JPH06211689A - Sedative and ataractic food - Google Patents

Abstract

PURPOSE:To obtain a sedative ataractic food comprising a peptide having sedative action. CONSTITUTION:This sedative or ataractic food is characterized by including a peptide expressed by formulas I and/or II or its salt. H-Leu-Lys-Lys-I le-Ser- Glh-Arg-Tyr-Gln-Lys-Phe-Ala-Leu-Pro-Gin-Tyr-OH... (I) and H-Val-Tyr-Gin-His- Gin-Lys-Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys-Thr-Lys-Val-Ile-Pro-Tyr-Va l-Arg- Tyr-R... (II) (R denotes OH or Leu-OH).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sedative and a tranquilizer food comprising a peptide isolated from a pepsin degradation product of α-casein or a salt thereof.

[0002]

As a peptide having a sedative effect, a hydrolyzate of casein is known (Japanese Patent Laid-Open No. 1821/1990).
No. 69 publication). However, what is being raised here is a mixture of peptides having a molecular weight of 1,100 to 5,400, and the sedative effect of a single fragment peptide has not been clarified.

[0003]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a sedative and tranquilizing food comprising a peptide or its salt isolated from a pepsin degradation product of casein.

[0004]

Means for Solving the Problems The above-mentioned object is to include a peptide represented by the following general formula (I) or the following general formula (II) or a salt thereof, and a sedative and tranquilizing food. Achieved by

H-Leu-Lys-Lys-Ile-Ser-Gln-Arg-Tyr-G ln-Lys-Phe-Ala-Leu-Pro-Gln-Tyr-OH (I)

H-Val-Tyr-Gln-His-Gln-Lys-Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys-Thr-Lys-Val-Ile-Pro-Tyr-Val -Arg-Tyr-R ... (II) (In the formula, R represents OH or Leu-OH)

In the present specification, amino acids, amino acid residues, amino acid derivatives, peptides and protecting groups are IUPAC-
Symbols recommended by the IUB Biochemistry Nomenclature Committee (Biochemistr
y, Volume 11, p. 1726, 1972, IUPAC Pure Appl., Volume 40, 31
(See p. 7, 1974) or symbols commonly used in the art, and the L symbols for L-amino acids and their residues are omitted. Such symbols are as follows, for example.

Val: Valine, Tyr: Tyrosine, Gl
n: glutamine, His: histidine, Lys: lysine, Ala: alanine, Met: methionine, Pro:
Proline, Trp: tryptophan, Ile: isoleucine, Thr: threonine, Arg: arginine, Le
u: leucine, Ser: serine, Phe: phenylalanine, Boc: t-butyloxycarbonyl, Br-
Z: p-bromobenzyloxycarbonyl, Cl-Z:
p-chlorobenzyloxycarbonyl, Bzl: benzyl, Mts: mesitylene-2-sulfonyl, Bom: benzyloxymethyl, CHO: formyl.

The peptide represented by the general formula (I) or a salt thereof is disclosed in JP-A-3-255094, and the peptide represented by the general formula (II) or a salt thereof is disclosed in It can be obtained by purification or synthesis from the production method disclosed in Japanese Patent Publication No. 3-255095, specifically, hydrolyzed casein. The obtained peptide can be directly used as a lyophilized product for the purpose of a sedative. There is no problem in terms of effect even if taken as it is or dissolved in a suitable beverage. Also,
It is also possible to preliminarily include it in gums, candy, beverages, etc., and ingest it as a food for tranquilization.

[0010]

The present invention will be described in more detail with reference to the following examples.

Example 1 (Production of sedative) The hydrochloride of the peptide represented by the above formula (I) was produced as follows. First, using a peptide synthesizer (Model 431A manufactured by Applied Biosystems),
It was synthesized at room temperature by the solid phase method.

Boc-Tyr (Br-Z) -Pam resin
Starting from 0.5 mmol, Boc-Leu, Boc-
Lys (Cl-Z), Boc-Ile, Boc-Ser
(Bzl), Boc-Gln, Boc-Arg (Mt
s), Boc-Tyr (Br-Z), Boc-Phe,
Using each protected amino acid of Boc-Ala and Boc-Pro, deprotection cleaning of N-terminal Boc Neutralization cleaning Condensation cleaning cleaning Unreacted N-terminal amino group acetylation cleaning,
Each step was repeated to sequentially extend the peptide chain on the resin from the C end.

After the final Boc-Leu condensation reaction,
After drying, 1.96 g of protected peptide-Pam-resin was obtained.

To 1 g of the obtained protected peptide-Pam-resin, 1 ml of thioanisole and 0.5 ml of ethanedithiol were added, and the mixture was stirred at room temperature for 10 minutes. Next, while cooling with ice, 10 ml of trifluoroacetic acid was slowly added, and after stirring for 10 minutes, 1 ml of trifluoromethanesulfonic acid was added dropwise and the temperature was returned to room temperature.
Stir for 25 minutes. After the reaction was completed, cold diethyl ether 100m
The flask was filled with l and stirred for 1 minute to precipitate the peptide and Pam-resin. This was filtered with a Polyflon filter PF060 (manufactured by Advantech) and washed with cold diethyl ether (-40 ° C). Approximately 30 peptides of trifluoroacetic acid were added to 300 ml of cold diethyl ether prepared in advance.
It was dissolved in ml, added dropwise, and precipitated again. This is 3 μm
It was filtered with a porous PTFE membrane (manufactured by Advantech), washed with cold diethyl ether (-40 ° C), and the peptide was dissolved in 2N acetic acid and freeze-dried. Crude peptide 1.15 g was obtained from the protected peptide-Pam-resin 1.96 g.

The obtained crude peptide was purified by high performance liquid chromatography under the following conditions.

[0016]

A fraction having a retention time of about 6.9 minutes was collected to obtain 0.63 g of the title peptide / trifluoroacetate salt.

Peptide trifluoroacetate 254.0 mg
Was dissolved in 10 ml of 0.1N hydrochloric acid and then freeze-dried. After drying, water 5
The procedure of adding ml and freeze-drying was repeated 3 times to obtain 219.0 mg of the title peptide / hydrochloride salt.

Test Example 1 (Spontaneous Motility Inhibition Test of Peptide of Example 1) The freeze-dried product of the hydrochloride salt of the peptide of formula (I) obtained in Example 1 was dissolved in purified water, and this solution was 20 ml / kg. In percentage of weight
Oral administration was carried out to ddY male mice (5 weeks old) (the dose of the sample was 100,200,500 mg / kg body weight). Control group mice were administered with purified water alone at a rate of 20 ml / kg body weight.

Next, the experimental animal momentum measuring device ACTY-303
(Biomedica) was used to measure the spontaneous locomotor activity of the mice. The mean and standard error of the locomotor activity of each group during 30 minutes from 30 minutes to 60 minutes after sample administration were calculated, and the significant difference between the locomotor activity of the sample-administered group and the control group A test was performed. The test results are shown in Table 1.

[0021]

[Table 1]

The peptide of the present invention significantly suppressed locomotor activity at a dose of 500 mg / kg body weight.

Example 2 (Production of sedative) The hydrochloride of the peptide represented by the above formula (I) was produced as follows.

Starting from 0.5 mmol of Boc-Leu-Pam resin, Boc-Val and Boc-Tyr (Br-
Z), Boc-Gln, Boc-His (Bom), B
oc-Lys (Cl-Z), Boc-Ala, Boc-
Met, Boc-Trp (CHO), Boc-Pro,
Boc-Ile, Boc-Thr (Bzl), Boc-
Using each protected amino acid of Arg (Mts), Example 1
Was synthesized by the solid-phase method in the same manner as.

After completion of the final Boc-Val condensation reaction,
After drying, 2.66 g of protected peptide-Pam-resin was obtained.

1 g of the protected peptide-Pam-resin obtained in a round bottom flask previously cooled to -5 to 0 ° C with a freezing agent was added to 0.2 g of ethanedithiol, 0.8 ml of m-cresol, 3 ml of dimethyl sulfide, 5 ml of trifluoroacetic acid and 1 ml of trifluoromethanesulfonic acid was added in this order, and the mixture was stirred at -5 to 0 ° C for 3 hours. This is polyflon filter PF060
(Manufactured by Advantech) and then washed with cold diethyl ether (-40 ° C). The solid on the filter was dried with a vacuum pump. To this dried product, 1 ml of thioanisole and 0.5 ml of ethanedithiol were added, and the mixture was stirred at room temperature for 10 minutes. Next, under ice-cooling, 10 ml of trifluoroacetic acid was slowly added, and after stirring for 10 minutes, trifluoromethanesulfonic acid was added.
After dropping 1 ml, the mixture was returned to room temperature and stirred for 25 minutes. After the reaction was completed, fill the flask with 100 ml of cold diethyl ether,
The mixture was stirred for 1 minute to precipitate the peptide and Pam-resin. This is filtered with a polyflon filter PF060,
It was washed with cold diethyl ether (-40 ° C). The peptide was dissolved in about 30 ml of trifluoroacetic acid in 300 ml of cold diethyl ether prepared in advance, added dropwise, and precipitated again. This was filtered with a 3 μm-pore PTFE membrane (manufactured by Advantech), washed with cold diethyl ether (-40 ° C.), the peptide was dissolved in 2N acetic acid, and then freeze-dried. Protected peptide-Pam-resin 2.
From 66 g, 1.6 g of crude peptide was obtained.

The obtained crude peptide was purified by high performance liquid chromatography under the following conditions. Column: R-355-15 ODS (50 x 500 mm,
Yamamura Chemical Co., Ltd.) Elution: Water-acetonitrile-trifluoroacetic acid (80:20:
0.1) to water-acetonitylyl-trifluoroacetic acid (70: 3
(0: 0.1) linear concentration gradient (concentration gradient is applied over 25 minutes.) Flow rate: 100 ml / min Detection: Absorbance at 235 nm Fraction with retention time of about 5.7 minutes was collected and the title peptide trifluoroacetate salt was collected. Obtained.

Peptide trifluoroacetate 245.7 mg
Was dissolved in 10 ml of 0.1N hydrochloric acid and then freeze-dried. After drying, 5 ml of water was added and freeze-drying was repeated 3 times to obtain 207.7 mg of the title peptide / hydrochloride.

Test Example 2 (Spontaneous Momentum Suppression Test of Peptide Hydrochloride of Example 2) A freeze-dried product of the hydrochloride of the peptide of Formula (II) obtained in Example 2 in which R is Leu-OH is purified water. DdY male mouse (5 weeks old) at a rate of 20 ml / kg body weight
Was orally administered (the dose of the sample was 100, 200, 500 mg / kg body weight). Control group mice were administered with purified water alone at a rate of 20 ml / kg body weight.

Next, the experimental animal momentum measuring device ACTY-303
(Biomedica) was used to measure the spontaneous locomotor activity of the mice. The mean value and standard error of the locomotor activity of each group for 30 minutes immediately after the administration of the sample were calculated, and a significant difference test was performed between the locomotor activity of the sample-administered group and the control group. The test results are shown in Table 2.

[0031]

[Table 2]

The peptide of the present invention is 200 mg / kg body weight and 500 m
A dose of g / kg body weight significantly suppressed locomotor activity.

Example 3 (Manufacture of chewing gum for tranquilization) A chewing gum for tranquilizing was manufactured with the composition shown below.

[0034]

[Table 3]

Preparation method Chewing gum base and starch syrup kept at 60 ° C. were put into a kneader and kneaded for 10 minutes, and 1/3 amount of powdered sugar and glucose were added for 5 minutes, and then 1/3 amount of powdered sugar. It was charged and kneaded for 5 minutes. Next, peppermint oil,
Powdered peppermint flavor and the peptide of the present invention
It was mixed with / 3 amount of powdered sugar and then added, and kneaded for 5 minutes to obtain a gum mix. This gum mix in the usual way,
It was extruded, rolled, cut, packaged and commercialized as plate gum.

Example 4 (Production of tranquilizing candy) A tranquilizing candy was produced with the composition shown below.

[0037]

[Table 4]

Manufacturing method Granulated sugar, starch syrup and a small amount of water were put into a preliminary dissolution pot and boiled once to completely dissolve the granulated sugar, and this mixture was pumped into a vacuum cooker at a vacuum degree of 4
After boiling at 60 mmHg and temperature of 130 ℃, put it in the take-out pot,
Transferred to a mixing kettle. Next, apple flavor, citric acid, and gardenia dye were added and mixed well, then spread on a cooling plate, and when the temperature reached 110 ° C. or lower, the peptide of the present invention was mixed to obtain candy mass. This candy mass was put into a corn roller by a conventional method, sized, stamped, and then packaged to produce a granular hard candy.

Example 5 (Production of sports beverage for tranquilization) [0039] A sports beverage for tranquilization was produced with the following composition.

[0040]

[Table 5]

Production Method All raw materials were dissolved in water, passed through a filter and heated to 85 ° C., and then cooled to 40 ° C. by a filling and winding cooler to produce a product.

[0042]

EFFECTS OF THE INVENTION Since the sedative of the present invention exhibits a sedative effect by oral ingestion and has low toxicity, it can be orally ingested arbitrarily without the need for a doctor as in the case of intravenous injection. It has the advantage of

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Claims (4)

[Claims] 1. A sedative comprising a peptide represented by the following general formula (I) or a salt thereof. H-Leu-Lys-Lys-Ile-Ser-Gln-Arg-Tyr-G In-Lys-Phe-Ala-Leu-Pro-Gln-Tyr-OH ... (I) 2. A food for tranquilization comprising the peptide according to claim 1 or a salt thereof. 3. A sedative containing a peptide represented by the following general formula (II) or a salt thereof. H-Val-Tyr-Gln-His-Gln-Lys-Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys-Thr-Lys-Val-Ile-Pro-Tyr-Val-Arg- Tyr-R ... (II) (In the formula, R represents OH or Leu-OH) 4. A food for tranquilization comprising the peptide according to claim 3 or a salt thereof.