JPH07224093A - Peptide - Google Patents

Abstract

PURPOSE:To provide new peptides originated from soybean protein, having specific amino acid sequences, exhibiting immunological activation actions such as a phagocytosis-stimulating action and an active oxygen production- stimulating action, and having new food and medicine uses. CONSTITUTION:The new peptide having an amino acid sequence of formula I is obtained by dissolving separated soybean protein in water, separating a soluble fraction by a centrifugal method, boiling the separated soluble fraction, adding trypsin to the treated fraction, adjusting the pH of the fraction to 7.6 with a 1N NaOH solution, digesting the fraction for 5 hrs at 37 deg.C, boiling the treated fraction for stopping the digestion, centrifuging the boiled solution, lyophilizing the separated supernatant, applying the obtained trypsin-digested product to an anion exchange column, collecting non-adsorbed fractions, allowing the collected fractions to pass through a hydrophobic column, and subsequently developing the treated fractions with an acetonitrile linear concentration gradient containing 0.1% trifluoroacetic acid. Further, the peptide is treated with an enzyme and subsequently fractionated by a chromatography to obtain new peptides of formulas II-X having immunological activation actions.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide having physiological activity and a pharmaceutical composition containing the peptide.

[0002]

2. Description of the Related Art It is known that peptides having various physiological activities are derived from food proteins. These peptides originating from foods can be expected to be safe for the living body. In general, many bioactive peptides derived from food proteins have an unexpected amino acid sequence unlike the endogenous bioactive peptides, and there are also multiple functions, so many have not been confirmed yet. It seems that there is. One of the physiologically active actions of peptides is a phagocytosis promoting action. This action is important as an initial reaction of the biological defense by neutrophils and macrophages when external foreign substances such as bacteria enter the living body. The present inventor previously identified Gln-Arg-Pro-Arg and His-Cys-Gln-Ar contained in soybean glycinin A _1a subunits as peptides that activate phagocytosis.
We have demonstrated that g-Pro-Arg enhances phagocytosis of macrophages and reported it as a novel peptide.

[0003]

After that, further research was conducted on peptides derived from soybean protein, and it was found that phagocytosis-promoting activity was newly added to soybean protein enzymatic degradation products, and other excellent physiological properties were obtained. We have found peptides with active effects. Further, the similar compound was also examined, and the present invention was completed by finding out that it has the same effect as the above peptide. Therefore, the present invention provides a novel peptide having the above-mentioned physiological activity and a novel food and pharmaceutical use of the peptide.

[0004]

The present invention relates to peptides of SEQ ID NOs: 1 to 10 represented by the following formula. SEQ ID NO: 1: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg SEQ ID NO: 2: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn -Lys-Pro-Gly SEQ ID NO: 3: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro SEQ ID NO: 4: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val -Asn-Lys SEQ ID NO: 5: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn SEQ ID NO: 6: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val SEQ ID NO: 7: Met- Ile-Thr-Leu-Ala-Ile-Pro SEQ ID NO: 8: Met-Ile-Thr-Leu-Ala-Ile SEQ ID NO: 9: Met-Ile-Thr-Leu-Ala SEQ ID NO: 10: Met-Ile-Thr-Leu Furthermore, the present invention relates to a pharmaceutical composition having an immune system activating action, which comprises the peptides represented by SEQ ID NOS: 1 to 10 and pharmaceutically acceptable salts thereof as active ingredients. The peptides represented by SEQ ID NOs: 1 to 10 have a sequence length of 13 to
4, each has a sequence type: amino acid topology: linear sequence type: peptide.

The peptide of SEQ ID NO: 1, which is an active ingredient of the present invention, is obtained by enzymatically hydrolyzing soybean protein, and the obtained digestion product is chromatographed by DEAE-cellulose column and further by octadesilyl (ODS) column and phenethyl column. High performance liquid chromatography (HP
It can be obtained by fractionating by LC).

The present inventors have found that the peptide of SEQ ID NO: 1 obtained by enzymatic decomposition of soybean protein has a phagocytosis-promoting action, and as a result of further research based on the peptide, the results of SEQ ID NO: 1 It was found that the peptides and the peptides of SEQ ID NOS: 2 to 10, which are analogs thereof, also have an action of promoting phagocytosis, and the peptides of SEQ ID NOS: 1 to 8 have an action of promoting active oxygen production.
Therefore, the peptides of SEQ ID NOS: 1 to 10 can be used as a pharmaceutical composition having an immune system activating action.

The peptide of the present invention can be obtained by the above-mentioned method to obtain the peptide of SEQ ID NO: 1, which can be further hydrolyzed and fractionated to obtain the peptides of SEQ ID NO: 2-10. The peptides of SEQ ID NOS: 1 to 10 can also be obtained by known methods.

[0008]

PRODUCTION EXAMPLES Examples of the production of the peptides of SEQ ID NOS: 1 to 10 of the present invention are shown below, but the invention is not limited to these examples.

Production Example 1 Preparation of peptide of SEQ ID NO: 1 by digestive enzyme A. The outline of the preparation method using digestive enzymes is shown in Table 1 below. Table-1 Outline of preparation method from soybean protein Soybean protein ↓ Trypsin digestion ↓ Column chromatography (DEAE cellulose column) ↓ HPLC (ODS-column) ↓ HPLC (phenethyl column) ↓ HPLC (ODS-column) ↓ Met-Ile-Thr -Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg

B. Preparation of peptide of SEQ ID NO: 1 from digest of soybean protein Dissolve 50 g of isolated soybean protein in 800 ml of water, 3000 rpm ×
The soluble fraction was separated by centrifugation for 20 minutes. Boil this for 30 minutes, add 500 mg of trypsin, and add 1N-NaOH to obtain pH =
It was adjusted to 7.6 and digested at 37 ° C. After 5 hours, the digestion was stopped by boiling, and the tryptic digest (solid content: 23.8 g) was obtained by freeze-drying the supernatant obtained by centrifugation at 10,000 rpm for 15 minutes. 100 mg of this is DEA
E-cellulose column (DE-52, Whatman, carrier 2 ml) was loaded and 20 mM Tris-HCl buffer (pH = 7.
When the non-adsorbed fraction was developed by 8) and 1 ml of the non-adsorbed fraction was collected, the active peptide was contained in the fraction eluted at a position of 2 to 3 ml. Next, this fraction was subjected to ODS-column (Co
smosil 5C ₁₈ -AR, 20 × 250mm, made by Nakarai Tesque),
Followed by a phenethyl column (4.7 x 250 mm, Develosil Ph
AT-5, manufactured by Nomura Chemical Co., Ltd., and linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid (1% / min)
). Elution of active peptides by these columns was carried out by using 33-34% acetonitrile (see FIG. 1),
The position was 36 to 37% (see FIG. 2). Further, this active fraction was subjected to ODS-column (Cosmosil 5C ₁₈ -AR, 4.6 × 150).
mm, manufactured by Nacalai Tesque, Inc., and developed with a linear concentration gradient (1% / min) of acetonitrile containing 10 mM phosphate buffer (pH = 7.4). Eluted as a single peak (see FIG. 3). When the structure was determined with a protein sequencer, the active peptide was Met-Ile-Thr-Leu-Ala-
It was found to be Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg.

Production Example 2 Preparation of peptide of SEQ ID NO: 1 by chemical synthesis method (Fmoc method) 0.3 g of Fmoc-Arg (Pmc) -resin having a substitution rate of 0.70 meq / g was added to S.
Set it in the reaction vessel of AM2 peptide synthesizer (Biosearch), add deblocking solution (piperidine: toluene: dimethylformamide (DMF) = 30: 35: 35) and stir to mix 9-fluorenylmethoxycarbonyl (Fmoc). ) Group was removed. This resin was added to dichloromethane (DCM): D
After washing with MF = 1: 1, 4-fold equivalents of Fmoc-Arg (Pmc) -resin of hydroxybenzotriazole (HOBT) and Fm
Coupling was performed by adding oc-Gly. After the reaction is completed, D
After washing with CM: DMF = 1: 1, Fmoc-Gly-Arg (Pmc) -resin was obtained. Similarly, synthesize up to the N-terminal Met,
Washed with CM and methanol, Met-Ile-Thr (Bzl) -Leu-Ala
A peptide of -Ile-Pro-Val-Asn (Trt) -Lys (Boc) -Pro-Gly-Arg (Pmc) -resin was obtained. Deprotection agent (trifluoroacetic acid: water: thioanisole: ethanediol: ethylmethylsulfide: phenol = 82: 5: 5: 3: 2:
3) was added and the mixture was allowed to stand at room temperature for 4 hours. The separated resin was then filtered, washed with ether and lyophilized to give crude Met-Il.
e-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg was obtained. This is an ODS-column (Cosmosil 5C ₁₈ -AR, 20 ×
250 mm, manufactured by Nacalai Tesque, Inc., and purified by HPLC with a linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid to obtain about 200 mg of a pure product. In the above, Pmc represents a 2,2,5,7,8-pentamethylchroman-6-sulfonyl group, Bzl represents a benzyl group, Trt represents a trityl group, and Boc represents a t-butoxycarbonyl group.

Production Example 3 Preparation of peptide of SEQ ID NO: 2 by chemical synthesis method (Fmoc method) 0.4 g of Fmoc-Gly-resin having a substitution rate of 0.55 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 12-residue peptide-resin. Deprotection agent (trifluoroacetic acid:
Ethanediol: anisole = 94: 1: 5),
It was left at room temperature for 1 hour. Hereinafter, in the same manner as in Production Example 2, about
150 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-
Lys-Pro-Gly was obtained.

Production Example 4 Preparation of peptide of SEQ ID NO: 3 by chemical synthesis method (Fmoc method) 0.3 g of Fmoc-Pro-resin having a substitution rate of 0.71 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 11-residue peptide-resin. Then, in the same manner as in Production Example 3, about 150 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro was prepared.
Got

Production Example 5 Preparation of peptide of SEQ ID NO: 4 by chemical synthesis method (Fmoc method) 0.4 g of Fmoc-Lys-resin having a substitution rate of 0.46 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 10-residue peptide-resin. Then, in the same manner as in Production Example 3, about 150 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys was obtained.

Production Example 6 Preparation of peptide of SEQ ID NO: 5 by chemical synthesis method (Fmoc method) 0.3 g of Fmoc-Asn-resin having a substitution rate of 0.70 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 9-residue peptide-resin. Then, in the same manner as in Production Example 3, about 150 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn was obtained.

Production Example 7 Preparation of Peptide of SEQ ID NO: 5 by Enzyme The peptide of SEQ ID NO: 5 can also be obtained by enzymatic hydrolysis of the peptide of SEQ ID NO: 1. The enzyme used is a metalloendopeptidase that specifically cleaves the peptide bond of Asn-Lys contained in the peptide of SEQ ID NO: 1,
Use asparaginyl endopeptidase or the like. An example of the preparation method is shown below, but the enzyme used is not limited to this example. SEQ ID NO: 1 peptide 20 mg
In addition, 500U of metalloendopeptidase (manufactured by Seikagaku Corporation)
100 mM glycine-NaOH buffer (pH = 10.0)
2 ml was added and incubated at 70 ° C. for 5 hours. After stopping the reaction, this solution was used for ODS-column (Cosmosil5C ₁₈ -AR,
4.6 x 150 mm) and purified by HPLC with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. The fraction eluted near 34% acetonitrile was freeze-dried to obtain about 10 mg of the pure peptide of SEQ ID NO: 5.

Production Example 8 Preparation of peptide of SEQ ID NO: 6 by chemical synthesis method (Fmoc) 0.4 g of Fmoc-Val-resin having a substitution rate of 0.52 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 8-residue peptide-resin. Thereafter, in the same manner as in Production Example 3, about 100 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro-Val was obtained.

Production Example 9 Preparation of peptide of SEQ ID NO: 7 by chemical synthesis method (Fmoc) 0.3 g of Fmoc-Pro-resin having a substitution rate of 0.71 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 7-residue peptide-resin. Thereafter, in the same manner as in Production Example 3, about 100 mg of pure Met-Ile-Thr-Leu-Ala-Ile-Pro was obtained.

Production Example 10 Preparation of peptide of SEQ ID NO: 8 by chemical synthesis method (Fmoc) 0.4 g of Fmoc-Ile-resin having a substitution rate of 0.55 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 6-residue peptide-resin. Thereafter, in the same manner as in Production Example 3, about 100 mg of pure Met-Ile-Thr-Leu-Ala-Ile was obtained.

Production Example 11 Preparation of peptide of SEQ ID NO: 9 by chemical synthesis method (Fmoc) 0.3 g of Fmoc-Ala-resin having a substitution rate of 0.70 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 5-residue peptide-resin. Thereafter, in the same manner as in Production Example 3, about 50 mg of pure Met-Ile-Thr-Leu-Ala was obtained.

Production Example 12 Preparation of peptide of SEQ ID NO: 10 by chemical synthesis method (Fmoc) 0.3 g of Fmoc-Leu-resin having a substitution rate of 0.58 meq / g was added to SAM2.
It was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in 2 to obtain a corresponding 4-residue peptide-resin. Thereafter, in the same manner as in Production Example 3, about 50 mg of pure Met-Ile-Thr-Leu was obtained.

Table 2 shows the amino acid compositions of the peptides of SEQ ID NOS: 1 to 10, the elution position of HPLC, and the Rf value of thin layer chromatography. The natural product (Production Example 1) and the synthetic product (Production Example 2) of SEQ ID NO: 1 were found to be the same product from the results. Therefore, in the following, studies are conducted using synthetic products.

[0023] In the above table, M is Met, I is Ile, T is Thr, L is Leu,
A is Ala, P is Pro, V is Val, D is Asp, K is Lys,
G represents Gly and R represents Arg. * 1 The value after hydrolysis with 6N-HCl at 110 ° C. Therefore, the peptide composition Asn is detected as Asp. * 2 ODS-column (Cosmosil 5C ₁₈ -AR, 4.6 x 150 mm
) Is used for measurement with a linear concentration gradient of acetonitrile. * 3 It was developed at room temperature using a Kiesel gel plate made by Merck. The composition of the developing solvent is butanol: acetic acid: pyridine: water = 15: 3: 10: 12.

[0024]

[Test Example] The pharmacological activity of the peptide of the present invention will be shown below by a test example. Test Example 1 Measurement of phagocytosis activity Test method: Physiological saline (PBS) containing phosphate buffer was added to human peripheral blood, and blood cells were washed by centrifugation at 1000 rpm for 5 minutes, and then 4 × with PBS. A suspension of 10 ⁶ cells / ml was prepared. 100 μl of this solution was taken, 10 μl of the peptide solution dissolved in PBS was added, the mixture was incubated at 37 ° C. for 10 minutes, and then obsonated with human peripheral blood.
10 µl of a fluorescent labeled latex bead solution of × 10 ⁸ cells / ml was added, and the mixture was further incubated for 5 minutes. Stop the reaction with PBS containing EDTA, separate the blood cells by centrifugation,
An ammonium chloride hemolyzing agent was added to this to cause hemolysis, and white blood cells were obtained. This was suspended in PBS containing ETDA and then measured by flow cytometry.

Results: Numerical values are shown as magnifications relative to the control (PBS).

Test Example 2 Measurement of Active Oxygen Production Test Method: After washing adult human peripheral blood with PBS,
Erythrocytes were hemolyzed and dissolved in HEPES-physiological saline buffer to prepare a 2 × 10 ⁶ / ml neutrophil suspension. This liquid 125μ
355 μl of HEPES-physiological saline buffer, 10 μl of 5 mM cytochrome-C solution, and 10 μl of an aqueous peptide solution were added to 1 l, and the mixture was incubated at 37 ° C. for 15 minutes. After 15 minutes, the incubation was stopped by quenching in ice. This solution was centrifuged, and active oxygen production was calculated from the absorbance of the supernatant at 550 nm and 468 nm. The amount of active oxygen produced was calculated by the following formula. Active oxygen production = (A ₁ −A ₂ ) × k where A ₁ is the absorbance at wavelength 550 nm A ₂ is the absorbance at wavelength 468 nm k is the coefficient (the coefficient when using a 1 cm cell for measurement is 95) .)

Results: The peptide concentration was 30 μM, and the numerical value was shown as the ratio to the control (water).

[Brief description of drawings]

FIG. 1 is a chart in which the absorbance of the component was measured when the active fraction obtained by loading the enzymatic hydrolyzate on a DEAE-cellulose column was eluted by HPLC with an ODS column.

FIG. 2 is a chart in which the absorbance of the component was measured when the active fraction of HPLC by the ODS column of FIG. 1 was eluted by HPLC by the phenethyl column.

FIG. 3 is a chart in which the absorbance of the component when the active fraction of HPLC by the phenethyl column of FIG. 2 is eluted by the neutral ODS column.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display area C07K 7/08 8318-4H C12P 21/06 9282-4B

Claims (4)

[Claims] 1. A peptide having the physiological activity of SEQ ID NOs: 1 to 10 represented by the following formula. SEQ ID NO: 1: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg SEQ ID NO: 2: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn -Lys-Pro-Gly SEQ ID NO: 3: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro SEQ ID NO: 4: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val -Asn-Lys SEQ ID NO: 5: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn SEQ ID NO: 6: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val SEQ ID NO: 7: Met- Ile-Thr-Leu-Ala-Ile-Pro SEQ ID NO: 8: Met-Ile-Thr-Leu-Ala-Ile SEQ ID NO: 9: Met-Ile-Thr-Leu-Ala SEQ ID NO: 10: Met-Ile-Thr-Leu 2. An immune system activating action which comprises, as an active ingredient, one or more selected from the group consisting of the peptides represented by SEQ ID NOS: 1 to 10 and pharmaceutically acceptable salts thereof according to claim 1. A pharmaceutical composition having. 3. The pharmaceutical composition according to claim 2, wherein the immune system activating action is a phagocytosis promoting action. 4. An active ingredient comprising one or two or more of the peptides of SEQ ID NOs: 1 to 8 and pharmaceutically acceptable salts thereof, wherein the immune system activating action is an active oxygen production promoting action. The pharmaceutical composition according to claim 2.