Peptides with dipeptidyl peptidase-IV inhibitory activity and their use

Abstract

To find out a novel component having dipeptidyl peptidase-IV inhibitory activity, and provide a dipeptidyl peptidase-IV inhibitor, a hyperglycemia inhibitor and an anti-diabetic agent.SOLUTION: Tetrapeptide Ile-Pro-Ala-Gly and tripeptide Val-Ala-Tyr included in hydrolysate of almond-derived protein has dipeptidyl peptidase-IV inhibitory activity, and can be used as a dipeptidyl peptidase-IV inhibitor, a hyperglycemia inhibitor and an anti-diabetic agent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a peptide having dipeptidyl peptidase-IV inhibitory activity, and also to a dipeptidyl peptidase-IV inhibitor, a blood glucose level suppressant, an antidiabetic agent, and the like, which utilizes the peptide. [Background technology]

[0002] In recent years, gastrointestinal hormones called incretins have been attracting attention in the treatment of type 2 diabetes. Incretins are secreted from the gastrointestinal tract following meal intake and act on pancreatic β cells to promote insulin secretion. They also inhibit the secretion of glucagon, which increases blood glucose levels, and protect and promote the proliferation of pancreatic β cells. Although incretins play an important role in lowering blood glucose levels, they are rapidly degraded and inactivated by dipeptidyl peptidase-IV (DPP-IV) in the blood. Therefore, the use of DPP-IV inhibitors has been considered a promising therapeutic strategy for type 2 diabetes. Therefore, various compounds with DPP-IV inhibitory activity have been identified.

[0003] For example, Patent Document 1 describes that a fluoropyrrolidone derivative with a specific structure can be used as a DPP-IV inhibitor to inhibit serine proteases such as DPP-IV. Patent Document 2 describes that a proline derivative with a specific structure can be used as a DPP-IV inhibitor. However, the DPP-IV inhibitors reported in Patent Documents 1 and 2 are chemically synthesized products that have never been consumed, and have the disadvantage that they cannot be used routinely as foods or the like.

[0004] Meanwhile, several DPP-IV inhibitors utilizing naturally occurring ingredients have also been reported. For example, Patent Document 3 describes that the tripeptide Met-Lys-Pro contained in casein hydrolysate has DPP-IV inhibitory activity. Patent Document 4 describes that a specific peptide contained in the water-soluble fraction of cheese has DPP-IV inhibitory activity. Patent Document 5 describes that the tripeptide X-Pro-Gln obtained by hydrolyzing gluten with an enzyme derived from ginger rhizome has DPP-IV inhibitory activity. Patent Document 6 describes that tea leaf protein hydrolysates have DPP-IV inhibitory activity. As described above, several DPP-IV inhibitors utilizing naturally occurring ingredients have been reported. However, from the perspective of increasing the variety of ingredients used as DPP-IV inhibitors, there is a need for the development of new DPP-IV inhibitors utilizing naturally occurring ingredients. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-63286 [Patent Document 2] Special Publication No. 2007-537231 [Patent Document 3] Japanese Patent Application Laid-Open No. 2016-136966 [Patent Document 4] Japanese Patent Application Laid-Open No. 2014-1223 [Patent Document 5] Japanese Patent Application Publication No. 2017-214334 [Patent Document 6] Japanese Patent Application Laid-Open No. 2017-43618 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to find a novel component having DPP-IV inhibitory activity and to provide a DPP-IV inhibitor, a blood glucose level suppressant, and an antidiabetic agent. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that the tetrapeptide Ile-Pro-Ala-Gly and the tripeptide Val-Ala-Tyr contained in an almond-derived protein hydrolysate have DPP-IV inhibitory activity. Based on this finding, further research has led to the completion of the present invention.

[0008] That is, the present invention provides the following aspects. Item 1. A tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly. Item 2. A tripeptide having the amino acid sequence Val-Ala-Tyr. Item 3. A DPP-IV inhibitor comprising the tetrapeptide according to Item 1 and / or the tripeptide according to Item 2. Item 4. The DPP-IV inhibitor according to Item 3, comprising an almond protein hydrolysate containing Ile-Pro-Ala-Gly and / or the tripeptide Val-Ala-Tyr as the tetrapeptide according to Item 1 and / or the tripeptide according to Item 2. Item 5. A food or drink for inhibiting DPP-IV, comprising the DPP-IV inhibitor according to Item 3 or 4. Item 6. A pharmaceutical product for inhibiting DPP-IV, comprising the DPP-IV inhibitor according to Item 3 or 4. Item 7. An agent for suppressing elevation of blood glucose level, comprising the tetrapeptide according to Item 1 and / or the tripeptide according to Item 2. Item 8. The blood glucose level elevation suppressant according to Item 7, comprising an almond protein hydrolysate containing Ile-Pro-Ala-Gly and / or the tripeptide Val-Ala-Tyr as the tetrapeptide according to Item 1 and / or the tripeptide according to Item 2. Item 9. A food or drink for suppressing an increase in blood glucose level, comprising the blood glucose level suppressor according to Item 7 or 8. Item 10. A pharmaceutical for suppressing elevated blood glucose levels, comprising the blood glucose level suppressing agent according to Item 7 or 8. Item 11. An antidiabetic agent comprising the tetrapeptide according to Item 1 and / or the tripeptide according to Item 2. Item 12. The antidiabetic agent according to Item 11, comprising an almond protein hydrolysate containing Ile-Pro-Ala-Gly and / or the tripeptide Val-Ala-Tyr as the tetrapeptide according to Item 1 and / or the tripeptide according to Item 2. Item 13. An antidiabetic food or drink comprising the antidiabetic agent according to Item 11 or 12. Item 14. An antidiabetic drug comprising the antidiabetic agent according to Item 11 or 12. [Effects of the Invention]

[0009] According to the present invention, DPP-IV is inhibited using a peptide derived from almonds, which is a natural product, and therefore it is possible to provide a DPP-IV inhibitor, a blood glucose level increase suppressant, and an antidiabetic agent that are highly safe and can be taken daily as food or beverages. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows the results of HPLC analysis of fractions obtained by purifying almond-derived protein hydrolysates, confirming the presence of Ile-Pro-Ala-Gly and Val-Ala-Tyr. The upper chromatogram shows the fraction obtained by the purification process, and the lower chromatogram shows a chromatographic standard. [Figure 2] FIG. 1 shows the results of measuring blood glucose levels over time after ingestion of an almond-derived protein hydrolysate containing Ile-Pro-Ala-Gly and Val-Ala-Tyr, followed by a glucose load (ingestion of an aqueous glucose solution). DETAILED DESCRIPTION OF THE INVENTION

[0011] 1. Peptides The peptides of the present invention are peptides having DPP-IV inhibitory activity, and specifically, a tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly (SEQ ID NO: 1) and a tripeptide having the amino acid sequence Val-Ala-Tyr.

[0012] In the present invention, amino acids are represented by three-letter symbols, with Ile representing isoleucine, Pro representing proline, Ala representing alanine, Gly representing glycine, Val representing valine, and Tyr representing tyrosine. Furthermore, in the present invention, the symbols for tetrapeptides and tripeptides indicate that the left end is the N-terminus and the right end is the C-terminus. Specifically, Ile-Pro-Ala-Gly indicates that Ile, Pro, Ala, and Gly are arranged in this order from the N-terminus to the C-terminus, and Val-Ala-Tyr indicates that Val, Ala, and Tyr are arranged in this order from the N-terminus to the C-terminus.

[0013] The method for producing the peptide of the present invention is not particularly limited, as long as it can produce a tetrapeptide and / or tripeptide having the amino acid sequence described above, and examples thereof include a method of hydrolyzing almond-derived proteins, biotechnology methods, chemical synthesis methods, etc. Among these, from the viewpoint of safety, etc., the method of hydrolyzing almond-derived proteins is preferred.

[0014] A method for producing the peptide of the present invention by hydrolyzing almond-derived proteins will be described below.

[0015] Almonds are the seeds (sweet almonds) of the almond (Prunus amygdalus Batsch) of the Rosaceae family. Almond-derived proteins can be obtained by extracting almonds. The almonds used as the raw material for the almond-derived proteins may be raw almonds, roasted almonds, or defatted almonds (e.g., defatted roasted almonds) produced during the almond oil production process. However, from the viewpoints of reducing production costs and effectively utilizing unused resources, defatted almonds are preferred.

[0016] Protein extraction from almonds can be carried out according to conventional methods, for example, by crushing or powdering almonds as necessary and then immersing them in an alkaline aqueous solution. The pH of the alkaline aqueous solution is, for example, 7 to 14, preferably 7.5 to 12, and more preferably 8 to 10. The alkaline aqueous solution is prepared by adding an alkaline agent such as sodium hydroxide or potassium hydroxide. The extraction temperature is, for example, 40 to 100°C, preferably 50 to 90°C, and more preferably 60 to 80°C. The extraction time is, for example, 0.5 hours or more, preferably 1 to 24 hours, and more preferably 2 to 12 hours.

[0017] The extract containing almond-derived protein thus obtained may be subjected to a solid-liquid separation, concentration, etc., if necessary, before being subjected to a hydrolysis treatment, or may be subjected to a hydrolysis treatment as it is.

[0018] The method for hydrolyzing the almond-derived protein is not particularly limited as long as Ile-Pro-Ala-Gly and / or Val-Ala-Tyr is produced, and any method such as protease treatment, acid treatment, or alkali treatment may be used, with protease treatment being preferred.

[0019] The type of protease used in the protease treatment is not particularly limited as long as it is capable of producing Ile-Pro-Ala-Gly and / or Val-Ala-Tyr, and specific examples include endopeptidases, exopeptidases, and combinations thereof. The origin of the protease is also not particularly limited, and examples include plant-derived proteases such as papain, bromelain, and ficin; proteases derived from various microorganisms; and animal-derived proteases such as pepsin, (chymo)trypsin, and cathepsin. Commercially available proteases can be used. Commercially available proteases include, for example, Papain W-40, Samoase PC10F, Neurase F3G, Pancreatin F, Protin SD-AY10, Protin SD-NY10, Protease A "Amano" SD, Protease M "Amano" SD, Protease P "Amano" 3SD, Bromelain F, and Proteax (all manufactured by Amano Enzyme Co., Ltd.); Orientase AY, Orientase 10NL, Orientase 90N, and Orientase 10NL. Examples of suitable antibodies include Tase OP, Nucleicin, Orientase 22BF, Bioprase SP-20FG, and Bioprase OP (all manufactured by HIBI Corporation); Sumiteam LP-G, Sumiteam FL-G, Sumiteam CP, Sumiteam FP-G, Sumiteam MP, Sumiteam BR, and Sumiteam BNP (all manufactured by Shin Nippon Chemical Industry Co., Ltd.); Aroase AP-10, Aroase XA-10, and Aroase NP-10 (all manufactured by Yakult Pharmaceutical Co., Ltd.); Denateam CPO PEPRICH (manufactured by Nagase ChemteX Corporation); MaxiPro PSP (manufactured by DSM); purified papain (manufactured by Mitsubishi Chemical Foods Corporation); and FormeaTL and FormeaCTL (all manufactured by Novozymes).

[0020] Among proteases, from the viewpoint of efficiently producing Ile-Pro-Ala-Gly and Val-Ala-Tyr from almond-derived proteins, preferred are papain, protease derived from Geobacillus stearothermophilus, and chymotrypsin, and more preferred are Papain W-40 (papain, manufactured by Amano Enzyme Co., Ltd.), Thermoase PC10F (protease derived from Geobacillus stearothermophilus, manufactured by Amano Enzyme Co., Ltd.), and FormeaCTL (chymotrypsin, manufactured by Novozymes).

[0021] These proteases may be used singly or in combination of two or more. In particular, the combined use of papain and the protease derived from Geobacillus stearothermophilus, or the combined use of papain and chymotrypsin, enables the efficient production of the tetrapeptide Ile-Pro-Ala-Gly and the tripeptide Val-Ala-Tyr from almond-derived proteins.

[0022] The protease treatment can be carried out by allowing the protease and the almond-derived protein to coexist in a solvent in which the protease reaction is possible. For example, the protease and the almond-derived protein may be added to the solvent in any order. However, from the viewpoint of reducing production costs, it is preferable to add the protease to the extract containing the almond-derived protein described above, a concentrate thereof, or a diluted solution thereof.

[0023] In the protease treatment, the amount of almond-derived protein added as a substrate is not particularly limited, but may be 10 to 300 g / L, preferably 25 to 200 g / L, and more preferably 50 to 100 g / L. The amount of protease added may be appropriately determined depending on the type of enzyme used, and may be, for example, 0.01 to 20 parts by mass, preferably 0.1 to 10 parts by mass, and more preferably 1 to 5 parts by mass of protease in total per 100 parts by mass of almond-derived protein.

[0024] The pH during protease treatment may be within a range in which the protease used can act. For example, when papain and / or a protease derived from Geobacillus stearothermophilus is used, the pH may be 7 to 14, preferably 7.5 to 12, and more preferably 8 to 10. When papain and / or chymotrypsin is used, the pH may be 7 to 14, preferably 7.5 to 12, and more preferably 8 to 10.

[0025] The temperature conditions for the protease treatment may be within a range in which the protease used can act. For example, when papain and / or a protease derived from Geobacillus stearothermophilus is used, the temperature may be 30 to 70°C, preferably 40 to 60°C, and more preferably 45 to 55°C. When papain and / or chymotrypsin is used, the temperature may be 30 to 70°C, preferably 40 to 60°C, and more preferably 45 to 55°C.

[0026] The treatment time for the protease treatment can be appropriately set, taking into consideration the amount of almond-derived protein used, the type and amount of enzyme, the pH and temperature during the protease treatment, etc., the time required to produce Ile-Pro-Ala-Gly and Val-Ala-Tyr, and can be, for example, 5 to 24 hours, preferably 10 to 22 hours, and more preferably 15 to 20 hours.

[0027] After the protease treatment, if necessary, the protease may be inactivated, residue may be removed by solid-liquid separation, and the product may be concentrated, dried, or the like.

[0028] The almond protein hydrolysate thus obtained contains Ile-Pro-Ala-Gly and / or Val-Ala-Tyr and may be used as is as the peptide-containing composition of the present invention. Alternatively, the obtained almond protein hydrolysate may be subjected to purification treatment such as ultrafiltration, adsorption resin treatment, chromatography, etc., as necessary, to increase the purity of the tetrapeptide Ile-Pro-Ala-Gly and / or tripeptide Val-Ala-Tyr, or may be converted into a purified product of the tetrapeptide Ile-Pro-Ala-Gly and / or tripeptide Val-Ala-Tyr, which may then be used as the peptide-containing composition of the present invention.

[0029] The use of the peptide of the present invention is not particularly limited, but since the peptide of the present invention has DPP-IV inhibitory activity, it can be suitably used as a DPP-IV inhibitor, an agent for suppressing blood glucose level elevation, an antidiabetic agent, etc. These uses will be described in detail in the section "2. DPP-IV inhibitors, agents for suppressing blood glucose level elevation, and antidiabetic agents."

[0030] 2. DPP-IV inhibitors, blood glucose suppressants, and antidiabetic agents The tetrapeptide Ile-Pro-Ala-Gly and / or the tripeptide Val-Ala-Tyr have DPP-IV inhibitory activity and can therefore be used as the active ingredient of a DPP-IV inhibitor.

[0031] Furthermore, since incretins promote insulin secretion and suppress the secretion of glucagon, which increases blood glucose levels, and DPP-IV inhibition can suppress the degradation of incretins, the tetrapeptide Ile-Pro-Ala-Gly and / or the tripeptide Val-Ala-Tyr can also be used as active ingredients in blood glucose level inhibitors or antidiabetic agents, which are used for the prevention or treatment of type 2 diabetes.

[0032] The DPP-IV inhibitor, blood glucose level increase suppressant, or antidiabetic agent of the present invention may contain at least one of the tetrapeptide Ile-Pro-Ala-Gly and the tripeptide Val-Ala-Tyr as an active ingredient, but it is preferable to contain both of these, as this will enable the agent to exhibit even more excellent DPP-IV inhibitory activity.

[0033] In the DPP-IV inhibitor, blood glucose level increase suppressant, or antidiabetic agent of the present invention, the tetrapeptide Ile-Pro-Ala-Gly and / or tripeptide Val-Ala-Tyr used as an active ingredient may be in a purified or unpurified state. For example, an almond protein hydrolysate containing the tetrapeptide Ile-Pro-Ala-Gly and / or the tripeptide Val-Ala-Tyr may be used in an unpurified state or in a state in which the purity of these peptides has been increased.

[0034] The DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is used by oral ingestion or administration. The intake or administration amount of the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is an amount effective for inhibiting DPP-IV in the body, and may be appropriately set depending on the product form used, etc., but for example, the daily intake or administration amount for an adult is 1 mg to 500 mg, preferably 5 to 250 mg, more preferably 10 to 100 mg, in terms of the total amount of the tetrapeptide Ile-Pro-Ala-Gly and / or the tripeptide Val-Ala-Tyr.

[0035] The DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is used by being incorporated into a product that is required to have a DPP-IV inhibitory effect, a blood glucose level increase inhibitory effect, or an antidiabetic effect. Products that can incorporate the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention are not particularly limited, as long as they are orally ingested or orally administered, and examples thereof include foods and beverages, pharmaceuticals, etc.

[0036] The dosage form of a product containing the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention may be any of solid, semi-solid, liquid, etc., and is appropriately selected depending on the type and use of the product. Products containing the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention may contain food materials, food additives, nutritional components, pharmaceutically acceptable base materials, pharmaceutically acceptable additives, pharmacological components, etc., depending on the type, dosage form, etc., within the range that does not impair the effects of the present invention.

[0037] When the DPP-IV inhibitor, blood glucose level increase suppressant, or antidiabetic agent of the present invention is used in the food and beverage field, the tetrapeptide Ile-Pro-Ala-Gly and / or the tripeptide Val-Ala-Tyr may be prepared into a desired form either directly or in combination with other food materials or additives, and provided as a DPP-IV inhibitory food and beverage, a blood glucose level increase suppressant, or an antidiabetic food and beverage. Examples of such foods and beverages include general foods and beverages, as well as foods with health claims (including foods for specified health uses, foods with nutrient functions, and supplements), and foods for patients. Foods for patients can be provided for patients who require DPP-IV inhibition, suppression of blood glucose level increase, or prevention or treatment of diabetes.The form of these foods and beverages is not particularly limited, but specific examples include sweets such as chocolate, cookies, biscuits, snacks, crackers, chewing gum, candy, caramels, mouth-refreshing sweets, rice crackers, etc.; Japanese sweets such as buns and dorayaki; energy bars or balanced nutritional foods containing dried fruit, nuts, dietary fiber, etc.; frozen sweets such as ice cream, ice milk, lacto ice cream, and frozen desserts; desserts such as yogurt, pudding, and jelly; bakery products such as bread, cookies, biscuits, pizza dough, pie dough, ice cream cones, monaka wafer wrappers, and cream puff wrappers; Western sweets such as sponge cake, chiffon cake, castella, madeleines, financiers, pound cake, roll cake, and pancakes; noodles such as pasta, udon, buckwheat, hiyamugi, somen, Chinese noodles, macaroni, rice vermicelli, and harusame; jams such as strawberry jam, apple jam, blueberry jam, and marmalade; curry, hayashi, stew, meat sauce, etc. Sauce bases such as sashimi and white sauce; rice bowl bases for Chinese rice bowls, beef bowls, oyakodon (chukadon), etc.; seasoning ingredients such as fried rice base, mapo tofu base, and kamameshi base; freeze-dried foods such as miso soup and soup; sauces and condiments such as fruit sauce, Chinese bean paste, kabayaki sauce, mitarashi dango sauce, white sauce, dressing, Worcestershire sauce, meat sauce, and Thousand Island dressing; processed meat products such as ham, sausage, and roast pork; fish sausage, fish ham, fish paste, kamaboko, chikuwa, These include fish paste products such as hanpen and satsuma-age; cereals (processed grain products); supplements such as capsules (soft capsules, hard capsules), tablets, granules, powders, jellies, and liposome preparations; and beverages such as soft drinks, milk drinks, lactic acid bacteria drinks, carbonated drinks, fruit juice drinks, vegetable drinks, vegetable and fruit drinks, powdered drinks, jelly drinks, coffee drinks, cocoa drinks, black tea drinks, green tea drinks, sports drinks, nutritional drinks, powdered drinks, energy drinks, non-alcoholic drinks, and alcoholic drinks.

[0038] Furthermore, when the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is used in the food and beverage field, the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention can be provided alone or in combination with other components as a food additive for inhibiting DPP-IV, a food additive for suppressing blood glucose level increase, or a food additive for antidiabetic purposes.

[0039] When the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is used in a food or beverage, the amount of the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent in the food or beverage may be appropriately set within a range that satisfies the aforementioned intake amount depending on the type and form of the food or beverage, but an example of such a range is the total amount of the tetrapeptide Ile-Pro-Ala-Gly and / or tripeptide Val-Ala-Tyr in the food or beverage of 0.0001 to 90% by mass, preferably 0.0005 to 75% by mass, and more preferably 0.001 to 50% by mass.

[0040] Furthermore, when the DPP-IV inhibitor, blood glucose level increase suppressant, or antidiabetic agent of the present invention is used in the pharmaceutical field, the tetrapeptide Ile-Pro-Ala-Gly and / or the tripeptide Val-Ala-Tyr may be prepared alone or in combination with other pharmacological ingredients, pharmaceutically acceptable bases, additives, etc. into a desired dosage form to provide the DPP-IV inhibitor, blood glucose level increase suppressant, or antidiabetic drug. The form of such a drug is not particularly limited, and specific examples include oral drugs such as drinks, tablets, pills, powders, fine granules, granules, capsules (including hard capsules and soft capsules), troches, chewable tablets, extracts (including soft extracts, dry extracts, etc.), jellies, syrups, and spirits.

[0041] When the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent of the present invention is used in a pharmaceutical product, the amount of the DPP-IV inhibitor, blood glucose level increase inhibitor, or antidiabetic agent in the pharmaceutical product may be appropriately determined within a range that satisfies the above-mentioned dosage depending on the type and dosage form of the pharmaceutical product, and examples of such amounts include a range in which the total amount of the tetrapeptide Ile-Pro-Ala-Gly and / or tripeptide Val-Ala-Tyr in the pharmaceutical product is 0.0001 to 90% by mass, preferably 0.0005 to 75% by mass, and more preferably 0.001 to 50% by mass. [Example]

[0042] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0043] Example 1: Preparation of almond protein hydrolysate 400 g of defatted roasted almond dry powder was added to 3600 g of water and thoroughly dispersed to prepare an almond-containing solution with a concentration of 10% by mass. Sodium hydroxide was added to this almond-containing solution to adjust the pH of the solution to 9.0, and the solution was heated and stirred at 70°C for 9 hours to obtain an almond protein extract.

[0044] The resulting almond protein extract was then cooled to 50°C and re-adjusted to pH 9.0 by adding sodium hydroxide. Then, 4 g each of Papain W-40 (Amano Enzyme) and Thermoase PC10F (Amano Enzyme) (0.1% by mass based on the almond-containing solution) were added, and the mixture was heated and stirred at 50°C for 18 hours to carry out the hydrolysis reaction. The enzymes were then inactivated by heating at 80°C for 30 minutes, and the hydrolysis reaction was stopped. The mixture was then cooled to room temperature. The hydrolyzed solution was centrifuged (15,000 × g, 4°C, 10 minutes) to recover the supernatant, which was then subjected to suction filtration to remove the residue. The filtered aqueous solution was freeze-dried to obtain approximately 200 g of hydrolyzate (lyophilized product).

[0045] Example 2: Identification of peptides with DPP-IV inhibitory activity 1. Isolation of peptides with DPP-IV inhibitory activity The hydrolysate (lyophilized product) obtained in Example 1 was dissolved in ultrapure water to a concentration of 5% by mass to prepare a hydrolysate-containing aqueous solution. 500 μL of this hydrolysate-containing aqueous solution was injected into a solid-phase extraction cartridge (Sep-Pak Plus Short C18 Cartridge, manufactured by Waters), and then 3 mL of ultrapure water was injected using a syringe. The extruded solution was discarded without being collected. Next, 1 mL of 10% by volume aqueous methanol solution was injected into the solid-phase extraction cartridge, and the extruded solution was discarded without being collected. Finally, 2 mL of 10% by volume aqueous methanol solution was injected into the solid-phase extraction cartridge, and the extruded solution was collected.

[0046] The collected solution was concentrated and dried, then dissolved in 200 μL of ultrapure water. The solution passed through a 0.45 μm centrifugal filter (5,000 rpm / min) was used as a sample to separate peptides using reverse-phase HPLC under the following separation conditions 1 and 2.

[0047] <Separation condition 1> Column: Inertsil ODS-3 (GL Sciences) Detection: UV 210nm Flow rate: 3mL / min Eluent A: Ultrapure water Eluent B: 50% acetonitrile in water Gradient: A linear gradient condition was set such that the ratio of eluent A was 100% and the ratio of eluent B became 50% after 133 minutes.

[0048] <Separation condition 2> Column: LiChrospher 100 RP-18 (Merck) Detection: UV 210nm Flow rate: 0.8mL / min Eluent A: Ultrapure water Eluent B: 50% acetonitrile in water Gradient: A linear gradient condition was set such that the ratio of eluent A was 100% and the ratio of eluent B became 100% after 20 minutes.

[0049] The DPP-IV inhibitory activity of the eluted fractions obtained under separation condition 1 was measured using a DPPIV drug discovery kit (BML-AK499, Enzo Life Sciences). A peptide with DPP-IV inhibitory activity was found in the eluted fraction with a retention time of 80 minutes. The peptide separated under these conditions was designated "peptide A."

[0050] Furthermore, the DPP-IV inhibitory activity of the eluted fractions obtained under separation condition 2 was measured using a DPPIV drug discovery kit (BML-AK499, Enzo Life Sciences). A peptide with DPP-IV inhibitory activity was found in the eluted fraction with a retention time of 12 minutes. The peptide separated under these conditions was designated "peptide B."

[0051] 2. Identification of the amino acid sequences of peptides A and B The amino acid sequences of peptides A and B, which showed DPP-IV inhibitory activity, were identified using a protein sequencer (PPSQ-53A, Shimadzu Corporation). As a result, peptide A was found to be a tripeptide consisting of Val-Ala-Tyr, and peptide B was found to be a tetrapeptide consisting of Ile-Pro-Ala-Gly.

[0052] 3. Confirmation of DPP-IV inhibitory activity of the tripeptide Val-Ala-Tyr and the tetrapeptide Ile-Pro-Ala-Gly 3-1. Test method The tripeptide Val-Ala-Tyr and the tetrapeptide Ile-Pro-Ala-Gly were synthesized and their DPP-IV inhibitory activity was measured using a DPP-IV drug discovery kit (BML-AK499, Enzo Life Sciences). Specific measurement conditions are as follows:

[0053] <Material> buffer solution :50mM Tris buffer (pH7.5) substrate solution:H-Gly-Pro-pNA (10 mM in DMSO) The substrate solution included in the kit was diluted 50-fold with the buffer solution and used. Enzyme solution 40 μL of the enzyme solution included in the Recombinant Human DPPIV / CD26 kit was diluted with the buffer solution to a total volume of 1011.5 μL. This diluted solution was further diluted 2-fold with the buffer solution and used. Sample solution The synthesized Val-Ala-Tyr and Ile-Pro-Ala-Gly were used after being appropriately serially diluted with ultrapure water.

[0054] <Method> The buffer solution, substrate solution, and sample solution were each added to a 96-well plate so that a total volume of 100 μL per well was determined according to Table 1. The 96-well plate was then inserted into a microplate reader (Infinite 200PRO, manufactured by Tecan) and pre-incubated at 37°C for 10 minutes. After pre-incubation, the enzyme solution according to Table 1 was added to the microplate reader. Subsequently, the reaction was continued at 37°C for 60 minutes, during which the increase in absorbance due to the cleavage of p-nitroaniline (pNA) from the substrate by DPP-IV was measured at 405 nm at 5-minute intervals.

[0055] [Table 1]

[0056] The DPP-IV inhibition rate at each concentration of Val-Ala-Tyr and Ile-Pro-Ala-Gly was calculated according to the following formula, and the IC 50 (the concentration that inhibits DPP-IV activity by 50%) was determined.

number

[0057] 3-2. Test results The results are shown in Table 2. As a result, it was confirmed that Val-Ala-Tyr and Ile-Pro-Ala-Gly have DPP-IV inhibitory activity, and the DPP-IV inhibitory activity of Ile-Pro-Ala-Gly is extremely high.

[0058]

Table 2

[0059] Example 3: Preparation of purified tripeptide Val-Ala-Tyr and tetrapeptide Ile-Pro-Ala-Gly The hydrolyzate obtained in Example 1 was subjected to a purification treatment using a resin to prepare a purified product with increased purity of Val-Ala-Tyr and Ile-Pro-Ala-Gly. Specifically, the hydrolyzate obtained in Example 1 was added to ultrapure water and mixed to prepare an aqueous solution. 1 mL of this aqueous solution was loaded onto a column (inner diameter 50 mm × length 200 mm, manufactured by BIORAD) filled with a resin (PrepC18, manufactured by Waters Japan). Next, two ethanol aqueous solutions with different concentrations were dropped onto the column to elute the peptides, and the eluate was fractionated. For each of the obtained fractions, HPLC analysis was performed under the following conditions using synthesized Val-Ala-Tyr and Ile-Pro-Ala-Gly as standards.

[0060] <HPLC Conditions> HPLC system: LC-2010 CHT (SHIMADZU) Column: Waters Xterra C18 (4.6 × 150 mm, 5 μm) Detection: UV 210 nm Flow rate: 800 μL / min Eluent A: 0.1% acetic acid aqueous solution Eluent B: 50% ethanol aqueous solution containing 0.1% acetic acid Gradient: Time (min) Solution A (%) Solution B (%) 0 - 4 96 4 4 - 20 60 40 Column temperature: 40°C Injection volume: 5 μL

[0061] As a result, a fraction containing both Val-Ala-Tyr and Ile-Pro-Ala-Gly was detected. The results of HPLC analysis of this fraction are shown in Figure 1. In addition, this fraction was concentrated and lyophilized to prepare a purified product containing Val-Ala-Tyr and Ile-Pro-Ala-Gly, which was used in Example 4 described below.

[0062] Example 4: Confirmation of DPP-IV inhibitory activity The DPP-IV inhibitory activity was measured for the hydrolysate obtained in Example 1 (containing 1.60 mg / g of Val-Ala-Tyr and 1.33 mg / g of Ile-Pro-Ala-Gly) and the purified product obtained in Example 4 (containing 5.10 mg / g of Val-Ala-Tyr and 4.30 mg / g of Ile-Pro-Ala-Gly). TM The measurement was carried out using Protease Assay (G8350, manufactured by Promega). The specific measurement conditions are as follows.

[0063] <Material> buffer solution 1M Tris-HCl (pH 8.0, Nippon Gene Co., Ltd.) was diluted 100-fold with ultrapure water to prepare 10 mM Tris-HCl (pH 8.0). substrate solution :DPPIV-Glo included in the kit TM A 10 mM substrate solution was prepared by adding 25 μL of ultrapure water to the substrate powder. Luciferin detection reagent : The Luciferin Detection Reagent powder included in the kit contains DPPIV-Glo TM The entire volume of 10 mL of buffer was added and mixed before use. Measurement reagents 20 μL of the substrate solution was added to 10 mL of the luciferin detection reagent and mixed thoroughly to adjust the substrate concentration to 20 μM. Enzyme solutionA 0.5 mg / mL solution of Dipeptidylpeptidase IV, human recombinant (manufactured by BioVision) was diluted 16,000-fold with the buffer solution and used. Sample liquid The hydrolysate obtained in Example 1 or the purified product obtained in Example 4 was prepared using ultrapure water to a concentration of 150 mg / mL. This aqueous solution was then mixed with an aqueous solution of dimethyl sulfoxide (DMSO, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) prepared to 10 vol% using ultrapure water in a ratio of 3:1 (so that the DMSO concentration was 2.5 vol%), and the mixture was used. Sample solution (negative control) DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to ultrapure water to a concentration of 2.5 vol%. Sample solution (positive control) : P32 / 98 (BML-PI142-0010, manufactured by Enzo Life Sciences) was added to 2.5 vol% DMSO to a concentration of 19.2 μM.

[0064] <Method> 5 μL of the measurement reagent, 1 μL of the sample solution, and 4 μL of the enzyme solution were added to each well of a 384-well plate and incubated at room temperature for 30 minutes. The 384-well plate was then inserted into a microplate reader (EnVision 2105, manufactured by PerkinElmer) and the luminescence intensity was measured. Using the medical statistical analysis software GraphPadPrism9 (manufactured by MDF), the luminescence intensity for the sample solution (negative control) was set to 100% and the luminescence intensity for the sample solution (positive control) was set to 0%, and the IC values ​​of the hydrolysate obtained in Example 1 and the purified product obtained in Example 4 were calculated. 50 (the concentration that inhibits DPP-IV activity by 50%) was determined.

[0065] The results are shown in Table 3. IC of the purified product obtained in Example 4 50was 0.6-fold compared to the hydrolysate obtained in Example 1. That is, it was confirmed that Val-Ala-Tyr and Ile-Pro-Ala-Gly, which have DPP-IV inhibitory activity, were concentrated by purification using the resin, resulting in higher DPP-IV inhibitory activity.

[0066] [Table 3]

[0067] Example 5: Confirmation test of the effect of suppressing postprandial blood glucose rise As mentioned above, the tripeptide Val-Ala-Tyr and the tetrapeptide Ile-Pro-Ala-Gly were found to have DPP-IV inhibitory activity in vitro. Therefore, a human study was conducted to confirm whether a composition containing Val-Ala-Tyr and Ile-Pro-Ala-Gly (the hydrolysate obtained in Example 1; containing 1.60 mg / g of Val-Ala-Tyr and 1.33 mg / g of Ile-Pro-Ala-Gly) has the effect of suppressing postprandial blood glucose elevation in the human body.

[0068] In accordance with the Declaration of Helsinki, the subjects were healthy adults (3 men, 3 women; average age 35 years) who provided consent after a thorough explanation of the purpose, methods, and results of this study. The study was conducted over two days. Furthermore, because it was necessary to measure fasting blood glucose levels during this study, subjects fasted from 10:00 PM the day before the study until the start of the study. They were only allowed to drink water until 1.5 hours before the start of the study, after which they were abstained from drinking water until the start of the study. The dietary content of the day before the study was not specified. During the study, exercise and activities that may affect blood glucose levels were restricted, and subjects were asked to remain at rest.

[0069] The test meal was a beverage prepared by suspending the hydrolysate (lyophilized product) obtained in Example 1 in 100 mL of water to achieve an intake of 5 g. The placebo meal was prepared by suspending the dried powder of defatted roasted almonds used as the raw material for the hydrolysate obtained in Example 1 in 100 mL of water to achieve a protein intake of 5 g. The reference meal for carbohydrate loading was a beverage prepared by dissolving glucose (D(+)-glucose, Fujifilm Wako Pure Chemical Industries, Ltd.) in 300 mL of water to achieve a carbohydrate intake of 50 g.

[0070] In this study, subjects ate a test meal or a placebo meal, and then ingested a reference meal 30 minutes later. Blood samples were taken immediately before ingesting the test meal or placebo meal (-30 minutes), immediately before ingesting the reference meal (0 minutes), and 30, 60, 90, and 120 minutes after ingesting the reference meal. Blood samples were taken by a doctor or nurse using an indwelling needle. Blood glucose levels were measured in the collected blood samples. Measurements were performed using a laboratory assay. TM Glucose (Fujifilm Wako Pure Chemical Industries, Ltd.) was used.

[0071] Figure 2 shows the time course of the average blood glucose levels of six subjects from immediately before ingestion of the test meal or placebo meal to 120 minutes after ingestion of the reference meal. As a result, it was confirmed that ingestion of the hydrolysate obtained in Example 1 had the effect of suppressing the rise in blood glucose levels immediately after sugar loading. In other words, it was revealed that the tripeptide Val-Ala-Tyr and the tetrapeptide Ile-Pro-Ala-Gly have the effect of suppressing postprandial blood glucose rise in the human body.

Claims

1. A tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly.

2. A dipeptidyl peptidase-IV inhibitor comprising a tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly and / or a tripeptide having the amino acid sequence Val-Ala-Tyr.

3. A dipeptidyl peptidase-IV inhibitor comprising an almond protein hydrolysate containing a tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly and / or a tripeptide having the amino acid sequence Val-Ala-Tyr.

4. A food or drink for inhibiting dipeptidyl peptidase-IV, comprising the dipeptidyl peptidase-IV inhibitor according to claim 2 or 3.

5. A pharmaceutical for inhibiting dipeptidyl peptidase-IV, comprising the dipeptidyl peptidase-IV inhibitor according to claim 2 or 3.

6. A blood glucose level increase inhibitor comprising the tetrapeptide according to claim 1.

7. An agent for suppressing blood glucose level elevation, comprising an almond protein hydrolysate containing a tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly and / or a tripeptide having the amino acid sequence Val-Ala-Tyr.

8. A food or drink for suppressing an increase in blood glucose level, comprising the blood glucose level increase suppressor according to claim 6 or 7.

9. A pharmaceutical for suppressing an increase in blood glucose level, comprising the blood glucose level increase suppressant according to claim 6 or 7.

10. An antidiabetic agent comprising the tetrapeptide of claim 1.

11. An antidiabetic agent comprising an almond protein hydrolysate containing a tetrapeptide having the amino acid sequence Ile-Pro-Ala-Gly and / or a tripeptide having the amino acid sequence Val-Ala-Tyr.

12. An antidiabetic food or drink comprising the antidiabetic agent according to claim 10 or 11.

13. An antidiabetic pharmaceutical product comprising the antidiabetic agent according to claim 10 or 11.

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