Pharmaceutical compositions containing dipeptides

Abstract

Providing a novel anti-inflammatory agent. An anti-inflammatory agent containing, as an active ingredient, a dipeptide 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.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical or food composition containing a dipeptide. [Background technology]

[0002] In an ultra-aging society, efforts to prevent and treat lifestyle-related diseases, such as metabolic diseases (e.g., diabetes, obesity) and cardiovascular diseases (e.g., arteriosclerosis), which are associated with chronic inflammation, are becoming increasingly important. In fact, IL-1β signaling inhibitors and NF-κB inhibitors have been developed for diabetes. In chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory cytokines (e.g., TNF-α, IL-1, and IL-6) are abnormally produced and secreted in large amounts, and inhibitors of these inflammatory cytokines are useful as effective anti-inflammatory agents. For example, antibody drugs such as infliximab are used as TNF-α inhibitors for the treatment of rheumatoid arthritis (Non-Patent Document 1). Furthermore, the antibody drug tricizumab is used as an IL-6 inhibitor (Non-Patent Document 2). Furthermore, protein preparations and antibody drugs such as anakinra, rilonacept, and canakinumab have been developed as IL-1 inhibitors (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Guidelines for TNF inhibitors in rheumatoid arthritis (RA) (Japan College of Rheumatology) [Non-patent document 2] Pharmaceutical Journal 129(6) 667-674(2009) [Non-patent document 3] Journal of the Japanese Society of Internal Medicine, Vol. 100, No. 10, pp. 2985-2990 (October 10, 2011) Summary of the Invention [Problem to be solved by the invention]

[0004] Against this background, the present inventors discovered that liver hydrolysates have excellent IL-1 production inhibitory effects and are useful as cytokine production inhibitors and anti-inflammatory agents, and previously filed a patent application (PCT / JP2018 / 006351). However, although liver hydrolysate is said to contain a large amount of amino acids and peptides, the actual active ingredients have not been identified. Therefore, an object of the present invention is to provide a new anti-inflammatory agent based on the cytokine production inhibitory effect. [Means for solving the problem]

[0005] Therefore, the present inventors have been investigating the development of a new anti-inflammatory agent, and have administered liver hydrolysates to animals to search for indigestible peptides that are transferred into the blood. They have also fractionated the peptides from the liver hydrolysates using various columns and evaluated their pharmacological effects. As a result, they have discovered dipeptides derived not from L-amino acids but from specific D-amino acids, and have found that these dipeptides have the effect of inhibiting the production of cytokines such as IL-1β and IL-6, thereby completing the present invention.

[0006] That is, the present invention provides the following [1] to

[15] .

[0007] [1] (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)H yp, (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)P An anti-inflammatory agent containing as an active ingredient a dipeptide selected from the group consisting of (D)ro, (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. [2] The anti-inflammatory agent according to [1], wherein 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. [3] (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, ( An inhibitor of inflammatory cytokine production, comprising as an active ingredient a dipeptide selected from 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. [4] The inhibitor of inflammatory cytokine production according to [3], wherein 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. [5] (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)Le An IL-1 production inhibitor or IL-6 production inhibitor comprising, as an active ingredient, a dipeptide selected from u-(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. [6] The IL-1 production inhibitor or IL-6 production inhibitor according to [5], wherein 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] (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)Hy p, (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, A food composition for improving inflammation, comprising as an active ingredient a dipeptide selected from (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. [8] The food composition according to [7], wherein 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. [9] (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)L A food composition for suppressing inflammatory cytokine production, comprising as an active ingredient a dipeptide selected from eu-(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.

[10] The food composition according to [9], wherein 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.

[11] (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)pGl u, (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, ( A food composition for suppressing IL-1 production or a food composition for suppressing IL-6 production, comprising as an active ingredient a dipeptide selected from 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.

[12] The food composition according to

[11] , wherein 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.

[13] (D)Ile-(D)Pro, (D)Leu-(D)Pro, (D)Pro-(D)Ile, (D)Pro-(D)Leu, (D)Val-(D)Pro, (D)Pro-(D)Val, for the production of anti-inflammatory agents, (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) Use of a dipeptide selected from Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D)Asp-(L)Val, (D)Asp-(L)Leu, and (D)Asp-(L)Phe. A dipeptide 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 for treating an inflammatory disease. A method for treating an anti-inflammatory disease, comprising administering an effective amount of a dipeptide 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.

Effects of the Invention

[0008] The dipeptides used in the present invention are derived from D-amino acids and are therefore indigestible, persist in the blood for a long period of time, and have inhibitory effects on IL-1 production and IL-6 production, making them useful as pharmaceuticals and food compositions for inhibiting cytokine production and as anti-inflammatory agents. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows the inhibitory effect of the hydrophobic peptide fraction on IL-6 production. [Figure 2] 1 shows the inhibitory effect of hydrophobic pyroglutamyl peptide fractions on IL-6 production. [Figure 3] 1 shows the inhibitory effect of the hydrophilic peptide fraction on IL-6 production. [Figure 4] 1 shows the inhibitory effect of hydrophilic pyroglutamyl peptide fractions on IL-6 production. [Figure 5] 1 shows the inhibitory effect of the hydrophobic peptide fraction on IL-1β production. [Figure 6] 1 shows the inhibitory effect of hydrophobic pyroglutamyl peptide fractions on IL-1β production. DETAILED DESCRIPTION OF THE INVENTION

[0010] The dipeptides that are the active ingredients of the anti-inflammatory agent of the present invention include (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)Hy and (D)Asp-(L)Phe. Among these, in terms of transfer into the bloodstream after oral administration, duration of action, and anti-inflammatory effect, dipeptides selected from (D)Asp-(D)Val, (D)Asp-(D)Ile-(L)Hyp, (D)Val-(L)Hyp, (D)Asp-(L)Ile, (D)Asp-(L)Val, (D)Asp-(L)Leu, (D)Asp-(D)Phe are preferred, with (D)Asp-(D)Leu and (D)Asp-(L)Leu being more preferred. Here, the notation (D) means that the amino acid is in the D configuration, and the notation (L) means that the amino acid is in the L configuration. Of the α- and β-forms of the (D)- or (L)-dipeptides, the β-form is more preferred.

[0011] The dipeptide can be produced by a conventional liquid-phase or solid-phase peptide synthesis method using (D) an amino acid as a starting material. For example, it can be produced by condensing an amino acid in which functional groups other than the α-amino group are protected with an amino acid in which functional groups other than the carboxy group are protected, or an amino acid in which the carboxy group has been activated and functional groups other than the carboxy group are protected, followed by removing the protecting groups. Examples of the protecting group for the amino group of the amino acid include a benzyloxycarbonyl group, a tert-butoxycarbonyl group, and a fluorenylmethoxycarbonyl group. Examples of the protecting group for the carboxy group include a tert-butyl group and a benzyl group. The condensation reaction can be carried out using a condensing agent such as N,N'-dicyclohexylcarbodiimide or dicyclohexylurea, an active ester method using nitrophenol or N-hydroxysuccinimide, or a mixed acid anhydride method. After the condensation reaction is complete, the protecting group is removed, and in the case of the solid phase method, the bond between the C-terminus of the peptide and the resin is further cleaved. The peptide is then purified according to a conventional method.

[0012] The dipeptide can be an acid addition salt or a base salt. Examples of acid addition salts include inorganic acid salts such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, and perchloric acid, and organic acid salts such as citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, and trifluoroacetic acid. Examples of base salts include alkali metal salts such as sodium, potassium, and lithium, and alkaline earth metal salts such as calcium and magnesium.

[0013] The peptide may be in the form of a solvate, such as a solvate of water (in the case of a hydrate), methanol, ethanol, isopropanol, or the like.

[0014] The dipeptide is indigestible, exhibits high blood translocation after oral administration, and exhibits excellent sustained activity. It has the effect of strongly suppressing the production of IL-1β and IL-6, which are inflammatory cytokines, and suppressing the mRNA expression of IL-1β and IL-6. Therefore, the dipeptide suppresses the production of inflammatory cytokines, such as IL-1 and IL-6, and is useful as a therapeutic agent for inflammatory diseases associated with inflammatory cytokines and as a food or beverage composition for alleviating inflammation. Examples of diseases associated with inflammatory cytokines, such as IL-1 and IL-6, include rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, sepsis, acute and chronic myeloid leukemia, osteoporosis, and lifestyle-related diseases.

[0015] The pharmaceutical composition of the present invention can be administered orally, transdermally, enterally, intravenously, etc., with oral administration being more preferred. Examples of formulations for oral administration include liquids, tablets, powders, fine granules, granules, capsules, etc., with liquids and tablets being preferred, and liquids being more preferred.

[0016] To prepare these oral preparations, excipients such as lactose, mannitol, corn starch, crystalline cellulose, etc., binders such as cellulose derivatives, gum arabic, gelatin, etc., disintegrants such as carboxymethylcellulose calcium, lubricants such as talc, magnesium stearate, etc., solubilizers such as nonionic surfactants, flavoring agents, sweeteners, stabilizers, pH adjusters, water, ethanol, propylene glycol, glycerin, etc. Furthermore, coating agents such as hydroxymethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, cellulose acetate phthalate, methacrylate copolymers, etc. may be used.

[0017] The pharmaceutical composition of the present invention may also contain other active ingredients, such as vitamin B1s, thiamine, thiamine nitrate, thiamine hydrochloride, fursultiamine, bisbentiamine, benfotiamine, thiamine disulfide, dicethiamine, thiamine propyl disulfide, and derivatives thereof, vitamin B2s, riboflavin and derivatives and salts thereof, vitamin B3s, niacin, nicotinic acid, nicotinamide and derivatives and salts thereof, vitamin B5s, panthenol, pantothenic acid and derivatives and salts thereof, vitamin B6s, pyridoxine and derivatives and salts thereof, and 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, caffeine, turmeric, milk thistle, dandelion, burdock, garlic, chrysanthemum, yarrow, gardenia, sesame, Panax notoginseng, asparagus, onion, chicory, medicinal salvia, artichoke, wolfberry, plants of the Fabaceae and Iridaceae families, mountain quail, passariño elba, sete sangria, Agametula japonicus, black tea, resveratrol, catechins, berberine, rosemary, bean extract, metformin, etc.

[0018] In addition to pharmaceuticals, the composition of the present invention can also be used as functional foods such as quasi-drugs, foods for specified health uses, sports drinks, drinks for rehabilitation, and pet foods.

[0019] The content of the dipeptide in the pharmaceutical or food composition of the present invention varies depending on the dosage form, but is usually preferably 0.001 to 10% by mass, more preferably 0.001 to 5% by mass. The daily dose of the dipeptide in the pharmaceutical or food composition of the present invention is preferably 10 mg to 1000 mg, more preferably 20 mg to 800 mg, and even more preferably 50 mg to 800 mg. [Example]

[0020] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

[0021] Example 1 (Fractionation of Liver Hydrolysate) (1) Fractionation of peptides and pyroglutamyl peptides A strong cation exchange resin (AG50) was packed into an Econo Column (2.5 x 20 cm) and equilibrated with 10 mM HCl. One gram of liver hydrolysate (Sample A) was dissolved in 20 mL of 10 mM HCl. This solution was applied to the resin, and 20 mL of the flow-through fraction was collected (flow-through fraction 1). Next, 20 mL of 10 mM HCl was applied to the resin, and 20 mL of the flow-through fraction was collected. This procedure was repeated 19 times (flow-through fractions 2–20). The absorbance (230 nm) of flow-through fractions 1–20 was measured to confirm peptide elution. Next, 20 mL of 50% ammonia solution was added to the resin, and 20 mL of adsorbed fraction was collected. This procedure was repeated 20 times (adsorbed fractions 1 to 20). The absorbance (230 nm) of adsorbed fractions 1 to 20 was measured to confirm the elution of the peptide. The flow-through fraction (pyroglutamyl peptide fraction) and the adsorbed fraction (peptide fraction) were concentrated under reduced pressure using an evaporator.

[0022] (2) Hydrophilic and hydrophobic fractions A solid-phase extraction column (Sep-Pak) was equilibrated with 10 mM HCl. The pyroglutamyl peptide fraction was passed through the column, and the flow-through fraction was eluted and collected (hydrophilic pyroglutamyl peptide fraction). Next, a 60% acetonitrile solution containing 10 mM HCl was passed through the column, and the adsorbed fraction was eluted and collected (hydrophobic pyroglutamyl peptide fraction). The peptide fraction was also subjected to these procedures. The four fractions obtained were freeze-dried.

[0023] Example 2 (1) Quantitative analysis of indigestible peptides in liver hydrolysates 1) 2.5 mg of liver hydrolysate was dissolved in 1 mL of 50 mM Tris-HCl. 2) Pancreatin (0.1 mg), leucine aminopeptidase (2.45 units), and carboxypeptidase (7.7 units) were added to 1), and the mixture was allowed to react at 37°C for 24 hours. 3) The enzyme was removed by ultrafiltration (10K). 4) 3) was passed through a strong cation exchange resin (AG50) packed in a spin column, and the flow-through fraction was collected (pyroglutamyl peptide fraction). 5) 200 μL of 3) (peptide fraction) and 4) (pyroglutamyl peptide fraction) were fractionated and collected by size-exclusion HPLC (SEC Fr. 35-44). 6) For the peptide fraction, SEC Fr.35-44 was evaporated to dryness and converted to AccQ. For the pyroglutamyl peptide fraction, SEC Fr.35-44 was used as is. 7) The structures of the indigestible peptides were determined by LC-MS / MS analysis. Column: Inertsil ODS-3 Eluent: 0.1% formic acid and 80% acetonitrile with 0.1% formic acid Analysis method: For peptide fractions, the AccQ fragment (m / z=171.1) is specifically detected (precursor ion scan analysis), and then the structure is estimated by MS / MS analysis. Standards are synthesized and identified and quantified by MRM analysis. For the pyroglutamyl peptide fraction, peaks are detected by total scan ion analysis, and then the structure is estimated by MS / MS analysis. Standards are synthesized and identified and quantified by MRM.

[0024] (2) Experiment on the transfer of peptides into the bloodstream when liver hydrolysate was administered to rats 1) A single dose of liver hydrolysate solution (10g / 60kg) was administered to male Wistar rats (6 weeks old). 2) Thirty and 60 minutes after administration, blood samples were collected from the abdominal vena cava under isoflurane anesthesia to obtain plasma. The digestive tract (duodenum to ileum) was then removed, and the luminal contents were extracted with 10 mL of saline. Three volumes of ethanol were added to the plasma and digestive tract contents (stored at -20°C until analysis). 3) Plasma and digestive tract contents were evaporated to dryness on ethanol and subjected to AccQ conversion. 4) The indigestible peptides identified in (1) were identified and quantified by MRM analysis. The dipeptide concentrations in the blood are shown in Table 1.

[0025] [Table 1]

[0026] 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 have good blood transfer properties following oral administration and persist in the blood for a long period of time.

[0027] Example 3 (Synthesis of dipeptide) 1) Add the reagents to a recovery flask in the following order and react with stirring (4°C, overnight). (i) H-Leu-OtBu HCl (ii) DMF (iii) TEA (iv) Boc-Asp(OtBu)-OH (Lα form) (v)HOBt (vi) EDL·HCl For other isomers, the following protected amino acids are used: Boc-D-Asp(OtBu)-OH (Dα form) Boc-Asp-OtBu (Lβ form) Boc-D-Asp-OtBu (Dβ form) 2) Remove the DMF using an evaporator. 3) Dissolve in ethyl acetate and transfer to a separatory funnel. 4) Add 5% sodium bicarbonate, stir, and remove the aqueous layer. (x2) 5) Add 10% citric acid, stir, and remove the aqueous layer. (x2) 6) Add saturated saline and stir, then remove the aqueous layer. 7) The ethyl acetate layer is collected and dehydrated by adding sodium hydrogen sulfate. 8) The ethyl acetate layer is collected by filtration and concentrated using an evaporator. 9) Add petroleum ether and dry the resulting precipitate. (If no precipitate forms, proceed to 10)) 10) Add 4M HCl / dioxane to the dried material and react with stirring (4°C, 24-48 hours). 11) Remove the 4M HCl / dioxane using an evaporator. 12) Diethyl ether is added, the resulting precipitate is ultrasonically crushed and washed, and the ethereal supernatant is removed with decane (x3). 13) Add diethyl ether and leave at 4°C overnight. 14) Diethyl ether is added, the resulting precipitate is ultrasonically crushed and washed, and the ethereal supernatant is removed with decane (x3). 15) Dry the precipitate.

[0028] Example 4 The number of RAW264.7 cells was 1.5 × 10 6 The cells were prepared and seeded on a dish and cultured overnight. Both the pre-fractionation sample and the filtrate fraction were diluted 20-fold with EMEM medium (final concentration: approximately 0.6 mg / mL). For the control, PBS was diluted 20-fold with EMEM medium instead of the sample. Each of the above samples (control, pre-fractionation, and filtrate fractions) diluted with medium was added to each dish group. After 24 hours of culture, lipopolysaccharide (LPS) was added to a final concentration of 1.0 μg / mL. After 3 hours of incubation, RNA was collected and cDNA synthesis was performed by reverse transcription. EEF1A1 and IL-6 were then measured by RT-PCR.

[0029] The results are shown in Figures 1 to 4. In LPS-stimulated RAW cells, the mRNA expression levels of inflammation-related genes were compared between the dipeptide group and the control group, in which PBS was added instead of dipeptide. IL-6 was significantly reduced in the hydrophobic peptide fraction and the hydrophobic pyroglutamyl peptide fraction, suggesting the anti-inflammatory effect of the dipeptide.

[0030] Example 5 The number of RAW264.7 cells was 1.5 × 10 6 The cells were prepared and seeded on a dish and cultured overnight. Both the pre-fractionation sample and the filtrate fraction were diluted 20-fold with EMEM medium (final concentration: approximately 0.6 mg / mL). For the control, PBS was diluted 20-fold with EMEM medium instead of the sample. Each of the above samples (control, pre-fractionation, and filtrate fractions) diluted with medium was added to each dish group. After 24 hours of culture, lipopolysaccharide (LPS) was added to a final concentration of 1.0 μg / mL. After 3 hours of incubation, RNA was collected and cDNA synthesis was performed by reverse transcription. EEF1A1 and IL-1β were then measured by RT-PCR.

[0031] The results are shown in Figures 5 and 6. In LPS-stimulated RAW cells, the mRNA expression levels of inflammation-related genes were compared between the dipeptide group and the control group, in which PBS was added instead of the dipeptide. IL-1β was significantly reduced in the hydrophobic peptide fraction, suggesting the anti-inflammatory effect of the dipeptide.

Claims

1. An anti-inflammatory agent containing as an active ingredient a hydrophobic peptide fraction of liver hydrolysate, wherein the liver hydrolysate is treated with pancreatin, leucine aminopeptidase and carboxypeptidase to produce (D)Asp-(D)Leu, (D)Asp-(D)Val, (D)Asp-(D)Phe, (D)Asp-(D)Ile, (D)Asp-(L)Leu, (D)Asp-(L)Val, (D)Asp-(L)Phe and (D)Asp-(L)Ile.

2. An inhibitor of inflammatory cytokine production, the active ingredient of which is a hydrophobic peptide fraction of liver hydrolysate, wherein the liver hydrolysate is treated with pancreatin, leucine aminopeptidase and carboxypeptidase to produce (D)Asp-(D)Leu, (D)Asp-(D)Val, (D)Asp-(D)Phe, (D)Asp-(D)Ile, (D)Asp-(L)Leu, (D)Asp-(L)Val, (D)Asp-(L)Phe and (D)Asp-(L)Ile.

3. An IL-1 production inhibitor or an IL-6 production inhibitor, the active ingredient of which is a hydrophobic peptide fraction of liver hydrolysate, wherein the liver hydrolysate is treated with pancreatin, leucine aminopeptidase and carboxypeptidase to produce (D)Asp-(D)Leu, (D)Asp-(D)Val, (D)Asp-(D)Phe, (D)Asp-(D)Ile, (D)Asp-(L)Leu, (D)Asp-(L)Val, (D)Asp-(L)Phe and (D)Asp-(L)Ile.

4. A food composition for improving inflammation, containing as an active ingredient a hydrophobic peptide fraction of liver hydrolysate, wherein the liver hydrolysate is treated with pancreatin, leucine aminopeptidase and carboxypeptidase to produce (D)Asp-(D)Leu, (D)Asp-(D)Val, (D)Asp-(D)Phe, (D)Asp-(D)Ile, (D)Asp-(L)Leu, (D)Asp-(L)Val, (D)Asp-(L)Phe and (D)Asp-(L)Ile.

5. A food composition for suppressing inflammatory cytokine production, comprising as an active ingredient a hydrophobic peptide fraction of liver hydrolysate, wherein the liver hydrolysate is treated with pancreatin, leucine aminopeptidase and carboxypeptidase to produce (D)Asp-(D)Leu, (D)Asp-(D)Val, (D)Asp-(D)Phe, (D)Asp-(D)Ile, (D)Asp-(L)Leu, (D)Asp-(L)Val, (D)Asp-(L)Phe and (D)Asp-(L)Ile.

6. A food composition for inhibiting IL-1 production or a food composition for inhibiting IL-6 production, the active ingredient of which is a hydrophobic peptide fraction from a liver hydrolysate, wherein the liver hydrolysate is treated with pancreatin, leucine aminopeptidase and carboxypeptidase to produce (D)Asp-(D)Leu, (D)Asp-(D)Val, (D)Asp-(D)Phe, (D)Asp-(D)Ile, (D)Asp-(L)Leu, (D)Asp-(L)Val, (D)Asp-(L)Phe and (D)Asp-(L)Ile.

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