TW202014197A - Pharmaceutical composition containing dipeptide - Google Patents

Abstract

Provided is a novel anti-inflammatory agent. This anti-inflammatory agent has as an active ingredient a dipeptide selected from among: (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, D)Pro-(D)Leu, (D)Val-(D)Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, (D)Val-(D)Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Ile-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)Pro, (D)Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D)Asp-(L)Val, (D)Asp-(L)Leu, and (D)Asp-(L)Phe.

Description

Pharmaceutical composition containing dipeptide

The invention relates to a pharmaceutical or food composition containing dipeptide.

With the super-aging society, the importance of prevention/treatment measures for lifestyle-related diseases such as chronic inflammation-related metabolic diseases such as diabetes and obesity and arteriosclerosis and other circulatory organ diseases is increasing. In fact, the development of IL-1β signal inhibition or NF-κB inhibition is developed for diabetes. In chronic inflammatory diseases represented by rheumatoid arthritis, abnormal production and secretion of large amounts of inflammatory cytokines such as TNF-α, IL-1, IL-6, and the inhibition of these inflammatory cytokines The medicine is useful as an excellent anti-inflammatory agent. For example, as TNF-α inhibitors, antibody medicines such as infliximab are used as therapeutic drugs for rheumatoid arthritis (Non-Patent Document 1). In addition, as an IL-6 inhibitory drug, an antibody drug tocilizumab (Non-Patent Document 2) is used. Furthermore, as IL-1 inhibitors, protein preparations such as anakinra, linaccept, and kanazumab, or antibody medicines have been developed (Non-Patent Document 3). [Prior Technical Literature] [Non-patent literature]

[Non-Patent Document 1] Guidelines for TNF Inhibitors for Rheumatoid Arthritis (RA) (Japanese Society of Rheumatology) [Non-Patent Literature 2] Pharmaceutical Journal 129(6) 667-674(2009) [Non-Patent Document 3] Journal of the Japanese Society of Internal Medicine Volume 100 No. 10 p. 2985-2990 (October 10, 2011)

[Problems to be solved by the invention]

Under this background, the present inventors found that liver hydrolysate has an excellent IL-1 production inhibitory effect, and is useful as a cytokine production inhibitor and anti-inflammatory agent, and applied for a patent application (PCT/JP2018/006351). However, although liver hydrolysate contains a large amount of amino acids or peptides, the actual effective ingredients have not yet been determined. Therefore, the object of the present invention is to provide a novel anti-inflammatory agent based on the inhibitory effect of cytokine production. [Technical means to solve the problem]

Therefore, in order to develop a novel anti-inflammatory agent, the present inventors conducted research, administered liver hydrolysate into animals, and explored indigestible peptides that migrated in the blood. In addition, for the liver hydrolysate, various columns were used to transfer peptides Distinguish and evaluate the efficacy of the drug. As a result, it was found that the dipeptide derived from the specific D body amino acid rather than the L body amino acid was found to have cytokine production inhibitory effects of IL-1β, IL-6, etc. To complete the present invention.

That is, the present invention provides the following [1] to [15].

[1] An anti-inflammatory agent selected from (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, (D)Pro-(D) Leu, (D)Val-(D)Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, (D)Val-(D) Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Ile-(L) Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D)Pro-(L )Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D)Asp-(L ) The dipeptide in Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe is the active ingredient. [2] The anti-inflammatory agent as described in [1], whose effective ingredients are selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D) Leu, ( D) Asp-(D)Phe, (D)Asp-(L)Val, (D)Asp-(L)Ile, (D)Asp-(L)Leu, and (D)Asp-(L)Phe By. [3] An inflammatory cytokine production inhibitor selected from (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, (D)Pro -(D)Leu, (D)Val-(D)Pro, (D)Pro-(D)Val, (D)Leu-(D)Glu, (D)Ile-(D)pGlu, (D)Val -(D)pGlu, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Ile -(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D) Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D) The dipeptide in Asp-(L)Val, (D)Asp-(L)Leu, and (D)Asp-(L)Phe is the active ingredient. [4] The inflammatory cytokine production inhibitor as described in [3], the effective component is selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D ) Leu, (D) Asp-(D) Phe, (D) Asp-(L) Val, (D) Asp-(L) Ile, (D) Asp-(L) Leu, and (D) Asp-( L) Phe. [5] An IL-1 production inhibitor or IL-6 production inhibitor selected from (D) Ile-(D) Pro, (D) Leu-(D) Pro, (D) Pro- (D) Ile, (D)Pro-(D)Leu, (D)Val-(D)-Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D )Hyp, (D)Val-(D)Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D )Phe, (D)Ile-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L )-Pro, (D)Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-( The dipeptide in L) Ile, (D) Asp-(L) Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe is the active ingredient. [6] The IL-1 production inhibitor or IL-6 production inhibitor as described in [5], the active ingredient is selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, ( D) Asp-(L) Leu, (D) Asp-(D) Phe, (D) Asp-(L) Val, (D) Asp-(L) Ile, (D) Asp-(L) Leu, and (D) Asp-(L)Phe. [7] An inflammation-improving food composition selected from (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, (D)Pro- (D)Leu, (D)Val-(D)Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, (D)Val- (D)Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Ile- (L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D)Pro -(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D)Asp -The dipeptide in (L) Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe is an active ingredient. [8] The food composition according to [7], wherein the effective ingredients are selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D) Leu, ( D) Asp-(D)Phe, (D)Asp-(L)Val, (D)Asp-(L)Ile, (D)Asp-(L)Leu, and (D)Asp-(L)Phe By. [9] An inflammatory cytokine production inhibiting food composition selected from (D) Ile-(D) Pro, (D) Leu-(D) Pro, (D) Pro-(D) Ile, (D )Pro-(D)Leu, (D)Val-(D)-Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, ( D) Val-(D)Hyp, (D) Asp-(D) Ile, (D) Asp-(D) Val, (D) Asp-(D) Leu, (D) Asp-(D) Phe, ( D)Ile-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D)Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, The dipeptide in (D) Asp-(L) Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe is the active ingredient. [10] The food composition according to [9], wherein the effective ingredients are selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D) Leu, ( D) Asp-(D)Phe, (D)Asp-(L)Val, (D)Asp-(L)Ile, (D)Asp-(L)Leu, and (D)Asp-(L)Phe By. [11] A food composition for inhibiting IL-1 production or a food composition for inhibiting IL-6 production, selected from (D) Ile-(D) Pro, (D) Leu-(D) Pro, (D )Pro-(D)Ile, (D)Val-(D)-Pro, (D)Pro-(D)Val, (D)Leu-(D)Glu, (D)Ile-(D)pGlu, ( D) Val-(D)pGlu, (D) Asp-(D) Ile, (D) Asp-(D) Val, (D) Asp-(D) Leu, (D) Asp-(D) Phe, ( D)Ile-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D)Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, The dipeptide in (D) Asp-(L) Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe is the active ingredient. [12] The food composition according to [11], wherein the effective ingredients are selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D) Leu, ( D) Asp-(D)Phe, (D)Asp-(L)Val, (D)Asp-(L)Ile, (D)Asp-(L)Leu, and (D)Asp-(L)Phe By. [13] Use of a dipeptide for the manufacture of anti-inflammatory agents and selected from (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D) Ile, (D)Pro-(D)Leu, (D)Val-(D)Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D) Hyp, (D)Val-(D)Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D) Phe, (D)Ile-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L) -Pro, (D)Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L ) Ile, (D) Asp-(L) Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe. [14] A dipeptide for the treatment of inflammatory diseases and is selected from (D) Ile-(D) Pro, (D) Leu-(D) Pro, (D) Pro-(D) Ile, (D )Pro-(D)Leu, (D)Val-(D)Pro, (D)Pro-(D)Val, (D)Leu-(D)Glu, (D)Ile-(D)pGlu, (D )Val-(D)pGlu, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D )Ile-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, ( D)Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, ( D) Asp-(L) Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe. [15] An anti-inflammatory disease treatment method, characterized in that an effective amount of (D) Ile-(D) Pro, (D) Leu-(D) Pro, (D) Pro-(D) is selected Ile, (D)Pro-(D)Leu, (D)Val-(D)-Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D )Hyp, (D)Val-(D)Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D )Phe, (D)Ile-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L )-Pro, (D)Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-( L) Dipeptide in Ile, (D) Asp-(L) Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe. [Effect of invention]

The dipeptide used in the present invention is indigestible because it is derived from D-body amino acids, exists in the blood for a long time, and has an inhibitory effect on IL-1 production and an inhibitory effect on IL-6 production, so it is inhibited as a cytokine production Pharmaceutical and food compositions for use and anti-inflammatory are useful.

The dipeptide as the active ingredient of the anti-inflammatory agent of the present invention is selected from (D) Ile-(D) Pro, (D) Leu-(D) Pro, (D) Pro-(D) Ile, (D) Pro -(D)Leu, (D)Val-(D)-Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, (D) Val-(D)Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D) Ile-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D )Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D ) Asp-(L)Val, (D)Asp-(L)Leu, and (D)Asp-(L)Phe dipeptides, of which, the migration and effect of blood administered orally continue From the viewpoint of sex and anti-inflammatory effects, it is preferably selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D) Leu, (D) Asp- (D) Phe, (D) Asp-(L) Val, (D) Asp-(L) Ile, (D) Asp-(L) Leu, and (D) Asp-(L) Phe dipeptide, More preferably, they are (D) Asp-(D) Leu and (D) Asp-(L) Leu. Here, the notation (D) means that the amino acid is D-body. Notation (L) means that the amino acid is L-body. Further, among the α body and β body of the (D) body or (L) body dipeptide, the β body is more preferable.

The above-mentioned dipeptide can be produced by (A) amino acid as a raw material by a general liquid-phase peptide synthesis method or a solid-phase peptide synthesis method. For example, it can be manufactured by condensing an amino acid that protects a functional group other than an α-amino group with an amino acid that protects a functional group other than a carboxyl group, or an amino acid that activates a carboxyl group and protects a functional group other than the carboxyl group After that, the protective group is detached. Here, as the protective group of the amine group of the amino acid, a benzyloxycarbonyl group, a third butoxycarbonyl group, a sulfenylmethoxycarbonyl group and the like can be mentioned. Examples of protecting groups for carboxyl groups include tertiary butyl and benzyl. For the condensation reaction, a method using a condensing agent such as N,N'-dicyclohexylcarbodiimide, dicyclohexylmethaneurea, an active ester method using nitrophenol, N-hydroxysuccinimide, or mixed acid anhydride Law etc. After the condensation reaction is completed, the protecting group is removed, but in the case of the solid-phase method, the bond between the C-terminal of the peptide and the resin is further cleaved. Furthermore, the above-mentioned peptides were purified according to a usual method.

The above-mentioned dipeptide may be an acid addition salt or a basic salt. Examples of acid addition salts include salts of inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, and perchloric acid; citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, p Toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, trifluoroacetic acid and other organic acid salts. Examples of basic salts include salts of alkali metals such as sodium, potassium, and lithium; salts of alkaline earth metals such as calcium and magnesium;

The above peptide may be a solvate. Examples of solvates include solvates such as water (in the case of hydrates), methanol, ethanol, and isopropanol.

The above-mentioned dipeptides are indigestible, have high blood migration after oral administration, and are excellent in persistence. They have a strong effect of inhibiting the production of IL-1β and IL-6, which is one of inflammatory cytokines, and inhibit IL. -1β and IL-6 mRNA expression. Therefore, the above-mentioned dipeptide suppresses the production of inflammatory cytokines typified by IL-1 and IL-6, and is useful as a therapeutic agent for inflammatory cytokine-related inflammatory diseases and a food and beverage composition for improving inflammation. Here, examples of diseases related to inflammatory cytokines such as IL-1 and IL-6 include rheumatoid arthritis, deformable arthropathy, inflammatory bowel disease, septicemia, acute and chronic osteocytic leukemia, and bone Porosity, lifestyle habits, etc.

The pharmaceutical composition of the present invention can be administered by oral administration, transdermal administration, enteral administration, intravenous administration, etc., and is more preferably oral administration. Examples of preparations for oral administration include liquids, lozenges, powders, fine granules, granules, and capsules. Liquids, lozenges, and liquids are more preferred.

In order to prepare such oral administration preparations, excipients such as lactose, mannitol, corn starch, crystalline cellulose, etc.; binders such as cellulose derivatives, gum arabic, gelatin; calcium carboxymethyl cellulose, etc. can be used Disintegrants; lubricants such as talc, magnesium stearate; dissolution aids such as nonionic surfactants; flavoring agents, sweeteners, stabilizers, pH adjusters, water, ethanol, propylene glycol, glycerin, etc. In addition, coating agents such as hydroxymethyl cellulose phthalate, hydroxypropyl methyl cellulose succinate, cellulose acetate phthalate, and methacrylate copolymer can also be used.

In addition, other active ingredients may be formulated in the pharmaceutical composition of the present invention. Examples of other active ingredients include: Vitamin B ₁ ; thiamine, thiamine nitrate, thiamine hydrochloride, furanthiamine, diphenyldithiamine, benfotiamine, thiamine disulfide, Cythiamin, thiamine propyl disulfide and their derivatives, vitamin B ₂ ; riboflavin and derivatives and their salts, vitamin B ₃ ; niacin, nicotinic acid, nicotine Acetamides and derivatives and their salts, vitamin B ₅ ; panthenol, pantothenic acid and derivatives and their salts, vitamin B ₆ ; pyridoxine and derivatives and their salts, vitamin B ₁₂ ; Cyanocobalamin and derivatives and their salts, other vitamins; Vitamin A, Vitamin C, Vitamin E, Vitamin K, Vitamin P, Diisopropylamine Dichloroacetate, Taurine, Chondroitin Sulfate, Royal Jelly, Coffee Cauliflower, turmeric, silybum, dandelion, dandelion, burdock, garlic, chrysanthemum, yarrow, gardenia, sesame, ginseng, asparagus, onion, chicory, medicinal sage, artichoke (artichoke) , Lycium barbarum, Leguminosae/Hydraceae plants, Phyllanthus, Erva De Passarinho, Balsam calyx, wild tung, black tea, resveratrol, catechins, berberine, rosemary, bean extract Substances, metformin, etc.

In addition to the pharmaceutical products, the composition of the present invention can also be used as functional foods such as quasi-drugs, foods for specific health care, sports drinks, beverages for rehabilitation, pet foods and the like.

The content of the above-mentioned dipeptide in the pharmaceutical composition or food composition of the present invention varies depending on the form of administration, and is usually preferably 0.001 to 10% by mass, and more preferably 0.001 to 5% by mass. In addition, the daily dose of the dipeptide in the pharmaceutical composition or food composition of the present invention is preferably 10 mg to 1000 mg, more preferably 20 mg to 800 mg, and still more preferably 50 mg to 800 mg. [Example]

Next, the present invention will be described in detail with examples, but the present invention is not limited by these examples.

Example 1 (differentiation of liver hydrolysate) (1) The distinction between peptide and pyroglutamine A strong cation exchange resin (AG50) was filled in the Econo Column (2.5×20 cm), and the resin was equilibrated with 10 mM HCl. Dissolve 1 g of liver hydrolysate (Sample A) in 20 mL of 10 mM HCl. This solution was added to the resin, and 20 mL flowing through the division part was recovered (flowing through the division part 1). Then, 20 mL of 10 mM HCl was added to the resin, and 20 mL of the divided portion was recovered and flowed. This operation is repeated 19 times (flowing through the divisions 2 to 20). The absorbance (230 nm) flowing through the division 1 to 20 was measured to confirm the dissolution of the peptide. Then, 20 mL of 50% ammonia solution was added to the resin, and 20 mL of the adsorption separation portion was recovered. This operation is repeated 20 times (adsorption divisions 1 to 20). The absorbance (230 nm) of the adsorption separation parts 1 to 20 was measured to confirm the dissolution of the peptide. An evaporator was used to concentrate the flow-through discrimination section (pyroglutamine peptide discrimination section) and the adsorption discrimination section (peptide discrimination section) under reduced pressure.

(2) The distinction between hydrophilicity and hydrophobicity The solid phase extraction column (Sep-Pak) was equilibrated with 10 mM HCl. The pyroglutamine distinguishing part was passed through the column, and the flow-through distinguishing part was eluted and recovered (hydrophilic pyroglutamine distinguishing part). Then, a 60% acetonitrile solution containing 10 mM HCl was passed through the column to dissolve the adsorption discrimination portion and recover it (hydrophobic pyroglutamine discrimination portion). These operations are also performed on the peptide distinguishing part. The obtained four kinds of differentiated portions were freeze-dried.

Example 2 (1) Quantitative experiment of indigestible peptides in liver hydrolysate 1) Dissolve 2.5 mg of liver hydrolysate in 1 mL of 50 mM Tris-HCl. 2) Add pancreatin (0.1 mg), leucine peptidase (2.45 unit), and carboxypeptidase (7.7 unit) to 1), and perform an enzyme reaction (37°C, 24 h). 3) Remove the enzyme by ultrafiltration (10 K). 4) Make 3) pass the strong cation exchange resin (AG50) packed in the rotating column to recover and flow through the distinguishing part (pyroglutamine distinguishing part). 5) By size exclusion HPLC (high performance liquid chromatography) classification, 200 μL of 3) (peptide discrimination section) and 4) (pyroglutamine peptide discrimination section) (SEC Fr.35 -44). 6) For the peptide discrimination part, SEC Fr. 35-44 was dried and solidified, and AccQ was made. For the discrimination of pyroglutamine, SEC Fr. 35-44 was used directly. 7) Determine the structure of indigestible peptides by LC-MS/MS analysis. Column: Inertsil ODS-3 Dissolution solution: 0.1% formic acid and 80% acetonitrile containing 0.1% formic acid Analysis method: After the peptide discrimination part is specifically detected (Precursor ion scan analysis), the fragment of AccQ (m/z=171.1), the structure is estimated by MS/MS analysis, and the standard is synthesized. Identification and quantification by MRM analysis. For the distinguished part of pyroglutamine, the peak was detected by Total scan ion analysis, the structure was estimated by MS/MS analysis, the standard was synthesized, and identified and quantified by MRM.

(2) Experiment of peptide migration in blood when liver hydrolysate is administered to rats 1) A single dose of liver hydrolysate aqueous solution (10 g/60 kg) was administered to a rat (Wister rat) (6 weeks old, male). 2) 30 minutes and 60 minutes after the administration, blood was collected from the great abdominal vein under isoflurane anesthesia to obtain plasma. Furthermore, the digestive tube (duodenum to ileum) was removed, and the contents of the lumen were dissolved out using 10 mL of physiological saline. Add 3 times the amount of ethanol to the plasma and the contents of the digestive tract (store at -20°C before analysis). 3) Dry and fix the ethanol in the plasma and digestive tube contents, and then accQ. 4) By MRM analysis, identification and quantification of the indigestible peptides identified in (1). Table 1 shows the concentration of dipeptide in blood.

[Table 1]

As a result, it was found that (D) Asp-(D) Val, (D) Asp-(D) Phe, (D) Asp-(D) Ile, (D) Asp-(D) Leu, (D) Asp-( L) Val, (D) Asp-(L) Ile, (D) Asp-(L) Leu, and (D) Asp-(L) Phe Lasts for a long time.

Example 3 (synthesis of dipeptide) 1) The reagents were added to the eggplant-shaped flask in the following order, and the reaction was carried out while stirring (4°C overnight). (i) H-Leu-OtBu・HCl (ii) DMF (iii) TEA (iv) Boc-Asp(OtBu)-OH(Lα body) (v)HOBt (vi)EDL・HCl In the case of other isomers, the following protected amino acids are used. Boc-D-Asp(OtBu)-OH(Dα body) Boc-Asp-OtBu (Lβ body) Boc-D-Asp-OtBu (Dβ body) 2) Use an evaporator to remove DMF. 3) Dissolve in ethyl acetate and transfer to a separatory funnel. 4) Add 5% sodium bicarbonate and stir to remove the water layer. (×2) 5) Add 10% citric acid and stir to remove the water layer. (×2) 6) Add saturated brine and stir to remove the water layer. 7) Recover the ethyl acetate layer and add sodium bisulfate for dehydration. 8) The ethyl acetate layer was recovered by filtration and concentrated using an evaporator. 9) Add petroleum ether to dry the resulting precipitate. (When there is no precipitation, proceed to 10)) 10) Add 4 M HCl/dioxane to the dried product, and stir while reacting (4°C, 24-48 h). 11) Remove 4 M HCl/dioxane with an evaporator. 12) Add diethyl ether, use ultrasound to crush and wash the obtained precipitate, and use decane to remove the ether supernatant. (×3) 13) Add diethyl ether and let stand (4°C overnight). 14) Add diethyl ether, crush and wash the obtained precipitate with ultrasound, and remove the ether supernatant with decane. (×3) 15) Dry the precipitate.

Example 4 The cell number of RAW264.7 cells was adjusted to 1.5×10 ⁶ cells/dish, sowed and cultured overnight. The pre-differentiated sample or filtrate differentiated portion was diluted together 20 times in EMEM medium (final concentration about 0.6 mg/mL). As a control, instead of the sample, PBS was diluted 20-fold in EMEM medium. Each of the above-mentioned samples (control/pre-differentiation/filtrate discrimination portion) diluted in the medium was added to each dish group. After culturing for 24 hours, lipopolysaccharide (LPS) was added so that the final concentration became 1.0 μg/mL. After standing for 3 hours, RNA was recovered, and cDNA synthesis was performed by reverse transcription reaction. Then, EEF1A1 and IL-6 were measured by RT-RCR.

The results are shown in Figs. 1 to 4. In RAW cells stimulated by LPS, the mRNA expression of inflammation-related genes was compared between the dipeptide group and the Cont group supplemented with PBS instead of the dipeptide. As a result, IL-6 was distinguished in the hydrophobic peptide distinguishing part and the hydrophobic pyroglutamine. The marked decrease in the distinguishing part suggests the inflammation-inhibiting effect of the dipeptide.

Example 5 The cell number of RAW264.7 cells was adjusted to 1.5×10 ⁶ cells/dish, and seeded and cultured overnight. The pre-differentiated sample or filtrate differentiated portion was diluted together 20 times in EMEM medium (final concentration about 0.6 mg/mL). As a control, instead of the sample, PBS was diluted 20-fold in EMEM medium. Each of the above-mentioned samples (control/pre-differentiation/filtrate discrimination portion) diluted in the culture medium was added to each dish group. After culturing for 24 hours, lipopolysaccharide (LPS) was added so that the final concentration became 1.0 μg/mL. After standing for 3 hours, RNA was recovered, and cDNA synthesis was performed by reverse transcription reaction. Subsequently, EEF1A1 and IL-1β were measured by RT-RCR.

The result is shown in FIGS. 5-6. In RAW cells stimulated by LPS, the mRNA expression levels of inflammation-related genes were compared between the dipeptide group and the Cont group to which PBS was added instead of the dipeptide. As a result, IL-1β significantly decreased in the hydrophobic peptide distinguishing part, suggesting the dipeptide The inflammation inhibitory effect.

Figure 1 shows the inhibitory effect of IL-6 produced by the hydrophobic peptide discriminating part. Figure 2 shows the inhibitory effect of IL-6 produced by the hydrophobic pyroglutamine peptide discrimination portion. Fig. 3 shows the inhibitory effect of IL-6 produced by the hydrophilic peptide discriminating part. Fig. 4 shows the inhibitory effect of IL-6 produced by the hydrophilic pyroglutamine distinguishing part. Fig. 5 shows the inhibitory effect of IL-1β produced by the hydrophobic peptide discrimination portion. Fig. 6 shows the inhibitory effect of IL-1β on the distinguishing part of hydrophobic pyroglutamine.

Claims (13)

An anti-inflammatory agent selected from (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, (D)Pro-(D)Leu, ( D)Val-(D)-Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, (D)Val-(D)Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Ile-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D)Pro-(L)Val , (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D)Asp-(L)Val , (D) Asp-(L) Leu, and (D) Asp-(L) Phe dipeptide is the active ingredient. As in the anti-inflammatory agent of claim 1, the active ingredient is selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D) Leu, (D) Asp- (D) Phe, (D) Asp-(L) Val, (D) Asp-(L) Ile, (D) Asp-(L) Leu, and (D) Asp-(L) Phe. An inflammatory cytokine production inhibitor selected from (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, (D)Pro-(D ) Leu, (D)Val-(D)-Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, (D)Val-( D)Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Ile-( L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D)Pro- (L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D)Asp- The dipeptide in (L) Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe is the active ingredient. For example, the inflammatory cytokine production inhibitor of claim 3, the effective ingredients are selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D) Leu, ( D) Asp-(D)Phe, (D)Asp-(L)Val, (D)Asp-(L)Ile, (D)Asp-(L)Leu, and (D)Asp-(L)Phe By. An IL-1 production inhibitor or IL-6 production inhibitor selected from (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, ( D)Pro-(D)Leu, (D)Val-(D)-Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, (D)Val-(D)Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Ile-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro , (D)Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile , (D) Asp-(L) Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe dipeptide is the active ingredient. If the IL-1 production inhibitor or IL-6 production inhibitor of claim 5, the active ingredient is selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp- (D) Leu, (D) Asp-(D) Phe, (D) Asp-(L) Val, (D) Asp-(L) Ile, (D) Asp-(L) Leu, and (D) Asp -(L)Phe. An inflammation-improving food composition selected from (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, (D)Pro-(D) Leu, (D)Val-(D)Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, (D)Val-(D) Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Ile-(L) Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D)Pro-(L )Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D)Asp-(L ) The dipeptide in Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe is the active ingredient. As in the food composition of claim 7, the effective ingredients are selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D) Leu, (D) Asp- (D) Phe, (D) Asp-(L) Val, (D) Asp-(L) Ile, (D) Asp-(L) Leu, and (D) Asp-(L) Phe. An inflammatory cytokine production inhibiting food composition selected from (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, (D)Pro- (D)Leu, (D)Val-(D)-Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, (D)Val -(D)Hyp, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Ile -(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D) Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D) The dipeptide in Asp-(L)Val, (D)Asp-(L)Leu, and (D)Asp-(L)Phe is the active ingredient. As in the food composition of claim 9, the effective ingredients are selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D) Leu, (D) Asp- (D) Phe, (D) Asp-(L) Val, (D) Asp-(L) Ile, (D) Asp-(L) Leu, and (D) Asp-(L) Phe. A food composition for inhibiting IL-1 production or a food composition for inhibiting IL-6 production selected from (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro- (D)Ile, (D)Val-(D)-Pro, (D)Pro-(D)Val, (D)Leu-(D)Glu, (D)Ile-(D)pGlu, (D)Val -(D)pGlu, (D)Asp-(D)Ile, (D)Asp-(D)Val, (D)Asp-(D)Leu, (D)Asp-(D)Phe, (D)Ile -(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D) Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D) The dipeptide in Asp-(L)Val, (D)Asp-(L)Leu, and (D)Asp-(L)Phe is the active ingredient. As in the food composition of claim 11, the effective ingredients are selected from (D) Asp-(D) Val, (D) Asp-(D) Ile, (D) Asp-(D) Leu, (D) Asp- (D) Phe, (D) Asp-(L) Val, (D) Asp-(L) Ile, (D) Asp-(L) Leu, and (D) Asp-(L) Phe. The use of a dipeptide used in the manufacture of anti-inflammatory agents and is selected from (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, ( D)Pro-(D)Leu, (D)Val-(D)Pro, (D)Pro-(D)Val, (D)Leu-(D)Hyp, (D)Ile-(D)Hyp, ( D) Val-(D)Hyp, (D) Asp-(D) Ile, (D) Asp-(D) Val, (D) Asp-(D) Leu, (D) Asp-(D) Phe, ( D)Ile-(L)Pro, (D)Leu-(L)Pro, (D)Pro-(L)Ile, (D)Pro-(L)Leu, (D)Val-(L)-Pro, (D)Pro-(L)Val, (D)Leu-(L)Hyp, (D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D) Asp-(L) Val, (D) Asp-(L) Leu, and (D) Asp-(L) Phe.

Images