JP2952830B2 - Antihypertensive - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an antihypertensive agent, and particularly to a medicament effective for the prevention and treatment of hypertension, which has been significantly increasing in recent years and for which measures are desired. Or, it relates to a hypotensive agent expected to be used for food.

(Prior Art) Hypertension has been increasing in recent years, and countermeasures are desired. Conventionally, many substances effective for prevention and treatment of hypertension are known. On the other hand, there is an increasing interest in functional foods that are useful for preventing diseases through daily intake of food.

From such a viewpoint, research for finding a substance having a blood pressure lowering action from highly safe foods or hydrolysates thereof has been actively conducted recently. Examples of this are endless, for example, ACE (angiotensin converting enzyme) derived from trypsin hydrolyzate of milk casein.
An antihypertensive agent obtained by isolating an inhibitor or further treating with a peptidase (JP-B-60-23085, JP-B-60-23086, JP-B-60-23086)
60-23087, JP-A-61-36226, JP-A-61-36227),
An antihypertensive agent containing a peptide obtained by hydrolyzing fish or soybean protein with a serine protease derived from a bacterium belonging to the genus Bacillus, a metal protease derived from the genus Bacillus, or a thiol protease derived from a plant, and a component of corn protein Val contained in γ-zein
An amino acid having a C-terminal amino acid sequence of Leu-Pro-Por obtained by hydrolysis with thermolysin from a repeating structural part having a basic unit of -His-Leu-Pro-Pro-Pro, and further obtained by enzymatic or acid degradation. Many proposals have been made, such as an ACE inhibitor containing a peptide having a polymerization degree of 3 to 5 as an active ingredient.

On the other hand, the presence of glutenin and gliadin as components to form gluten in wheat protein, glutenin is insoluble in ethanol and has the property of dissolving in dilute acid.When hydrolyzing this with the enzyme chymotrypsin, Ser-Gln −
It is known that the octapeptide Gln-Gln-Gln-Pro-Pro-Phe is produced in large quantities (Z. Lebensm Unters For
sch 187: 27-34, (1988)).

(Problems to be Solved by the Invention) The present invention provides an antihypertensive agent which has an excellent blood pressure lowering effect, 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. The purpose is to provide.

(Means for Solving the Problems) As a result of various searches for substances having an antihypertensive action, the present inventors found that glutenin in wheat protein, one of the most inexpensive and most common food proteins, was converted to the enzyme chymotrypsin. Certain peptides obtained by hydrolysis in the above, further, in the hydrolysis treatment, the component that yields the highest yield is Se
It has been found that the octapeptide r-Gln-Gln-Gln-Gln-Pro-Pro-Phe has a particularly strong hypotensive effect,
The present invention has been completed.

That is, the present invention according to claim 1 is a hypotensive agent comprising a peptide having a specific molecular weight range obtained by hydrolyzing wheat glutenin with chymotrypsin as an active ingredient. Se produced in large quantities in
It is a hypotensive agent containing an octapeptide, r-Gln-Gln-Gln-Gln-Pro-Pro-Phe, as an active ingredient.

 Hereinafter, the present invention will be described in detail.

The wheat glutenin used in the present invention is obtained, for example, by suspending a fine powder of gluten in a 50-70% (v / v) aqueous ethanol solution and leaving it to stand. It can be prepared by a known method such as extraction with 0.2% sodium hydroxide, followed by neutralization and precipitation with acetic acid.

The enzyme chymotrypsin used is an enzyme secreted into the small intestine from the pancreas of a vertebrate, and a commercially available product derived from bovine pancreas or the like can be used as it is.

The hydrolysis of glutenin by chymotrypsin is usually carried out simply in water or in a buffer (eg Tris-HCl buffer,
(Phosphate buffer). The concentration of glutenin in the treatment solution may be any as long as it is within a range in which the reaction can be stirred and mixed, but desirably, the concentration is in the range of 2 to 20% (w / v), which facilitates stirring.

The amount of oxygen chymotrypsin to be added depends on its titer, but is usually 0.01% by weight or more per protein, preferably
0.1 to 10% by weight is suitable. The pH and temperature of the reaction solution may be around the optimal pH of chymotrypsin and around the optimal temperature.
The temperature is suitably 6 to 10, more preferably 7 to 8, and the temperature is suitably 20 to 70 ° C, more preferably 30 to 50 ° C. The pH during the reaction is adjusted with an aqueous solution of sodium hydroxide, hydrochloric acid or the like, if necessary.

The reaction time is not constant because it varies depending on factors such as the substrate concentration, the amount of enzyme added, the reaction temperature, and the reaction pH, but is usually about 1 to 50 hours.

The hydrolysis reaction can be stopped by heating the hydrolyzate or changing the pH by adding an organic acid such as citric acid or malic acid, an inorganic acid such as hydrochloric acid or phosphoric acid, or an alkali such as sodium hydroxide or potassium hydroxide. , Etc., and filtration of the enzyme by an ultrafiltration membrane or the like, according to a known method.

The chymotrypsin hydrolyzate of wheat glutenin referred to in the present invention, wherein the content of the fraction having a molecular weight of 10,000 or less is 30% by weight or more based on solid content, wheat glutenin is as described above Molecular weight obtained by separating (eg, by ultrafiltration, gel filtration, etc.) a hydrolyzate obtained by hydrolysis or a liquid obtained by solid-liquid separation (eg, centrifugation, filtration, etc.) of this as required. Or 10,0
Liquids containing the following fractions or concentrates thereof (eg, concentrates, spray-dried products, freeze-dried products, fluidized-bed dried products, etc.) are included. In the present invention, the solid content based on the solid content means a dried product obtained by evaporating and drying water, and more specifically, a product dried at 105 ° C. for 4 hours.

The hydrolyzate of the present invention exerts an excellent blood pressure lowering effect when administered orally or parenterally (intravenous injection, intraluminal injection, etc.). Ser-Gln-Gl obtained by fractionation from this
The octapeptide n-Gln-Gln-Pro-Pro-Phe and its salt show a particularly strong blood pressure lowering effect.

One method for separating the above octapeptide from the glutenin hydrolyzate is gel filtration (for example, using Sephadex G-25), and cation exchange resin (for example, DO
The fraction containing the octapeptide is subjected to WEX 50WX2), and the fraction containing the octapeptide is applied to an anion exchange resin (for example, using Durrum DAX2-20) to separate a single octapeptide.

The organic chemical synthesis methods include a liquid phase method and a solid phase method.
In the former, an amino acid protected with a Boc (tertiary butyloxycarbonyl) group or the like is dissolved in DMF (dimethylformamide), and is dissolved overnight in the presence of DCC (dicyclohexylcarbodiimide) and HOBt (1-hydroxybenzotriazole). The reaction is carried out, and the carboxy terminal is sequentially bonded. In the latter, a peptide synthesizer (Model 430A) manufactured by Applied Biosystems,
Amino acids are sequentially extended on a PAM (phenylacetamidomethyl) resin, and the resin and various protecting groups are cleaved with hydrogen fluoride or the like to obtain a target octapeptide.

The wheat gliadin hydrolyzate of claim 1 of the present invention and the peptide of claim 2 (hereinafter, sometimes collectively referred to as the “substance of the present invention”) may be used as they are, or usually at least one of them. Pharmaceutical compositions are added and used as pharmaceutical compositions. All of these can be administered parenterally (intravenous injection, rectal administration, etc.) or orally to mammals including humans, and can be produced in a form suitable for each administration method.

Acute toxicity of a substance of the present invention are all by LD ₅₀ (rat oral)> 5g / kg.

In addition, since the substance of the present invention does not adversely affect the whole even when collected in large amounts, it has a function of lowering blood pressure and preventing hypertension by itself or by adding various nutrients or the like to food or drink. It may be eaten as a functional food or health food.

(Example) Next, the present invention will be described with reference to examples.

Example 1 Wheat gluten (manufactured by Kanto Chemical Co., Ltd.) is 60% of 20-fold volume
(V / v) Dispersed in an aqueous ethanol solution and left for 5 hours with occasional stirring. The supernatant was removed by centrifugation, and the insoluble matter was added with 5 volumes of 60% (v / v) aqueous ethanol solution,
Stirred for hours. After this operation was repeated once, 10 times the volume of 0.2% sodium hydroxide was added to the separated insoluble material, and the mixture was stirred and extracted for 2 hours. After removing the insoluble matter by centrifugation, this was neutralized with acetic acid. The precipitate was dispersed in 5 volumes of water, and then washed twice by centrifugation, followed by lyophilization to obtain a dried glutenin.

100 g of glitenin (solid content) obtained as above is added to pure water 2
And chymotrypsin (derived from bovine pancreas manufactured by Kanto Chemical Co., Ltd.) was added thereto, followed by stirring at 50 ° C. for 15 hours.
During the reaction, the pH was maintained at 8.0 by appropriately adding 2N sodium hydroxide. Next, 1 g of chymotrypsin was further added and reacted at 50 ° C. for 24 hours while maintaining the pH at 8.0. After the reaction, the reaction solution was heated at 105 ° C. for 5 minutes in an autoclave to inactivate the enzyme.

The obtained hydrolyzate was centrifuged at 5,000 rpm for 10 minutes, and the supernatant was subjected to ultrafiltration using Amicon PM-10 (ultrafiltration membrane, molecular weight cut off 10,000, Amicon), and the filtrate was filtered. Was freeze-dried to obtain 94.2 g (solid content) of a fraction concentrate.

The molecular weight distribution of the present hydrolyzate was measured as follows.
That is, a standard product having a known molecular weight was previously passed through a column (1.3 φ × 820 cm) 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 2,147 bacitracin 1,411 oxytocin 1,007 glycylglycylalanine 203 Next, from the gel filtration pattern obtained by flowing the membrane permeated fraction under the same conditions, the membrane permeated fraction is substantially 200 to 5,000 in molecular weight.
, And it was apparent that it contained almost no peptide having a molecular weight of more than 5,000 and less than 200.

Example 2 Ser-Gln-Gln-Gln-Gln from glutenin hydrolyzate
Preparation of -Pro-Pro-Phe 500 mg of the hydrolyzate obtained in Example 1 was dissolved in 0.1 M N-ethylmorpholineacetic acid (pH 6.5), and Sephadex G-2
Eluting with the same solvent after adding to the column of 5 Superfine. Fractions having a large blood lowering action were collected and concentrated under reduced pressure. The column processing conditions in this case are as follows.

Column φ3 × 100 cm Elution solvent 0.1 M N-ethylmorpholine acetic acid (pH 6.5) Flow rate 25 ml / hr Next, the active fraction fractionated by the Sephadex G25 was converted to a cation exchange resin column (Dowex 50WX2 200 ~
400mesh) and fractionated as follows.

Column φ0.9 × 110cm Elution solvent 0.2M pyridine-acetate buffer (pH3.1) 100ml Same as above 300ml buffer and 1.0M pyridine-acetate buffer (pH4.
2) Linear concentration gradient elution with 300 ml 1.0 M pyridine-acetate buffer (pH 5.0) 150 ml Flow rate 32 ml / hr The fraction with high blood pressure lowering fractionated by the above cation exchange resin chromatography was concentrated, and the following conditions were applied. Was applied to a column of an anion exchange resin Durrum DAX2-20.

Column 0.9φ × 60cm Elution solvent N-ethylmorpholine 15ml, α-picoline 20ml, pyridine 10ml and water 955ml buffer solution adjusted to pH 9.4 with acetic acid 20ml Buffer solution adjusted to pH 8.4 from solution of the above composition Solution 30 ml Buffer solution prepared by adjusting the solution of the above composition to pH 6.5 40 ml 0.5 M acetic acid 60 ml 2.0 M acetic acid 100 ml Flow rate 20 ml / hr Next, the active fraction obtained above was separated by Sephadex.
Add to the LH-20 column and desalinate.

 The processing conditions in this case are as follows.

Column 2.0φ × 60cm Elution solvent Pure water flow rate 0.4ml When the sample desalted as described above is dried under reduced pressure, a white powder substance is obtained.

This substance was dissolved in 6N hydrochloric acid, heated at 110 ° C. for 24 hours under vacuum, and analyzed by an amino acid analyzer. The following results were obtained.

Amino acid molar ratio to serine Ser (serine) 1.00 Glu (glutamic acid) 4.20 Pro (proline) 2.15 Phe (phenylalanine) 1.10 Further, the white powder sample was subjected to Edman degradation by an automatic peptide structure analyzer. The eight generated PTH amino acids were identified by high performance liquid chromatography and the primary sequence of the amino acids was determined. Ser-Gln-Gln-Gln-Gln
It was confirmed to have a structure of -Pro-Pro-Phe.

Example 3 Ser-Gln-Gln-Gln-Gln-Pro-Pro-P by synthetic method
he preparation Applied Biosystems peptide synthesizer in (430A type) of 0.5 mmol of _{Boc-L-Phe-OCH 2} -PAM resin and each 2 mM Boc-L-Pro, Boc- L-Pro, Bo
c-L-Gln, Boc-L-Gln, Boc-L-Gln, Boc-LG
ln and Bor-L-Ser were attached, and L-Ser-L-Gln-L-Gln-L-Gln-L-Gln
The -L-Pro-L-Pro- L-Pre-O-OH 2 -PAM were synthesized. Next, the synthetic peptide resin was introduced into a hydrogen fluoride treatment apparatus manufactured by Peptide Research Laboratories, 1.5 ml of anisole was added, and then 10 ml of liquid hydrogen fluoride was introduced. -20 ° C for 30 minutes, 0 ° C
After reaction for 30 minutes, hydrogen fluoride was removed under reduced pressure, and the peptide was washed three times with anhydrous ether and chloroform alternately, then dissolved in 60 ml of 2N acetic acid and lyophilized. By this method, Ser-Gln-Gln-Gln-Gln-Pro-Pro-Phe white powder
213 mg were obtained.

For the structural analysis of this peptide, amino acid analysis after hydrolysis with 6N hydrochloric acid (by Hitachi L-8500 type amino acid analyzer), mass spectrometry (by JEOL JMX-DX303 type), amino acid sequence analysis (Shimadzu protein sequencer PSQ −
1 system). Table 1 shows the results
Shown in

Test Example 1 Blood pressure lowering effect of chymotrypsin hydrolyzate of glutenin Spontaneously hypertensive rats (SHR) of 10 weeks old (Japan Rat Co., Ltd., ♂, body weight 240 to 280 g, 6 animals per group) were used. Dissolve the sample in physiological saline, 2g / kg as active ingredient
-Body weight was administered intraperitoneally. Saline was intraperitoneally administered as a control.

Blood pressure was measured using a non-invasive sphygmomanometer TK-350 (manufactured by Unicom) for rats and mice.
It was measured by the ff method. Table 2 shows the results.

As shown in Table 2, administration of glutenin chymotrypsin hydrolyzate showed a significant hypotensive effect over 1 to 5 hours.

Test Example 2 Peptide "Ser-Gln-Gln-Gln-Gln-Pro-Pro-Ph
e) Blood pressure lowering effect The test was carried out in the same manner as in Test Example 2 except that the dose of the peptide was 150 mg / kg-body weight. Table 3 shows the results.

As seen in Table 3, the peptides Ser-Gln-Gln-Gln
By administration of -Gln-Pro-Pro-Phe, a remarkable blood pressure lowering effect was observed for 1 to 5 hours after the administration.

(Effects of the Invention) According to the present invention described in claims 1 and 2, a blood pressure effect agent having excellent effects is provided by using wheat glutenin which is an inexpensive protein material as a material.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) A61K 38/00, 35/78 CA (STN)

Claims (2)

(57) [Claims] 1. An antihypertensive agent comprising, as an active ingredient, a chymotrypsin hydrolyzate of wheat glutenin, wherein the hydrolyzate having a molecular weight of 10,000 or less has a content of 30% by weight or more on a solid basis. 2. The peptide Ser-Gln-Gln-Gln-Gln-Pro-
An antihypertensive comprising Pro-Phe and salts thereof as an active ingredient.