JPH0873374A - Medicinal composition having promoting action on tnf secretion - Google Patents

Abstract

PURPOSE: To obtain a peptide safely usable in the field of foods and medicines due to its activities in promoting phagocytosis, effects on promotion of active oxygen production and TNF secretion and its availability from soybean proteins which are natural products. CONSTITUTION: This medicinal composition having promoting actions on the secretion of a TNF(tumor necrosis factor) contains one or more selected from the group consisting of peptides represented by sequence Nos. 1 and 2 of the following formulas and pharmaceutically permissible salts thereof as an active ingredient: sequence No. 1: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn and sequence No. 2: Met-Ile-Thr-Leu.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art It is already known that peptides having various physiological activities are derived from food proteins. Since they are of food origin, these peptides 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.

The present inventors have previously derived Gln-Arg-Pro-Arg (QRPR) derived from soybean glycinin A _1a subunit.
And His-Cys-Gln-Arg-Pro-Arg (HCQRPR) have neutrophil accumulation, macrophage phagocytosis-promoting activity, TNF (tumor necrosis factor) secretion-promoting, and antitumor effect, etc. It was reported that the peptide has a novel peptide (Japanese Patent Laid-Open Nos. 6-92867 and 6-1479).
4 publication). Following this, Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gl derived from soybean conglycinin
It was reported that y-Arg (hereinafter abbreviated as MITLAIPVNKPGR) and its related peptides were immune system activating peptides having phagocytosis promoting activity and active oxygen production promoting effect (Japanese Patent Application No. 6-377).
No. 07).

[0004]

Then, the present inventors
A peptide having an immune system activating action obtained by enzymatic decomposition of soybean protein: When the physiologically active action of a peptide related to MITLAIPVNKPGR was examined, the peptides of SEQ ID NOs: 1 and 2 described later among the analogues had a TNF secretion promoting action. I found a thing. TN
F is known to express a cell-killing activity and a cell growth-suppressing activity on tumor cells. Therefore, promotion of TNF secretion is expected to have an antitumor effect and enhance the immunity of the host. Therefore, the present invention provides a novel food and pharmaceutical use of the above-mentioned physiologically active peptide.

[0005]

The present invention is intended to provide a novel food and pharmaceutical use for the following two peptides, the sequences of which are as follows: SEQ ID NO: 1: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn
(Hereinafter, abbreviated as MITLAIPVN) SEQ ID NO: 2: Met-Ile-Thr-Leu (hereinafter, abbreviated as MITL) Therefore, the present invention is effective for the peptides of SEQ ID NOS: 1 and 2 and pharmaceutically acceptable salts thereof. The present invention relates to a pharmaceutical composition having an immune system activating action, particularly a TNF secretion promoting action, as a component. Each of the peptides represented by SEQ ID NOS: 1 and 2 has the following characteristics: sequence type: amino acid topology: linear sequence type: peptide.

SEQ ID NOs: 1 and 2 which are the active ingredients of the present invention
The peptide can be obtained by enzymatically hydrolyzing soybean protein. More specifically, in the case of enzymatic hydrolysis of soybean protein, it can be prepared by further enzymatically degrading the peptide of MITLAIPVNKPGR consisting of the amino acid of 13 residues reported above. It can also be chemically synthesized by.

The present inventors have found that the peptides of SEQ ID NOs: 1 and 2 obtained by enzymatic degradation of soybean protein have an action of promoting phagocytosis, and as a result of further research, they also have an action of promoting TNF secretion. It was a finding. Therefore, the peptides of SEQ ID NOS: 1 and 2 also show a phagocytosis promoting action, and further, the peptide of SEQ ID NO: 1 also has an active oxygen production promoting action. Therefore, the peptides of SEQ ID NOS: 1 and 2 can be used as a pharmaceutical composition having an immune system activating action. These peptides can be converted into a pharmaceutically acceptable salt according to a conventional method, and can be prepared into a pharmaceutical composition together with carriers, auxiliaries and the like which are commonly used in the pharmaceutical industry.

[0008]

PRODUCTION EXAMPLES Examples of the production of the peptides of SEQ ID NOS: 1 and 2 of the present invention are shown below, but the invention is not limited to these examples.

Reference Example MITL from digested soybean protein
Preparation of AIPVNKPGR (SEQ ID NO: A) peptide 50 g of isolated soybean protein was dissolved in 800 ml of water, and 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 the active peptide by these columns was at 33-34% and 36-37% acetonitrile, respectively. Furthermore, this active fraction was added to the ODS-column (Co
smosil 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). Elutes as a single peak at 34-35% acetonitrile. When the structure was determined with a protein sequencer, the active peptide was Met-Il.
It was found to be e-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg.

Production Example 1 Preparation of Peptide of SEQ ID NO: 1 by Enzyme The peptide of SEQ ID NO: 1 can be obtained by enzymatic hydrolysis of soybean protein. In this example, the peptide of SEQ ID NO: A was enzymatically hydrolyzed. Prepared by decomposition. The enzyme used is a metalloendopeptidase that specifically cleaves the peptide bond of Asn-Lys contained in the peptide of SEQ ID NO: A,
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. Peptide of SEQ ID NO: 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: 1.

Production Example 2 Preparation of peptide of SEQ ID NO: 1 by chemical synthesis method (Fmoc method) 0.3 g of Fmoc-Asn (Trt) -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 times equivalents of Fmoc-Asn (Trt) -resin hydroxybenzotriazole (HOBT) and Fmoc
-Val was added for coupling. After the reaction, DC
It was washed with M: DMF = 1: 1 to obtain Fmoc-Val-Asn (Trt) -resin. Similarly, synthesize up to the N-terminal Met and
M, washed with methanol and washed with Met-Ile-Thr (tBu) -Leu-Ala-I
A le-Pro-Val-Asn (Trt) -resin was obtained. Deprotection agent (trifluoroacetic acid: ethanediol: anisole = 94:
1: 5) was added and left at room temperature for 1 hour. The separated resin was then filtered, washed with ether and freeze-dried to obtain crude Me
t-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn was obtained. This is O
When loaded on a DS-column (Cosmosil 5C ₁₈ -AR, 20 × 250 mm, manufactured by Nacalai Tesque) and purified by HPLC using a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid, about 150 mg of pure product was obtained. It was In the above, Trt represents a trityl group and tBu represents a tertiary butyl group. OD of the obtained synthetic MITLAIPVN (20 μg)
FIG. 1 shows a chart in which the absorbance was measured when loaded on an S column (Cosmosil 5C ₁₈ -AR, 4.6 × 150 mm) and eluted with a linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid.

Production Example 3 Preparation of peptide of SEQ ID NO: 2 by chemical synthesis method (Fmoc method) 0.3 g of Fmoc-Leu-resin having a substitution rate of 0.58 meq / g was added to SAM2.
The peptide was set in a reaction vessel of a peptide synthesizer (Biosearch) and synthesized in the same manner as in Production Example 2 to obtain a corresponding 4-residue peptide-resin. Thereafter, in the same manner as in Production Example 2, about 50
mg of pure Met-Ile-Thr-Leu was obtained. Obtained synthetic MI
TL (100 μg) was transferred to an ODS column (Cosmosil 5C ₁₈ -AR,
FIG. 2 shows a chart in which the absorbance was measured when it was loaded on a linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid and loaded on a 4.6 × 150 mm) column.

Table 1 shows the amino acid composition of the peptides of SEQ ID NOs: 1 and 2, 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.

[0014] 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.

[0015]

[Test example]

Test Example: Effect on TNF production by macrophages Six-week-old male ICR mice were orally administered with 300 nmol of the peptide of the present invention, and after 3 hours, 0.3 mg of bicyanil (OK-432), a killed streptococcal cell, was intravenously administered. It was administered internally. Two hours later, blood was collected, and the toxicity to L-929 cells contained in the serum was measured by radioimmunoassay. Table 2 shows the results. This peptide exhibited almost the same activity as QRPR reported so far.

[0016] Note 1) QRPR is Gln-Arg-Pro-Arg peptide 2) PBS is phosphate buffer solution

[0017]

The peptides of SEQ ID NOs: 1 and 2 of the present invention have a phagocytosis promoting activity, an active oxygen production promoting effect, and a TNF (tumor necrosis factor) secretion promoting activity, and are obtained from a natural soybean protein. Therefore, it can be safely used in the fields of food and medicine.

[Brief description of drawings]

FIG. 1 is a chart showing the absorbance of the component when it was eluted by HPLC with an ODS column of synthetic MITLAIPVN.

FIG. 2 is a chart in which the absorbance of the component was measured when it was eluted by HPLC using an ODS column of synthetic MITL.

Claims (3)

[Claims] 1. A peptide represented by the following formulas: SEQ ID NO: 1 and 2: SEQ ID NO: 1: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn SEQ ID NO: 2: Met-Ile-Thr-Leu And a pharmaceutical composition having a TNF (tumor necrosis factor) secretion-promoting activity, which comprises one or more selected from the group consisting of pharmaceutically acceptable salts thereof as an active ingredient. 2. The pharmaceutical composition according to claim 1, wherein the active ingredient is the peptide represented by SEQ ID NO: 1: Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn or a pharmaceutically acceptable salt thereof. Stuff. 3. The pharmaceutical composition according to claim 1, wherein the active ingredient is the peptide represented by SEQ ID NO: 2: Met-Ile-Thr-Leu or a pharmaceutically acceptable salt thereof.