 0.5M. (b) Both fractions having high inhibitory activity are treated with a molecular sieve (□). For example, pass through a column filled with Bio-Kel P-2 manufactured by Bio-Rad), elute and separate several fractions with distilled water, and collect the fractions with even higher inhibitory activity (c ) Both fractions recovered in (b) above were subjected to high performance liquid chromatography (for example, 0DS-1207 manufactured by Tosoh Corporation).
), and the adsorbed components were removed using 0.1% trifluoroacetic acid aqueous solution (liquid A) and 0.
When eluted using a linear concentration gradient eluent, which is a mixture with a 1% trifluoroacetic acid aqueous solution (solution B), the concentration of solution B in the mixture increases linearly from 0% to 100%.
A high absorption peak appears in the eluent fraction in which the concentration of acetonitrile is approximately 20 to 22%, and the ACE inhibitory activity of this fraction is measured and recovered. (d) If necessary, the step of (c) above. Repeat (e)
(d) The solvent is removed from the fraction obtained in step (drying, etc.) to recover a white solid, and (f) the amino acid sequence of the product obtained as the white solid is It is examined using a gas phase protein sequencer (PSQ-1 system), etc., and confirmed to be a tripeptide consisting of 1ie-Ala-Pro.

Furthermore, when the tripeptide of the present invention is produced by chemical synthesis, the following method can be employed, for example.

Chemical synthesis method for tripeptide of the present invention Synthesis is carried out using a peptide synthesizer [Biolynx 4170 manufactured by Pharmacia (Sweden)].

Specifically, after condensing Fmoc/florin to a polyamide resin, removing the Fmoc group to liberate the terminal amino group, condensing Fmoc/alanine to this free amino group, and then removing the Fmoc group, Further, Fmoe/inleucine is condensed and the Fmoc group is removed to form a peptide protected with the resin. 95% of this
- After separating the resin by reacting it with an aqueous solution of refluoroacetic acid at room temperature for 60 minutes, the resin is filtered off. After distilling off the dribble-oacetic acid aqueous solution under reduced pressure, the residue was reduced to 0. lfi acetic acid and the solution was subjected to high performance liquid chromatography (OD
Highly pure Ile-Ala-Pro is isolated by removing impurities through a 100% pure Ile-Ala-Pro.

The ACE inhibitor of the present invention can be administered to humans and various animals, and significant blood pressure lowering and elevation suppression can be achieved by administering a small amount.

A suitable dosage of the ACE inhibitor of the present invention varies depending on various conditions such as the age, body weight, sex, symptoms, and type of animal of the human or animal to be administered.

The ACE inhibitor of the present invention can be administered either orally or parenterally, and can be administered alone or together with solid carriers or liquid carriers commonly used in the pharmaceutical industry. Alternatively, it can be used in combination with or mixed with other drugs. Further, the dosage form can be any form such as a tablet, pill, granule, capsule, powder, aqueous solution, or injection.

Furthermore, the ACE inhibitor of the present invention can be added to food or feed or administered together with the L-11e-L-A derived from natural protein.
la-L-Pro is preferred.

The present invention will be specifically explained below by giving examples, but the present invention is not limited thereto.

Example 5g of gliadin, a component of wheat gluten, was added to 0.03N
After dispersing and dissolving in 100 ml of hydrochloric acid, add distilled water to make a total volume of 200 ml, and add 6 IN hydrochloric acid to adjust the pH to 2.
．． After adjusting to 0, add pepsin (manufactured by Sigma, USA) 50
00 units were added and reacted at 37°C for 15 hours. Next, 4.
After adjusting to 4, albartic proteinase of Aspergillus origin (Protease M manufactured by Ohno Pharmaceutical Co., Ltd.)
1ooUnits were added and reacted at 45°C for 5 hours. Next, the pI (6.
After adjusting the temperature to 0, the mixture was heated at 90"C for 20 minutes to inactivate the enzyme and precipitate undissolved substances. After cooling to room temperature, the solids were separated and removed by centrifugation at 100 OOG for 20 minutes. The clear liquid was collected and freeze-dried to obtain 3.8 g of a peptide mixture.

After dissolving 500 mg of the peptide mixture obtained above in 50 rrlQ of 5 mM acetate buffer, the pH was adjusted to 3.5 with IN hydrochloric acid.

This was transferred to an ion exchange chromatography column with a diameter of 16 mm and a length of 200 mm filled with 40 mQ of sp-choyopear J 5505 manufactured by Tosoh Corporation.
After passing through the chromatographic column at a flow rate of Q/min, the components adsorbed on this chromatographic column were washed with 120 ml of an eluent (2 to 1
The column was eluted with stream 1 at a flow rate of mQ 1 min.
Both fractions having high inhibitory activity were obtained at a NaCl aqueous solution concentration of 0.2 to 0.3M, and these were collected separately.

Next, the above fraction was transferred to Biogel P- manufactured by Bioland.
Column packed with 200mff of 2 (column diameter 16mm
, a length of 100,100 O at a flow rate of 0.33 mff/min to perform molecular sieving treatment, and then elute with distilled water to collect a fraction with high inhibitory activity therein.

The above fraction was passed through a high performance liquid chromatograph EODS-120T manufactured by Tosoh Corporation at a flow rate of 1 mff7 minutes, and the adsorbed components were extracted with a 0.1% trifluoroacetic acid aqueous solution (liquid A) and acetonitrile containing 50%. 0.1% to do
Elute by passing a linear concentration gradient eluent, which is a mixture with an aqueous trifluoroacetic acid solution (solution B) in which the concentration of solution B in the mixture increases linearly from 0% to 100%, at a flow rate of 1 mff 7 minutes. Then, the concentration of acetonitrile will be 20-22%.
Since the eluent fraction had high inhibitory activity, this fraction was collected and this high performance liquid chromatography treatment was repeated again.

The solvent was removed from the obtained fraction by drying to obtain 200% of a white solid.
μg was collected. When the amino acid sequence of this white solid was examined using a gas-phase protein sequencer (PSQ-1 system) manufactured by Takafusa Seisakusho, L-11e, L-Ala and L-Pro were released sequentially from the N-terminus. I've been doing it. From this, the formula) (-Llie -L-Ala
It was confirmed that it is a tripeptide represented by -L-Pro .OH.

The ACE inhibitory activity of the tripeptide prepared above and known ACE inhibitory peptides was measured by the method below, and the results are shown in the table below.

Method for measuring ACE inhibitory activity of peptides Place 50 μQ of sample solution in a test tube, add 1 unit of ACE solution (manufactured by Sigma, USA, rabbit lung-derived ACE) to 50 μQ of water.
Add 20 μg (dissolved in m12).

After keeping at 37°C for 5 minutes, the substrate (5mM Hip-H
isLeu: pH 8,3) and heated at 37°C for 30
0.3M aqueous sodium hydroxide solution I
Add mQ to stop the reaction. Add 100 μQ of a fluorescent reagent ortho-phthalaldehyde solution and react for 10 minutes at room temperature. Next, add 200 ma of 3N hydrochloric acid and dilute 50 times with distilled water. After about 30 minutes, use a spectrofluorometer to measure excitation wavelength 3.
00nm, fluorescence intensity at fluorescence wavelength 490nm (A)
Measure. Instead of the sample solution, 50 μQ of distilled water is treated in the same manner and the fluorescence intensity (B) is measured.

Inhibitory activity is determined by BA/B.

The inhibitory activity was measured in the same manner as above by changing the concentration of the sample solution, and the concentration that inhibited the activity by 50% was determined, and this was determined as IC. It is shown closed.

[ACE inhibitory activity of peptide (IC,.)] Peptide
IC5° (μM) H-L-11e-L-Ala-L-Pro ・OH (invention) 2.7 Bradykinin potentiator B6.4
Bradykinin Potentiator C29.0 From the results in the table above, the ACE inhibitor of the present invention can be used at a much lower concentration than the known ACE inhibitors Bradykinin Potentiator B and C, that is, when used in a very small amount, IC. It can be seen that the ACE inhibitory activity is very high.

[Effects of the invention] The ACE inhibitor of the present invention can inhibit ACE with extremely small doses.
By inhibiting the activity of H, lowering of blood pressure and suppression of increase in blood pressure can be achieved.

Furthermore, since the ACE inhibitor of the present invention is a white water-soluble powder, it can be administered very easily either by oral administration or parenteral administration, either as it is or dissolved in water.

Moreover, the novel tripeptide of the present invention
-Pro is a low-molecular-weight compound with an extremely simple structure consisting of just a sequence of three amino acids, so it can be easily produced by chemical synthesis, and it is well absorbed in the body when administered. Shows high blood pressure lowering effect.

Claims (1)

[Scope of Claims] 1) A peptide represented by the following formula Ile-Ala-Pro and a salt thereof. 2) An angiotensin converting enzyme inhibitor containing a peptide represented by the following formula Ile-Ala-Pro or a salt thereof as an active ingredient.