JP2016175862A - Novel composition and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel use method for Myctophidae fish and a novel DPP-IV inhibitor.SOLUTION: The present invention relates to a composition obtained by treating fish bodies and/or fish meal with protease, the composition comprising L-R of 10 μg/kg or more.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a dipeptidyl peptidase IV (DPP-IV) inhibitor derived from the scorpionidae. The present invention further relates to methods for preventing and / or treating conditions and diseases involving DPP-IV, including type II diabetes. It also relates to the use for producing pharmaceuticals, dietary supplements, beverages or foods.

  Dipeptidyl peptidase IV (DPP-IV) is a serine aminodipeptidase that specifically hydrolyzes the dipeptide X-Pro or X-Ala (X is any amino acid residue) from the amino terminus of polypeptides and proteins. It is distributed in a very wide range of tissues in the body (intestinal tract, kidney, liver, lung, pancreas, spleen, placenta, bone marrow and other epithelial cells and endothelial cells) and is considered to be involved in various in vivo reactions. It has been. The expression pattern is expressed on the cell surface of the tissue as a transmembrane type, and also exists in a body fluid (eg, serum, plasma, cerebrospinal fluid, joint fluid, semen, etc.) as a soluble type lacking the transmembrane domain ( Non-patent documents 1, 2).

  The number of patients with type II diabetes, which is a chronic metabolic disease due to the failure to regulate blood glucose levels appropriately due to insulin secretion and insulin resistance from pancreatic β cells, has been on the rise. It is estimated that the number of patients worldwide exceeds 360 million (Non-patent Document 3). Therefore, measures against type II diabetes have been examined from various approaches.

  Among a number of approaches, incretin (for example, GLP-1 and GIP) which are intestinal hormones has attracted attention as a target factor. After ingestion, incretin is secreted from gastrointestinal endocrine cells (L cells), which are present in the large intestine from the ileum, and promotes the secretion of insulin from pancreatic β cells, thereby suppressing a rapid increase in blood glucose level. . However, since incretin is rapidly degraded, the half-life in vivo is very short (Non-patent Document 4).

  DPP-IV is known to degrade incretin and convert it into an inactive form. Substances that inhibit DPP-IV are a rapid increase in postprandial blood glucose levels by extending the in vivo half-life of incretin. It is considered effective for the prevention and treatment of type II diabetes (Non-patent Document 3).

  DPP-IV is the same molecule as CD26, which is known as a T cell activation molecule, and has been reported to be involved in various immune responses, particularly autoimmune diseases (see, for example, Non-Patent Document 3). It is known that the expression of CD26 increases with the activation of T cells, and its expression is particularly high in memory T cells and T helper (Th) 1 cells that play an important role in immune memory. A substance that inhibits DPP-IV (CD26) is considered to be useful for the treatment of autoimmune diseases and rejection seen during organ transplantation (Non-patent Documents 5 and 6).

  It is known that DPP-IV (CD26) is highly expressed in cancer cells (for example, lung adenocarcinoma, thyroid cancer, leukemia). In T-cell lymphoma cells, it has been reported that DPP-IV (CD26) promotes cancer progression by promoting cell adhesion and tumorigenicity (Non-patent Document 7). From this, it is thought that the substance which inhibits DPP-IV (CD26) is useful for the prevention and treatment of cancer.

  DPP-IV is involved in the metabolism of various neuropeptides, hormones, growth factors, cytokines and chemokines, and is thought to be involved in the onset and exacerbation of various diseases by changing the activity of these factors. ing. For example, DPP-IV cleaves the peptide sequence of chemokine SDF-1 (Stomal cell-derived factor-1), thereby eliminating its activity and negatively regulating the migration and survival of hematopoietic stem cells and hematopoietic progenitor cells. (Non-patent Document 8). Similarly, DPP-IV negatively regulates the proliferation and survival of hematopoietic stem cells and hematopoietic progenitor cells by cleaving GM-CSF, G-CSF, IL-3, and EPO peptide sequences and reducing their activity. (Non-patent Document 9), a substance that inhibits DPP-IV is useful for improving the reduction in immunity seen during radiochemotherapy for hematopoietic stem cell transplantation and cancer treatment. it is conceivable that.

  Furthermore, DPP-IV is associated with psoriasis (Non-Patent Document 10), benign prostatic hyperplasia (Non-Patent Document 11), HIV infection (Non-Patent Document 12), osteoporosis (Non-Patent Document 13), and gastrointestinal disorders (Non-Patent Document 14). ), Infertility (patent document 1), cancer (non-patent documents 15, 16), inflammatory disease (non-patent document 17), autoimmune disease (non-patent document 18), cardiovascular disease (non-patent document 19), Since the relationship with diseases such as kidney diseases (Non-patent Document 20) and mental disorders (Non-Patent Documents 21 and 22) has been reported, substances that inhibit DPP-IV are useful for the prevention and treatment of these diseases. It is considered useful.

  Several compounds having a DPP-IV inhibitory action have been proposed, and vildagliptin (patent document 2), fern gliptin (patent document 3), alogliptin (patent document 4) and the like have been put into practical use as pharmaceuticals. However, since these are synthetic compounds, they are not sufficient in terms of safety and side effects. Therefore, it is strongly desired to provide a natural product-derived component that can be used as a pharmaceutical or food material, has high safety, and can be ingested for a long period of time.

  From such a viewpoint, a food protein hydrolyzate containing a peptide having a DPP-IV inhibitory action has been proposed. For example, cheese water-soluble fraction (Patent Document 5), collagen and gelatin hydrolyzate (Patent Documents 6 and 7), milk protein hydrolyzate (Patent Documents 8 and 9), rice bran protein hydrolyzate (Patent Document 10) Corn protein hydrolyzate (Patent Document 11), β-lactoglobulin hydrolyzate (Patent Document 12). However, these proposals are not sufficient in terms of raw material supply amount and raw material cost.

  The stag beetle is a general term for fish belonging to the order of Mycotopiformae (Myctophylformes), and is composed of 240 species of 32 genera. It is characteristic to have a light emitter on the surface of the body. Distributed in the oceans around the world, it lives in the deep sea near the seabed from the middle layer offshore.

  Despite the abundance of resources, there is almost no fishery around the world for the stag beetle. In Japan, although some regions consume a small amount of food, they are considered to be one of the unused marine resources and are expected to be used effectively.

  Concerning the nutritional value and health functionality of the stag beetle fish, EPA and DHA are rich in EPA and DHA as well as anti-protein hydrolysates in sardine (Benthosema pterotum), sardine sardine (Diaphus suborbitalis) and sardine sardine (Diaphus watasei). Although it has been reported to have an oxidative activity, there have been few reports on its health functionality (Non-patent Documents 23 and 24).

WO2000 / 056296 publication International Publication No. 2000/34241 Pamphlet International Publication No. 2003/04998 Pamphlet Japanese Patent No. 4068118 Japanese Patent No. 4915833 Japanese Patent No. 5176964 Japanese Patent No. 5612131 Japanese Patent No. 5068174 JP2013-184962A JP2013-40111A JP 2011-182742 A JP 2011-144167 A

FEBS J.M. 2010 Mar; 277 (5): 1126-1144. Blood. 2013 Jul; 122 (2): 161-169 Proc Nutr Soc. 2014 Feb; 73 (1): 34-46 J Clin Invest. 2007 Jan; 117 (1): 24-32. Front Biosci. 2008 Jan; 13: 2299-2310 Trends Immunol. 2008 Jun; 29 (6): 295-301 Cancer Res. 2005 Aug; 65 (15): 6950-6756 J Immunol. 2002; 169: 7000-7008 Nat Med. 2012 Dec; 18 (12): 1786-1796 J Cell Physiol. 1992 May; 151 (2): 378-385. Eur J Clin Chem Clin Biochem. 1992 Jun; 30 (6): 333-338. Science. 1993 Dec; 262 (5142): 2045-2050. Clin Chem. 1988 Dec; 34 (12): 2499-2501. Endocrinology. 2000 Nov; 141 (11): 4013-4020 Br J Cancer. 1999 Mar; 79 (7-8): 1042-1048. J Andol. 2000 Mar; 21 (2): 220-226 J Immunol. 2001 Feb; 166 (3): 2041-2048. Science. 1993 Jul; 261 (5120): 466-469. Curr Diab Rep. 2014 Mar; 14 (5): 483 Curr Opin Nephrol Hypertens. 2014 Jan; 23 (1): 54-60 Peptides. 2007 Feb; 28 (2): 257-268. Physiol Res. 2008 Oct; 57 (3): 443-449. J Oleo Sci. 2014; 63 (5): 461-470 Food Res Int. 2012; 46: 118-126

  The present invention provides a novel utilization method of a sardine eagle and a novel DPP-IV inhibitor.

(1) A composition obtained by treating fish and / or fish meal with a proteolytic enzyme, comprising 10 μg / kg or more of leucine-arginine.
(2) The composition according to (1), wherein the fish body and / or fish meal is derived from a sea sardine.
(3) The composition according to (1) or (2), wherein the proteolytic enzyme is an enzyme selected from the group consisting of neutral protease, papain, endo-type protease / exo-type protease, or a mixture thereof.
(4) The composition according to any one of (1) to (3), wherein the IC50 value (50% inhibitory concentration) of DPP-IV inhibitory activity is 10 mg / ml or less in terms of solid content.
(5) A DPP-IV inhibitor comprising the composition according to any one of (1) to (4) as an active ingredient.
(6) A DPP-IV inhibitor containing leucine-arginine as an active ingredient.
(7) The DPP-IV inhibitor according to (5) or (6), wherein leucine-arginine is derived from an sardine.
(8) Diabetes, psoriasis, benign prostatic hyperplasia, HIV infection, osteoporosis, gastrointestinal tract disorder, infertility comprising the composition of (1) to (4) or the DPP-IV inhibitor of (5) to (7) as an active ingredient Prevention and / or prevention of graft-versus-host disease (GVHD) or graft survival at the time of hematopoietic, cancer, inflammatory disease, autoimmune disease, cardiovascular disease, kidney disease, mental illness, and / or hematopoietic stem cell transplantation therapy Remedy.
(9) Leucine by adding 0.1% to 1.2% by mass of a proteolytic enzyme and decomposing at 30 ° C. to 80 ° C. for 60 minutes to 24 hours with respect to the total mass of fish body and / or fish meal A method for producing an arginine-containing composition.
(10) A method of increasing the leucine-arginine content by treating fish and / or fish meal with a proteolytic enzyme.

(11) A method of inhibiting DPP-IV using leucine-arginine.
(12) Diabetes, psoriasis, prostatic hypertrophy, HIV infection, osteoporosis, gastrointestinal disorders, infertility, cancer, inflammatory diseases, autoimmune diseases, cardiovascular diseases, kidney diseases, mental diseases using leucine-arginine, and A method of preventing and / or treating graft versus host disease (GVHD) or graft survival delay during hematopoietic stem cell transplantation therapy.
(13) Use of the composition of (1) to (4), the DPP-IV inhibitor or leucine-arginine of (5) to (7) for the manufacture of a therapeutic agent for diabetes.

  ADVANTAGE OF THE INVENTION According to this invention, the utilization method and DDPIV inhibitor of a sardine sardine are obtained.

FIG. 1 is a diagram showing DDP-IV inhibitory activity by various enzyme treatments. FIG. 2 is a graph showing the relationship between enzyme concentration and DDP-IV inhibitory activity. FIG. 3 is a diagram showing DDP-IV inhibitory activity by the combined treatment of various enzymes. FIG. 4 is a diagram showing the influence of enzyme treatment temperature and pH on DDP-IV inhibitory activity. FIG. 5 is a graph showing the relationship between enzyme concentration, treatment time, and DDP-IV inhibitory activity. FIG. 6 is a diagram showing the DDP-IV inhibitory activity when fish endogenous enzymes or samoses are inactivated. FIG. 7 is a graph showing the relationship between fractionation by gel filtration chromatography and its DDP-IV activity. FIG. 8 is a graph showing the relationship between the fraction obtained by reversed-phase HPLC of the gel filtration active fraction and the DDP-IV activity of the fraction. FIG. 9 is a diagram showing re-fractionation by reverse phase HPLC and DDP-IV activity of the fraction. FIG. 10 is a diagram showing the DDPIV inhibitory activity of LR. FIG. 11 is a diagram showing the retention time by ion chromatography and the results of mass spectrometry.

  In the present specification, “saddle eagle (bare coral)” is a general term for fish belonging to the order of the stag beetle. Included in the stag beetle are: squirrel hawkfish, black-tailed hawkfish, black-tailed swordfish, black-tailed hawkfish, black-tailed hawkfish , Gantenhadaka, senhadaka, tokurihadaka, kurohadaka, mayoihadaka, kanmurihadaka, tsunohadaka, agarihadaka, chibihadaka, takahadaka, bouhadaka, hanarehadaka, tsukushihadaka, hirohadaka, hakuhaka In the present invention, the stag beetle is used, but preferably Iwadaka and Senhadaka.

  The present invention is characterized by increasing the component having DDP-IV inhibitory activity by proteolytically degrading fish and / or fish meal (powder obtained by pulverizing fish), preferably fish sardine fish and / or fish meal with a proteolytic enzyme. To do. Here, as the proteolytic enzyme, an endogenous one may be used as it is, or a composition containing a proteolytic enzyme may be added and processed from the outside. The “composition obtained by treating fish and / or fish meal with a proteolytic enzyme” of the present invention is treated with only an endogenous proteolytic enzyme, or added externally in addition to the endogenous proteolytic enzyme. This includes all those treated with both proteolytic enzymes and those treated by adding a composition containing proteolytic enzymes from the outside after inactivating endogenous proteolytic enzymes.

  As used herein, “fish” refers to the whole or part of a fish that is raw or has been subjected to a treatment such as heating, freezing, or drying. For example, raw fish, frozen fish, freeze-dried fish, boiled fish Fish body, boiled fish body, sun-dried fish body, machine-dried fish body and parts thereof. “Fishmeal” refers to powdered fish. Powdering can be performed using a mortar and pestle, a Waring blender, a mill, a cutter, or the like.

  The composition of the present invention has DPP-IV inhibitory activity. The IC50 value (50% inhibitory concentration) of the DPP-IV inhibitory activity of the composition of the present invention is 10 mg / mL or less, more preferably 5 mg / mL or less, more preferably 2 mg / mL or less, particularly preferably solid content concentration. Preferably it is 1.2 mg / mL or less, and most preferably 0.6 mg / mL or less. In this specification, DPP-IV may be expressed as DPPIV, but they are the same.

  Incretin promotes insulin secretion and suppresses glucagon secretion, and thus has a function of lowering blood sugar during hyperglycemia after meals. Since DPP-IV degrades incretin, incretin is usually degraded in a short time. Since inhibition of DPP-IV with a DPP-IV inhibitor can suppress the degradation of incretin, the DPP-IV inhibitor is used as a therapeutic agent for diabetes, particularly as a therapeutic agent for type II diabetes.

  Since the composition and / or leucine-arginine of the present invention has an effect of inhibiting DPP-IV, it can be used as a therapeutic agent for diabetes.

  The proteolytic enzyme preferably includes, but is not limited to, an enzyme selected from the group consisting of neutral protease, papain or endo-type protease / exo-type protease, or a mixture thereof. In short, any proteolytic enzyme capable of degrading proteins and increasing the leucine-arginine content can be used.

  The amount of enzyme used cannot be generally stated depending on the titer of each protease, but it is sufficient that the amount of enzyme used is proteolytic activity of 30,000 units / g or more.

  Preferred enzyme specific activities include purified papain 400,000-1,000,000 units, orientase 5N 50,000 units / g or more (CFA method), Sumiteam LP 50,000 units / g or more, Sumiteam FP 50,000 Units or more (CFA method), Coclase P 48,000-62,000 units / g, Alcalase 2.4 AU / g or more, Samoaze PC10F 90,000 units / g or more (Forin method), Flavorzyme 1,000 LAPU / g or more (Leucine aminopeptidase unit).

  In the enzyme treatment, for example, it is preferable to add 0.1% to 1.2% by mass of proteolytic enzyme with respect to the total mass of fish and / or fish meal, and to treat at 30 ° C. to 80 ° C. for 1 to 24 hours. Not limited to. In short, the enzyme concentration and treatment conditions may be determined so that the leucine-arginine content becomes the required concentration.

  The concentration of leucine-arginine in the composition obtained by protease treatment of fish and / or fish meal is preferably 10 μg / kg, more preferably 100 μg / kg, still more preferably 500 μg / kg, still more preferably 1 mg. / Kg, particularly preferably 3 mg / kg, most preferably 10 mg / kg.

  The protease originally contained in the sardine may be inactivated or may be used as it is without being inactivated.

  The composition of the present invention is characterized by rich umami and strong taste. Therefore, it can be used as a single product (single food) of a food claiming individuality. Examples of the single food include sprinkles, green tea paste, dashi, seasoning, baby food, and feed (including pet food).

  Moreover, since the composition of the present invention has a natural taste and texture with an appropriate texture, it can be used as an additive that maintains harmony with other ingredients. Additives include instant foods such as instant noodles, cup noodles, instant soups, instant miso soup and soup, retort foods, canned seasonings (including soups), microwave foods, etc .; frozen foods such as Frozen foods, semi-cooked frozen foods, cooked frozen foods, etc .; flour foods such as bread, macaroni, spaghetti, noodles, okonomiyaki, takoyaki flour, fried powder, cake mix, bread crumbs, etc .; Processed livestock products include, for example, canned fish ham and sausages, fish paste products, canned fish, canned livestock, pastes, livestock ham and sausages, etc .; processed agricultural foods include canned agricultural products, pickles, boiled beans, dried agricultural products As a basic and complex seasoning, for example, soy sauce, miso, dashi, sauces, mirin, cooked mi Box, curry Motorui, sauce acids, dressings, noodle soup include, but spices like not limited thereto.

  Examples of beverages that can be used in the present invention include carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks with fruit juice, fruit drinks, fruit drinks with fruits, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, Examples include, but are not limited to, powdered beverages, concentrated beverages, sports beverages, nutritional beverages, alcoholic beverages, and other favorite beverages.

  Examples of confectionery that can be used in the present invention include caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pie, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, and the like. I can't.

  The composition of the present invention is characterized by its deliciousness with timelessness and elongation.

  The composition of the present invention may contain other components that are usually contained in conventional food additives in any amount as long as the flavor is not impaired. Although it does not specifically limit as another component, For example, an excipient | filler is mentioned. The excipient is not particularly limited, and examples thereof include sugars and sugar alcohols and gums. Although it does not specifically limit as carbohydrate, For example, a monosaccharide, an oligosaccharide, and a polysaccharide are mentioned. The monosaccharide is not particularly limited, and examples thereof include glucose, galactose, mannose, and fructose. Although it does not specifically limit as oligosaccharide, For example, lactose, sucrose, and maltose are mentioned. The polysaccharide is not particularly limited, and examples thereof include starch, pectin, and dextrin. The sugar alcohol is not particularly limited, and examples thereof include maltitol, mannitol, sorbitol, and xylitol. Although it does not specifically limit as gums, For example, guar gum and xanthan gum are mentioned. In the food additive of the present invention, the content of these components can be set arbitrarily by those skilled in the art.

  The present invention also provides a DPP-IV inhibitor containing leucine-arginine as an active ingredient. A DPP-IV inhibitor can be used as a prophylactic / therapeutic agent for diabetes as described above. In addition to diabetes, the DPP-IV inhibitor of the present invention includes, for example, psoriasis, prostatic hypertrophy, HIV infection, osteoporosis, gastrointestinal disorders, infertility, cancer, inflammatory diseases, autoimmune diseases, cardiovascular diseases, It can also be used as a prophylactic and / or therapeutic agent for kidney disease, mental illness, and / or graft versus host disease (GVHD) or graft survival delay during hematopoietic stem cell transplantation treatment, but is not limited thereto. The therapeutic agent may be a medicine.

  In the preparation of the present invention, the content of leucine-arginine varies depending on the form of the preparation, but is usually 0.0001% by mass or more, preferably 0.001% by mass or more as the amount of leucine-arginine relative to the whole preparation, Preferably it is about 0.01 mass% or more. As long as it can be formulated, the upper limit may be 100% by mass, more preferably 95% by mass or less, and still more preferably 90% by mass or less.

  The dose varies depending on the type of leucine-arginine, administration route, symptoms, patient age, and the like. For example, when orally administered to an adult patient with diabetes, about 0. 0 as leucine-arginine per kg body weight per day. 3 to 300 mg, more preferably about 1 to 100 mg, can be divided into 1 to several doses.

  The composition of the present invention may further contain a carrier (at least one component selected from a binder, an excipient, and a disintegrant).

  Examples of the excipient include sugar alcohols such as D-mannitol, D-sorbitol, erythritol, and xylitol, sugars such as lactose, glucose, fructose, sucrose, and powdered reduced maltose water candy, crystalline cellulose, powdered cellulose, starches ( Potato starch, corn starch, etc.), dextrin, β-cyclodextrin, carmellose sodium, hydrous silicon dioxide, silicon dioxide, precipitated calcium carbonate, anhydrous calcium hydrogen phosphate, magnesium oxide, titanium oxide, calcium lactate, aluminate metasilicate Examples thereof include magnesium, synthetic hydrotalcite, talc and kaolin. As the excipient, saccharides, crystalline cellulose, starches, anhydrous calcium hydrogen phosphate and the like are often used. The amount of the excipient used may be 1 to 100 parts by weight, preferably 3 to 75 parts by weight, more preferably about 5 to 50 parts by weight with respect to 100 parts by weight of the total amount of the active ingredient and the adsorbent. . In addition, since the 1st and / or 2nd adsorption agent can be functioned as an excipient | filler, an excipient | filler is not necessarily required. Therefore, the amount of excipient used is 0 to 30 parts by weight, preferably about 3 to 20 parts by weight (for example, 5 to 10 parts by weight) with respect to 100 parts by weight of the total amount of the active ingredient and the adsorbent. Also good.

  Examples of the binder include cellulose derivatives such as methyl cellulose (MC), ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl pyrrolidone (povidone), Examples include vinylpyrrolidone copolymer (copolyvidone), acrylic polymer, gelatin, gum arabic, pullulan, pregelatinized starch, agar, tragacanth, sodium alginate, propylene glycol ester alginate, sucrose, and the like. As the binder, cellulose derivatives (MC, HPC, HPMC, etc.), povidone and the like are often used. The amount of the binder used is 1 to 50 parts by weight, preferably 3 to 30 parts by weight, more preferably 5 to 25 parts by weight (for example, 5 to 15 parts by weight) with respect to 100 parts by weight of the total amount of the active ingredient and the adsorbent. Part) degree.

  Examples of the disintegrant include carmellose, carboxymethylcellulose calcium (carmellose calcium), croscarmellose sodium, low-substituted hydroxypropylcellulose (L-HPC), crospovidone, starches (such as corn starch), hydroxypropyl starch, Examples include partially pregelatinized starch, alginic acid, and bentonite. As the disintegrant, carmellose calcium, croscarmellose sodium, low-substituted hydroxypropylcellulose (L-HPC), crospovidone, starches and the like are often used. The amount of the disintegrant used is 1 to 30 parts by weight, preferably 3 to 25 parts by weight, more preferably 5 to 20 parts by weight (for example, 5 to 15 parts by weight) with respect to 100 parts by weight of the total amount of the active ingredient and the adsorbent. Part) degree.

  The composition of the present invention may contain other carriers or additives such as lubricants (stearic acid, magnesium stearate, calcium stearate, talc, sucrose fatty acid ester, glycerin fatty acid ester, hydrogenated oil, polyethylene glycol, dimethylpolysiloxane. Antioxidants (dibutylhydroxytoluene (BHT), propyl gallate, butylhydroxyanisole (BHA), tocopherol, citric acid, etc.); preservatives (paraoxybenzoates, etc.); colorants (Turmeric extract, riboflavin, carotene solution, tar dye, caramel, titanium oxide, bengara, etc.); surfactant (polyoxyethylene hydrogenated castor oil, glyceryl monostearate, sorbitan fatty acid ester (sorbitan monostearate, Sorbitan nolaurate), polyoxyethylene polyoxypropylene, polysorbates, sodium lauryl sulfate, macrogol, sucrose fatty acid ester, etc.); fluidizing agents (magnesium stearate, calcium stearate, light anhydrous silicic acid, talc, water content) Plasticizer (triethyl citrate, polyethylene glycol, triacetin, cetanol, etc.); sweetener or corrigent (sucrose, mannitol, D-sorbitol, xylitol, aspartame, acesulfame potassium, sucralose, ascorbic acid, stevia, Menthol, licorice crude extract, simple syrup, etc.); flavoring agents or fragrances (menthol, ginger oil, etc.); pigments, cooling agents, preservatives or preservatives, wetting agents, antistatic agents, etc. That. These carriers or additives can be used alone or in combination of two or more.

[Dosage form]
The form of the preparation (pharmaceutical composition) of the present invention is not particularly limited, and various solid preparations (oral solid preparations) such as powders, granules (fine granules or granules), pills, tablets, capsules, chewables. It may be a lock. The composition is often in the form of a granulated product (powder, granule) or tablet, and the granule includes fine granules and granules.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

(Test Example 1)
The measurement of DPP-IV inhibitory activity was performed according to the following method. 20 μl of sample solution or ultrapure water was added to each well of a 96-well black plate, and the total amount was adjusted to 160 μl using 100 μl of 200 mM sodium phosphate buffer (pH 8.0) and 40 μl of ultrapure water. Thereafter, 20 μl of human DPP-IV solution and 20 μl of 0.5 mM Gly-Pro-MCA (Glycyl-L-proline 4-methyl commentary-7-amide) were added (total amount 200 μl) and stirred at 37 ° C. for 1 The enzyme reaction was performed for a time. Moreover, the well which added the ultrapure water instead of the human DPP-IV solution was made into the control group. The DPP-IV enzyme reaction was measured using a fluorescence microplate reader (Flex Station II, manufactured by Molecular Devices), and the amount of AMC (7-amino-4-methylcoumarin) released by the DPP-IV enzyme reaction was measured (excitation). Wavelength is 360 nm, measurement wavelength is 440 nm). The DPP-IV inhibitory activity was calculated by calculating the relative activity of the well to which the sample was added, with the activity of the well to which ultrapure water was added instead of the sample being 100%.

Example 1
100 g of fish of Iwadaka and Senhadaka, 200 ml of water and Sumiteam FP (manufactured by Shinnippon Chemical Co., Ltd.), orientase 5N (manufactured by HBI Enzymes), purified papain F (manufactured by Asahi Food & Healthcare) or Flavorzyme (manufactured by Novozymes) 0.3 g) was added, and the enzyme treatment was performed at 50 ° C. for 1, 2 or 3 hours with stirring. Thereafter, the enzyme was inactivated by heating at 90 ° C. for 5 minutes. The obtained enzyme degradation product is centrifuged for 15 minutes (10000 × g) to remove the precipitate, and then the supernatant is filtered through diatomaceous earth (manufactured by Advantech, Filter paper 5C) to obtain a filtrate from which insoluble substances have been removed. It was. This filtrate was freeze-dried to obtain a powder of hydrolyzed protein. This powder was dissolved in ultrapure water to a concentration of 10 mg / ml, and DPP-IV inhibitory activity was measured according to the description in Test Example 1 (sample final concentration 1 mg / ml). The results are shown in FIG. Both Iwadaka and Senhadaka fish protein hydrolysates showed good DPP-IV inhibitory activity. Moreover, the intensity of DPP-IV inhibitory activity did not vary greatly depending on the type of enzyme and the treatment time.

(Example 2)
After freeze-drying 50 g of Iwadaka fish body as it was, fish powder was obtained using a small food grinder. To 125 mg of this powder, 1 ml of water and 0.375 mg of proteolytic enzyme were added. For enzymatic degradation, Sumiteam FP, Orientase 5N, Flavorzyme, Purified Papain F, Sumiteam LP (manufactured by Shin Nippon Chemical Industry Co., Ltd.), Coclase P (manufactured by Mitsubishi Chemical Foods), Alcalase (manufactured by Noboenzymes), Samoaase PC10F ( One or two types (manufactured by Amano Enzyme) were used, and the reaction was carried out at 55 ° C. all day and night (14 hours). Thereafter, the enzyme was inactivated by heating at 90 ° C. for 5 minutes. The obtained enzyme degradation product was centrifuged (10000 × g) for 15 minutes to remove precipitates, thereby obtaining a liquid from which insoluble substances were removed. This liquid was freeze-dried to obtain powder of hydrolyzate of iwahadaka protein. This powder was dissolved in ultrapure water, and DPP-IV inhibitory activity was measured as described in Test Example 1. The results are shown in FIGS. A single enzyme treatment showed good DPP-IV inhibitory activity regardless of the type, and the activity was dependent on the sample concentration (FIG. 2). In the treatment with the two enzyme combinations, DPP-IV inhibitory activity was observed under any conditions, but no synergistic effect of the treated enzyme on the inhibitory activity was observed (FIG. 3).

(Example 3)
According to the description in Example 2, powder was obtained from lyophilized fish. After adding 20 ml of water and 3.75 g of Samoaase PC10F to 1.25 g of this powder, pH (pH 4.0 to pH 9.0) was adjusted using 1N hydrochloric acid and 1N sodium hydroxide. These mixed solutions were enzymatically decomposed under constant temperature conditions (30 ° C. to 80 ° C.) for 24 hours. After completion of the reaction, the enzyme was inactivated by heating at 90 ° C. for 5 minutes. After adding diatomaceous earth to the obtained enzyme degradation product and stirring, the precipitate was removed by centrifuging (5000 × g) for 15 minutes to obtain a liquid from which insoluble substances were removed. This liquid was freeze-dried to obtain powder of hydrolyzate of iwahadaka protein. This powder was dissolved in ultrapure water, and DPP-IV inhibitory activity was measured as described in Test Example 1. The results are shown in FIG. Regarding the pH and temperature of the solution, DPP-IV inhibitory activity was observed under any conditions. In particular, a strong DPP-IV inhibitory activity was observed in samples decomposed at a low pH and a high temperature range (for example, treatment at 70 ° C at pH 5.0).

Example 4
According to the description in Example 2, 200 ml of water was added to 10 g of powder obtained from lyophilized fish of Iwadaka, and then adjusted to pH 5.0 using 1N hydrochloric acid. Samoaase PC10F was added to this solution in a final concentration range of 0% to 1.2% with respect to the raw material, and enzymatic decomposition was performed while stirring at a temperature of 70 ° C. The reaction time was 0 to 24 hours. After the reaction, the enzyme was inactivated by heating at 90 ° C. for 15 minutes. After adding diatomaceous earth to the obtained enzyme degradation product and stirring, the precipitate was removed by centrifuging (5000 × g) for 15 minutes to obtain a liquid from which insoluble substances were removed. This liquid was freeze-dried to obtain powder of hydrolyzate of iwahadaka protein. This powder was dissolved in ultrapure water, and DPP-IV inhibitory activity was measured as described in Test Example 1. The results are shown in FIG. It was found that a degradation product having strong DPP-IV inhibitory activity was obtained depending on the enzyme concentration and reaction time.

(Example 5)
The endogenous proteolytic enzyme contained in the fish body was inactivated by heating the Iwadaka fish body at 90 ° C. for 30 minutes. Thereafter, according to the description in Example 2, the sardine fish was freeze-dried, and then a fish powder was obtained using a small food grinder. As a comparative control, a sample that was not subjected to enzyme inactivation by heat treatment was also prepared. After adding 200 ml of water to 10 g of this powder, the pH was adjusted to 5.0 with 1N hydrochloric acid. 0.03 g of Samoaze PC10F was added to this solution. As a comparative control, a sample to which no Samoa PC10F was added was also prepared. The reaction was carried out for 24 hours while stirring at 70 ° C. After the reaction, the enzyme was inactivated by heating at 90 ° C. for 15 minutes. The resulting enzyme degradation product was centrifuged (10000 × g) for 15 minutes to remove the precipitate, and then the supernatant was filtered through diatomaceous earth to obtain a filtrate from which insoluble substances had been removed. The obtained filtrate was lyophilized to be powdered. This powder was dissolved in ultrapure water, and DPP-IV inhibitory activity was measured as described in Test Example 1. The results are shown in FIG. Regarding the presence or absence of inactivation of the endogenous proteolytic enzyme contained in the fish body, very weak DPP-IV inhibitory activity was observed in the sample with inactivation, whereas the activity in the sample without inactivation was remarkably increased. From the enhancement, it was speculated that the peptide which is a degradation product of the protein is important for the DPP-IV inhibitory activity. On the other hand, regardless of the presence or absence of inactivation of the endogenous proteolytic enzyme contained in the fish body, the sample treated with protein by Samoaase PC10F showed remarkable DPP-IV inhibitory activity.

(Example 6)
In order to identify a DPP-IV inhibitory active ingredient, the smeltfish fish protein hydrolyzate of Example 2 was subjected to gel filtration fractionation. 3 g of Iwadaka fish protein hydrolyzate was dissolved in 10 ml of 0.1 M acetic acid, treated with a 0.2 μm filter, and 1 ml thereof was injected into a gel filtration column. For the fractionation, an HPLC system (AKTA Pure; GE Healthcare Life Science) was used, and a Sephadex G25 Medium column (Φ1.6 × 70 cm) was used at 25 ° C. The mobile phase was fractionated using 0.1 M acetic acid at a flow rate of 0.3 ml / min, and detection was performed at UV 214 nm and UV 280 nm. After the injection, the eluate was collected at intervals of 10 minutes between 100 and 650 minutes. Each sample was dried with a centrifugal evaporator (EC-2000; Sakuma), redissolved in 200 μl of ultrapure water, and tested for DPP-IV inhibitory activity. The results are shown in FIG. All samples showed broad inhibitory activity, but some fractions were particularly active. In this way, it is also possible to fractionate the active ingredient.

(Example 7)
In the gel filtration fraction obtained in Example 6, Fraction 20 (25 μl) and Fraction 21 (25 μl), which showed remarkable DPP-IV inhibitory activity, were mixed at a ratio of 1: 1, and then filtered with a 0.45 μm filter. The total amount (50 μl) was injected onto the column. The column used was JUPITER Proteo 90A 4.6 * 250 mm (Phenomenex) at 40 ° C. Elution was performed with a gradient of 0 → 80% acetonitrile (0.1% TFA) / 100 minutes at a flow rate of 1 ml / min. Detection was performed at UV 214 nm. Samples were taken at 1 minute intervals. After drying with a centrifugal evaporator, it was redissolved in 50 μl of ultrapure water and tested for DPP-IV inhibitory activity. The results are shown in FIG. All samples showed broad inhibitory activity, but some fractions were particularly active. In this way, it is also possible to fractionate the active ingredient in detail.

(Example 8)
Among the reverse-phase HPLC fractions obtained in Example 7, the fraction having a strong DPP-IV inhibitory activity (Fraction 19) was further purified, and the DPP-IV inhibitory activity was tested for each fraction. As a result, a purified fraction having strong DPP-IV inhibitory activity was obtained (FIG. 9). As a result of analyzing the sample by mass spectrum (MS), dipeptide Leucine-Arginine (LR) was identified (FIG. 11).

Example 9
The dipeptide LR estimated by MS was synthesized and dissolved in distilled water, and the DPP-IV inhibitory activity was tested as described in Test Example 1. As a comparative control, Alanine-Alinene (AA) and Arginine-Isolucine (RI), which are known not to show DPP-IV inhibitory activity, were also tested. The results are shown in FIG. LR showed DPP-IV inhibitory activity depending on the concentration. On the other hand, AA and RI did not show DPP-IV inhibitory activity in the same concentration range. Thus, it can be seen that LR is one of the active ingredients in the DPP-IV inhibitory activity of the sardine fish hydrolyzate.

Claims (10)

  A composition obtained by treating fish and / or fish meal with a proteolytic enzyme, comprising 10 μg / kg or more of leucine-arginine.   The composition according to claim 1, wherein the fish and / or fish meal is derived from a sardine.   The composition according to claim 1 or 2, wherein the proteolytic enzyme is an enzyme selected from the group consisting of neutral protease, papain or endo-type protease / exo-type protease, or a mixture thereof.   The composition according to any one of claims 1 to 3, wherein the IC50 value (50% inhibitory concentration) of DPP-IV inhibitory activity is 10 mg / mL or less in terms of solid concentration.   A DPP-IV inhibitor comprising the composition according to claim 1 as an active ingredient.   A DPP-IV inhibitor comprising leucine-arginine as an active ingredient.   The DPP-IV inhibitor according to claim 6, wherein the leucine-arginine is derived from a sardine.   Diabetes, psoriasis, prostatic hypertrophy, HIV infection, osteoporosis, gastrointestinal tract disorder, infertility, cancer, inflammatory disease comprising the composition according to claims 1 to 4 or the DPP-IV inhibitor according to claims 5 to 7 as an active ingredient Prophylactic and / or therapeutic agents for autoimmune disease, cardiovascular disease, kidney disease, mental illness, and / or graft versus host disease (GVHD) or graft survival delay during hematopoietic stem cell transplantation therapy.   A leucine-arginine-containing composition obtained by adding 0.1% by mass to 1.2% by mass of a proteolytic enzyme and decomposing at 30 to 80 ° C. for 1 to 24 hours with respect to the total mass of a fish body and / or fish meal. Manufacturing method.   A method of increasing leucine-arginine content by treating fish and / or fish meal with a proteolytic enzyme.