JPH09262094A - New peptide or its salt - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new peptide of a physiologically active substance isolated from hemoglobin having ileum contracting action and immune promoting activity. SOLUTION: This peptide or its salt contains an amino acid sequence represented by the formula X-ValValLeuAlaArg (X is represented by formula I when hemoglobin is derived from cow, formula II when the hemoglobin is derived from pig and formula III when the hemoglobin is derived from horse and at least one amino acid may successively be lost from the amino acid sequence on N terminal side among amino acid sequences of formulas I to III). The peptide or its salt is obtained by enzynmatically decomposing hemoglobin of edible animals.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide having ileal contractile action and immunostimulatory activity or a salt thereof.

[0002]

2. Description of the Related Art It is known that proteins contained in foods have various physiological activities as well as nutritional effects. For example, by subjecting casein, which is a milk protein, to enzymatic degradation, it is possible to obtain a peptide having a blood pressure lowering action, as disclosed in JP-B-60-23085 and JP-B-60-230.
It is disclosed in Japanese Patent Publication No. 86, Japanese Patent Publication No. 60-23087 and the like.

Recently, it has been confirmed that peptides obtained by enzymatically decomposing various proteins have various physiological activities, and the amino acid sequences of food-derived proteins include:
It is estimated to have potential physiological functions. Hemoglobin is a protein contained in the blood of animals and is contained in various types of edible meat. Since hemoglobin is a protein derived from food, isolation of physiologically active substances from hemoglobin is expected.

[0004]

The object of the present invention is to provide a novel peptide having an ileal contractile action and an immunostimulatory activity.

[0005]

Means for Solving the Problems Based on the above-mentioned problems, the present inventors have conducted intensive studies focusing on the amino acid sequence of hemoglobin of edible animals (cattle, pigs, horses, etc.),
By enzymatically degrading hemoglobin, a novel peptide having physiological activity is found from these amino acid sequences,
The present invention has been completed.

That is, the present invention is a peptide consisting of 5 to 12 amino acid sequences containing the following amino acid sequence: Val Val Leu Ala Arg on the C-terminal side, or a salt thereof.

Furthermore, the present invention provides the following formula I: X-Val Val Leu Ala Arg (I) (X is a hydrogen atom or any amino acid sequence represented by SEQ ID NOs: 2 to 4 or the N-terminal of the amino acid sequence. It represents an amino acid sequence in which at least one amino acid is cleaved from the side)) or a salt thereof.

Hereinafter, the present invention will be described in detail. The novel peptide of the present invention is a β of bovine, porcine and equine hemoglobin.
It is present in the primary structure of the chain and corresponds to residues 105 to 116 of the amino acid sequence of 146 hemoglobins. The peptide of the present invention is composed of 5 to 12 amino acids containing the following amino acid sequence: Val Val Leu Ala Arg (SEQ ID NO: 1) on the C-terminal side, and has an ileal contractile activity or an immunostimulatory activity function. Further, the peptide of the present invention can be represented by the following formula I: X-Val Val Leu Ala Arg (I).

In formula I, the amino acid sequence represented by Val Val Leu Ala Arg (SEQ ID NO: 1) is common to bovine, porcine and equine. On the other hand, X in the formula I is represented by SEQ ID NO: 2 for bovine hemoglobin, SEQ ID NO: 3 for porcine hemoglobin, and SEQ ID NO: 4 for equine hemoglobin.

Furthermore, in the peptide of the present invention, at least one amino acid may be sequentially deleted from the amino acid at the N-terminal side in the amino acid sequences represented by SEQ ID NOs: 2 to 4. For example, in the amino acid sequence represented by SEQ ID NO: 2 (bovine hemoglobin), one amino acid on the N-terminal side (leucine) may be deleted, and two amino acids on the N-terminal side (leucine and leucine) may be deleted. It may be deleted, three N-terminal amino acids (leucine, leucine, and glycine) may be deleted, and further all seven amino acids may be deleted. When 7 amino acids are deleted, X is a hydrogen atom.

In the present specification, amino acids, peptides,
Abbreviations for protecting groups, etc. shall be in accordance with the provisions of IUB or IUPAC or the symbols customary in the field, examples of which are given below. In addition, if there are optical isomers of amino acids, L-
The body shall be indicated. Bzl: benzyl group Boc: tert-butoxycarbonyl group TFA: trifluoroacetic acid DMF: dimethylformamide DCM: dichloromethane Tos: p-toluenesulfonyl group The method for obtaining the peptide of the present invention includes natural purification and chemical synthesis. .

(1) Purification from nature Blood was collected from cattle, horses and pigs while adding sodium citrate, and hemoglobin was obtained by continuously centrifuging immediately after blood collection, and heat-treated with a trypsin-like enzyme. Etc. to deactivate the enzyme. Next, a crude peptide solution is obtained by centrifuging the sample solution after inactivation. Next, the target peptide can be purified by subjecting the crude peptide solution to reverse phase chromatography, hydrophobic chromatography, ion exchange chromatography, or the like, or by using in combination.

(2) Purification by chemical synthesis The peptide of the present invention can also be obtained by chemical synthesis. When chemical synthesis is performed, it can be synthesized by a conventional means for peptide synthesis. For example, azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, carboimidazole method, redox method, DCC-
The additive (HONB, HOBt, HOSu) method and the like can be exemplified ("The Peptide", Volume 1 (1966) [Sch
by Reder and Luhke, Academic Press, New York, US
A.] or “Peptide Synthesis” [Izumiya et al., Maruzen Co., Ltd. (1
975)]).

By chemical synthesis, a peptide consisting of 5 to 12 amino acid sequences containing the amino acid sequence represented by Val Val Leu Ala Arg (SEQ ID NO: 1) at the C-terminal side can be obtained.

For example, by synthesizing an amino acid containing any of the sequences of SEQ ID NOs: 2 to 4 at the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1, a peptide consisting of a total of 12 amino acid sequences is synthesized. You can As long as the resulting peptide has an ileal contractile activity or an immunostimulatory activity function, the peptide is included in the present invention.

Further, the peptide of the present invention comprises synthesizing a peptide containing an amino acid in which at least one amino acid is sequentially deleted from the amino acid on the N-terminal side in the amino acid sequences represented by SEQ ID NOS: 2 to 4 above. Can also be obtained by For example, in the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 of formula I (bovine hemoglobin), one amino acid (leucine) on the N-terminal side of the sequence represented by SEQ ID NO: 2 is deleted. One in which two amino acids on the N-terminal side (leucine and leucine) have been deleted,
It is possible to obtain those in which three amino acids (leucine, leucine and glycine) on the N-terminal side are deleted, and those in which all seven amino acids are deleted.

When performing the above chemical synthesis, both solid phase synthesis method and liquid phase synthesis method can be applied. In the above various methods, it is desirable to protect the side chain functional groups of amino acids having side chain functional groups, such as arginine. In this case, a commonly used protecting group (for example, Boc) can be used for protection, and the protecting group may be removed after the reaction is completed.

(3) Amino acid sequence analysis The peptide obtained as described above can be identified by sequence analysis using an amino acid sequencer and amino acid composition. Further, the peptide of the present invention forms a salt with acetic acid, hydrochloric acid and the like. The present invention also includes these salts. The novel peptide thus obtained has an ileal contractile action and an immunoreactive action in guinea pigs and the like.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to these examples.

[Example 1] Preparation of peptide of the present invention derived from bovine hemoglobin Hemoglobin was prepared by continuously centrifuging blood collected from cattle while adding sodium citrate. Next, trypsin was added to an aqueous solution dissolved so that the concentration of bovine hemoglobin was 30 mg / ml to 300 μg / ml, and dissolved, and the pH was adjusted to 7.8 with NaOH. Keep this solution at 37 ° C for 5 hours and then at 100 ° C.
Hold for 10 minutes to inactivate the enzyme. Then, centrifuge (1
The supernatant was obtained at 2,000 rpm for 5 minutes, and 1.2 ml of this supernatant was
Attached to PLC. The HPLC is a Cosmosil 5C ₁₈ column (250 x 20 m
m, manufactured by Nacalai Tesque, Inc., and eluted at a flow rate of 10 ml / min under a linear concentration gradient condition of 1% / min with a concentration gradient of 0-60% acetonitrile (containing 0.1% TFA).

Collect the desired peptide fractions, then
velosil PhA-T-5 (150 × 4.6mm column; Nomura Chemical Co., Ltd.)
Was subjected to HPLC with the same apparatus and eluted under the same concentration gradient condition at a flow rate of 1 ml / min to obtain 20 μg of the peptide of the present invention. When the obtained peptide was analyzed by a peptide sequencer, it had a sequence represented by SEQ ID NO: 5 (where X in Formula I is represented by SEQ ID NO: 2). The amino acid analysis is shown below. Asp (1) 1.00, Gly (1) 1.00, Ala (1) 1.00, Val (4) 4.0
0, Leu (4) 4.00, Arg (1) 1.00

Example 2 Preparation of Peptide of the Present Invention by Chemical Synthesis It was synthesized by a SAM2 peptide synthesizer (manufactured by Bioserch) according to the protocol of the same. That is, 0 per 1 g.
2g of acyloxymethyl resin bound with 3mmol of 12th protected amino acid Boc-Arg (Tos) -OH was set in the reaction vessel of the peptide synthesizer, and 45% (v / v) TFA and 2.5% (v / v) anisole were set. The Boc group was removed by contacting with a deblocking solution containing 52.5% (v / v) DCM for 20 minutes. 10% (v / v) after washing with DCM
The resin was neutralized with DCM containing diisopropylethyleneamine and washed with DCM. After that, it contains 4.0mmole of 11th protected amino acid Boc-Ala-OH and 4.0mmole of diisopropylcarbodiimide (each 6.7 times theoretical equivalent).
The reaction was carried out in a mixed solution of 20 ml of DCM and DMF for 2 hours at room temperature.
Boc-Ala-Arg (Tos) -PAM resin was obtained by sequentially washing with DMF and DCM.

In the following, the protected amino acids shown below are used in order.
The 10th to 1st amino acids were coupled.

[0024] 10% on protected peptide resin coupled as above
After adding anhydrous hydrogen fluoride containing anisole and reacting at 0 ° C. for 1 hour, hydrogen fluoride was distilled off and washing with ether was performed. 10 from the resulting mixture of peptide and resin
Extraction of peptide with% acetic acid and freeze-drying about 350m
g of crude peptide was obtained.

After dissolving the crude peptide in 0.1% TFA, it was dissolved in an octadecyl silica (ODS) column (Cosmosil 5C ₁₈ , 250 × 20 m).
m; manufactured by Nakarai Tesque Co., Ltd.) with a high performance liquid chromatograph (M600 type, manufactured by Nippon Waters Co., Ltd.)
A linear gradient of acetonitrile containing 0% TFA (0-5
The target peptide was obtained at 0% / 50 min and developed at 10 ml / min). The obtained peptide had the sequence represented by SEQ ID NO: 5.

The amino acid analysis is shown below. Asp (1) 1.01, Gly (1) 1.00, Ala (1) 1.02, Val (4) 4.1
0, Leu (4) 3.90, Arg (1) 0.98 High Performance Liquid Chromatography Column: TSKgel ODS-120T (4.6 × 250 mm) Flow rate: 1.0 ml / min Eluent: 25% CH ₃ CN-0.01N HCl Retention time: 13.6 Minute

[Example 3] Preparation of peptide of the present invention by chemical synthesis In the coupling described in Example 2, the sixth amino acid "Boc-Leu-OH" was replaced with "Boc-Ile-OH". The same operation was performed to obtain the target peptide (amino acid sequence represented by SEQ ID NO: 6).

[Example 4] Preparation of peptide of the present invention by chemical synthesis In the coupling described in Example 2, the 7th amino acid "Boc-Val-OH" was replaced with "Boc-Ala-OH". The same operation was performed to obtain the target peptide (amino acid sequence represented by SEQ ID NO: 7).

Example 5 Preparation of Peptide of the Present Invention by Chemical Synthesis From the amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 5 by the same method as the method for preparing the peptide by chemical synthesis described in Example 2. And a truncated peptide (SEQ ID NOS: 8 to 13) in which one amino acid was sequentially deleted from the N-terminal side of the 12 amino acid sequence represented by SEQ ID NO: 5 (Table 1) .

[0030]

[Table 1]

[Test Example 1] A pharmacological test example which supports that the novel peptide obtained as described above has a guinea pig ileal contractile action and an immunoreactive action is shown below.

(1) Ileal contractile action: A method of longitudinal ileal muscle slices extracted from guinea pigs weighing 300 to 400 g, written by Takagi et al. (Takagi et al., Pharmacological experiment,
94-99, 1972, Nanzan-do) and processed. One end of the specimen was connected to an isometric transducer via a thread, the other end was fixed in a Manugus tube with an internal volume of 2 ml, and the tension of muscle contraction was recorded electrically.

The peptide concentration (EC ₅₀ ) required to induce contraction of 50% of the maximum contraction was determined for the peptides obtained in each of the above Examples. As a positive control, complement C3
a (70-77) (manufactured by Backchem, Switzerland) was used. (EC ₅₀ =
1.67 μM) The results are shown in Table 2.

[0034]

[Table 2]

The ileal contractile action of each peptide obtained in Example 5 was tested in the same manner as above. The results are shown in Table 3. In addition, "Peptide No." in Table 3
Corresponds to “Peptide No.” in Table 1.

[0036]

[Table 3]

(2) Immunoreactive action Measurement of phagocytosis (phagocytosis) activity In order to examine the phagocytic action of human leukocytes on latex beads, the following test was conducted. Physiological saline (PBS) containing phosphate buffer was added to human peripheral blood, and after centrifugation at 1,000 rpm for 5 minutes to wash blood cells, 4 × 10 ⁶ cells / ml with PBS
A blood cell suspension was prepared. Take 100 μl of this solution and
10 μl of peptide (amino acid sequence represented by SEQ ID NO: 5; synthetic) solution dissolved in BS was added, incubated at 37 ° C. for 10 minutes, and then opsonized with human peripheral blood 4 × 10 ⁸ cells / ml Fluorescent labeled latex bead solution 10 μl
Was added and the mixture was further incubated for 5 minutes. The reaction was stopped with PBS containing EDTA, blood cells were separated by centrifugation, and an ammonium chloride hemolyzing agent was added to this to cause hemolysis to obtain leukocytes. This was suspended in PBS containing EDTA and then measured by flow cytometry.

The results are shown in Table 4. In Table 4, the numerical value (relative phagocytic activity) is shown as the activity when the control (PBS) is set to 100.

[0039]

[Table 4]

The immunoreactive action of each peptide obtained in Example 5 was also tested in the same manner as above. The results are shown in Table 3. In Table 3, relative phagocytic activity is control
The activity is shown when (PBS) is set to 100. Table 3 and Table 4
From this, it was found that the peptide of the present invention has an effect of enhancing human immune activity.

[0041]

INDUSTRIAL APPLICABILITY The present invention provides a novel peptide. The peptide of the present invention is extremely useful because it can be used as an agent for preventing microbial psoriasis, an immunostimulant, a functional food, a milk powder for childcare and the like.

[0042]

[Sequence list]

SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 2 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 3 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 4 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 5 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 6 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 7 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 8 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 9 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 10 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 11 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 12 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 13 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display area A61K 38/00 ABD A61K 37/18 ABD

Claims (2)

[Claims] 1. A peptide comprising an amino acid sequence of 5 to 12 containing the following amino acid sequence: Val Val Leu Ala Arg on the C-terminal side, or a salt thereof. 2. The following formula I: X-Val Val Leu Ala Arg (I) (X is a hydrogen atom or any amino acid sequence represented by SEQ ID NOs: 2 to 4 or an amino acid from the N-terminal side of the amino acid sequence. A peptide comprising an amino acid sequence represented by the above, which represents an amino acid sequence cleaved by at least one, or a salt thereof.