JPH0614794A - Immunostimulant composition composed of soybean protein-derived peptide - Google Patents

Abstract

PURPOSE:To provide the subject immunostimulant composition composed of a soybean protein-derived peptide. CONSTITUTION:The immunostimulant composition containing a peptide represented by the formula: His-Cys-Gln-Arg-Pro-Arg (His, Cys, Gln, Arg and Pro represent respectively histidine, cysteine, glutamine, arginine and proline) or its salt as the active component and a method for producing the peptide by the enzymatic hydrolysis method. The above-mentioned peptide exhibits a, promotion effect on accumulation of neotrophil, productivity of active oxygen and productivity of tumor necrosis factor and an antitumor effect.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a pharmaceutical composition containing a physiologically active peptide derived from soybean protein as an active ingredient.

[0002]

2. Description of the Related Art It is known that some peptides derived from food proteins have various physiological activities, and various studies have been conducted because they are expected to be safe for living organisms due to food origin. Phagocytosis (phagocytosis) is known as one of them. When external foreign substances such as bacteria enter the living body, phagocytosis of the foreign substances by macrophages and neutrophils is important as the initial reaction of biological defense. Peptides that activate this phagocytosis include immunoglobulin-derived tuftsin (Thft-Lys-Pro-Arg) and rigin (Rigi).
n: Gly-Gln-Pro-Arg) is known. The present inventor has previously demonstrated using a synthetic peptide that a peptide contained in a soybean glycinin A _1a subunit having a structure similar to these: Gln-Arg-Pro-Arg enhances phagocytic ability of macrophages. And reported it as a novel peptide (Japanese Patent Laid-Open No. 1-249800).

[0003] However, physiologically active peptides derived from food proteins have many substances which show a surprising structure-activity relationship as compared with endogenous physiologically active peptides, and many peptides having multiple functions. It is said that its function cannot be predicted from its structure.

[0004]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention As a result of further research on peptides derived from food proteins, the present inventors have found peptides having further excellent physiological activity in addition to the action of enhancing phagocytic ability of macrophages. Completed the invention. Therefore, the present invention is intended to provide a novel pharmaceutical use of the peptide.

[0005]

The present invention provides the following formula I: His-Cys-Gln-Arg-Pro-Arg (I) (wherein His is histidine, Cys is cysteine, Gln is glutamine, and Arg is arginine. And Pro represent proline) or a pharmaceutically acceptable salt thereof as an active ingredient. The present inventors have found that the peptide represented by the above formula I obtained by enzymatic decomposition of soybean protein enhances neutrophil accumulation, active oxygen production and tumor necrosis factor production, and has an inhibitory action on ascites cancer. I found a thing.

Peptide which is the active ingredient of the present invention: His-Cy
s-Gln-Arg-Pro-Arg (hereinafter abbreviated as HCQRPR) can be synthesized by a conventionally known peptide chemical synthesis method. In addition, it is considered that the protein can be obtained by enzymatic hydrolysis from a protein containing the amino acid sequence of the above formula I, such as soybean protein, but its method has not been known. The present inventors have found a new production method by the enzymatic hydrolysis method. Therefore, the present invention also provides a novel method for producing the above peptide.
According to the enzymatic hydrolysis method of the present invention, soybean protein is digested with trypsin, and the resulting digest is octadecylsilyl (OD).
S) column and phenyl column for high performance liquid chromatography (HPLC).

[0007]

PRODUCTION EXAMPLES Next, production examples of the present invention will be shown, but the present invention is not limited to these examples.

Production Example 1: Chemical synthesis of His-Cys-Gln-Arg-Pro-Arg 2 g of Boc-Arg (Tos) -resin (0.3 meq / g, manufactured by Biosearch) was used as SAM2 (manufactured by Biosearch). Set in the reaction vessel of the peptide synthesizer, and use the deblocking solution (45% trifluoroacetic acid, 2.5% anisole, 2% ethanedithiol,
The Boc group was removed by stirring in 50.5% dichloromethane) for 25 minutes. The resin was washed successively with dichloromethane, dichloromethane containing 10% diisopropylethylamine and dichloromethane, then 10 ml of 0.4M Boc-Pro in dimethylformamide solution and 10 ml of diisopropylcarbodiimide in methylene chloride solution were added and stirred at room temperature for 2 hours. And coupled with Boc-Pro to give Boc-Pro-Arg (Tos) -resin. Similarly, Boc-Arg (Tos), Boc-Gln, Boc-Cys (MB
zl) and Boc-His (Tos) are sequentially coupled, and Boc-His
A (Tos) -Cys (Bzl) -Gln-Arg (Tos) -Pro-Arg (Tos) resin was obtained. Note that when coupling Boc-Gln, Boc-Gln
0.6M 1-Hydroxybenzotriazole was added to the dimethylformamide solution to prevent the formation of nitrile.

The resin was deprotected by stirring in about 20 ml of liquid hydrogen fluoride containing 10% anisole for 1 hour at 0 ° C. After removing hydrogen fluoride under reduced pressure, the resin was washed with ether, the peptide was extracted with 15% acetic acid, and freeze-dried to obtain crude His-Cys-Gln-Arg-Pro-Arg. further
In the presence of 0.1% trifluoroacetic acid, an ODS column (Cosmo
200 mg of pure product was obtained by high performance liquid chromatography using sil 5C ₁₈ AR, manufactured by Nacalai Tesque. In the above, His is histidine, Cys is cysteine, Gln is glutamine, Arg is arginine, Pro is each amino acid residue of proline, Boc is t-butoxycarbonyl group, and Toc is tosyl group.

Production Example 2: His-Cys-Gln-Ar by enzymatic digestion
Preparation of g-Pro-Arg (HCQRPR) The outline of the preparation method is shown schematically below.

[0011]

Isolation process: Hi from soybean protein digest
Isolation of s-Cys-Gln-Arg-Pro-Arg (HCQRPR) 2 g of the isolated soybean protein was dissolved in 32 ml of water to adjust the pH to 7.0, and the insoluble matter was removed by centrifugation at 3000 rpm for 30 minutes. After boiling for 30 minutes, 16 mg of trypsin was added, and digestion was carried out at 37 ° C for 5 hours. After boiling for another 10 minutes, 10,000 rpm.,
The 10-minute centrifugation supernatant was used as a trypsin digest. 2-mercaptoethanol was added to the above digest to give 1%,
An ODS-column (Cosmosil 5C ₁₈ , 20 × 250 mm, equilibrated with 50 mg protein equivalent amount thereof by 0.1% trifluoroacetic acid,
Loaded on Nacalai Tesque), and linear gradient of acetonitrile containing 0.1% trifluoroacetic acid (1% / 10ml)
/ Min). HCQRPR is contained in the fraction eluted with 16 to 17% acetonitrile, but since other peptides also coexist, HCQRPR is added to the phenyl column (Cosmosil
5Ph, 4.6 × 250mm, manufactured by Nacalai Tesque, then cyanopropyl column (Cosmosil 5CN, 4.6 × 250m)
m, manufactured by Nacalai Tesque, Inc.) to obtain HCQRPR. Both columns were developed by a linear concentration gradient (1% / 1 ml / min) of acetonitrile containing 0.1% trifluoroacetic acid.

HCQRPR was eluted from the phenyl and cyanopropyl columns with 13% and 4.7% acetonitrile, respectively. The yield of HCQRPR from the isolated soybean protein was 0.06%. The absorbance chart of the fraction of the soybean protein trypsin digested by the ODS-column is shown in FIG. 1, the chart of the fractionation of the ODS-column fraction by the phenyl column is shown in FIG. 2, and the fraction of the phenyl column fraction by the cyanopropyl column is shown. An image chart is shown in FIG. HCQRP is added to the fraction indicated by A in each chart.
R is recovered.

[0014]

TEST EXAMPLE Each action of the peptide of the present invention will be described below. Test Example 1: Accumulation of Neutrophils 7-week-old male ddY mice were intraperitoneally administered with 3 mg of the peptide, and one day later, the intraperitoneal cells were collected and stained with Diffquick (Midori Cross). The percentage of neutrophils was calculated by microscopy. The results are shown in Table 1.

[0015]

Test Example 2: Measurement of phagocytosis 3 weeks of ddY male mice were intraperitoneally administered with 3 mg of the peptide, and 3 days after the intraperitoneal cells were collected, a paraffin emulsion containing oil red was added in an amount of 1/10. In addition, the reaction is started, and after 5 minutes, ice-cooled physiological saline is added to stop the reaction. After centrifugation, paradioxane is added to the pellet to extract the dye taken up by the cells. The dye amount is 524 nm and 6
The amount of phagocytosis was evaluated based on the difference measured from the absorbance at 00 nm. The absorbance at 600 nm is a correction for turbidity.

[0017]

Test Example 3: Measurement of active oxygen production capacity Mechanisms of active oxygen production and measurement are shown in Schemes 1 and 2. As shown in Scheme 1, active oxygen is absorbed by HM in phagocytes.
Oxygen is reduced and produced by NADPH supplied from the P pathway. Since active oxygen quantitatively reduces cytochrome C in a test tube, the amount of reduction was measured by measuring the absorbance (OD) with an absorptiometer to determine the amount of active oxygen production.

[0019]

[Table 1]

[0020]

[Table 2]

As shown in Scheme 2, cells are obtained by opsonizing zymosan (yeast cell wall) which is a foreign substance,
Start the reaction by adding cytochrome C, and after 15 minutes, put it in ice water to stop the reaction. After centrifugation, the absorbance of the supernatant was measured at 550 nm and 468 nm, and the amount of active oxygen production was calculated from the difference.
The calculation formula is as follows. [O ₂ ⁻ ] = A / ε · 2 × 10 ⁻³ · 500/400 [μmol / 2 × 10 ⁶ · 15 min] From ε = 0.0245 = 102 · A [nmol / 2 × 10 ⁶ · 15 min] In addition, A : Absorbance results are shown in Table 3.

[0022]

Test Example 4 Measurement of Elevating Effect of Tumor Necrosis Factor (TNF) Level 7-week-old male C3H / Hc mice were intravenously or orally administered with the peptide, and 3 hours later, 0.3 mg of picibanil (OK) was administered.
-432) was administered intravenously. After 2 hours, blood was collected and TNF level in serum was measured by radioimmunoassay. The results are shown in Table 4. The peptides of the present invention are particularly effective in intravenous administration.

[0024]

Test Example 5: Inhibitory activity against ascites cancer Cancer 3 mg of peptide and 1 × 10 ⁵ L-1210 cells were intraperitoneally administered to 6-week-old male DBA / 2 mice, and the mice died of ascites cancer. The number of days until was measured. 5 or 6 for 1 group
I made it The survival days are shown in Table 5, and the survival rate is shown in FIG.

[0026] Although the difference from the control (physiological saline) is not so large, it shows the same level of action as tuftsin, which is said to have an ascites cancer suppressing action.

Test Example 6: Immunity-enhancing action A group of 10 mice was intraperitoneally administered with a peptide dissolved in physiological saline, and 1 hour later, Candida albicans was killed 10 days after the control group. Rate is
The number of bacteria was 90 to 100%, which was intravenously inoculated, and the immunopotentiating effect was determined by the number of surviving 10 days later. The results are shown in Table 6.

[0028]

Test Example 7: Immune-restoring action A group of 10 mice was intraperitoneally administered with a peptide dissolved in physiological saline on the 1st, 3rd and 5th days. Furthermore, the immunosuppressant cyclophosphamide 25 mg / Kg is orally administered on the 2nd, 4th and 6th days. One hour after the last dose of cyclophosphamide, Candida albicans
Lbicans) was inoculated intravenously with a number of bacteria so that the lethality rate after 10 days was 90 to 100% in the control group, and the immune recovery effect was determined by the survival number after 10 days. The results are shown in Table 7.

[0030]

[0031]

EFFECTS OF THE INVENTION As can be seen from the above results, the peptides of the present invention are superior to tuftsin and QRPR in phagocytic activity and tumor necrosis factor level-elevating action, and have various immune system activating actions. It can be used as a pharmaceutical composition for the above diseases. Further, according to the enzymatic hydrolysis method of the present invention, the obtained peptide is derived from a food protein, so that safety can be expected sufficiently. In use, the peptide of the present invention can be orally or intravenously prepared by itself or in a suitable dosage form together with pharmaceutically used carriers and auxiliaries.

[Brief description of drawings]

FIG. 1 is an ODS-column fraction absorbance chart of a soy protein trypsin digest.

FIG. 2 is a chart of the absorbance of ODS-column fractions obtained by phenyl column fractionation.

FIG. 3 is a chart of the absorbance of a phenyl column fraction obtained by a cyanopropyl column fraction.

FIG. 4 is a graph showing an inhibitory effect (survival rate) on ascites cancer.

Claims (4)

[Claims] 1. The following formula I: His-Cys-Gln-Arg-Pro-Arg (I) (wherein His is histidine, Cys is cysteine, Gln is glutamine, Arg is arginine and Pro is proline). An immune system activating composition comprising a peptide represented by: or a salt thereof as an active ingredient. 2. The pharmaceutical composition according to claim 1, wherein the immune system activating action is an action of enhancing neutrophil accumulation ability, active oxygen production ability and tumor necrosis factor production ability. 3. The pharmaceutical composition according to claim 1, wherein the immune system activating action is a carcinostatic action. 4. A soybean protein is digested with trypsin, and the resulting digest is fractionated by high performance liquid chromatography using an octadecylsilyl (ODS) column and a phenyl column. The following formula I: His-Cys- A method for producing a peptide represented by Gln-Arg-Pro-Arg (I) (wherein, His is histidine, Cys is cysteine, Gln is glutamine, Arg is arginine, and Pro is proline).