JP2805032B2 - Angiotensin converting enzyme inhibitor - Google Patents

Description

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

[Prior art] It is well known that the onset of hypertension is closely related to the renin-angiotensin system, and this renin-angiotensin system includes an angiotensin converting enzyme (EC3.4.15.1, hereinafter ACE) plays an important role. In this case, ACE is angiotensin I (Asp-Arg-Val-Tyr-Tyr-) in which angiotensinogen secreted in the liver is degraded by the enzyme renin produced in the kidney.
It acts on Ile-His-Pro-Phe-His-Leu and converts it to angiotensin II (Asp-Arg-Val-Tyr-Ile).
-His-Pro-Phe). And this angiotensin II contracts the vascular wall smooth muscle to increase blood pressure,
Furthermore, it acts on the adrenal cortex to promote aldosterone secretion. In addition, the enzyme kallikrein present in plasma decomposes a protein called kininogen to produce bradykinin which dilates blood vessels and lowers blood pressure, and this bradykinin is decomposed and inactivated by the action of ACE. Thus, on the one hand, ACE produces a pressor peptide (angiotensin II),
On the other hand, it degrades antihypertensive peptides (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).

Many synthetic substances such as captopril (D-2-methyl-3-mercaptopropanoyl-L-proline) are known as ACE inhibitors, including several peptide inhibitors obtained from snake venom, Of these, captopril has already been put to practical use as an oral antihypertensive. In recent years, ACE inhibitors have also been found in microorganisms and various foods, and their practical use as antihypertensive agents has been studied.

Also, AC derived from trypsin hydrolyzate of milk casein
It has been proposed to isolate an E inhibitor or treat it further with a peptidase and use it as a hypotensive agent (JP-B-60-23085, JP-B-60-23086, JP-B-60-2308).
7, JP-A Nos. 61-36226 and 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 No. H10-163,837). 62-169732).

On the other hand, corn protein converts prolamin to 50-60.
%, Which contains 35-40% glutelin, and the main component, prolamin, is called zein. Zein is classified into three types, α, β, and γ (J. Cereal Sci. 5 , 117 (1987)).
γ-zein contains a repeating structure having a basic unit of Val-His-Leu-Pro-Pro-Pro (Nucleic Acid
s Res. 13 (5), 1493 (1985)).

[Problems to be Solved by the Invention] There is always a 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.

Therefore, the present invention provides an angiotensin converting enzyme inhibitor which has an excellent angiotensin converting enzyme inhibitory activity, is extremely safe, can be supplied inexpensively and in large quantities, and is useful not only as a pharmaceutical product but also as a functional food. With the goal.

[Means for Solving the Problems] As a result of various searches for substances having ACE inhibitory activity, the present inventors have found that zein in corn protein, which is the most inexpensive and most common food protein, has a certain degree of hydrolysis by a specific protease. The inventor has found that the hydrolyzate has angiotensin converting enzyme inhibitory activity, and that the above problem can be solved by using the hydrolyzate.

That is, the present invention relates to an angiotensin converting enzyme inhibitor containing, as an active ingredient, a hydrolyzate of zein with thermolysin or papain, and a peptide having a molecular weight of 200 to 5,000 and a content of 30% by weight or more on a solid basis as an active ingredient. I will provide a.

Hereinafter, the present invention will be described in detail. The zein used in the present invention may be α-zein, β-zein or γ-zein alone or a mixture of 2 or 3. These zeins may be commercially available, or may be separated from corn protein obtained in the process of producing corn starch, or separated from the corn protein by a known method (Plant Ph.
ysiol., 80 , 623 (1986)) may be α-, β-, or γ-zein. Reference Examples 1 and 2 show production examples of γ-zein and β-zein, respectively.

The enzyme used in the present invention is thermolysin or papain.

Next, the conditions for the hydrolysis are not particularly limited as long as a hydrolyzate containing at least 30% by weight (hereinafter abbreviated as%) of the peptide having a molecular weight of 200 to 5,000 with respect to the total hydrolyzed solid is obtained. There is no.

Specifically, the substrate concentration may be any value as long as it can be stirred and mixed at the time of the reaction, but it is preferably carried out at a protein concentration within a range of 2 to 20%, which facilitates stirring. The amount of the enzyme to be added varies depending on the titer of the enzyme to be used, but is usually 0.01% or more per protein, preferably 0.1 to 10%. Although the pH and temperature of the reaction vary depending on each enzyme, the respective optimum pH and the vicinity of the optimum temperature may be used. Thermolysin has a pH of 6 to 9, and a temperature of 30 to 70 ° C.
8. A temperature of 30-60 ° C is appropriate. The reaction time varies depending on the type of the enzyme, the amount added, the reaction temperature and the reaction pH, and is not constant, but is usually about 1 to 40 hours.

The hydrolysis reaction is stopped by heating the reaction mixture or changing the pH of the enzyme by an organic acid such as citric acid or malic acid, or an inorganic acid such as hydrochloric acid or phosphoric acid, or a pH change caused by the addition of an alkali such as sodium hydroxide or potassium hydroxide. It can be carried out according to a known method such as inactivation, filtration of an enzyme by an ultrafiltration membrane or the like.

The reaction mixture has an excellent ACE inhibitory action as it is, but can be used after removing solids by a known solid-liquid separation method such as centrifugation or filtration. The hydrolyzate, with or without removing solids, can be used as a liquid as it is, or, if necessary, after decolorization, desalting, etc., is subjected to known drying such as spray drying or freeze drying. Depending on the method, it can be used as a powder. By the above-described operation, the content of the peptide having a molecular weight of 200 to 5,000 is 30% by weight or more based on the total hydrolyzed solid, and zein (α
-, Β-, and γ-zein each alone or an arbitrary mixture thereof). As is clear from the above description, the hydrolyzate may be solid or liquid.

The hydrolyzate of the present invention may be one obtained by cutting a high molecular weight portion and / or a low molecular weight portion (amino acid or the like) by ultrafiltration, gel filtration or the like. For example, a transmembrane fraction obtained by ultrafiltration of the hydrolysis-completed solution of α-zein using a membrane having a cut-off molecular weight of 10,000 using a membrane having a molecular weight cut-off of 10,000, substantially contains peptides having a molecular weight of more than 5,000 and peptides and amino acids having a molecular weight of less than 200. Although hardly contained, such a fraction or a purified or powdered product thereof can also be used as an active ingredient of the ACE inhibitor of the present invention.

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

Formulations as injections usually include a sterile aqueous solution. Formulations 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.

Oral formulations are formulated in a form suitable for absorption by the gastrointestinal tract. Tablets, capsules, granules, fine granules and powders are commonly used pharmaceutical auxiliaries such as binding (syrup, gum arabic,
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, carboxymethyl cellulose, etc.), wetting agents, (sodium lauryl sulfate, etc.). 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 oral solutions may contain conventional additives such as preservatives (methyl or propyl p-hydroxybenzoate, sorbic acid, and the like).

Although the amount of the hydrolyzate in the ACE inhibitor of the present invention can be variously changed, it is usually 1 to 1 as the hydrolyzate (solid).
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 hydrolyzate does not add toxicity. For example, the acute toxicity of the hydrolyzate (high molecular weight cut) obtained in Examples 1 and 2
LD ₅₀ (rat, oral administration)> 5 g / kg.

Further, since the hydrolyzate of the present invention has an advantage that it does not adversely affect the living body even when ingested in large amounts, it is used as it is, or it is added with various nutrients and the like, or is contained in food or drink to lower blood pressure, It may be eaten as a functional food or health food having a function of preventing hypertension. That is, for example, by adding nutrients such as various vitamins and minerals,
For example, liquid foods such as nutritional drinks, soy milk, and soups, solid foods of various shapes, and powders can be used as they are or added to various foods. The content and intake of the present hydrolyzate in the ACE inhibitor of the present invention as such functional foods and health foods may be the same as those in the above-mentioned pharmaceuticals.

[Examples] Next, the present invention will be described with reference to Examples. In Examples,% indicates% by weight.

Example 1 α-zein (Sigma) was adjusted to 50% in a concentration of 2%.
In addition to Tris-HCl (pH 8.0), trypsin, chymotrypsin (both from P-L Biochemicals), subtilisin Carlsberg (Sigma) or papain (Sigma) to a concentration of 0.2% was added at 37 ° C. The enzyme reaction was performed for 20 hours.

Separately, 50 mM Tris-HCl (pH 8.0) containing 5 mM calcium chloride
Combination with 0.2% Thermolysin (Sigma), 0.0
Using a combination of 57N hydrochloric acid and 0.2% pepsin (PL Biochemical) or a combination of 10 mM borate (pH 12.0) and 0.2% alkaline protease (Toyobo Co., Ltd.) An enzymatic reaction was performed in the same manner as described above.

The enzyme reaction solution was subjected to ultrafiltration (using Millipore's Morkat, molecular weight cut off 10,000), and the fraction that passed through the membrane was collected.

Next, the ACE inhibitory activity of each of the above fractions was measured by the following method. First, 5 g of rabbit langacetone powder was dissolved in 50 ml of 0.1 M sodium borate buffer (pH 8.3), centrifuged at 40,000 G for 40 minutes, and the supernatant was further treated with the above buffer. Diluted 5 times,
An angiotensin converting enzyme solution was obtained.

0.03 ml of the above-mentioned fraction was placed in a test tube, and 0.25 ml of hypryl histidylleucine (final concentration 5 m) was used as a substrate.
M, containing 300 mM NaCl), 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 then 1.5 ml of ethyl acetate was added.The absorption value at 228 nm of hypoprilic acid extracted in ethyl acetate was measured, and this was defined as the enzyme activity. . Under these conditions, the absorbance at 228 nm when the inhibitor of the present invention was not contained was approximately 0.35.

A plurality of such experiments were performed, and the inhibition rate was calculated by the following equation.

A: Absorption value at 228 nm when no inhibitor was contained B: Absorption value at 228 nm when an inhibitor was added The inhibitor concentration I ₅₀ at an inhibition rate of 50% was determined.

In the case of thermolysin and papain, the ratio of the peptide having a molecular weight of 200 to 5,000 and the ratio of the peptide having a molecular weight of 200 to 5,000 with respect to the total amount of α-zein before hydrolysis were examined by the following methods.

That is, a standard product having a known molecular weight was passed through a column (13 mm × 820 mm) of Sephadex G-25 (fine) (solvent: 0.1 M ammonium acetate), and the relationship between the molecular weight and the elution position was determined. The used standards and their molecular weights are as follows.

Ribonuclease A 13,700 aprotinin 6,500 dynorphin A 2,147 bacitracin 1,411 oxytocin 1,007 glycylglycylalanine 203 Next, from the gel filtration pattern obtained by flowing the above-mentioned membrane-passing fraction under the same conditions, the membrane-passing fraction is either thermolysin or papain. When the enzyme (1) was used, the peptide was substantially composed of peptides having a molecular weight of 200 to 5,000, and it was revealed that the peptide contained more than 5,000 peptides, less than 200 peptides, and almost no amino acids. Next, by dividing the amount of nitrogen (by the micro-Kjeldahl method) in the transmembrane fraction obtained for thermolysin and papain by the amount of nitrogen of α-zein before decomposition, a peptide having a molecular weight of 200 to 5,000 relative to the starting α-zein was obtained. The ratio was determined.

 The results obtained are summarized in Table 1.

Example 2 β-zein (prepared in Reference Example 2) or γ-zein (prepared in Reference Example 1) was added to 5% to a concentration of 2%.
In addition to 50 mM Tris-HCl (pH 8.0) containing M calcium chloride,
Thermolysin (Sigma) was added to a concentration of 0.2%, and an enzyme reaction was performed at 37 ° C for 20 hours.

The ACE inhibitory activity (I ₅₀ ) of the obtained enzyme reaction solution was determined in the same manner as in Example 1 using the fraction obtained by subjecting the obtained enzyme reaction solution to ultrafiltration in the same manner as in Example 1. 50 μg / ml for zein, γ
-In the case of zein, the ratio of a peptide having a molecular weight of 200 to 5,000 to solid zein (based on solid content) is 110 µg / ml and β-
It was 75% for zein and 58% for γ-zein.

Example 3 Intravenous Injection The transmembrane fraction (lyophilized product) obtained in Example 1 was used for 20-
The filtrate is dissolved in 100-fold (volume / weight) sterile physiological saline and aseptically filtered through a filter (pore size: 0.45 μm) to give an injection.

Membrane-passing fraction obtained in Example 1 (lyophilized product) 5 parts Hydroxypropylcellulose 1 part Lactose 12.9 parts Potato starch 1 part Magnesium stearate 0.1 part Prepare 20 parts of a 60% ethanol aqueous solution containing 1 part of hydroxypropylcellulose. Then, 5 parts of the present hydrolyzate and 12.9 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. Tablets are made with a tablet machine.

Reference Example 1 Preparation of γ-zein The preparation was performed according to the method of Esen (J. Cereal Sci. 5 , 117 (1987)). Ground corn (ordinary seed dent corn) 10
To 0 g, 5 times the volume of 60% isopropyl alcohol water containing 1% 2-mercaptoethanol was added, and the mixture was stirred at 60 ° C. for 2 hours to extract all zein fractions. 3,000g of mixture
And centrifuged for 10 minutes at the same volume.
An aqueous solution of sodium acetate was added, and the mixture was adjusted to pH 6 with a small amount of acetic acid and allowed to stand at 4 ° C overnight to precipitate α- and β-zein. 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 lyophilized to give a pale yellow powder. As a result, 0.4 g of γ-zein was obtained.

Reference Example 2 Preparation of β-Zein The preparation was performed according to the method of Esen (the same reference as Reference Example 1).

The mixture of α- and β-zein precipitated in Reference Example 1 was collected, and 5 times the volume of 60% isopropyl alcohol water containing 2% 2-mercaptoethanol was added to redissolve both α- and β-zein. 3 times the amount of isopropyl alcohol was added to precipitate only α-zein. 10 at 3,000G
The supernatant was collected by centrifugation for minutes and then dialyzed and freeze-dried in the same manner as in the case of γ-zein to obtain 0.6 g of β-zein as a pale yellow powder.

[Effects of the Invention] According to the present invention, it is possible to provide an ACE inhibitor inexpensively and in large quantities from zein in corn protein, which is the most common food protein.

Since the present ACE inhibitor is derived from food protein, it is extremely safe even when ingested in large quantities, and therefore does not show any side effects.

An ACE inhibitor containing a hydrolyzate of zein exhibits a higher ACE inhibitory activity than the ACE inhibitor containing a hydrolyzate of a fish protein or a soybean protein described in JP-A-62-169732.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 9/99 C12P 21/06 C12P 21/06 A61K 37/18 ABU (72) Inventor Shinsuke Miyoshi 2-chome, Hinode, Funabashi City, Chiba Prefecture No. 20-2 Akira Sansan Dormitory (72) Inventor Fumio Fukui 1-2117 Nakadai, Narita-shi, Chiba Examiner Seiko Tamura (56) References Patent 2662682 (JP, B1) (58) Fields investigated ( Int.Cl. ⁶ , DB name) A61K 38/01-38/58 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. Angiotensin converting enzyme inhibitor comprising as an active ingredient a hydrolyzate of zein by thermolysin or papain, wherein the hydrolyzate having a molecular weight of 200 to 5,000 and a peptide content of 30% by weight or more on a solid basis is contained as an active ingredient. Agent.