JPH04187643A - Hypotensor - Google Patents

Abstract

PURPOSE:To obtain a hypotensor expected to be useful as a pharmaceutical agent or food additives effective for the prevention and treatment of hypertension by hydrolyzing wheat glutenin with chymotrypsin and using the obtained peptide having a molecular weight falling within a specific range as an active component. CONSTITUTION:Wheat glutenin is hydrolyzed with chymotrypsin at pH 6-10 (preferably 7-8) and 20-70 deg.C (preferably 30-50 deg.C). The hydrolyzed liquid or a liquid separated from the hydrolyzed liquid by solid-liquid separation is fractionated by ultrafiltration, get-filtration, etc., to obtain a hydrolyzed product containing >=30wt.% (in terms of solid) of a fraction having a molecular weight of <=10,000. The octapeptide of formula produced as a major product by the above hydrolysis treatment exhibits especially strong antihypertensive action.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to antihypertensive agents, and in particular to pharmaceuticals effective for the prevention and treatment of hypertension, which has been on the rise in recent years and for which countermeasures are desired. Or it relates to antihypertensive agents that are expected to be used in food.

(Prior Art) Hypertension has been on the rise in recent years, and countermeasures are desired. Many substances have been known to be effective in preventing and treating hypertension. On the other hand, interest in functional foods that help prevent diseases through daily food intake is increasing.

From this point of view, research has been actively conducted recently to find substances that have a blood pressure lowering effect from highly safe foods or their hydrolysates.

There are too many examples to list. For example, an ACE (anno-otenone-converting enzyme) inhibitor derived from a tryptic hydrolyzate of milk casein may be isolated, or it may be further treated with peptidase to produce an antihypertensive agent (especially Kosho 60-23
No. 085, No. 60-23086, No. 60-23087
JP-A-61-36226, JP-A No. 61-36227), obtained by hydrolyzing fish protein or soybean protein with serine protease derived from Bacillus bacteria, metalloprotease derived from Bacillus genus, or thiol protease derived from plants. An antihypertensive agent containing a peptide as an active ingredient, contained in γ-zein, a component of corn protein.
Val-His-Leu-Pro-Pro-Pr.

The C-terminal amino acid sequence, Leu-Pro-Pr, is obtained by hydrolyzing the repeating structure with a basic unit of
Many proposals have been made, including ACE inhibitors containing peptides with an amino acid polymerization degree of 3 to 5 as active ingredients.

On the other hand, glutenin and glutinin exist in wheat protein as gluten-forming components, and glutenin has the property of being insoluble in ethanol and soluble in dilute acid, and when hydrolyzed with the enzyme chymotrypsin, Ser-Gl
n-Gln-Gln-Gln-Pro-Pro-Phe
It is known that a large amount of octapeptide is produced (Z, Lebensm tlnters Fors
ch 187: 27-34° (1988no.

(Problems to be Solved by the Invention) The present invention provides a hypotensive agent that 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 medicine but also as a functional food. The purpose is to provide.

(Means for Solving the Problems) As a result of various searches for substances that have antihypertensive effects, the present inventors discovered that glutenin in wheat protein, which is the cheapest and most common food protein, was synthesized using the enzyme chymotrypsin. A certain peptide obtained by hydrolysis with
r-Gin-Gln-Gln8Gin-Pro-Pro
They discovered that the octapeptide Phe has a particularly strong antihypertensive effect, and completed the present invention.

That is, the present invention according to claim 1 is an antihypertensive agent containing as an active ingredient a peptide having a specific molecular weight range obtained by hydrolyzing wheat glutenin with chymotrypsin, and claim 2
The present invention described herein is based on Se, which is produced in large amounts in the hydrolysis solution.
r-Gln-Gln-Gln-Gln-Pro-Pro
-Phe is an antihypertensive agent containing an octapeptide as an active ingredient.

The present invention will be explained in detail below.

Wheat glutenin used in the present invention can be obtained by, for example, suspending fine gluten powder in a 50-70% (V/V) ethanol aqueous solution, leaving it to stand, and then separating the undissolved matter by an appropriate means such as centrifugation. It can be prepared by a known method such as extraction with % sodium hydroxide, followed by neutralization with acetic acid and precipitation.

The enzyme chymotrypsin used is an enzyme secreted from the pancreas of animals to the small intestine, and a commercially available product originating from bovine pancreas or the like can be used as is.

Hydrolysis of glutenin by chymotrypsin is usually
It is carried out simply in water or in a buffer (eg Tris-HCl buffer, phosphate buffer). The concentration of glutenin in the treatment solution is
Any amount may be used as long as reaction specific mixing is possible, but it is preferable to use a range of 2 to 20% (w/v) so that stirring is easy.

The amount of the enzyme chymotrypsin added varies depending on its potency, but is usually 0.01% by weight or more, preferably 0.61 to 10% by weight, based on protein. p[ of the reaction solution
(The temperature should be around the optimal pH 1 of chymotrypsin, and the pH is more preferably 7-8 than 6-10.
The temperature is preferably 20 to 70°C, more preferably 30 to 50°C. I)Fl during the reaction is adjusted by using an aqueous sodium hydroxide solution, hydrochloric acid, etc., if necessary.

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

The hydrolysis reaction can be stopped by heating the hydrolyzate or by 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. This can be carried out according to known methods, such as inactivation of the enzyme using a filtrate, separation of the enzyme using an ultrafiltration membrane, etc.

The chymotryptic hydrolyzate of wheat glutenin referred to in the present invention, in which the content of the fraction with a molecular weight of 10,000 or less is 30% by weight or more on a solid basis, includes wheat glutenin as described above. The hydrolyzed liquid obtained by hydrolysis or solid-liquid separation (if necessary)
A liquid obtained by fractionating (e.g., ultrafiltration, gel filtration, etc.) a liquid obtained by centrifugation, filtration, etc.) containing a fraction with a molecular weight of 10,000 or less, or a concentrate thereof (e.g., concentrated liquid, spray-dried product, freeze-dried product, fluidized bed-dried product, etc.). In the present invention, the solid content on a solid content basis means a dried product obtained by evaporating water and drying, or more specifically, refers to a product dried at 105° C. for 4 hours.

The above hydrolyzate of the present invention can be administered orally or parenterally (intravenous injection, intraperitoneal injection, etc.) to provide excellent results.
Ser-Gi, which exhibits a two-sided pressure-lowering effect, is present in large amounts in this hydrolyzate and can be obtained without fractionation.
n-Gln-Gln-Gln-Pro-Pro-Phe
The octapeptide and its salts exhibit particularly strong hypotensive effects.

One method for fractionating the above-mentioned octapeptide from the glutenin hydrolyzate is gel filtration (e.g. using Sephadex G-25) and cation exchange resin (e.g. D
OWEX 50W
0 use) etc. to separate single octapeptides.

In addition, as organic chemical synthesis methods, liquid phase method and solid phase method 2N
In the former, an amino acid protected with a Boc (ternary butyloxycarbonyl) group, etc. is dissolved in DMF (dimethylformamide), and DCC (nonoclohequinolcarphonoimite) and HOBt (1-hydroquinobenzotriazole) are dissolved in DMF (dimethylformamide). ) in the presence of 200 mg/kg for a day and night to sequentially connect from the carboquino terminal side. In addition, the latter uses a peptide synthesizer (430) manufactured by Applied Bionostems.
Amino acids are sequentially extended on a PAM (phenylacetamide methyl) resin using a method such as Type A), and the resin and various protective groups are cleaved using hydrogen fluoride or the like to obtain the desired octapeptide.

The wheat gliadin hydrolyzate of claim 1 of the present invention and the peptide of claim 2 (hereinafter, both may be collectively referred to as the "substance of the present invention") may be used as is or usually with at least one pharmaceutical agent. Add auxiliary agents and use as a pharmaceutical composition.

All of these are parenteral (intravenous injection, rectal administration, etc.)
Alternatively, it can be administered orally to mammals including humans, and can be formulated into a form suitable for each administration method.

The acute toxicity of all substances of the invention is LD 5o (oral administration in rats)>59/9.

Furthermore, since the substance of the present invention does not have any adverse effects on the living body even when ingested in large quantities, it can be used as it is, with various nutrients added to it, or incorporated into food and drink to exert blood pressure lowering and hypertension prevention functions. It can be eaten as a moisturizing bifunctional food or a health food.

(Example) Next, the present invention will be explained by examples.

Example 1 Wheat gluten (manufactured by Kanto Kagaku Co., Ltd.) 20 times 8 60%
(v/v) It was dispersed in an aqueous ethanol solution and allowed to stand for 5 hours with occasional stirring. Remove the supernatant by centrifugation, and add 60% (v/v) ethanol aqueous solution 5 to undissolved matter.
The mixture was stirred for 1 hour. After repeating this operation once more, 8 lO times 0.2% sodium hydroxide was added to the separated insoluble matter, and extraction was performed with stirring for 2 hours. After removing insoluble matter by centrifugation, this was neutralized with acetic acid. A washing operation in which the precipitate was dispersed in 58 volumes of water and then centrifuged was performed twice, and then freeze-dried to obtain a dried product of glutenin.

0 g (solid content) of glutenin IO obtained above was added to 2 ml of pure water.
Chymotrypsin (derived from Tsun Pancreas, manufactured by Kanto Kagaku Co., Ltd.) was added thereto, and the mixture was stirred and reacted at 50° C. for 15 hours. During the reaction, 2N sodium hydroxide was added dropwise to adjust the pH.
was kept at 8.0. Then add another 1 g of chymotrypsin.
Further, the reaction was carried out at 50° C. for 24 hours while keeping the pH at 8.0. After the reaction, the reaction solution was placed in an autoclave at 105
The enzyme was inactivated by heating at ℃ for 5 minutes.

The obtained hydrolyzate was centrifuged at 5,000 rpm for 10 minutes, and the supernatant was collected using Aiicon PM-1
0 (ultrafiltration membrane, molecular weight cutoff 10,000, Amicon), and the filtrate was freeze-dried to obtain a fraction concentrate of 94.29 (solid content).

The molecular weight distribution of this hydrolyzate was measured as follows. That is, a Sephadex G-25 (fine) column (
A standard product of known molecular weight was applied in advance to 1,3φX HOcz) to determine the relationship between molecular weight and elution position (solvent: 0.1M ammonium acetate). The standard products used and their molecular weights are as follows.

Ribonuclease A 13,700 Aprotinin 6.5QO Guinolphin
2.147 Bacitracin
1.411 Oxyton 1,00
7glycylglycylalanine 203 Next, from the gel filtration pattern obtained by passing the membrane-permeable fraction under the same conditions, it was found that the membrane-permeable fraction had a molecular weight of substantially 200 to 5. It was clear that it consisted of OQO peptides and contained almost no peptides with a molecular weight of more than 5,000, as well as peptides and amino acids with a molecular weight of less than 200.

Example 2 Ser-Gln-Gln-G from glutenin hydrolyzate
Preparation Example 1 of ln-Gln-Pro-Pro-Phe
500 μ of the hydrolyzate obtained in 0. Sephadex G-2 dissolved in IMN ethylmorpholinoacetic acid (pH 6,5)
5 Superfine column and eluted with the same solvent. The fractions that have a large blood pressure lowering effect are collected and concentrated under reduced pressure. Note that the column processing conditions in this case are as follows.

Column φ3xlOOc Shoulder Elution solvent 0.1M N-ethylmorpholine acetic acid (pH
6,5) Flow rate 25 IQ/hr Next, the active fraction fractionated with Sephadex G-25 was subjected to a cation exchange resin column (Dowex 50
WX2200-400mesh) column, and fractionation was performed as follows.

Column φ0.9 x 110cs + elution solvent 00
．． 2M pyrinone-acetate buffer (pH 3,1) 100
+u2 ■Linear concentration gradient elution with the same buffer 300xQ and 1.0M pyridine-acetate buffer (pH 4,2) 3[11]a+ff■1. OM pyridine-acetate buffer (pH 5,0) 150 passes Flow rate 32 passes/hr The fraction with a large blood pressure lowering effect fractionated by the above cation exchange resin chromatography was concentrated, and further anion exchange resin DurrumDAX2-20 was used under the following conditions.
column.

Column 0.9φ Buffer solution 30 adjusted to PF18.4
1. Buffer solution prepared by adjusting the above composition to pH 6.5 40xQ 00.5M acetic acid 60 Pass. ■2. OM acetic acid 1001 Flow rate 20IQ/hr Next, the active fraction obtained above was treated with Sephadex L.
Add to H-20 column and desalt.

The processing conditions in this case are as follows.

Column: 20φX 60cm Elution solvent: Pure water Flow rate: 0.4ff (! When a sample desalted by surface writing is dried under reduced pressure, a white powder substance is obtained.

When this substance was dissolved in 6N hydrochloric acid and heated under vacuum for 24 hours on IIO'C, it was analyzed using an amino acid analyzer, and the following results were obtained.

Amino acid Molar ratio to serine 5et (serine) 100Glu (glutamic acid)
4.20Pro (Proline) 21
5Phe (phenylalanine) 110 Further, the white powder sample was subjected to Edman degradation using an automatic peptide structure analyzer. The eight PTH amino acids produced were identified by high performance liquid chromatography and the primary sequence of the amino acids was determined.
It was confirmed that it has the structure n-Pro-Pro-Phe. Example 3 Synthesis of Ser-Gln-Gln-Gln-Gl
Preparation of n-Pro-Pro-Phe Bebutite synthesis equipment manufactured by Applied Pionostems (
430A) with 0.5 mmol of Boc-L-Phe-0
-CH, -PAM resin and 2 mmol each of Boc-L-
Pro, Boc-L-Pro, Boc-L-Gin
, Boa-L-Gln, Boc-L-Gln, B
Attach oc-L-Gln and Boc-L-Ser, and
By anhydrous symmetry method by CC, L-3er-L-Gln
-L-Gln-L-Gln-L-Gln-L-Pro-
L-Pro-L-Phe-0-CH, -PAM was synthesized. Next, the above synthetic peptide resin was introduced into a hydrogen fluoride treatment equipment manufactured by Peptide Institute, and after adding Anisole 15 Pass, 10z (l) of liquid hydrogen fluoride was introduced. -20℃ 30
After 30 min of reaction at 0 °C, the hydrogen fluoride was removed under reduced pressure and the peptide was washed with anhydrous ether and chloroform alternately for 3 min.
It was washed twice, then dissolved in 2N acetic acid 60 toge and lyophilized. By this method, Ser-Gln-Gln-Gln-
Gln-Pro-Pro-Phe white powder 213+y
I got it.

In order to analyze the structure of this peptide, we performed amino acid analysis (by Hitachi L-8500 amino acid analyzer) after hydrolysis with 6N hydrochloric acid, mass spectrometry (by JEOL JMX-DX 303 model), and amino acid sequence Fr (Ryuri Protein Noc. (by Encer PSQ-1 system) were carried out respectively. The results are shown in Table 1.

Gln-Gln-Pro-Glu(4,3) 9
59 (M+H)Pro-Phe Pro(2
,2) Decomposition analysis test example! Antihypertensive effect of chymotrypsin hydrolyzate of glutenin The animals used were IO-week old spontaneously hypertensive rats (SHR) (Nippon Rat Co., Ltd., male, 6 rats in a 9.1 group, weight 240-28). The sample was dissolved in physiological saline, and 29 kg/kg of the active ingredient was intraperitoneally administered. As a control, physiological saline was administered intraperitoneally for 1 day.

Blood pressure is measured using a non-invasive sphygmomanometer for rats and mice TK-35i)
(manufactured by Unicom), and T
It was measured by the a1l-Cuff method. The results are shown in Table 2.

Table 2 Control area 190±6 195”5 197:7 190
+7 1! to: 8 administration area 1) 20 (1+8 185i
9*Ig4i7*184f101F38+5 Note 1) Hydrolyzate 2ti/ky single body weight administration group unit is zxHg (Heiheiken standard deviation) *marked has a risk rate of 5% and is significantly different from the control group.As seen in Table 2. , administration of chymotribunone hydrolyzate of glutenin had a significant hypotensive effect over a period of 1 to 5 hours.

Test Example 2 Peptide rSer-Gin-Gin-Gln-Gln
The test was conducted in the same manner as in Test Example 2, except that the dose of the hypotensive peptide of -Pro-Pro-PheJ was I 50 zg/kg body weight. The results are shown in Table 3.

Table 3 Control area 19 North 4202 ± 8196 ± 8197 Sat 719
As shown in Table 1 and Table 3, peptide Ser-Gln-Gl
By administering n-Gln-Gln-Pro-Pro-Phe, a significant blood pressure lowering effect was observed for 1 to 5 hours after administration.

(Effect of the invention) Claim! and 2 EC! According to the present invention described above, a blood pressure effecting agent having excellent effects is provided by using wheat glutenin, which is an inexpensive protein raw material, as a raw material.

Patent applicant: Showa Sangyo Co., Ltd.

Claims (2)

[Claims] (1) An antihypertensive agent containing as an active ingredient a chymotrypsin hydrolyzate of wheat glutenin in which the content of fractions with a molecular weight of 10,000 or less is 30% by weight or more on a solid basis. (2) Peptide Ser-Gln-Gln-Gln-Gl
A hypotensive agent containing n-Pro-Pro-Phe and its salts as active ingredients.