Pentapeptide and compositions containing said peptide

Abstract

To provide novel means that inhibits DPP-4.SOLUTION: The present invention provides a pentapeptide composed of Val-Tyr-Thr-Pro-Gly, and a composition containing the peptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a specific pentapeptide (Val-Tyr-Thr-Pro-Gly) and a composition containing said peptide. [Background technology]

[0002] The incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are hormones involved in maintaining glucose homeostasis. DPP-4 (Dipeptidyl Peptidase-4, EC3.4.14.5) is an enzyme that decomposes incretins. Inhibiting DPP-4 suppresses the decomposition of incretins, thereby increasing the concentrations of GLP-1 and GIP in the blood and promoting insulin secretion. Therefore, inhibiting DPP-4 improves blood glucose control (especially suppressing blood glucose rise) and is effective in preventing or treating diabetes (especially type 2 diabetes). DPP-4 inhibitors (e.g., sitagliptin) have already been marketed as drugs for treating type 2 diabetes. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2017 / 217535 [Patent Document 2] JP 2017-043618 A [Patent Document 3] JP 2016-003225 A Summary of the Invention [Problem to be solved by the invention]

[0004] The objective of the present invention is to provide a novel means for inhibiting DPP-4. [Means for solving the problem]

[0005] The present inventors found that a pentapeptide consisting of Val-Tyr-Thr-Pro-Gly has DPP-4 inhibitory activity, and through further improvements, have completed the present invention.

[0006] The present invention encompasses, for example, the subject matter described in the following paragraphs. Section 1. A pentapeptide consisting of Val-Tyr-Thr-Pro-Gly. Section 2. A composition comprising a pentapeptide consisting of Val-Tyr-Thr-Pro-Gly. Section 3. Item 3. The composition according to Item 2, which is for inhibiting DPP-4, improving glucose metabolism, suppressing an increase in blood glucose level, or for treating diabetes. Section 4. Item 4. The composition according to item 2 or 3, which is an oral composition. Section 5. Item 5. The composition according to any one of Items 2 to 4, which is a food composition or a pharmaceutical composition. Section A-1. A method for inhibiting DPP-4, improving glucose metabolism, suppressing blood glucose level increase, or preventing or treating diabetes, comprising the step of administering to a subject a pentapeptide consisting of Val-Tyr-Thr-Pro-Gly, or a composition containing the pentapeptide and a pharma- ceutical or food hygienically acceptable carrier. Section B-1. A pentapeptide consisting of Val-Tyr-Thr-Pro-Gly, or a composition comprising the pentapeptide and a pharma- ceutical or food hygienically acceptable carrier, for use in inhibiting DPP-4, improving glucose metabolism, suppressing blood glucose level increase, or preventing or treating diabetes. Section C-1. Use of a pentapeptide consisting of Val-Tyr-Thr-Pro-Gly in the manufacture of a medicine or food for inhibiting DPP-4, improving glucose metabolism, suppressing blood glucose level increase, or preventing or treating diabetes. Effect of the Invention

[0007] DPP-4 activity can be efficiently inhibited, and as a result, effects such as improving sugar metabolism, suppressing an increase in blood glucose level, and antidiabetic effects (preferably preventing or treating diabetes) can be obtained. [Brief description of the drawings]

[0008] [Figure 1] 3 shows a chart obtained by HPLC analysis of a rice bran enzyme (thermolysin) treated composition. [Diagram 2] The peaks indicated by the arrows in FIG. 1 were fractionated and further analyzed by HPLC, and a chart was obtained as shown below. [Diagram 3] The chart shown here was obtained by separating the peak indicated by the arrow in Figure 2 and then analyzing it by HPLC. The arrow indicates the peak containing the VYTPG peptide. [Figure 4] The results of investigating the DPP-4 inhibitory activity of the VYTPG peptide using the kit are shown below. The lower the fluorescence intensity, the stronger the DPP-4 inhibitory activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Each embodiment of the present invention will be described in more detail below. The present invention preferably includes a specific pentapeptide, a composition containing the pentapeptide, a method for administering the pentapeptide to a subject, and uses of the pentapeptide, but is not limited thereto, and the present invention includes all of the disclosures herein.

[0010] The composition encompassed by the present invention contains a specific pentapeptide (Val-Tyr-Thr-Pro-Gly). In the present specification, the composition may be referred to as the "composition of the present invention." In addition, in the present specification, the pentapeptide consisting of the amino acid sequence Val-Tyr-Thr-Pro-Gly may be referred to as the "VYTPG peptide."

[0011] The VYTPG peptide can be prepared by chemical synthesis using known peptide synthesis methods. Examples of peptide synthesis methods include the azide method, the acid chloride method, the acid anhydride method, the mixed acid anhydride method, the DDC method, the active ester method, the carboimidazole method, and the oxidation-reduction method. These peptide synthesis methods can be performed by either solid-phase synthesis or liquid-phase synthesis.

[0012] In the above-mentioned peptide synthesis method, it is preferable to protect the amino group, carboxy group, and / or side chain functional group (e.g., the guanidino group of arginine (Arg)) with a protecting group. The protecting group is not particularly limited and any known protecting group can be used, such as benzyloxycarbonyl group (Cbz), tert-butoxycarbonyl group (Boc), fluorenylmethoxycarbonyl group (Fmoc), benzyl group (Bz), p-toluenesulfonyl group (p-Ts), etc.

[0013] In addition, after synthesizing the VYTPG peptide by the above-mentioned peptide synthesis method, if necessary, the peptide can be purified by a known method and used as the VYTPG peptide.

[0014] Furthermore, the VYTPG peptide can also be produced by treating rice bran with thermolysin. In other words, a composition obtained by treating rice bran with thermolysin (rice bran thermolysin-treated composition) contains the VYTPG peptide. Thus, the rice bran thermolysin-treated composition may be purified, for example, by HPLC to obtain the VYTPG peptide, or the rice bran thermolysin-treated composition itself or a composition obtained using the rice bran thermolysin-treated composition may be used as the composition of the present invention.

[0015] In addition, when subjecting rice bran to the thermolysin treatment, the rice bran used is not particularly limited, but is preferably defatted rice bran. In addition, defatted rice bran defatted by pressing (pressed defatted rice bran) is preferable. In particular, pressed defatted rice bran obtained by heating rice bran (raw rice bran) obtained after polishing to inactivate lipase and further performing a pressing process is preferable. In addition, it is preferable to use defatted rice bran with a lipid content of 5 to 20 mass%. Defatted rice bran with a lipid content in this range is preferably obtained by pressing defatting (particularly pressing defatting after lipase inactivation treatment).

[0016] The lipase inactivation treatment is carried out for the purpose of preventing oxidation deterioration of rice bran (raw rice bran) containing about 18 to 20% by mass of lipids. Usually, the raw rice bran is heated and roasted at about 70 to 130°C. The pressing treatment is carried out by a known pressing method, for example, pressing the rice bran that has been heated and roasted to about 100 to 115°C with a low-temperature continuous press (for example, the Miracle Chamber sold by Technosigma Co., Ltd.). It is preferable to press until the lipid content in the defatted rice bran after pressing is about 5 to 20% by weight. In addition, a drying treatment may be carried out for the purpose of reducing the moisture content of the rice bran before the lipase inactivation treatment and the defatted rice bran after oil extraction. Furthermore, it is preferable that the defatted rice bran obtained by the pressing treatment is further crushed and classified by a known method to be made into a powder form. In this case, the average particle size of the powder is preferably 5 to 200 μm, more preferably 10 to 150 μm, and most preferably 40 to 100 μm. As described above, the lipid content in the defatted rice bran is preferably 5 to 20% by weight, more preferably 7 to 15% by weight. The above-mentioned commercially available defatted rice bran can also be purchased and used. An example of such commercially available defatted rice bran is Hybref (manufactured by Sunbran Co., Ltd.).

[0017] Thermolysin used in the present invention is a known enzyme, and its EC number is 3.4.24.27. Thermolysin can be used, for example, by purchasing a commercially available product. For example, it can be purchased from Peptide Institute Co., Ltd. (Thermolysin) or Amano Enzyme Co., Ltd. (Thermoase).

[0018] The thermolysin treatment conditions for obtaining a rice bran thermolysin-treated composition are not particularly limited as long as the activity of thermolysin can be obtained, and can be set appropriately. For example, rice bran and thermolysin can be mixed in water, and reacted for about 10 to 30 hours with gentle stirring at about 37°C. After the treatment, thermolysin may be inactivated. The inactivation treatment of thermolysin can be performed, for example, by heating (for example, treatment at 100°C for about 10 to 20 minutes). After the treatment, purification may be performed, for example, by filtering and recovering the filtrate.

[0019] Since the VYTPG peptide has a DPP-4 inhibitory effect, the composition of the present invention containing the VYTPG peptide can be used as a composition for inhibiting DPP-4, and further as a composition for improving sugar metabolism, a composition for suppressing an increase in blood glucose level, an antidiabetic composition (particularly a composition for preventing or treating diabetes), etc. As described in detail below, the composition of the present invention can contain, for example, a pharmacologically or food hygienically acceptable carrier, etc. In addition, the composition of the present invention can be preferably used as, for example, an oral composition as an intake form. In addition, the composition of the present invention can be preferably used as, for example, a pharmaceutical composition or a food composition.

[0020] In this specification, the food composition includes not only ordinary fresh or processed food compositions, but also compositions that are widely consumed as food and beverages, such as food additive compositions such as salt substitutes, sweeteners, and additives for beverages, and food ingredient compositions such as food premixes for commercial and household use. In particular, when used as a food composition, it can be preferably used as a food composition that clearly indicates its action or effect on blood sugar on the product label. Specifically, for example, food compositions that clearly indicate its action or effect on sugar metabolism improvement, suppression of blood sugar rise, prevention of diabetes, etc., and food compositions that clearly indicate that the food composition is intended for people with high blood sugar levels or people who are concerned about blood sugar on the product label can be mentioned.

[0021] In this specification, the pharmaceutical composition includes drugs and quasi-drugs. When the composition of the present invention is used as a pharmaceutical composition, the composition of the present invention may contain other components as necessary in addition to the VYTPG peptide. The other components are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include pharma- ceutically acceptable bases, carriers, and / or additives (e.g., solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, etc.). The blending amount of the other components can be appropriately set depending on the purpose.

[0022] When the composition of the present invention is used as a pharmaceutical composition, the administration form is not particularly limited, and examples thereof include oral administration, intravascular administration (particularly intravenous injection), etc. Among these, oral administration is preferred.

[0023] When the composition of the present invention is used as a pharmaceutical composition, its dosage form is not particularly limited, and examples thereof include tablets such as orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, and dissolving tablets; troches; powders; suspensions; emulsions; elixirs; lemonades; syrups; lotions; granules; capsules such as hard capsules and soft capsules; creams; ointments; suppositories; poultices, tapes, microneedles, transdermal / mucosal administration agents such as iontophoresis and electroporation; and aerosols. These forms can be prepared by a conventional method by combining the VYTPG peptide with other components as necessary.

[0024] When the composition of the present invention is used as a pharmaceutical composition, the amount of VYTPG peptide in the pharmaceutical composition is not particularly limited as long as the above-mentioned effect of VYTPG peptide is exerted, and can be appropriately determined.

[0025] In addition, when the composition of the present invention is used as a pharmaceutical composition, the dosage, administration interval, administration subject, etc. are not particularly limited and can be set appropriately. For example, the dosage can be set appropriately depending on the age, sex, weight, health condition, and other conditions of the administration subject. In addition, for example, the administration interval may be once or multiple times a day (preferably 2 to 3 times), or once or multiple times every few days to weeks. In addition, the administration subject may be humans, and non-human mammals such as pets or livestock (for example, dogs, cats, cows, horses, sheep, llamas, rats, mice, etc.).

[0026] In particular, when the subject of administration is a human, the amount of VYTPG peptide administered (ingested) per day for an adult is, for example, preferably about 10 to 300 μg, more preferably about 15 to 200 μg, even more preferably about 20 to 100 μg, even more preferably about 30 to 80 μg, still more preferably about 35 to 70 μg, and particularly preferably about 40 to 60 μg.

[0027] The administration (ingestion) period is not particularly limited, but is preferably 3 to 12 weeks or more (3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more), and more preferably 9 to 12 weeks or more. In particular, when the daily VYTPG peptide dosage is about 30 to 80 μg, it is preferably 5 to 12 weeks or more, and when it is 10 μg or more but less than 30 μg, or more than 80 μg but not more than 300 μg, it is preferably 9 to 12 weeks or more.

[0028] When the subject of administration is a non-human mammal, the administration form, dosage form, dosage amount, administration interval, etc. can be appropriately determined with reference to the case where the subject of administration is a human.

[0029] When the composition of the present invention is used as a food composition, the composition of the present invention may contain other ingredients as necessary in addition to the VYTPG peptide. The other ingredients are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include bases, carriers, additives that are acceptable from the standpoint of food hygiene, and other ingredients and materials that can be used as foods.

[0030] When the composition of the present invention is used as a food composition, its intake form is oral intake.In this case, the form of the composition of the present invention is not particularly limited, and it can be used as general food, health functional food, or special purpose food.For example, it can be a specific health food, a nutritional functional food, a functional food, a food for sick people, a food for people with difficulty swallowing, a dietary supplement, a nutritional supplement, a hospital food, a nursing food, a processed food, a beverage, etc.These can be prepared by a conventional method. In addition, the dosage form of the food composition may be, for example, a hard capsule, a soft capsule, a supplement, a chewable tablet, a beverage, a powdered beverage, a granule, a film, or the like. In addition, when used as a food or drink, for example, a beverage such as a tea-based beverage, a sports beverage, a beauty beverage, a fruit juice beverage, a carbonated beverage, an alcoholic beverage, a soft drink, a jelly beverage, a concentrated type beverage diluted with water, hot water, carbonated water, or the like, a powder or granule dissolved or suspended in water or hot water, or the like, a dry solid such as a tablet, a tablet confectionery, a jelly, a snack, a baked confectionery, a fried confectionery, a cake, a chocolate, a gum, a candy, a gummy, or the like, a soup, a noodle, a rice dish, a cereal, or the like. Among these, in normal life, a supplement type, a chewable tablet, a one-shot drink type, or the like is preferred, and when taken for the purpose of enhancing the effect of exercise, a beverage such as a sports drink is also preferred. In particular, when the product is intended as a health food, dietary supplement, or nutritional supplement, it is preferable that the product be in the form of, for example, granules, capsules, tablets or pills (including chewable tablets), beverages (powdered beverages, energy drinks, etc.), jellies, etc., in order to facilitate continuous intake.

[0031] When the composition of the present invention is used as a food additive composition or a food ingredient composition, the form is not particularly limited, and examples thereof include liquid, powder, flake, granule, and paste. More specifically, examples thereof include seasonings (sweeteners, salt substitutes, soy sauce, vinegar, miso, sauces, ketchup, dressings, spices, herbs, etc.), flakes (furikake, rice cooking additives, etc.), barbecue sauce, roux paste (curry roux paste, etc.), and premixed food ingredients.

[0032] When the composition of the present invention is used as a food composition, the amount of VYTPG peptide in the food composition is not particularly limited as long as the above-mentioned effects of VYTPG peptide are exerted, and can be set appropriately.

[0033] In addition, when the composition of the present invention is used as a food composition, the intake amount, intake interval, intake subject, etc. are not particularly limited and can be set appropriately. For example, the intake amount can be set appropriately depending on the age, sex, weight, health condition of the subject, and other conditions of the intake subject. For example, the intake interval, when the above-mentioned amount is taken, may be once or multiple times a day (preferably 2 to 3 times), or once or multiple times every few days to weeks. In addition, the intake subject may be humans, and non-human mammals such as pets or livestock (for example, dogs, cats, cows, horses, sheep, llamas, rats, mice, etc.).

[0034] In particular, when the subject of administration is a human, the VYTPG peptide intake amount is, for example, preferably about 10 to 300 μg per day for an adult, more preferably about 15 to 200 μg, even more preferably about 20 to 100 μg, even more preferably about 30 to 80 μg, still more preferably about 35 to 70 μg, and particularly preferably about 40 to 60 μg.

[0035] The intake period is not particularly limited, but is preferably 3 to 12 weeks or more (3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more), and more preferably 9 to 12 weeks or more. In particular, when the daily intake of VYTPG peptide is about 30 to 80 μg, it is preferably 5 to 12 weeks or more, and when it is 10 μg or more but less than 30 μg, or more than 80 μg but less than 300 μg, it is preferably 9 to 12 weeks or more.

[0036] There are no particular limitations on the human subject to intake, but it is preferable for the human subject to intake to be a human who has a desire or need for improved sugar metabolism, suppression of blood sugar level rise, or prevention of diabetes, etc.

[0037] When the subject of intake is a non-human mammal, the form, intake amount, intake interval, etc. can be appropriately determined with reference to the case when the subject of intake is a human.

[0038] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present invention includes all arbitrary combinations of the constituent elements described in this specification.

[0039] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present invention above may be combined in any way to specify the subject matter included in the present invention. In other words, the present invention includes all subject matter consisting of all combinations of the combinable characteristics described in this specification. EXAMPLES

[0040] The present invention will be described in more detail below, but the present invention is not limited to the following examples. Production Example 1: Synthesis of pentapeptide by Fmoc method The pentapeptide Val-Tyr-Thr-Pro-Gly was synthesized by solid phase synthesis using the Fmoc method. The resulting VYTPG peptide was purified by HPLC and sequenced using a protein sequencer, confirming that it was a pentapeptide Val-Tyr-Thr-Pro-Gly.

[0041] Production Example 2: Synthesis of pentapeptide by the Boc method We requested the synthesis of a pentapeptide consisting of Val-Tyr-Thr-Pro-Gly by the Boc method from Peptide Institute, Inc. The delivered VYTPG peptide was confirmed to be a pentapeptide consisting of Val-Tyr-Thr-Pro-Gly by RP-HPLC, mass spectrometry and amino acid analysis.

[0042] Production Example 3: Purification of pentapeptide from enzymatically treated rice bran First, rice bran was enzymatically treated to obtain an enzymatically treated composition as described below.

[0043] Thermolysin (Peptide Institute, Inc.; Code 3504) was used as the enzyme.

[0044] The rice bran used was Hybref (manufactured by Sunbran Co., Ltd.). Hybref is pressed defatted rice bran that has been defatted by squeezing rice bran.

[0045] First, rice bran was mixed with ultrapure water to a concentration of 30 g / L, and thermolysin was added to the mixture to a concentration of 0.4 g / L. The mixture was then incubated in a 37°C incubator for 20 hours while stirring at 250 rpm using a stirrer. The mixture was then immersed in a boiling water bath for 15 minutes to inactivate the enzyme. The mixture was centrifuged at 15°C and 7150 rpm (8000 x g) for 15 minutes, and the pH of the resulting supernatant was adjusted to 7.0 using NaOH, filtered using filter paper No. 2 (manufactured by Advantec), and the resulting filtrate was freeze-dried. The freeze-dried product was used as a rice bran enzyme-treated composition. The rice bran enzyme-treated composition may be hereinafter referred to as "HB-T".

[0046] Next, the VYTPG peptide was purified from HB-T as follows.

[0047] An aqueous solution of HB-T adjusted to 50 mg / ml with ultrapure water was fractionated using a preparative HPLC system (1525 binary pump, 2487 spectrometer). A C18 (Cosmosil 5C18-AR-II 4.6 x 250 mm) column was used. The HPLC conditions were as shown in Table 1 below. The HPLC chart shown in Figure 1 was obtained, and peak 53 was fractionated. In Figure 1, peak 53 is indicated by an arrow.

[0048] [Table 1]

[0049] Peak 53 was fractionated, concentrated and centrifuged, and dissolved in 400 μl of ultrapure water. 100 μl of the fraction was fractionated under the analytical HPLC conditions shown in Table 2 below. A PE column (Cosmosil 5PE-MS 4.6×250 mm) was used. This operation gave the chart shown in FIG. 2. Peak 53-13 (indicated by an arrow in FIG. 2) in the chart was fractionated, concentrated and centrifuged, and dissolved in 100 μl of ultrapure water. The sample was again fractionated by analytical HPLC. The injection volume was 50 μl, and a CN column (Cosmosil 5CN-MS 4.6×250 mm) was used. This operation gave the chart shown in FIG. 3. Peak 533 (indicated by an arrow in FIG. 3) in the chart was purified. The purified sample obtained was analyzed by a mass spectrometer and a protein sequencer, and was found to be a VYTPG peptide.

[0050] [Table 2]

[0051] From the above studies, it was confirmed that VYTPG peptide is produced by treating rice bran with thermolysin.

[0052] Example 1: Measurement of DPP-4 inhibitory activity DPP-4 inhibitory activity measurement kit: DPP4 Activity Fluorometric Assay Kit (BioVision) was used to measure the DPP-4 inhibitory activity of VYTPG peptide. This kit is a kit for measuring the enzyme activity of DPP-4 by a fluorometric method, and more specifically, it is a kit for examining DPP-4 activity by measuring the fluorescence intensity generated by cleavage of a labeled substrate (H-Gly-Pro-AMC: DPP4 substrate) bound to AMC (7-Amino-4-Methyl Coumarin) by DPP-4. As can be seen from this, when DPP-4 activity is inhibited, the fluorescence intensity decreases.

[0053] The VYTPG peptide or sitagliptin (DPP-4 inhibitor: positive control included in the kit) synthesized in the above Production Example 1 was diluted with the DPP4 assay buffer included in the kit, and 25 μl was dispensed into a 96-well plate, and 50 μl of enzyme mix was added and incubated at 37° C. for 10 minutes. Then, 25 μl of reaction mix (23 μl of DPP4 assay buffer and 2 μl of DPP4 substrate) was added to each well, and the fluorescence intensity was measured using a plate reader (37° C. / excitation wavelength 360 nm, fluorescence wavelength 460 nm). In the above dilution procedure, the VYTPG peptide was diluted to 500 μM or 5000 μM when 25 μl of the above reaction mix was added to make the sample volume in the well 100 μl, and sitagliptin was diluted to 0.3 μM when 40 μl of the same reaction mix was added to make the sample volume in the well 100 μl.

[0054] The same procedure was also performed on samples containing 0 μM VYTPG peptide (i.e., DPP4 assay buffer only) and 5,000 μM of a dipeptide consisting of the amino acid sequence Tyr-Tyr (YY peptide: synthetic product), and the fluorescence intensity was measured.

[0055] The results are shown in Figure 4.

Claims

1. A pentapeptide consisting of Val-Tyr-Thr-Pro-Gly.

2. A composition containing a pentapeptide consisting of Val-Tyr-Thr-Pro-Gly (excluding rice bran thermolysin-treated compositions).

3. The composition according to claim 2, which is for inhibiting DPP-4, improving glucose metabolism, suppressing an increase in blood glucose level, or for antidiabetic purposes.

4. The composition of claim 2 or 3, which is an oral composition.

5. The composition according to any one of claims 2 to 4, which is a food composition or a pharmaceutical composition.

Images