JP2005247765A - Cytostatic agent for vascular smooth muscle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a cytostatic agent for vascular smooth muscle derived from natural materials and provide a substance containing the material. <P>SOLUTION: This cytostatic agent contains a natural tripeptide Ile-Val-Tyr (IVY) or a substance containing the tripeptide as an active component. The IVY can be separated from hydrolyzed wheat embryo and a hydrophobic adsorption resin containing the hydrolyzed embryo as an IVY-containing substance, for example, from an ODS (resin, YMC company (R)) fraction. It can be separated also from an ODS fraction of a hydrolyzed fish meat such as sardine meat. The IVY and the IVY-containing substance can be used as a food and drink as well as a pharmaceutical. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a vascular smooth muscle cell proliferation inhibitor, and more specifically, to a novel and useful vascular smooth muscle cell proliferation inhibitor containing a specific natural tripeptide and / or its content as an active ingredient. Is.

  First, the present inventors have carried out heat denaturation treatment of fish meat, inactivated autodigestive enzyme, hydrolyzed with proteolytic enzyme, inactivated enzyme, and then separated and processed by ACE (Angiotensin I-). The inventors have succeeded in developing a novel peptide α-1000 having an inhibitory activity (converting enzyme: angiotensin I converting enzyme) and have already obtained a patent right (see Patent Document 1).

On the other hand, the present invention has been found based on this useful new finding, for the first time that a natural peptide, particularly a specific tripeptide derived from nature (Ile-Val-Tyr) has an inhibitory action on vascular smooth muscle cell proliferation. However, it has not been known at all that the present tripeptide has such an excellent physiological action.
Japanese Patent No. 3117777

  The present invention was made for the development of natural product-derived drugs and functional foods, particularly from the viewpoint of safety, and the fish-derived peptide α- which was successfully developed by the present inventors previously. It was made for the purpose of newly developing a novel peptide having excellent physiological action by paying attention again to 1000 excellent physiological actions.

  As a result of examinations from various directions to achieve the above object, the present inventors have focused on wheat germ, purified and fractionated the hydrolyzate, and detailed physiological functions of various fractions. As a result, the inventors have found for the first time that the tripeptide Ile-Val-Tyr (isoleucil-valyl-tyrosine, hereinafter sometimes referred to as IVY) has an action of suppressing the proliferation of vascular smooth muscle cells.

  In addition to isolated IVY, screening of substances containing IVY, separation of high-IVY components, and methods of acquisition were also investigated, and IVY may be obtained from hydrolyzate of fish peptides in addition to wheat germ. As a result of various studies based on the findings for the first time and these useful new findings, the present invention has finally been completed.

  That is, the present invention is based on the first discovery of a new physiological action called vascular smooth muscle cell growth inhibitory action for the tripeptide IVY, and suppresses vascular smooth muscle cell proliferation containing IVY and / or its inclusion as an active ingredient. It also relates to preventive drugs or foods.

  In the present invention, tripeptide IVY is used as an active ingredient. As IVY, purified and isolated ones can be used, and various substances containing IVY, particularly natural products derived substances, should be used. Can do. As the substance, as a result of the study by the present inventors, it has been found that, for example, a processed product of wheat germ or a processed product of fish meat is suitable.

  Here, the treated product refers to a product obtained by one or more of various treatments commonly used for separation and purification of natural products such as amino acids, peptides, proteins, and the following treatments are exemplified. : Dialysis, enzyme hydrolysis, acid hydrolysis, degreasing, ion exchange resin, hydrophobic adsorption resin, chromatography and others.

  In the present invention, an IVY-containing substance derived from a natural product can also be used as an active ingredient. Examples of the substance include a processed wheat germ product and a processed fish meat product, and peptides WB-1, WB- derived from wheat germ. 2 and fish-derived peptides Y-2, SY, SY-MD and the like are advantageously used. If desired, separation and purification means such as chromatography can be repeated or appropriately combined to increase the IVY concentration or fractionate the IVY fraction. First, the processing of wheat germ will be described in detail.

  Wheat germ is dissolved in deionized water as it is without being defatted or without being defatted and homogenized, and then hydrolyzed with an enzyme after or after the α-amylase treatment. Degreasing may be performed in accordance with a conventional method, for example, using a solvent such as n-hexane or diethyl ether at 50 to 90 ° C. for about 1 to 10 hours.

  The α-amylase treatment may be performed by mixing wheat germ homogenate and α-amylase and, for example, the addition amount of α-amylase is 0.1 to 10% by weight, preferably 0.5 to 5% by weight, Usually, it may be about 1 to 3% by weight, and it may be treated at 35 to 39 ° C., 10 minutes to 24 hours, preferably 1 to 10 hours, usually 3 to 5 hours.

  For enzymatic hydrolysis, various proteases are used in amounts of 0.1 to 5%, preferably 0.3 to 2%, and treated at pH and temperature suitable for each for 1 to 10 hours, preferably about 3 to 7 hours. Good. Examples of proteases include the following (in parentheses indicate treatment pH and temperature): pepsin, protease YP-SS (trade name of Yakult) (pH 2 to 4, usually about 3; 30 to 50 ° C., usually Chymotrypsin, trypsin (pH 7-9, usually around 8; 30-50 ° C., usually around 40 ° C.); alkaline protease (pH 8-10, usually around 9; 40-60 ° C., usually around 50 ° C.). Commercially available products can be used as appropriate as proteases.

  After completion of the enzymatic hydrolysis, after adjusting the pH to 6-8, boiling and heat treatment for about 10-20 minutes, followed by centrifugation (7,000-15,000 rpm, 10-20 minutes) Filter the clear.

  In the above, the wheat germ hydrolyzate (peptide WB-1) obtained by subjecting the defatted wheat germ to α-amylase treatment and then alkaline protease treatment has a high IVY amount, and the raw wheat germ is a natural product. Some variation was observed, but 20 mg / 100 g or more, and 30 mg / 100 g or more is sufficiently expected. In fact, after degreasing the wheat germ, the peptide WB-1 obtained by treating with 3% α-amylase for 3 hours and then with 0.5% alkaline protease for 8 hours (that is, peptide WB− which is a hydrolyzate of wheat germ) In 1), it was confirmed that IVY contained 21.6 ± 3.3 mg / 100 g.

  Activated charcoal is added to the centrifuge supernatant filtrate of the wheat germ hydrolyzate (peptide WB-1) obtained as described above, and then applied to a hydrophobic adsorption resin (for example, ODS resin), and then 5 to 20%, preferably 7 to 15%. Elution with an ethanol aqueous solution is performed, and the obtained elution fraction is defined as a high IVY-containing substance (peptide WB-2). The IVY content is 120 mg / 100 g or more, and depending on the raw wheat germ, it is sufficiently expected to have a content of 160 mg / 100 g or more. In fact, the hydrolyzate (peptide WB-1) obtained by treating wheat germ with 3% α-amylase for 3 hours and defatting and then treating with 0.5% alkaline protease for 8 hours was applied to an ODS column, and 10% It was confirmed that peptide WB-2 (that is, wheat germ hydrolyzate ODS fraction: peptide WB-2), which is a fraction eluted with ethanol, contained 143 ± 16.7 mg / 100 g of IVY. As the hydrophobic adsorption resin, ODS resin, for example, YMC, ODS-AQ120-S50 (trade name, manufactured by YMC), for example, SEPABEADS SP207 (trade name, manufactured by Mitsubishi Chemical Corporation) can be used.

  The chromatographic fraction (WB-2) of the wheat germ hydrolyzate eluted in this way can be used as a sufficiently high IVY-containing composition, but the IVY content can be further increased. Non-limiting examples are given below.

After adding deionized water to the chromatographic fraction (dried product) such as the ODS fraction obtained as described above, this was applied to a column packed with a cation exchange resin (for example, AG50W-X8 (H ⁺ ) (trade name of Bio-Rad)). Wash with water and then elute with 0.1-5M, preferably 0.5-2M aqueous ammonia to obtain the elution fraction (AG50W peptide).

  Although it was confirmed that the IVY content of this fraction (AG50W peptide) was significantly increased to 240 mg / 100 g or more, it can be sufficiently increased to 280 mg / 100 g or more. Further, according to this method, the yield is increased 2 to 3 times as compared with, for example, 1 k Macrosep centrifugal concentration (Filtron, USA). This fraction can also be lyophilized and stored.

  This fraction has a high IVY content and is more suitable for use as an active ingredient of the medicament according to the present invention, but is further purified by known purification processes such as chromatography (hereinafter sometimes referred to as chromatography). It is also possible to do.

  For example, the AG50W fraction can be purified by HPLC treatment using an appropriate combination of Cosmosil 5C18-ARIL and Cosmosil 5 Ph (trade name of Nacalai Tesque) column, and isolated into each peptide to isolate IVY.

The physicochemical properties of the peptide WB-1 of the present invention are shown below.
(Physicochemical properties of peptide WB-1)
(A) Melting point (darkening point): 121.3 to 160.5 ° C
(B) Specific rotation: [α] _D ²⁰ = + 269 ° ± 5 ° (measured concentration; 2.11 mg / 5 ml-water)
(C) Liquidity (pH): 6.8 to 8.4 (concentration 0.03%)
(D) Physiological properties: Contains tripeptide Ile-Val-Tyr and has an inhibitory effect on vascular smooth muscle cell proliferation.
(E) Infrared absorption spectrum: FIG.
(F) Ultraviolet absorption spectrum: FIG.

The physicochemical properties of the peptide WB-2 of the present invention are shown below.
(Physicochemical properties of peptide WB-2)
(A) Melting point (darkening point): 155.2-205.7 ° C
(B) Specific rotation: [α] _D ²⁰ = + 213 ° ± 5 ° (measured concentration; 2.31 mg / 5 ml-water)
(C) Liquidity (pH): 6.8 to 8.0 (concentration 0.05%)
(D) Physiological properties: Contains tripeptide Ile-Val-Tyr and has an inhibitory effect on vascular smooth muscle cell proliferation.
(E) Infrared absorption spectrum: FIG.
(F) Ultraviolet absorption spectrum: FIG.

  Next, a processed fish meat product will be described in detail as another example of the IVY-containing material. As described above, the present inventors have succeeded in increasing the IVY concentration for the first time by containing the tripeptide IVY in wheat germ, and variously treating it. As a result of continuing research, after the fish-derived peptide α-1000 previously developed by the present inventors was applied to a hydrophobic adsorption resin ODS column, IVY was contained in a high concentration in the fraction eluted with 10% ethanol aqueous solution. As a result of intensive research, we succeeded in developing processed fish meat (peptide Y-2) with a high IVY content, and processed fish meat (peptide SY, SY-MD, etc.) by other processing. ) Also confirmed that IVY was contained in a high concentration, and succeeded in completing the invention from the aspect of fish-derived peptides.

  First, peptide Y-2 can be produced as follows. As a raw material for peptide stock, that is, peptide Y-2, a fish-derived peptide is used, and after subjecting it to a hydrophobic adsorption resin, 5-20, preferably 8-17, more preferably about 15 V / V% ethanol aqueous solution. By elution, peptide Y-2 is obtained. Alternatively, it may be eluted with water before elution with an aqueous ethanol solution and then with an aqueous ethanol solution. The elution fraction thus obtained contains IVY, and this fraction (peptide Y-2) can be advantageously used as an active ingredient in the present invention.

In addition to peptide Y-2, as a raw material or origin of SY or SY-MD, that is, a peptide stock solution, for example, an aqueous solution of peptide α-1000 can be used.
Peptide α-1000 can be produced by subjecting fish meat to heat denaturation, hydrolyzing it with neutral or alkaline protease treatment, and then inactivating the enzyme according to a conventional method such as heating, followed by separation treatment. Details are described below.

  Peptide α-1000 is produced from seafood as a raw material, and can be produced according to, for example, Japanese Patent No. 3117777. First, the seafood is processed by a meat collector, deboner, etc. to separate the fish meat quality. To do. The raw material is preferably as fresh as possible. The separated fish meat may be divided into about 10 kg of surimi and used for the next treatment as it is. However, it is rapidly frozen by blowing cold air at −20 to −50 ° C., for example, about −30 ° C., and −20 to − You may preserve | save at 25 degreeC and you may decide to use this suitably as needed.

  As seafood, red fish such as sardines, horse mackerel, tuna, skipjack, saury, mackerel; white fish such as flounder, thailand, kiss, konoshiro, cod, herring, yellowtail; cartilage fish such as shark, ray; Freshwater fish such as yamame trout; deep-sea fish such as sharks and salamanders, shrimp, crabs, octopuses, and mites can be used as appropriate.

  After the meat is minced, the seafood is pulverized by a pulverizer or the like, ½ amount to 20 times the amount of the raw material weight, preferably the equivalent amount to 10 times the amount of water, and then heat-treated, In addition to inactivating the self-digesting enzyme and killing bacteria, the protein is thermally denatured to increase the efficiency of the subsequent enzyme reaction. As heating conditions, all conditions can be used as long as such an effect is exerted, but for example, 65 ° C. or higher and 2 to 60 minutes, preferably 80 ° C. or higher and 5 to 30 minutes are preferable. .

  Next, an alkaline agent such as ammonia water or an aqueous solution of sodium hydroxide (potassium hydroxide) is added to adjust the pH to an appropriate value for the proteolytic enzyme used (for example, in the case of alkaline protease, pH 7.5 or higher, preferably 8). Above), the temperature is also set to an appropriate enzyme temperature (depending on the enzyme used, 20 to 65 ° C., 35 to 60 ° C., preferably 40 to 55 ° C. in the case of alkaline protease), and 30 minutes after adding the proteolytic enzyme Treatment is performed for 30 hours (30 minutes to 25 hours, preferably 1 to 17 hours in the case of alkaline protease).

  As the proteolytic enzyme, all enzymes can be used alone or in combination as long as they are capable of degrading proteins under neutral or alkaline conditions. Its origin can be found in microorganisms in addition to animals and plants, and in addition to pepsin, renin, trypsin, chymotrypsin, papain, bromelain, bacterial protease, filamentous fungal protease, actinomycete protease, etc. can be widely used. These enzymes are usually commercially available. However, unpurified enzymes, enzyme-containing culture fluids, and solid or liquid enzyme-containing materials such as sputum should also be used as required. Can do. The amount of enzyme added may be about 0.1% to 5.0%.

  Next, neutralization treatment is performed if necessary, followed by holding at a temperature of 70 ° C. (preferably 80 ° C.) or higher for 2 to 60 minutes (preferably 5 to 30 minutes) to inactivate the enzyme and subsequent separation Make good. After heat deactivation treatment, coarse separation is performed with a vibe screen or the like, and if necessary, diector treatment is performed, followed by ultracentrifugation treatment to remove suspended matters and precipitates.

  Next, the mixture is filtered using a filter aid such as diatomaceous earth (for example, celite), and the filtrate is treated with activated carbon (0.05 to 20 W / V%, preferably 0.1 to 10 W / V%, 20 to 65 ° C, (Preferably 25 to 60 ° C., 15 minutes to 4 hours, preferably 30 minutes to 2 hours) to deodorize, decolorize and purify.

  This is concentrated under reduced pressure (0 to 50 ° C.) or other conventional methods (to about 30 Bx), and if necessary, it is centrifuged again (ultra) or filtered to obtain a peptide stock solution. The peptide stock solution thus obtained is sterilized (by UHTST or other conventional method) and then used as a product (α-1000 (liquid)) filled in a container. If desired, the product can be further concentrated or diluted in reverse, or powdered to about 60 mesh by a conventional method such as spray drying or freeze drying, and filled into a container such as a bag ( α-1000 (powder)). For these products, store liquids in a refrigerated or frozen state, and store powders in a dry and dark place.

  The liquid, pasty or powdery peptide thus obtained is α-1000.

  The physicochemical properties of peptide α-1000 (spray dry powder) are as shown below.

Physicochemical properties of peptide α-1000 (powder) (A) molecular weight;
200-10,000 (by Sephadex G-25 column chromatography)
(B) Melting point: colored at 119 ° C. (decomposition point)
(C) Specific rotation [α] _D ²⁰ = −22 °
(D) solubility in solvents;
Easily soluble in water; hardly soluble in ethanol, acetone and hexane.
(E) distinction between acidic, neutral and basic;
Neutral pH 6.0-8.0 (10% solution)
(F) Appearance, components;
White powder; moisture 5.14% (heat drying under reduced pressure); protein 87.5% (Kjeldahl method, nitrogen / protein conversion factor 6.25); lipid 0% (Soxhlet extraction method); ash content 5.0% (directly) Ashing method).
(G) Characteristics;
It is a peptide derived from fish meat, obtained by inactivating a self-digesting enzyme by heating and hydrolyzing with a proteolytic enzyme.
(H) amino acid composition;
As shown below.

(Table 1)
Amino acid composition of peptide α-1000 (powder) ―――――――――――――――――
Analysis test item Result (%)
―――――――――――――――――
All amino acids arginine 3.34
Lysine 6.86
Histidine 3.34
Phenylalanine 2.33
Tyrosine 2.01
Leucine 6.35
Isoleucine 3.27
Methionine 2.26
Valine 4.16
Alanine 5.17
Glycine 3.59
Proline 2.15
Glutamic acid 12.35
Serine 3.30
Threonine 3.70
Aspartic acid 8.36
Tryptophan 0.32
Cystine 0.47
Total amount 73.33
―――――――――――――――――
Analysis method: Amino acid automatic analysis method (however, cystine was measured by hydrolyzing hydrochloric acid after performing formic acid oxidation treatment. Tryptophan was measured by high performance liquid chromatography.)

  Peptide α-1000 prepared in this way is liquid as it is, and in the case of powder, it is hydrated, passed through ODS resin and other hydrophobic adsorptive resin, washed with water, etc., and loaded with the peptide stock solution. Then, after elution with water, 5 to 20%, preferably 11 to 19%, more preferably 13 to 18% ethanol aqueous solution is added and elution with ethanol aqueous solution is performed to obtain peptide Y-2. As the resin, any resin can be used as long as it is a hydrophobic adsorptive resin, and the above-described commercially available resins can also be used as appropriate.

  The Y-2 fraction of the fish purified peptide thus obtained (that is, peptide Y-2) contains 100 μg / 100 g or more of IVY, and the presence of 150 μg / 100 g or more is sufficiently expected. . In fact, the hydrolyzate obtained by treating sardine fish meat with 0.7% alcalase for 17.5 hours was applied to the ODS column, and the second half of the water-eluted fraction and the Y-2 fraction were combined with the 15% ethanol-eluted fraction. As a result of analysis using an amino acid sequencer, it was confirmed that (Surden peptide Y-2, which is an enzyme-degraded purified product derived from sardine meat) contained 120 μg / 100 g of IVY.

The physicochemical properties of peptide Y-2 used as an active ingredient in the present invention are shown below.
(Physicochemical properties of peptide Y-2)
(A) Molecular weight: 200 to 10,000 (by ASAHIPAK GS-320 high performance liquid chromatography) (FIG. 5)
(B) Melting point: Colors and decomposes at 138 ° C.
(C) Specific rotation [α] _D ²⁰ = −40 °
(D) Solubility in solvents: Easily soluble in water, but hardly soluble in ethanol, acetone and hexane.
(E) Distinguishing between acidic, neutral and basic: neutral pH 5.0 to 8.0 (10% solution)
(F) Appearance of substance: white to light yellow powder.
Component (G): moisture 2.72% (atmospheric pressure drying method); protein 87.25% (Kjeldahl method, nitrogen / protein conversion factor 6.25); lipid 0% (Soxhlet extraction method); ash content 0.20 % (Direct ashing method)
(H) Physiological properties: Contains the tripeptide Ile-Val-Tyr and has an inhibitory effect on vascular smooth muscle cell proliferation.
(I) Infrared absorption spectrum: FIG.
(J) UV absorption spectrum: FIG.
(K) Amino acid composition: as shown in Table 2 below. The analysis method is an amino acid automatic analysis method (Shimadzu LC-6A system).

(Table 2)
Amino acid composition of peptide Y-2 ―――――――――――――――――
Amino acid Analysis value (%)
―――――――――――――――――
Aspartic acid 10.97
Threonine 4.10
Serine 2.90
Glutamic acid 12.52
Glycine 4.91
Alanine 5.06
Valine 6.20
Methionine 2.55
Isoleucine 5.55
Leucine 9.47
Tyrosine 2.96
Phenylalanine 4.75
Histidine 2.83
Lysine 10.07
Arginine 7.50
―――――――――――――――――

Furthermore, the present inventors paid attention to the usefulness of the above-mentioned peptide Y-2, and conducted research on peptides obtained by treating fish meat with proteolytic enzymes (for example, peptide α-1000). The derived peptide was treated with a hydrophobic adsorption resin (for example, ODS resin) and subjected to three steps of elution with water, elution with ethanol, and elution with water. After the first elution with water, 11-18V / V% ethanol The useful new knowledge that IVY in fish peptide was collect | recovered in large quantities in the aqueous solution elution fraction and the last water elution fraction was acquired.
Thus, the mixture after the first water elution fraction, the 11-18 V / V% ethanol aqueous solution elution fraction and the last water elution fraction has not only a high IVY content, but also a low bitterness and a good taste. It was confirmed that the peptide was excellent and stable, and was a completely new functional peptide. The peptide was identified as a novel peptide, and named peptide SY.
Furthermore, in the present invention, when only the above 11-18 V / V% ethanol aqueous solution elution fraction was isolated and examined, the Na content was about 0.1 to 0.2% (in peptide SY, Na was 1 to 3). %) And a new peptide with extremely low Na content was obtained, and this fraction was named peptide SY-MD.

In the present invention, research was conducted for the purpose of continuously recovering peptides containing as much Ile-Val-Tyr as possible using peptide α-1000 as a raw material.
As a result, after α-1000 was treated with ODS resin, water was added to obtain a part of the fraction eluted with water (the later fraction), and then an aqueous ethanol solution was continuously added. A fraction eluted with an aqueous ethanol solution is obtained. At this time, since the water used for water elution (1) partially remains, the ethanol concentration is 11 to 18 V / V%, preferably 14 to 16 V / V. It has been found that about V% is suitable.

  Furthermore, in obtaining the peptide SY, setting of the starting point for fractionation of the subsequent fraction of the target water elution (1), its end point (ie, the starting point of ethanol elution), The end point (starting point of water elution (2)) and the end point are identified or determined by fractionation time, salinity concentration, Bx, UV absorption measurement at wavelength 280 nm and monitoring, respectively. The manufacturing method of SY was also established, and based on these useful new findings, as a result of further research, the present invention was finally completed.

  That is, the present invention relates to a vascular smooth muscle cell proliferation inhibitor characterized by comprising a peptide SY or SY-MD containing the tripeptide IVY as an active ingredient, and has the following physicochemical properties: It is as follows.

(Physicochemical properties of peptide SY)
(A) Molecular weight: 200 to 10,000 (by ASAHIPAK GS-320 high performance liquid chromatography); FIG.
(B) Melting point: Colored and decomposed at 138 ± 3 ° C.
(C) Solubility in solvent: Easily soluble in water, but hardly soluble in ethanol, acetone and hexane.
(D) Appearance property: White to light yellow powder (E) Liquid property (pH): 4.0 to 6.0
(F) Ingredient: Moisture 1 to 5 W / W% (normal pressure heating drying method)
Protein 84-94 W / W% (micro Kjeldahl method)
; Lipid 0.5W / W% or less (Soxhlet extraction method)
; Ash content 4 ± 2W / W% (direct ashing method)
; Na 1-3 W / W% (atomic absorption photometry)
(G) Physiological properties: Contains the tripeptide Ile-Val-Tyr and has an inhibitory effect on vascular smooth muscle cell proliferation.
(H) Infrared absorption spectrum: FIG.
(I) UV absorption spectrum: FIG.
(J) Specific rotation: [α] _D ²⁰ = −40 ° to −51 °

(K) Main amino acid composition: As shown in Table 3 below, the analysis method is based on an automatic amino acid analysis method (Shimadzu LC-6A system).

(Table 3)
Amino acid composition of peptide SY ――――――――――――――――――――
Amino acid Analysis value (%)
――――――――――――――――――――
Aspartic acid 8.0-9.2
Glutamic acid 9.5 to 12.0
Valine 4.5-5.5
Methionine 2.5-3.8
Isoleucine 4.5-5.2
Leucine 7.3-8.5
Tyrosine 3.4-4.8
Phenylalanine 4.5-5.5
Histidine 3.0-3.8
Lysine 6.5-7.8
Arginine 5.0-6.0
――――――――――――――――――――

(Physicochemical properties of peptide SY-MD)
(A) Molecular weight: 200 to 10,000
(B) Melting point: Colored and decomposed at 138 ± 3 ° C.
(C) Solubility in solvent: Easily soluble in water, but hardly soluble in ethanol, acetone and hexane.
(D) Appearance property: White to light yellow powder (E) Liquid property (pH): 4.0 to 6.0
(F) component: moisture 2-6 W / W% (normal pressure heating drying method)
: Protein 90-98 W / W% (micro Kjeldahl method)
: Quality 0.5W / W% (Soxhlet extraction method)
: Ash content 3.0 W / W% or less (direct ashing method)
: Na 0.1-0.2 W / W% (atomic absorption photometry)
(G) Physiological properties: Contains the tripeptide Ile-Val-Tyr and has an inhibitory effect on vascular smooth muscle cell proliferation.
(H) Infrared absorption spectrum: FIG.
(I) Ultraviolet absorption spectrum: FIG.

  Peptide SY can be produced as follows. That is, a peptide stock solution, that is, an enzyme-treated fish meat (for example, peptide α-1000), which is a raw material for peptide SY, is added as it is in the case of a liquid, or in the case of a powder, to an ODS resin or other hydrophobic adsorptive resin. Then, the “stock solution loading” of FIG. 13 is performed to start the manufacturing process.

That is, a peptide component obtained by elution fractionation treatment with a hydrophobic adsorbent resin using a peptide stock solution, in which the elution fractionation treatment uses water, an ethanol aqueous solution, and water in this order as a developing solution in the elution pattern illustrated in FIG. The following fractions of water elution (1) obtained by each developing solution (1), 11-18V / V% ethanol elution fraction (FIG. 13 shows elution with 15% ethanol) and water elution ( 2) The peptide SY is produced by fractionating and mixing the fractions.
(1) Subsequent fraction of water elution (1): using water as a developing solution, starting from the time when the amount of sodium (Na) in the peptide (peptide SY) of water elution (1) is 1 to 3 g / 100 g. Fraction to the final fractionation point of the fraction after water elution (1) in which the amount of water is substantially 0 g / 100 g.
(2) Eleven to 18 V / V% ethanol elution fraction: Next, an ethanol aqueous solution with a concentration of 11 to 18 V / V% is used as a developing solution, and the elution amount of the peptide passes the peak and is reduced to about half of the peak. The fraction to be made. (The peptide SY-MD was obtained by isolating only this fraction.)
(3) Water elution (2) Fraction: Next, a fraction obtained by using water as a developing solution until the elution fraction treatment reaches the end point.

  Then, as described above, in (2), only the fraction obtained until the peptide elution amount passes the peak and is reduced to about half of the peak using the aqueous ethanol solution as the developing solution is isolated and collected. Peptide SY-MD containing peptide Ile-Val-Tyr can be obtained, and peptide Y-2 can also be obtained from (2) above or (2) and (1) above.

The method for producing these peptides will be described in detail below with reference to FIG. That is, the peptide SY can be fractionated and produced from the elution fraction described above. One example of elution patterns of various eluates is shown in FIG.
As shown in FIG. 13, the peptide SY according to the present invention adds, for example, α-1000 to a hydrophobic adsorption resin (stock solution loading) and then elutes with water (water elution (1)), and the water elution (1). This is produced by mixing the following fraction, the 11-18 V / V% ethanol-eluted fraction eluted with 11-18 V / V% ethanol solution, and the water-eluted (2) fraction eluted with water. be able to. Setting of the sampling start time of peptide SY fraction, setting of eluate switching timing, etc. are based on at least one measured value of Bx, salinity, UV (280 nm absorption), Na, or based on fractionation time What is necessary is just to determine suitably, It is also possible to monitor these items suitably, such as real time, and to implement by a computer.

For example, in the elution pattern of FIG. 13, the fractionation start time point of the subsequent fraction of peptide SY in water elution (2) can be determined by measuring the salinity value as follows.
i) Taking from 0 minutes after the start of water elution is not preferable because the Na content becomes 4 g / 100 g or more, which may result in a high Na material and increase blood pressure.
ii) When the fractionation time is 20 minutes after the start of elution with water, the Na content is within the allowable range of 1 to 3 g / 100 g.
iii) If the subsequent fractionation is started, the Na content is further reduced, but this time, the salt content is too low, so that guanine contained in the peptide SY tends to precipitate during concentration, which is not preferable. .
iv) Therefore, the fractionation start time was 20 minutes after the start of water elution, and the amount of Na was set to around 1 to 3 g / 100 g.
The final fractionation point of the water elution (1) fraction was the point at which the Na amount was substantially 0 g / 100 g.

  Next, 11-18 V / V% ethanol aqueous solution is added instead of water from this point, and the addition of the ethanol aqueous solution is stopped when the peptide elution amount passes the peak and is reduced to about half of the peak, and obtained here. Fractions were designated as 11-18 V / V% ethanol elution fractions. (A peptide SY-MD containing almost no Na is obtained by isolating only the 11-18 V / V% ethanol elution fraction.)

  The time when the addition of the 11-18 V / V% ethanol aqueous solution is stopped and switched to the addition of water is also the start point of water elution (2), but the UV absorption value at a wavelength of 280 nm indicating the total peptide amount rapidly decreases. The end point may be a time when the UV absorption value becomes zero and a steady state is entered. The fraction obtained here was used as the water elution (2) fraction.

The obtained (1) water elution (1) post-fraction, (2) 11-18V / V% ethanol elution fraction, and (3) water elution (2) fraction were separated separately or sequentially. The mixture obtained is the peptide SY of the present invention.
Thus, the fraction from the water-eluted (1) fraction in the middle of the fraction to the water-eluted (2) fraction through the 11-18 V / V% ethanol-eluted fraction is related to the present invention. It can be obtained as peptide SY (indicated as Surden peptide SY in FIG. 13).
Further, “15% ethanol elution” in FIG. 13 corresponds to “peptide SY-MD”.

  Peptides SY, SY-MD, and Y-2 are tripeptides (isoleucyl-valyl-tyrosine: Ile-Val-Tyr or IVY) first confirmed by the present inventors as one of the main bodies of vascular smooth muscle cell proliferation inhibitory peptides. In particular, peptide SY-MD does not contain the fraction after water elution (1), so it remains a bit bitter, but not only the taste is greatly improved, but Na is also added. It contains very little, so it is very useful for people who cannot take Na.

That is, the “stock solution loading” portion has a strong taste, but also contains some fish odor derived from the raw material, and has a high Na content. On the other hand, the post-fraction of water elution (1) has little fishy odor derived from the raw material and has a very good taste.
Therefore, by incorporating the post-fraction of water elution (1), a peptide material “Peptide SY” that can recover a large amount of IVY from peptide SY-MD and has excellent taste and stability can be obtained. It is.

  Peptides WB-1, WB-2, Y-2, SY and SY-MD according to the present invention are all natural product-derived substances containing the tripeptide IVY, and have excellent vascular smooth muscle cell proliferation inhibitory action. In addition, since there is no problem with regard to safety, it can also be used as a vascular smooth muscle cell growth inhibitor or a peptide for foods for specified health use for the purpose of inhibiting the growth. Therefore, this peptide can be used as a food additive such as a seasoning or a food for fortification or as an animal feed additive, and for the prevention of vascular diseases, and in some cases, treatment for vascular diseases because of its unique physiological activity. Or widely used as an infusion, health food, clinical nutrition food, or the like.

  When used as a food, the peptide can be added as it is, or used in combination with other foods or food ingredients as appropriate. Moreover, when using as a pharmaceutical, it can administer orally or parenterally. In the case of oral administration, for example, in accordance with conventional methods, tablets, granules, powders, capsules, powders, drinks can be prepared. For parenteral administration, for example, injection preparations, It can be used as drops, suppositories and the like. Of course, it is needless to say that the purified tripeptide IVY can also be used as a medicine or food (including beverages in the present invention) as described above.

  It has been confirmed for the first time that IVY suppresses the growth of normal human aortic vascular smooth muscle cells, and a vascular smooth muscle cell growth inhibitor containing IVY as an active ingredient has been developed. Furthermore, a substance containing IVY (for example, derived from wheat germ) Peptides WB-1, WB-2, or fish-derived peptides Y-2, SY, SY-MD, etc.) have also been confirmed to be effective, and the same growth inhibitors containing these peptides as active ingredients have also been developed. It was done.

  IVY and substances containing it (for example, the above-mentioned various peptides) have no safety problems (in fact, although 500 mg / day was forcibly administered orally to rats, 10 days later) Acute toxicity was not observed.) Since it has excellent taste as well as medicinal properties, it should be used not only as a growth inhibitor but also as a food such as peptides for specified health foods for the purpose of inhibiting the growth. Can do.

  In addition, IVY and substances containing it have an excellent vascular smooth muscle cell proliferation inhibitory action, so arteriosclerosis, diabetic vascular disorders, vascular thickening after angioplasty and arterial bypass surgery, and vascular reconstruction Prevention and / or treatment of various diseases such as vascular restenosis, arteriosclerotic disease, membrane proliferative nephritis, diabetes, peripheral arterial occlusion, etc., cell proliferative vascular inflammation resulting from organ transplantation and vascular injury It is also expected to be used as a medicine for food and food.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(1) Enzymatic hydrolysis of wheat germ After the wheat germ was treated with n-hexane at 80 ° C. for 5 hours for defatting, 10 g of the wheat germ was dissolved in 100 ml of deionized water and homogenized, and then α-amylase was treated with 1.0 W. / W% was added and treated at 37 ° C. for 5 hours. Next, protease was added at 1.0 W / W% and treated at the pH and temperature described below for 5 hours to obtain the protein yields shown below.

(Table 4)
Enzymatic hydrolysis of wheat germ ――――――――――――――――――――――――――――――――――――
Enzyme pH Temperature (℃) Yield
(Mg protein ml ^-1 )
――――――――――――――――――――――――――――――――――――
Pepsin 3.0 40 27.3
Protease YP-SS 3.0 40 20.9
Chymotrypsin 8.0 40 26.3
Trypsin 8.0 40 36.2
Alkaline protease 9.0 50 29.2
――――――――――――――――――――――――――――――――――――

  In the above, the wheat germ hydrolyzate obtained after degreasing the wheat germ, treated with 3% α-amylase for 3 hours, and treated with 0.5% alkaline protease for 8 hours was named peptide WB-1. Peptide WB-1 contained IVY of 21.6 ± 3.3 mg / 100 g.

  After the pH of the protease hydrolyzate was adjusted to 7.0, the enzyme was inactivated by boiling for 15 minutes and centrifuged (10,000 rpm, 15 minutes), and the resulting supernatant was filtered with a filter paper. Filtered.

(2) ODS separation of wheat germ hydrolyzate After adding 5 g of activated carbon to 100 ml of the obtained filtrate, this was applied to a hydrophobic adsorbent resin YMC ODS-AQ 120-S50 chromatographic column (3.5 × 13 cm), Elute sequentially with 500 ml of ethanol aqueous solutions of various concentrations (0, 10, 25, 50, 99.5% ethanol aqueous solution), and elute fractions F-1, F-2, F-3, F-4, and F-5, respectively. Fractionated and dried.

  The F-2 fraction obtained above (namely, the fraction obtained by subjecting peptide WB-1 to an ODS column and eluting with 10% ethanol water) was named peptide WB-2. Peptide WB-2 contained 143 ± 16.7 mg / 100 g of IVY.

(3) Separation of wheat germ hydrolyzate AG 50W-X8 20 mg (dry product) of the ODS fraction (F-2: peptide WB-2) obtained above was dissolved in 6 ml of deionized water, and this was dissolved in cation exchange resin AG 50W-X8. After applying to the (H ⁺ ) column, it was washed with 10 ml of water. And after eluting with 10 ml of 1.0 M NH ₄ OH aqueous solution, this was freeze-dried. The obtained AG 50W-X8 fraction contained 258 ± 15.1 mg / 100 g of IVY, and it was confirmed that the activity of wheat germ hydrolyzate was further enhanced by purification with ODS and AG50W.

(4) Purification and isolation by HPLC The AG50W-X8 1.0M NH ₄ OH elution fraction of the wheat germ hydrolyzate obtained above was subjected to 4-step HPLC (Shimadzu LC-9A) to purify IVY, Isolation was performed.

First step: Using a Cosmosil 5 Ph column, HPLC was performed while increasing the concentration of acetonitrile (CH ₃ CN) in 0.1% trifluoroacetic acid (TFA) to a linear gradient of 5 to 35% (flow rate 0.5 ml / min). ) As a result, two peaks I and II were obtained.

  Second step: Using a Cosmosil 5C18-ARIL column, elution was performed with acetonitrile (5 to 35% solution) to obtain 8 peaks from peak I and 6 peaks from peak II.

  Third step: Reverse phase column treatment of the second step was performed. The eluate was also the same as in the second step.

  Fourth step: Elution with Cosmosil 5 Ph and acetonitrile (5 to 35% solution), and by this final HPLC retention time, 10 peaks from peak I and 6 peaks from peak II are isolated, Each peptide was isolated. IVY was identified as the peak with a retention time of 43.7 minutes for peak II.

(Production of peptide α-1000)
Fresh sardines were processed with a deboner and minced. Divided fish meat is divided into 10kg surimi, rapidly frozen at -30 ° C or lower, crushed by a pulverizer, added with an equal amount of water, sent to a tank, and heated at 100 ° C for 10 minutes Then, the autolytic enzyme was inactivated and heat denatured. Next, aqueous ammonia was added to adjust the pH to 9.5.
A 0.1% solution of a commercially available alkaline protease product was added thereto, and the mixture was kept at 50 ° C. for 17.5 hours for enzymatic degradation. The enzyme was then inactivated by boiling for 15 minutes.
This was passed through a vibe screen (150 mesh), treated with a jet at 5000 rpm, then treated with a sharp press centrifuge (15000 rpm), and filtered using diatomaceous earth as a filter aid to obtain a peptide stock solution.

  Activated carbon was added to the peptide stock solution obtained above at 1 W / V%, stirred at 30 ° C. for 60 minutes, and then filtered to obtain a filtrate. This was concentrated under reduced pressure (20 ° C.) according to a conventional method and then UHTST sterilized according to a conventional method to obtain an α-1000 (liquid) product, which was further spray-dried according to a conventional method to obtain a particle size of 60 mesh. Α-1000 (powder) products were obtained and each of these products was stored frozen.

(Production 1 of peptide Y-2)
Sardine meat was digested with 0.7% alcalase for 17.5 hours, and the resulting hydrolyzate was applied to an ODS column, and the latter half of the water-eluted fraction was combined with the next 15% ethanol-eluted fraction. This fraction was designated as peptide Y-2. Peptide Y-2 contained 120 μg / 100 g of IVY.

(Production of peptide Y-2 2)
26.2 L of deionized water was added to 800 ml of sardine peptide α-1000 (liquid) produced in Example 2 (Brix45, protein content 29.6%), and this was added to an ODS resin (YMC ODS-AQ120-S50) column ( 1.5 × 50 cm) to adsorb the peptide, rinse with deionized water, and then sequentially elute with 27 L of 0%, 10%, 25%, 50%, 99.5% aqueous ethanol. Fractions Y-1, Y-2, Y-3, Y-4, and Y-5 were obtained. Among these, the Y-2 fraction was concentrated at 40 ° C. to remove ethanol, and lyophilized to obtain a sardine purified peptide (Y-2). This Y-2 fraction contained IVY about 2-3 times α-1000.

(IVY extraction from sardines)
Obtain 24.4 mg of the Surden Peptide Y-2 produced in (Production 1 of Peptide Y-2) of Example 3 and dissolve it in 3 ml of ultrapure water. The obtained chromatogram is shown in FIG. Here, from the chromatogram obtained by subjecting 100 μl of a solution of 535 μg IVY + 0.56 mg Y−2 / ml to HPLC condition 1, it was found that IVY was eluted around 32 minutes under this HPLC condition.

  In order to further promote the separated fraction, 0.8 ml of Y-2 solution was subjected to HPLC condition 1, and the fraction eluted at 31 to 33 minutes considered to contain IVY was collected. FIG. 15 shows a chromatogram obtained by drying and dissolving in 1 ml of ultrapure water and subjecting all of them to HPLC condition 2. From the IVY peak position of the chromatogram obtained by subjecting the mixture of IVY and Y-2 to HPLC condition 2, FIG. 15 (▼) is the IVY peak, and IVY is α-in the Sarden peptide Y-2. It was found that there are about three times 1000.

(Condition 1)
1min 2% CH ₃ CN
43min 35% CH ₃ CN
48min 35% CH ₃ CN
53min 2% CH ₃ CN
Flow rate 0.4ml / min
Wavelength 220nm
Column 5C18-ARIL (Waters)

(Condition 2)
10min 16.1% CH ₃ CN / 0.1% TFA
48min 35% CH ₃ CN / 0.1% TFA
53min 35% CH ₃ CN / 0.1% TFA
58min 16.1% CH ₃ CN / 0.1% TFA linear gradient analysis flow rate, wavelength, column are the same as condition 1

(Production of peptide SY)
5 g of sardine peptide α-1000 (powder) produced in Example 2 was dissolved in 500 ml of deionized water to obtain a stock solution, and a hydrophobic adsorption resin SEPABEADS SP207 (Mitsubishi Chemical Corporation) column (3.5 × 13 cm) To fill the column with α-1000 solution (stock load), then add water, 15% ethanol aqueous solution, then water, 500 ml each according to the elution pattern of FIG. All fractions, ie, the fraction after water elution (1), the 15% ethanol elution fraction, and the water elution (2) fraction, were mixed and lyophilized to obtain 2.1 g of peptide SY (powder). Obtained. The Na content of peptide SY was 1.45 W / W%. (By atomic absorption photometry)

(Production of peptide SY-MD)
5 g of peptide α-1000 (powder) obtained in Example 2 was dissolved in 500 ml of deionized water to obtain a stock solution, and a hydrophobic adsorption resin SEPABEADS SP207 (manufactured by Mitsubishi Chemical Corporation) column (3.5 × 13 cm) The column was filled with α-1000 solution (stock solution loading), and only the 15% ethanol-eluted fraction was isolated and fractioned out of all fractions of the saden peptide SY shown in the elution pattern of FIG. This was freeze-dried to obtain 1.7 g of peptide SY-MD (powder). The Na content of peptide SY-MD was 0.124 W / W%. (By atomic absorption photometry)

(Drink production)
―――――――――――――――――――――――
100ml drink recipe ------------
Fructose dextrose liquid sugar 4.5g
1g sugar alcohol
Sour seasoning 0.2g
Fragrance 0.13g
Sweetener (stevia) 0.03g
Caramel color 0.02g
Peptide SY (powder) 0.5g
(Obtained in Example 5)
Purified water Constant volume to 100 ml -----------
50ml drink formula list ――――――――――――――――――――――――
Fructose dextrose liquid sugar 10g
Fragrance 0.3g
Acidulant 0.16g
Sweetener (stevia) 0.015g
Peptide SY (powder) 0.5g
(Obtained in Example 5)
Purified water Constant volume to 50 ml -----------
30ml drink formula list ――――――――――――――――――――――――
Fructose dextrose liquid sugar 5g
Fragrance 0.25g
Sour seasoning 0.1g
Sweetener (stevia) 0.015g
Peptide SY (powder) 0.5g
(Obtained in Example 5)
Purified water Constant volume to 30 ml -----------

  The above blend was mixed and dissolved at 60 ° C. and then subjected to plate sterilization at 128 ° C. for 10 seconds. Next, 100 ml, 50 ml, and 30 ml brown bottles having been thoroughly washed were filled at 90 ° C., allowed to cool at room temperature, and then rapidly cooled in a running water tank to produce a drink.

(Manufacture of tablets)
Tablets were produced with the following formulation.
500 g of peptide SY (powder) obtained in Example 5, 356 g of reduced maltose starch syrup, 100 g of crystalline cellulose, 40 g of sucrose fatty acid ester and 4 g of sweetener (stevia) were mixed, and this mixture was compressed by a compression tablet machine to be uncoated tablet (250 mg × 4000 pieces) were prepared. This uncoated tablet was coated with 7.5 mg of shellac solution, and 4000 tablets containing 500 mg of peptide SY (powder) per 4 tablets were produced.

  In the same manner as described above, drinks and tablets were produced using the peptides WB-1 and WB-2 obtained in Example 1, the peptide Y-2 obtained in Example 3, and the peptide SY-MD obtained in Example 6. did.

(Vascular smooth muscle cell proliferation inhibition test)
About natural tripeptide Ile-Val-Tyr (IVY) discovered from wheat germ and fish protein, the effect | action with respect to a vascular smooth muscle cell was examined, and it confirmed that IVY had the effect | action which suppresses the proliferation.

(1) Test As normal cells, normal human aortic vascular smooth muscle cells (VSMC, Cyro AOSMC; Sanko Junyaku Co., Ltd.) passage number 6-11 are used, and as chemically synthesized IVY, tripeptide IVY is used. Cell proliferation was measured, and cytotoxicity tests and responsiveness to angiotensin II stimulation were also measured.

Cell proliferation measurement: 1.5 ml of VSMC cultured to a confluence in SmGM serum medium containing 5% FBS was seeded in a 24-well plate at a concentration of 1 × 10 ⁵ cells / ml. 1le-Val-Tyr was added to the serum medium to a concentration of 10, 50, and 100 μM, and the cells were cultured in a CO ₂ incubator at 37 ° C. for 5 days. The number of cells was stained with Trypan Blue, and the number of viable cells was counted with a hemocytometer. In addition, what added sterilized water instead of Ile-Val-Tyr was set as control.

Cytotoxicity test and responsiveness to angiotensin II stimulation: VSMC cultured to confluence in SmGM serum medium containing 5% FBS was diluted with serum-free medium to a concentration of 1 × 10 ⁵ cells / ml, and added to a 96-well plate. 0.1 ml was seeded. After pre-culturing at 37 ° C. for 24 hours in a CO ₂ incubator, 10 μl of 1 mM Ile-Val-Tyr solution was added and further incubated for 48 hours. A control to which the same amount of sterilized water was added was used as a control. Next, a solution (Cell Counting) containing WST-8 (2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium) which is a tetrazolium salt. After adding 10 μl of Kit-8, Doujin Chemical Co., Ltd. to the medium, cytotoxicity was evaluated from the amount of water-soluble formazan (450 nm) generated by a color reaction at 37 ° C. for 3 hours in a CO ₂ incubator. In addition, cell growth of Ile-Val-Tyr was determined by determining the change in cell number (Cell Counting Kit-8) accompanying the addition of Ile-Val-Tyr (10, 100, 1000 μM) in the presence of 1 μM angiotensin II after preculture. The inhibition mechanism was evaluated.

(2) Results
It showed additive effects of Ile-Val-Tyr to cytostatic 16 for VSMC proliferation for 5 days by serum culture. As a result, Ile-Val-Tyr inhibited the growth of VSMC in a concentration-dependent manner, and a growth inhibitory effect (P <0.05) of about 33% was observed when 100 μM was added. There are no reports on the development of the inhibitory effect of VSMC on the growth of peptides, and this result is the first demonstration that Ile-Val-Tyr has a preventive effect on blood pressure increase due to contraction of aortic blood vessels. It was.

Cytotoxicity and Cell Growth Inhibitory Mechanism FIG. 17 shows the amount of WST-8 incorporated into cells in the presence of 1 mM Ile-Val-Tyr. As a result, since no significant reduction in uptake was observed compared to the control, IIe-Val-Tyr did not have any toxic effect on VSMC cells, that is, expression of cytostatic activity was not observed in cells. It turned out not to be due to toxicity. On the other hand, the addition of angiotensin II, which exhibits a strong vasoconstrictive action in the renin-angiotensin system, which is a pressor metabolism system, to the serum-free medium clearly promoted the growth of VSMC. It was shown again that they were involved.

  Thus, when the amount of WST-8 taken up by angiotensin II stimulation in the presence of Ile-Val-Tyr was examined, no effect was observed (FIG. 18). This suggested that the VSMC cell growth inhibitory effect of Ile-Val-Tyr (FIG. 16) was not expressed by an antagonistic action on the angiotensin II receptor.

(3) Conclusion 1) It was confirmed that Ile-Val-Tyr has an action of inhibiting the contraction of human aortic vascular smooth muscle in vivo.
2) It was shown that the expression of the vasorelaxant action by Ile-Val-Tyr was not due to the antagonistic action of angiotensin II.

The infrared absorption spectrum of peptide WB-1 is shown. The ultraviolet absorption spectrum of peptide WB-1 is shown. The infrared absorption spectrum of peptide WB-2 is shown. The ultraviolet absorption spectrum of peptide WB-2 is shown. The molecular weight distribution by the gel filtration of peptide Y-2 is shown. The infrared absorption spectrum of peptide Y-2 is shown. The ultraviolet absorption spectrum of peptide Y-2 is shown. The molecular weight of peptide SY is shown. The infrared absorption spectrum of peptide SY is shown. The ultraviolet absorption spectrum of peptide SY is shown. The infrared absorption spectrum of peptide SY-MD is shown. The ultraviolet absorption spectrum of peptide SY-MD is shown. It is a graph of the elution pattern of peptides SY and SY-MD. It is the chromatogram of HPLC (condition 1) of peptide Y-2. It is the chromatogram of HPLC (condition 2) of peptide Y-2. The influence of IVY on VSMC grown for 5 days in 5% FBS medium is shown. Shown is the cytotoxicity of IVY against VSMC. The cells were treated with serum-free (Basal) or serum-free + 1 mM IVY for 48 hours, and the relative uptake rate of WST-8 into the cells was used for toxicity evaluation. Figure 4 shows the effect of IVY on VSMC uptake of WST-8 in response to angiotensin II. In the presence or absence of 1 μM angiotensin II, cells were treated with serum-free (Basal), serum-free + 1 mM IVY for 48 hours.

Claims (18)

  A vascular smooth muscle cell proliferation inhibitor comprising the tripeptide Ile-Val-Tyr as an active ingredient.   A vascular smooth muscle cell proliferation inhibitor characterized by comprising a substance derived from a natural product and containing a tripeptide Ile-Val-Tyr as an active ingredient.   The growth inhibitor according to claim 2, wherein fish meat or wheat germ is used as a natural product.   The growth inhibitor according to claim 3, wherein the natural product is subjected to at least one of a degreasing treatment, an enzyme treatment, an ion exchange resin treatment, a hydrophobic adsorption resin treatment, and a chromatography treatment.   Peptide WB-1 obtained by degreasing and α-amylase treatment of wheat germ and then protease treatment, or a peptide obtained by further adsorbing this peptide on a peptide-adsorbing resin and then eluting it with an aqueous ethanol solution The growth inhibitor according to any one of claims 2 to 4, wherein WB-2 is an active ingredient. Peptide WB-1 having the following physicochemical properties.
(A) Melting point (darkening point): 121.3 to 160.5 ° C.
(B) Specific rotation: [α] _D ²⁰ = + 269 ° ± 5 ° (measured concentration; 2.11 mg / 5 ml-water)
(C) Liquidity (pH): 6.8 to 8.4 (concentration 0.03%)
(D) Physiological properties: Contains tripeptide Ile-Val-Tyr and has an inhibitory effect on vascular smooth muscle cell proliferation.
(E) Infrared absorption spectrum: FIG.
(F) Ultraviolet absorption spectrum: FIG. Peptide WB-2 having the following physicochemical properties.
(A) Melting point (darkening point): 155.2-205.7 ° C
(B) Specific rotation: [α] _D ²⁰ = + 213 ° ± 5 ° (measured concentration; 2.31 mg / 5 ml-water)
(C) Liquidity (pH): 6.8 to 8.0 (concentration 0.05%)
(D) Physiological properties: Contains tripeptide Ile-Val-Tyr and has an inhibitory effect on vascular smooth muscle cell proliferation.
(E) Infrared absorption spectrum: FIG.
(F) Ultraviolet absorption spectrum: FIG.   A vascular smooth muscle cell proliferation inhibitor comprising the peptide WB-1 according to claim 6 or the peptide WB-2 according to claim 7 as an active ingredient.   Peptide Y-2 containing tripeptide Ile-Val-Tyr, which is a fraction eluted with 8-17 V / V% ethanol aqueous solution after adsorbing peptide stock solution on peptide adsorption resin An inhibitor of vascular smooth muscle cell proliferation. The growth inhibitor according to claim 9, wherein peptide Y-2 has the following physicochemical properties.
(Physicochemical properties of peptide Y-2)
(A) Molecular weight: 200 to 10,000 (by ASAHIPAK GS-320 high performance liquid chromatography)
(B) Melting point: Colored and decomposed at 138 °.
(C) Specific rotation [α] _D ²⁰ = −40 °
(D) Solubility in solvents: Easily soluble in water, but hardly soluble in ethanol, acetone and hexane.
(E) Distinguishing between acidic, neutral and basic: neutral pH 5.0 to 8.0 (10% solution)
(F) Appearance of substance: white to light yellow powder.
Component (G): moisture 2.72% (atmospheric pressure drying method); protein 87.25% (Kjeldahl method, nitrogen / protein conversion factor 6.25); lipid 0% (Soxhlet extraction method); ash content 0.20 % (Direct ashing method)
(H) Physiological properties: Contains the tripeptide Ile-Val-Tyr and has an inhibitory effect on vascular smooth muscle cell proliferation.
(I) Infrared absorption spectrum: FIG.
(J) UV absorption spectrum: FIG. The peptide stock solution is a peptide component obtained by elution fractionation treatment with a peptide adsorption resin, and the elution fractionation treatment uses water, an ethanol aqueous solution, and water in this order as a development solution. The following fraction obtained by water elution (1) obtained by the following steps, 11-18V / V% ethanol elution fraction and water elution (2) fraction were separated and mixed, and the tripeptide Ile-Val- An inhibitor of vascular smooth muscle cell proliferation, comprising a peptide SY containing Tyr as an active ingredient.
(1) Subsequent fraction of water elution (1): Water is used as a developing solution, and the amount of sodium from the time when the amount of sodium (Na) in the entire eluted peptide fraction (peptide SY) becomes 1 to 3 g / 100 g. Is the fraction up to the final fractionation point of the post-fraction of water elution (1) in which is substantially 0 g / 100 g.
(2) Ethanol elution fraction: Next, a fraction obtained by using an ethanol aqueous solution having a concentration of 11 to 18 V / V% as a developing solution until the peptide elution amount passes the peak and is reduced to about half of the peak.
(3) Water elution (2) Fraction: Next, a fraction obtained by using water as a developing solution until the elution fraction treatment reaches the end point. The growth inhibitor according to claim 11, wherein the peptide SY has the following physicochemical properties.
(Physicochemical properties of peptide SY)
(A) Molecular weight: 200 to 10,000 (by ASAHIPAK GS-320 high performance liquid chromatography)
(B) Melting point: Colored and decomposed at 138 ± 3 ° C.
(C) Solubility in solvent: Easily soluble in water, but hardly soluble in ethanol, acetone and hexane.
(D) Appearance property: White to light yellow powder (E) Liquid property (pH): 4.0 to 6.0
(F) Ingredient: Moisture 1 to 5 W / W% (normal pressure heating drying method)
Protein 84-94 W / W% (micro Kjeldahl method)
; Lipid 0.5% (Soxhlet extraction method)
; Ash content 4 ± 2% (direct ashing method)
; Na 1-3 W / W% (atomic absorption photometry)
(G) Physiological properties: Contains the tripeptide Ile-Val-Tyr and has an inhibitory effect on vascular smooth muscle cell proliferation.
(H) Infrared absorption spectrum: FIG.
(I) UV absorption spectrum: FIG.   Only the fraction obtained by using the aqueous ethanol solution in (2) of claim 11 as a developing solution until the peptide elution amount passes the peak and is reduced to about half of the peak is isolated and dispersed, and the tripeptide Ile-Val is obtained. -Vascular smooth muscle cell proliferation inhibitor containing peptide SY-MD containing Tyr as an active ingredient. The growth inhibitor according to claim 13, wherein the peptide SY-MD has the following physicochemical properties.
(Physicochemical properties of peptide SY-MD)
(A) Molecular weight: 200 to 10,000
(B) Melting point: Colored and decomposed at 138 ± 3 ° C.
(C) Solubility in solvent: Easily soluble in water, but hardly soluble in ethanol, acetone and hexane.
(D) Appearance property: White to light yellow powder (E) Liquid property (pH): 4.0 to 6.0
(F) component: moisture 2-6 W / W% (normal pressure heating drying method)
: Protein 90-98 W / W% (micro Kjeldahl method)
: Quality 0.5W / W% (Soxhlet extraction method)
: Ash content 3.0W / W% (Direct ashing method)
: Na 0.1-0.2 W / W% (atomic absorption photometry)
(G) Physiological properties: Contains the tripeptide Ile-Val-Tyr and has an inhibitory effect on vascular smooth muscle cell proliferation.
(H) Infrared absorption spectrum: FIG.
(I) Ultraviolet absorption spectrum: FIG.   The peptide stock solution is an aqueous solution of peptide α-1000 obtained by heat-denaturing fish meat, hydrolyzing it with neutral or alkaline protease treatment to inactivate the enzyme, and then separating it. The growth inhibitor according to any one of claims 9 to 14. The growth inhibitor according to claim 15, wherein the peptide α-1000 has the following physicochemical properties.
(Physicochemical properties of peptide α-1000)
(A) Molecular weight:
200-10,000 (by Sephadex G-25 column chromatography)
(B) Melting point: colored at 119 ° C. (decomposition point)
(C) Specific rotation:
[Α] _D ²⁰ = −22 °
(D) Solubility in solvents:
Easily soluble in water; hardly soluble in ethanol, acetone and hexane.
(E) Distinguishing between acidic, neutral and basic:
Neutral (f) appearance, ingredients:
White powder: moisture 5.14% (heat drying under reduced pressure); protein 87.5% (Kjeldahl method, nitrogen / protein conversion factor 6.25); lipid 0% (Soxhlet extraction method); ash content 5.0% (directly) Ashing method).
(G) Characteristics:
A peptide derived from fish meat, obtained by inactivating a self-digesting enzyme by heating and hydrolyzing with a proteolytic enzyme, and contains the tripeptide Ile-Val-Tyr.   A food for suppressing or preventing vascular smooth muscle cell proliferation, comprising a natural substance-derived substance containing a tripeptide Ile-Val-Tyr as an active ingredient.   Peptide WB-1 according to claim 6, peptide WB-2 according to claim 7, peptide Y-2 according to claim 9 or 10, peptide SY according to claim 11 or 12, claim 13 or 14. For suppressing vascular smooth muscle cell proliferation, comprising at least one peptide selected from the peptide SY-MD according to 14, or containing at least one peptide selected from these peptides Or preventive food.

Images