JP2873327B2 - Angiotensin converting enzyme inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an angiotensin converting enzyme inhibitor, and more particularly to angiotensin which is expected to be useful as a pharmaceutical or food useful for the prevention and treatment of hypertension, which has been increasing in recent years and for which measures are desired. The present invention relates to a converting enzyme inhibitor.

[0002]

2. Description of the Related Art It is well known that the onset of hypertension is closely related to the renin-angiotensin system, and this renin-angiotensin system is related to angiotensin converting enzyme (EC 3.4.15.1). , Below AC
E also plays an important role. In this case A
CE is angiotensin I (Asp-Arg-Val-Tyr-Ile-Hi) in which angiotensinogen secreted in the liver is degraded by the enzyme renin produced in the kidney.
s-Pro-Phe-His-Leu), which is converted to angiotensin II (Asp-Arg).
-Val-Tyr-Ile-His-Pro-Phe)
Is converted to And this angiotensin II has a function of contracting vascular wall smooth muscle to increase blood pressure, and furthermore, acts on the adrenal cortex to promote secretion of aldosterone. In addition, the enzyme kallikrein present in plasma degrades a protein called kininogen to produce bradykinin, which dilates blood vessels and lowers blood pressure. This bradykinin is degraded and inactivated by the action of ACE. Thus, ACE produces, on the one hand, a pressor peptide (angiotensin II) and, on the other hand, breaks down the antihypertensive peptide (bradykinin), and consequently pushes blood pressure upward. Therefore, by suppressing this enzyme activity, it is possible to prevent an increase in blood pressure (lower blood pressure).

As ACE inhibitors, there are several peptide inhibitors obtained from snake venom and captopril (D-2-methyl-3-mercaptopropanoyl-L-).
Many synthetic substances such as proline) are known, of which captopril has already been put to practical use as an oral antihypertensive agent. In recent years, microorganisms and various foods
A CE inhibitor has been found, and its practical use as a hypotensive agent is being studied.

[0004] It has also been proposed to isolate an ACE inhibitor derived from trypsin hydrolyzate of milk casein or to further treat it with peptidase and use it as a hypotensive agent (Japanese Patent Publication No. 60-23085; 60
No. 23086, No. 60-23087, JP-A-61-1986.
No. 36226, No. 61-36227). Recently, it has been proposed to use a hydrolyzate of a fish protein or a soybean protein with a serine protease derived from a bacterium belonging to the genus Bacillus, a metal protease derived from a bacterium belonging to the genus Bacillus, or a thiol protease derived from a plant as a blood pressure lowering agent (Japanese Patent Application Laid-Open (JP-A) no. No. 62-169732).

On the other hand, corn protein contains 50 to 60% prolamin and 35 to 40% glutelin, and the main component prolamin is called zein. Zein is divided into three types, α, β, and γ (J. Ce).
real Sci. 5 , 117 (1987)). Val-His-Leu-Pro-Pro- in γ-zein
A repeating structure having Pro as a basic unit is included (Nucleic Acids Res. 13 (5),
1493 (1985)).

[0006]

There is a constant need for new and useful antihypertensive agents and thus angiotensin converting enzyme inhibitors. In addition, not only pharmaceutical products but also functional foods for preventing various symptoms such as hypertension through daily ingestion are required these days. Accordingly, the present invention provides a specific oligopeptide angiotensin converting enzyme inhibitor which has an excellent angiotensin converting enzyme inhibitory action and a blood pressure lowering action, is extremely high in safety, and can be used not only as a pharmaceutical product but also as a functional food. The purpose is to do.

[0007]

Means for Solving the Problems The present inventors have searched various substances having ACE inhibitory activity and found that γ-zein in corn protein, which is an inexpensive and most common food protein, is hydrolyzed with a specific protease. It has been found that a certain peptide obtained by decomposition has angiotensin converting enzyme inhibitory activity, and that the above problem can be solved by using the peptide. That is, the present invention provides an angiotensin converting enzyme inhibitor containing Leu-Pro-Pro as an active ingredient.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The γ-zein used in the present invention can be separated from corn protein obtained from corn or corn protein obtained in the process of producing corn starch by a conventional method (for example, Plant Physiol., Ltd.). 80 , 623 (1
986)). Reference Example 1 shows a preparation example of γ-zein.

The hydrolysis in the present invention is performed by the following steps. a) γ-zein is first subjected to thermolysin hydrolysis to produce Val-His-Leu-Pro-Pro-Pro. That is, first, γ-zein is dissolved in an alkaline solution such as an aqueous sodium hydroxide solution, and ultrafiltration removes solubilized low molecular impurities. Then p as needed
Γ-zein is denatured by, for example, treatment at H10 to 12 and a temperature of 80 to 100 ° C for 5 minutes to 1 hour to make thermolysin easier to work. At this time, the low molecular weight fraction is removed by ultrafiltration.

Then, the pH is adjusted to around neutral with hydrochloric acid or the like,
The pH is adjusted to 6 to 9 with a Ca-containing buffer, and the temperature is adjusted to 30 to
While maintaining at 80 ° C., thermolysin is added and the enzyme reaction is performed for 1 to 40 hours. 0.005 to 0.01 as buffer
M CaCl-containing 0.05M Tris-HCl buffer (pH 8
To 8.5) and the like are preferably used. The appropriate amount of thermolysin used is 0.1 to 10 parts by weight per 100 parts by weight of the substrate. The reaction is terminated by, for example, adding an acid such as hydrochloric acid to inactivate the enzyme to a pH of 3 or less.

After the reaction, the enzyme solution is subjected to ultrafiltration to recover a filtrate containing low molecules passing therethrough. After neutralizing the filtrate with an aqueous alkali solution such as sodium hydroxide, the filtrate is concentrated and subjected to column chromatography, for example, Sephadex LH-20 column chromatography. The elution pattern of each fraction by HPLC is synthesized Val-His-Leu- Pro-Pro
Val-His by comparison with that of Pro
-A solution containing Leu-Pro-Pro-Pro is obtained. This product can be further purified by column chromatography, for example, SP-Toyopearl 650S cation exchange chromatography, reverse phase HPLC and the like.

B) Next, Val-His-Leu-Pr
o-Pro-Pro is hydrolyzed with leucine aminopeptidase to produce His-Leu-Pro-Pro-Pro or Leu-Pro-Pro-Pro. This enzymatic reaction is usually carried out in a buffer having a pH of 6 to 9 for 30 to 60.
C. for 1 to 24 hours. 0.05M Mg as buffer
0.1M Tris-HCl (pH 8.6) containing Cl is used. The amount of leucine aminopeptidase used is 100 substrates.
The suitable amount is 0.1 to 10 parts by weight with respect to parts by weight.

The reaction is terminated, for example, by heating at 100 ° C. for 5 minutes. The target compound can be isolated and purified from the reaction completed solution by column chromatography, for example, reverse phase HPLC. The target substance can be tracked by comparing the HPLC elution patterns of the synthetic peptides as in the case of a).

C) Next, Val-His-Leu-Pr
o-Pro-Pro, His-Leu-Pro-Pro
-Remove one of each C-terminal Pro by hydrolyzing Pro or Leu-Pro-Pro-Pro with carboxypeptidase C or subject to mild acid hydrolysis. This enzymatic reaction is usually carried out in a buffer at pH 4-7 at 30-60 ° C.
Perform for ~ 24 hours. As the buffer, a 0.1 M citrate buffer or the like is used. The amount of carboxypeptidase C used is suitably 0.1 to 10 parts by weight per 100 parts by weight of the substrate. The reaction is terminated, for example, by heating at 100 ° C. for 5 minutes.

The acid hydrolysis is usually carried out using an acid such as hydrochloric acid having a concentration of 0.1 to 6 N at a temperature of 80 to 120 ° C. for 5 to 120 minutes. The reaction is terminated by neutralization with an aqueous sodium hydroxide solution or the like. In any case, the target compound can be isolated and purified from the reaction-terminated liquid by column chromatography, for example, reverse phase HPLC. The target substance can be tracked by comparing the HPLC elution patterns of the synthetic peptides as in the case of a). Step b) is performed as necessary. In the case where the steps b) and c) are performed together, any one of the steps may be performed first.

The degree of polymerization of the amino acid obtained as described above is 3 to
5 show ACE inhibitory activity. When these peptides are used as an ACE inhibitor, these peptides may be contained alone, or may be contained as a mixture in any ratio, and further, other peptides derived from the hydrolyzate, amino acids May be contained as a minor component.

The peptide is used as it is or usually in the form of a pharmaceutical composition with at least one pharmaceutical auxiliary.
The peptide of the present invention can be administered parenterally (that is, intravenous injection, rectal administration, etc.) or orally to mammals including humans, and can be formulated into a form suitable for each administration method.

The preparation form as an injection usually includes a sterile aqueous solution. Preparations in the above form also include buffering agents / pH regulators (sodium hydrogen phosphate, citric acid, etc.), tonicity agents (sodium chloride, glucose, etc.), preservatives (methyl parahydroxybenzoate, propyl p-hydroxybenzoate, etc. ) And other pharmaceutical auxiliaries other than water. The preparation can be sterilized by filtration through a bacteria-retaining filter, by mixing a bactericide into the composition, or by irradiating or heating the composition. The pharmaceutical can also be manufactured as a sterilized solid composition and used after dissolving in sterilized water or the like at the time of use.

[0019] Oral formulations are formulated in a form suitable for absorption by the gastrointestinal tract. Tablets, capsules, granules, fine granules and powders are commonly used as formulation aids such as binders (syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone, hydroxypropylcellulose, etc.), excipients (lactose) , Sugar, corn starch, calcium phosphate, sorbite, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch,
Carboxymethylcellulose) and wetting agents (such as sodium lauryl sulfate). Tablets can be coated by conventional methods. The oral solution can be made into an aqueous solution or the like or a dry product.
Such an oral solution is prepared by using a conventional additive such as a preservative (p-
Methyl or propyl hydroxybenzoate, sorbic acid, etc.).

Although the amount of the present peptide in the ACE inhibitor of the present invention can be variously changed, it is usually 1 to 100% (w /
w) is appropriate. The dose of the present ACE inhibitor is suitably 0.5 to 500 mg / kg / day as an active ingredient. The acute toxicity of any of the peptides of the present invention was LD
(Rat, oral administration)> 5 g / kg.

The peptide of the present invention has an advantage that it does not adversely affect the living body even when ingested in a large amount. Therefore, the peptide has a blood pressure lowering effect as it is, or it is added to various nutrients or the like, or contained in food or drink. It may be eaten as a functional food or health food having a function of preventing hypertension. That is, for example, nutrients such as various vitamins and minerals are added and used, for example, liquid foods such as nutritional drinks, soy milk, soup and solid foods of various shapes, and further used as a powder or as it is or added to various foods. Can also.
The content and intake of the present peptide in the ACE inhibitor of the present invention as such functional foods and health foods may be the same as in the above-mentioned pharmaceuticals.

[0022]

Next, the present invention will be described with reference to examples. In Examples,% indicates% by weight. Example 1 Preparation of each oligopeptide and ACE inhibitory activity a) Val-His-Leu-Pro-Pro-Pr
Preparation of o 0.5 g of γ-zein was dispersed in 25 ml of distilled water, and 1N
The pH was adjusted to 12 with NaOH to dissolve γ-zein. Then, ultrafiltration using Amicon PM-10 (fraction molecular weight: 10,000) as an ultrafiltration membrane was performed to remove solubilized low molecular impurities. PH 12 Na in the internal solution
OH was added to make the total volume 25 ml, and the mixture was heated at 100 ° C. for 30 minutes to denature γ-zein. In order to remove the fraction degraded by this treatment, the filtrate was again subjected to the same ultrafiltration as described above.

0.25 volume of 0.05M Ca
0.25 M Tris HCl buffer containing Cl (pH 8.5)
And kept at 37 ° C., followed by addition of 18 mg of thermolysin (Sigma). 40 hours later, p with 1N HCl
The reaction was stopped by adjusting to H1.7 and subjected to the same ultrafiltration as described above to recover passing low molecules. This is 1NN
After neutralization with aOH, the concentrated solution was concentrated and applied to Sephadex LH.
The mixture was added to a -20 column and eluted with distilled water (elution conditions: column height 70 cm, inner diameter 1.6 cm, sample addition amount 2 ml, flow rate 33 ml / hr).

The elution pattern by HPLC was examined using a small amount of each fraction, and the synthesized Val-His-Leu-Pro
-A fraction having a peak at the same position as the elution position shown by Pro-Pro was collected (HPLC elution conditions: Column Waters Radial PAK C-8, 10 µm,
Sample addition volume 5 μl, flow rate 1 ml / min, elution phosphate buffer (10 mM KHPO, 50 mM NaSO, pH
3.0): acetonitrile = 2: 3, detection UV210n
m).

This fraction was added to an SP-Toyopearl 650S column equilibrated with 5 mM buffer (pH 4.0),
Elution was performed with a linear gradient of 0 to 0.3 M NaCl.
Val-His-Leu-Pro-Pro synthesized by LC
-A fraction having a peak eluted at the same position as Pro was collected (SP-Toyopearl elution condition: column height 20c)
m, inner diameter 1.6cm, flow rate 100ml / hr, elution 5m
0-0.3 M Na containing M acetate buffer (pH 4.0)
Cl. HPLC elution conditions are the same as described above).

The collected fractions were concentrated and subjected to HPLC to synthesize Val-His-Leu-Pro-Pro-Pro.
Only the peak at the same position as above was collected, adsorbed on a reverse-phase silica gel column SepPAK C-18 (Waters) washed with HCl at pH 2 and mixed salts were removed with HCl at pH 2, and then eluted with methanol. Amino acid analysis was performed (HPLC elution conditions at the time of fractionation: only 25 μl of sample was added, and the other conditions were the same as in the first case). For amino acid analysis above, the sample was dissolved in 6N HCl,
After heating at 24 ° C. for 24 hours, the measurement was performed with an amino acid analyzer.

As a result, the molar ratio, where Leu was 1, was Val
1.3, His 1.2, Leu1, Pro 3.1
And Val-His-Leu-Pro-Pro-P
ro could be collected. The result of mass spectrometry was 659 (M
+1), consistent with the expected molecular weight of the peptide.

B) Preparation of Leu-Pro-Pro-Pro Leucine aminopeptidase (Boehringer Mannheim Yamanouchi) (5 mg / ml liquid) was added to 0.05M MgC
The resultant was dissolved in 800 μl of 0.1 M Tris-HCl (pH 8.6) containing 1 l to prepare an enzyme solution. 300 μM Val-His-L
eu-Pro-Pro-Pro 50 μl and enzyme solution 20
0 μl was mixed and reacted at 37 ° C. for 23 hours. After the reaction, the reaction solution was synthesized by HPLC from Leu-Pro-Pro.
A peak eluted at the same position as that of Pro was collected and subjected to amino acid analysis (HPLC elution conditions: pH of the phosphate buffer used was set to 2.5, and the amount of sample added was 10 μm).
The same as the condition in the first case except that it was 1.) As a result L
The molar ratio where eu is 1 is Val 0.16, Leu
1, His 0.19, Pro 2.51, Leu
-Pro-Pro-Pro could be recovered.

C) Preparation of Leu-Pro-Pro 6.3 mM Leu-Pro-Pro-Pro 200
μl and 200 μl of 12N HCl were mixed and subjected to a hydrolysis reaction at 100 ° C. for 10 minutes. Subsequently, a fraction having a peak (3.18 mm) at the same position as that of the synthetic Leu-Pro-Pro was recovered by elution by HPLC. (Elution conditions: Column Merck Lichrosorb RP-
Select B 5 μm, flow rate 1 ml / min, elution
Phosphate buffer (pH 2.5): acetonitrile = 5:
1, detection UV 210 nm).

D) Val-His-Leu-Pro-P
Preparation of ro Obtained from Val-His-Leu-Pro-Pro-Pro in the same manner as in c) above.

E) Measurement of ACE inhibitory activity The ACE inhibitory activity of each peptide obtained as described above was measured as follows. That is, first, 5 g of rabbit lang acetone powder was dissolved in 50 ml of 0.1 M sodium borate buffer (pH 8.3), and 40,000 was dissolved.
G, centrifugation under the conditions of 40 minutes, and the supernatant was further diluted 5-fold with the above buffer to obtain an angiotensin converting enzyme solution.

0.03 ml of each peptide solution was placed in a test tube, and 0.25 ml of hypril histidylleucine (final concentration 5 mM, NaCl 300 mM) was used as a substrate.
Was added, and then 0.1 ml of the above-mentioned angiotensin converting enzyme solution was added, followed by reaction at 37 ° C. for 30 minutes. Thereafter, 0.25 ml of 1N hydrochloric acid was added to stop the reaction, and 1.5 ml of ethyl acetate was added. The absorption value of hyprilic acid extracted in ethyl acetate was measured at 228 nm, and the enzyme activity was measured. And Under the above conditions, the absorption value at 228 nm when the inhibitor of the present invention was not contained was approximately 0.3.
It was 5. A plurality of such experiments were performed, and the inhibition rate was calculated by the following equation.

[0033]

(Equation 1)

A: 228 nm absorption value without an inhibitor B: 228 nm absorption value with an inhibitor added The inhibitor concentration I at an inhibition rate of 50% was determined.
Table 1 shows the results.

[0035]

[Table 1]

Example 2 Blood pressure lowering effect of Leu-Pro-Pro Wistar male rats weighing 200 g (5 rats per group) were anesthetized by intraperitoneal administration of urethane 1.5 g / kg. , The common carotid artery pressure was continuously recorded via a transducer (SCK-590, Nihon Kohden). Leu-Pro-Pro dissolved in physiological saline was administered from the lower leg vein.
Five and 35 minutes later, 100 ng / kg of angiotensin I (human sequence, Sigma) was repeatedly administered, and the change in mean blood pressure before and after was measured. As a control, one to which physiological saline was administered was used. Table 2 shows the results. The peptide effectively suppressed the angiotensin I-induced hypertension 5 minutes after administration, and its action was continued even after 35 minutes.

[0037]

[Table 2]

Example 3 Intravenous Injection Leu-Pro-Pro was dissolved in a 20 to 100-fold (volume / weight) sterile physiological saline solution, and aseptically filtered through a filter (pore size: 0.45 μm). And

Example 4 Tablets Leu-Pro-Pro 7 parts Hydroxypropyl cellulose 1 part Lactose 10.9 parts Potato starch 1 part Magnesium stearate 0.1 part Prepare 20 parts of a 60% ethanol aqueous solution containing 1 part of hydroxypropyl cellulose. Then, 7 parts of the present peptide and 10.9 parts of lactose were added, and the mixture was sufficiently kneaded. The mixture was dried under reduced pressure, and 1 part of potato starch and 0.1 part of magnesium stearate were added to the obtained dried product and mixed. And tablet making with a tableting machine.

Reference Example 1 Preparation of γ-zein The method of Esen (J. Cereal Sci. 5 , 11)
7 (1987)). 60% isopropyl alcohol water containing 1% 2-mercaptoethanol in 100 g of ground corn (normal dent corn)
A double volume was added, and the whole zein fraction was extracted by stirring at 60 ° C. for 2 hours. The mixture was centrifuged at 3,000 G for 10 minutes, an equal volume of distilled water and 0.02 volume of 3M aqueous sodium acetate solution were added to the supernatant, adjusted to pH 6 with a small amount of acetic acid, and allowed to stand at 4 ° C. overnight, and α And β-zein were precipitated.
Then, the mixture was centrifuged at 3,000 G for 10 minutes, the supernatant was freeze-dried, the dried substance was dispersed in a small amount of distilled water, dialyzed against distilled water using a dialysis tube, and then freeze-dried.
0.4 g of γ-zein was obtained as a pale yellow powder.

[0041]

According to the present invention, there is provided an ACE inhibitor having an excellent ACE inhibitory action and an antihypertensive action. Since the ACE inhibitor of the present invention is derived from food proteins, it is extremely safe even when ingested in large amounts, and therefore does not show any side effects.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12P 21/06 A61K 37/18 (72) Inventor Noboru Tomizuka 1-3-1 Higashi, Tsukuba City, Ibaraki Pref. 72) Inventor Shinsuke Miyoshi 2-20-2, Hinode, Funabashi-shi, Chiba Pref.Shinsan Ryo (72) Inventor Fumio Fukui 1-2-117, Nakadai, Narita-shi, Chiba Examiner Seiko Tamura (58) Field (Int. Cl. ⁶ , DB name) A61K 38/55 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. An angiotensin converting enzyme inhibitor comprising Leu-Pro-Pro as an active ingredient.