Cyclic peptide, method for producing the same, and use thereof

Abstract

To provide a new functional component derived from a protein resource and useful for health and beauty.SOLUTION: A composition for promoting expression of a nicotinamide phosphoribosyltransferase (NAMPT) gene comprising one selected from cyclo(Pro-X), salts thereof, and precursors thereof, where X is preferably one selected from Gly, Val, Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, and Tyr, and more preferably one selected from Gly, Val, and Ala.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cyclic peptide, a method for producing the same, and its use as a food or medicine. [Background technology]

[0002] Cyclic dipeptides have been reported to exhibit a variety of physiological activities. For example, Patent Document 1 describes a melanin-concentrating hormone receptor antagonistic composition containing, as an active ingredient, a cyclic dipeptide or a salt thereof, the cyclic dipeptide or salt thereof comprising one or more cyclic dipeptides selected from the group consisting of cycloarginyl leucine [Cyclo(Arg-Leu)], cycloglutaminyl arginine [Cyclo(Gln-Arg)], cycloleucyl lysine [Cyclo(Leu-Lys)], etc.

[0003] Although cyclic dipeptides can be composed of various amino acids, Patent Document 2 describes an agent for improving the moisturizing function of skin, excluding pharmaceuticals used in the treatment of inflammatory diseases, which contains a cyclic dipeptide whose constituent units are amino acids or a salt thereof as an active ingredient, and which contains one or more amino acids selected from the group consisting of cycloglycylhydroxyproline [Cyclo(Gly-Hyp)], cyclohydroxyprolyl glutamic acid [Cyclo(Hyp-Glu)], cycloglycylglycine [Cyclo(Gly-Gly)], cycloprolyl hydroxyproline [Cyclo(Pro-Hyp)], and cycloprolyl alanine [Cyclo(Pro-Ala)]. Patent Document 3 describes a composition for suppressing the renin-angiotensin system, which contains as an active ingredient a cyclic dipeptide or a salt thereof having amino acids as constituent units, wherein the cyclic dipeptide or salt thereof contains one or more selected from the group consisting of cycloaspartylphenylalanine [Cyclo(Asp-Phe)], cycloglutamylphenylalanine [Cyclo(Glu-Phe)], cyclolysylproline [Cyclo(Lys-Pro)], and cycloglutamyltyrosine [Cyclo(Glu-Tyr)]. Patent Document 4 describes a composition for stimulating TRPV1, which contains as an active ingredient a cyclic dipeptide or a salt thereof having amino acids as constituent units, wherein the cyclic dipeptide or salt thereof contains one or more selected from the group consisting of cycloaspartylphenylalanine [Cyclo(Asp-Phe)], cycloalanylproline [Cyclo(Ala-Pro)], cycloprolylvaline [Cyclo(Pro-Val)], and the like. Patent Document 5 describes a method for regenerating neurons and glial cells lost as a result of damage due to injury or disease, which comprises administering to a subject cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine) or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG)), or a combination thereof, in an amount effective to regenerate new neurons and glial cells.Patent Document 6 describes a method for preventing or treating COVID or COVID-19, which comprises providing at least one pharmaceutical composition containing one or more components selected from a group of components including cycloprolylglycine.

[0004] Furthermore, cyclic dipeptides can be produced by various methods. Patent Document 7 describes a method for producing a cyclic dipeptide, which comprises heating an aqueous solution containing at least one of a linear dipeptide and a linear tripeptide, each containing at least one proline or hydroxyproline as a constituent, at a pressure of 0.5 MPa or less to obtain a cyclic dipeptide containing at least one proline or hydroxyproline as a constituent. Patent Document 8 also describes It is described that a heat-treated collagen peptide obtained by subjecting a collagen peptide to high-temperature, high-pressure treatment at a temperature of 100°C or higher and a pressure of 0.101 MPa or higher contains a cyclic dipeptide and can be used as an active ingredient in an anti-obesity composition.

[0005] Elastin, a protein present alongside connective tissue components such as collagen in skin, blood vessels, lungs, tendons, and other tissues, plays a role in maintaining tissue structure and providing elasticity that allows tissues to adapt to various morphological changes. Elastin provides skin with elasticity and firmness, and its loss can cause wrinkles and sagging skin. Therefore, elastin and its hydrolyzed peptides are used in topical and oral skin preparations with the expectation of improving skin elasticity. For example, Patent Document 9 describes a topical skin preparation containing a fish skin-derived hydrolyzed elastin solution and one or more components selected from cell-activating components and anti-inflammatory components. Patent Document 10 describes an oral skin aging prevention and improvement agent characterized by containing two or more mucopolysaccharides, collagen and / or elastin or their partial hydrolyzates, and coenzyme Q10 as active ingredients. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication WO2017 / 014212 (Patent No. 6687619) [Patent Document 2] International Publication WO2016 / 063901 (Patent No. 5456876, Patent No. 6374579) [Patent Document 3] International Publication WO2017 / 002838 (Patent No. 6684277) [Patent Document 4] International Publication WO2017 / 014120 (Patent No. 6666912) [Patent Document 5] International Publication WO2019 / 222339 (Patent Publication No. 2021-524864) [Patent Document 6] International Publication WO2021 / 216385 (Patent Publication No. 2023-526754) [Patent Document 7] Patent No. 5456876 [Patent Document 8] International Publication WO2017 / 014149 (Patent No. 6786490) [Patent Document 9] Japanese Patent Application Laid-Open No. 2001-72572 [Patent Document 10] Japanese Patent Application Laid-Open No. 2006-143671 Summary of the Invention [Problem to be solved by the invention]

[0007] It would be desirable to discover new functional ingredients useful for health and beauty from proteinaceous resources. [Means for solving the problem]

[0008] The present inventors discovered unique peptides in the blood after elastin ingestion and discovered that such peptides have various functions, leading to the completion of the present invention.

[0009] The present invention provides the following: [1] A composition for promoting nicotinamide phosphoribosyltransferase (NAMPT) gene expression, comprising any one selected from cyclo(Pro-X), its salts, and its precursors. [2] The composition described in 1, which further has the function of promoting SIRT1 gene expression. [3] The composition according to 1 or 2, wherein X is any one selected from Gly, Val, Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, and Tyr. [4] The agent according to any one of 1 to 3, wherein X is any one selected from Gly, Val, and Ala. [5] The composition described in any one of 1 to 4, wherein the composition comprises a precursor of cyclo(Pro-X), and the precursor of cyclo(Pro-X) is included as elastin or a fragment thereof. [6] An oral composition for producing a cyclic peptide in the blood, comprising a non-cyclic polypeptide. [7] The composition described in 6, wherein the polypeptide is elastin, and the elastin is a porcine-derived elastin peptide. [8] The composition described in 6 or 7, wherein the elastin is a peptide consisting of the sequence set forth in SEQ ID NO: 1. [9] The composition of any one of items 6 to 8, wherein the cyclic peptide is cyclo(Pro-X).

[10] The composition of any one of items 6 to 9, wherein X is any one selected from Gly, Val, Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, and Tyr.

[11] The composition of any one of items 6 to 10, wherein X is any one selected from Gly, Val, and Ala. [Effects of the Invention]

[0010] One aspect of the present invention provides a composition for promoting nicotinamide phosphoribosyltransferase (NAMPT) gene expression. Ingestion of such a composition activates SIRT1, 3, and 6 in the skin of a subject, which is expected to have an anti-aging effect. [Brief explanation of the drawings]

[0011] [Figure 1] Stimulation of NAMPT gene expression by cyclic dipeptides [Figure 2] Stimulation of SIRT1 gene expression by cyclic dipeptides [Figure 3] Elastin sequence DETAILED DESCRIPTION OF THE INVENTION

[0012] In the context of the present invention, amino acids may be represented by three-letter or one-letter symbols, which are well known and clear to those skilled in the art.

[0013] 1. Composition One aspect of the present invention relates to a composition containing, as an active ingredient, any one selected from cyclo(Pro-X), its salts, and precursors thereof. "As an active ingredient" means that the ingredient is used in the composition and is identified as contributing to a specific purpose through labeling, etc. In functional food products, the active ingredient is sometimes referred to as a functional ingredient (an ingredient that contributes to a specific health purpose (excluding those related to reducing disease risk)).

[0014] [Cyclic peptides] In the present invention, the term "cyclic peptide" refers to a peptide having a cyclized structure formed by the formation of an amide bond between the N-terminus and C-terminus of a peptide chain formed by peptide bonds between amino acids. Cyclic dipeptides are distinguished from linear peptides. Cyclic peptides are called cyclic dipeptides, cyclic tripeptides, etc. depending on the number of amino acid residues that make up the cyclic portion. In the present invention, the term "amino acid" may refer to an amino acid residue. Furthermore, unless otherwise specified, it does not matter whether the amino acid is in the L-form or the D-form. When a peptide is derived from a protein derived from a living organism, the amino acids contained therein are usually in the L-form.

[0015] The cyclic dipeptide contained in the composition of this embodiment is represented by cyclo(Pro-X). Pro is the amino acid at one end, proline. X represents any amino acid at the other end. For example, cyclo(Pro-Ala) and cyclo(Ala-Pro) represent the same cyclic dipeptide.

[0016] Proline itself has a special structure and is therefore located in a special position in the structure of proteins (proline brings about a special structure) (Nippon Noveikagaku Kaishi Vol. 70, No. 10, pp. 1095-1101, 1996). The area before and after proline in proteins is also special in terms of enzymatic degradation, so peptides with proline at the end are more likely to be produced, and peptides containing proline themselves are thought to be less susceptible to degradation.

[0017] In one aspect, X is a neutral amino acid, specifically any one selected from Gly, Val, Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, and Tyr, and preferably any one selected from Gly, Val, Ala, Ile, and Leu, because such a structure is expected to have the desired function described below.

[0018] In a particularly preferred embodiment, X is any one selected from Gly, Val, and Ala. That is, in a particularly preferred embodiment, the composition contains at least one selected from cyclo(Pro-Gly), cyclo(Pro-Val), and cyclo(Pro-Ala) as an active ingredient. According to the studies of the present inventors, these cyclic peptides not only exert the intended function but also have a resistant to decrease in blood concentration.

[0019] Cyclic peptides are more lipid-soluble than linear dipeptides, which have polar carboxyl or amino groups exposed at the molecular termini, and therefore have superior gastrointestinal and membrane permeability compared to linear dipeptides.

[0020] In one embodiment, the active ingredient may be a salt of a cyclic dipeptide. The salt is a salt acceptable for use as a food or pharmaceutical. Such salts include inorganic salts and organic salts, such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, hydrochloride, sulfate, nitrate, phosphate, acetate, citrate, maleate, malate, oxalate, lactate, succinate, fumarate, propionate, formate, benzoate, picrate, benzenesulfonate, and trifluoroacetate. Although the present invention is sometimes described herein using a cyclic dipeptide as an example, those skilled in the art can understand the description by applying it to a salt of a cyclic dipeptide as appropriate.

[0021] Cyclic dipeptides can be produced by a variety of methods. For example, they can be synthesized by synthesizing a linear peptide using an appropriate method and then cyclizing the linear peptide. Generally, peptide cyclization is carried out by synthesizing an activated ester of the linear peptide with a protected terminal amino group, then removing the protecting group from the terminal amino group, and adding the resulting activated ester of the linear peptide to a large amount of solvent. This method can also be applied to cyclic dipeptides. Another method involves heating a dipeptide in an organic solvent while removing water by distillation (JP Patent Publication No. 2003-531197). Another method for synthesizing cyclic peptides with any amino acid sequence is known, in which linear peptides are dehydrated and cyclized using high-temperature, high-pressure water (JP Patent Publication No. 2003-252896). Also known are methods for producing cyclic dipeptides containing at least one proline or hydroxyproline component by heating an aqueous solution containing a linear dipeptide at a pressure of 0.5 MPa or less (Patent Document 7 cited above), and methods for producing cyclic dipeptides by treating collagen peptides at high temperature and high pressure (Patent Document 8 cited above).

[0022] Furthermore, cyclic peptides are not limited to cyclic dipeptides, and can be produced in the blood by orally ingesting a protein such as elastin or a non-cyclic polypeptide that is a fragment thereof, as will be described later.

[0023] [Precursor of Cyclo(Pro-X)] In one embodiment, the composition contains a precursor of cyclo(Pro-X) as an active ingredient. In the context of the present invention, a precursor of cyclo(Pro-X) refers to a substance that can generate cyclo(Pro-X) in the blood when orally ingested. An example of a precursor of cyclo(Pro-X) is acyclic elastin or a fragment thereof.

[0024] Elastin is a protein found in skin, blood vessels, lungs, tendons, and other connective tissue components such as collagen. Approximately 95% of the amino acid residues in elastin are nonpolar amino acids, and amino acid residues abundant in elastin include Pro, Gly, Val, and Ala. Elastin characteristically contains desmosine and isodesmosine. It also has a lower hydroxyproline content than collagen. The sequence listing shows the amino acid sequences of bovine, porcine, and chicken elastins, respectively, as SEQ ID NOs: 1 to 3.

[0025] When acyclic elastin or a fragment thereof, which is a precursor of cyclo(Pro-X), is used as the active ingredient, the animal species and organ from which the elastin or a fragment thereof is derived are not particularly limited. It may be derived from bovine origin, such as bovine nuchal ligament, or from fish, such as fish skin or bulbus arteriosus. Because elastin or a fragment thereof is derived from a mammal like humans and there is no risk of BSE, it is preferable that the elastin or a fragment thereof be derived from porcine origin, such as the porcine aorta.

[0026] In a preferred embodiment, the active ingredient is an elastin-degrading peptide, which is an enzymatic hydrolysis product of porcine aortic blood vessel-derived elastin. Particularly preferred are elastin-degrading peptides in which the desmosine and / or isodesmosine content in the amino acid composition (molar ratio) is 0.06% or more, and the ratio (molar ratio) of the sum of the desmosine content and the isodesmosine content to the hydroxyproline content is 0.1 or more, more preferably 0.22 or more, and even more preferably 0.27 or more. Such elastin-degrading peptides can be produced by the method described in JP-A-2008-74846.

[0027] [Function / Application] The composition of this embodiment is suitable for promoting the expression of the nicotinamide phosphoribosyltransferase (NAMPT) gene. NAMPT plays an important role in the synthesis of NMN (β-nicotinamide mononucleotide) in vivo. NMN is a synthetic intermediate of NAD+ (nicotinamide adenine dinucleotide). In recent years, it has been known that NMN controls the activity of the longevity gene SIRT (sirtuin) through its conversion to NAD+ in vivo.

[0028] In the present invention, unless otherwise specified, "expression of the NAMPT gene" refers to the synthesis of mRNA, a transcription product, or the synthesis of a protein, a translation product, based on the information encoded in the gene. Enhancement of gene expression can be confirmed by detecting and quantifying the production of the transcription product and its translation into protein. Enhancement of NAMPT gene expression is sometimes referred to as activation of NAMPT.

[0029] Whether or not a certain component promotes the expression of the NAMPT gene can be determined, for example, by confirming whether or not the expression of the NAMPT gene is promoted when the component is added compared to when the component is not added.

[0030] Promoting NAMPT gene expression in vivo is expected to increase NMN production, and therefore the composition of this embodiment is suitable for promoting NMN production in vivo.

[0031] When orally ingested, the active ingredient, a cyclic dipeptide, is transported throughout the body via the bloodstream and can exert its function at the destination. Therefore, the tissues and cells in which the active ingredient promotes the expression of the NAMPT gene are not particularly limited. In one embodiment, the active ingredient can promote the expression of the NAMPT gene in the skin and blood vessels.

[0032] Promoting NAMPT gene expression activates one of the NAD+-dependent anti-aging genes SIRT1, SIRT3, and SIRT6, enabling the treatment of skin diseases and conditions. The SIRT1 gene is known to be involved in UVB-induced DNA damage repair (J Photochem Photobiol B. 2021 Aug:221:112238. doi: 10.1016 / j.jphotobiol.2021.112238. Epub 2021 Jun 12.8), wrinkle and pigmentation improvement, mitochondrial activation, and ceramide synthesis. The SIRT3 gene is known to be involved in scavenging reactive oxygen species, pigmentation improvement, and whitening. The SIRT6 gene is known to be involved in DNA double-strand break repair, cell aging inhibition, and hair graying prevention. Examples of diseases or conditions that can be improved by promoting NAMPT gene expression include loss of elasticity, wrinkles, pigmentation, and sagging skin.

[0033] In the present invention, the term "treatment" for a disease or condition includes reducing the risk of onset, delaying onset, prevention, treatment, and halting or delaying progression. Treatment includes medical procedures performed by physicians with the aim of treating a disease, as well as non-medical procedures performed by persons other than physicians, such as nutritionists (registered dietitians, public health nurses, midwives, nurses, clinical laboratory technicians, beauty consultants, estheticians, food manufacturers, and food sellers. Treatment also includes the administration or recommendation of specific foods, dietary guidance, health guidance, nutritional guidance (including nutritional guidance necessary for the medical treatment of injured or sick people, and nutritional guidance for maintaining and promoting health), school lunch management, and guidance necessary for improving nutrition related to school lunches.

[0034] The composition of this embodiment can be ingested by humans (individuals) and non-human animals, and can be administered to healthy subjects and subjects with a disease or condition. Non-human animals include companion animals (e.g., dogs, cats) and laboratory animals (e.g., mice, rats, hamsters). The subject is preferably a human, and more preferably a human who desires or needs to receive any of the above-mentioned treatments.

[0035] The age and sex of the subject to be ingested with the composition of this embodiment are not particularly limited. In the case of humans, the composition of this embodiment can be ingested by children (under 15 years old), adults (15 years old or older), middle-aged and elderly people (40 to 65 years old), elderly people (65 years old or older), men, and women. In the present invention, the term "ingestion" is sometimes used in the case of both a food composition and a pharmaceutical composition, but this can be replaced with "administration."

[0036] [Composition] (Dosage / Dosage) The composition of this embodiment can be taken orally.

[0037] Those skilled in the art can appropriately design the intake amount depending on the age, body weight, sex, disease or condition of the subject, etc. The daily intake amount of the active ingredient of the composition can be, for example, 1 mg / day, preferably 10 mg / day, more preferably 20 mg / day or more, and even more preferably 30 mg / day or more. It may also be 50 mg / day or more, 75 mg / day or more, 100 mg / day or more, or 200 mg / day or more. In either case, it can be 1,000 mg / day or less. In either case, the lower limit can be 800 mg / day or less, preferably 600 mg / day or less, more preferably 400 mg / day or less, and even more preferably 300 mg / day or less.

[0038] The above-mentioned daily intake amount may be taken in one dose or in multiple doses.

[0039] The content of the active ingredient in the composition of this embodiment can be appropriately designed by a person skilled in the art, but can be, for example, 1,000 mg / 100 g or more, preferably 1,500 mg / 100 g or more, more preferably 2,000 mg / 100 g or more, more preferably 2,500 mg / 100 g or more, more preferably 3,000 mg / 100 g or more, and even more preferably 3,500 mg / 100 g or more. In any case, it can be 50,000 mg / 100 g or less, preferably 40,000 mg / 100 g or less, more preferably 30,000 mg / 100 g or less, and even more preferably 20,000 mg / 100 g or less.

[0040] The composition of this embodiment can be administered repeatedly to a subject, and can be administered continuously for a long period of time. The period is not particularly limited, and may be, for example, 3 days or more, 1 week or more, 1 month or more, 3 months or more, or 6 months or more. To ensure sufficient efficacy, the composition may be administered continuously for 1 week or more, 12 weeks or more, or about 3 months.

[0041] (Other ingredients) The composition of the present embodiment may contain ingredients other than the active ingredient, as long as the intended effect can be achieved. The other ingredients may be various additives acceptable for pharmaceuticals or foods. Examples of various additives acceptable for pharmaceuticals or foods include excipients, antioxidants (antioxidants), flavorings, seasonings, sweeteners, coloring agents, thickening and stabilizing agents, color formers, bleaching agents, fungicides, gum bases, bittering agents, enzymes, glazing agents, acidulants, emulsifiers, strengthening agents, manufacturing agents, binders, tonicity agents (isotonic agents), buffers, solubilizers, preservatives, stabilizers, and coagulants.

[0042] The other ingredients may be functional ingredients or nutritional ingredients other than the active ingredient. Examples of other ingredients include imidazole dipeptides other than the active ingredient, amino acids (e.g., branched-chain amino acids, ornithine, β-alanine), unsaturated fatty acids (e.g., EPA, DHA), vitamins (e.g., vitamin A, vitamin B1, vitamin B2, vitamin C, vitamin D), minerals (e.g., calcium, iron, magnesium, zinc), glucosamine, chondroitins, collagen, type II collagen peptide, honey, black vinegar, protein, carbohydrates, lipids, dietary fiber, etc.

[0043] (form) Examples of the form of the composition of this embodiment include powders, fine granules, granules, tablets, capsules, liquid preparations (including elixirs, lemonades, syrups, emulsions, suspensions, solutions, and drinks), and gel preparations.

[0044] In one embodiment, the composition is a food composition. In the context of the present invention, the term "food" includes not only solids but also liquids, such as beverages. Food also includes foods for specified health uses (FOSHU), functional foods, foods with functional claims, and other foods (sometimes referred to as general foods, health foods, supplements, etc.). Food also includes therapeutic foods, dietary diets, ingredient-modified foods, reduced-salt foods, nursing care foods, reduced-calorie foods, diet foods, sports foods, and ingredients therefor.

[0045] More specific examples of the food composition include supplements (tablets, granules, drinks), beverages, jelly drinks, confectioneries, processed meat products, processed seafood products, processed vegetables, side dishes, seasoning compositions, and food additives.

[0046] (others) The composition of this embodiment may be labeled with information about its intended use, the function of the active ingredient, and recommendations for administration to specific subjects. Labeling may be direct or indirect. Examples of direct labeling include information on the product itself, its packaging, container, label, tag, etc., while examples of indirect labeling include advertising and promotional activities via places or means such as websites, stores, exhibitions, signs, bulletin boards, newspapers, magazines, television, radio, mail, and email.

[0047] [Manufacturing method] The composition of this embodiment can be manufactured using various known techniques. The process of adjusting the active ingredient to a predetermined concentration can be applied at various stages of the manufacturing process. Those skilled in the art can appropriately design the manufacturing process taking into account the solubility, stability, volatility, etc. of the active ingredient.

[0048] 2. Production of cyclic peptides in the blood The present embodiment relates to producing a cyclic peptide from a non-cyclic polypeptide. In one aspect, an oral composition for producing a cyclic peptide in blood, comprising a non-cyclic polypeptide, is provided.

[0049] The acyclic polypeptide contained in the composition of this embodiment is acyclic (also referred to as linear or chain-like) and has a structure in which two or more amino acids are linked by peptide bonds. The number of amino acids constituting the polypeptide is not particularly limited, as long as it can be broken down in the body when orally ingested and generate a cyclic peptide in the blood, and can be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, or 30 or more. When the acyclic polypeptide is a mixture of multiple polypeptides, such as an enzymatic hydrolyzate of a protein, at least one polypeptide may be composed of the above number of amino acids, and the average (which may be a weight average or a number average) of the mixture may be the above number.

[0050] The acyclic polypeptide contained in the composition of this embodiment may be derived from animals or plants. Examples of raw materials for the polypeptide include, but are not limited to, meat, placenta, milk, dairy products, seafood, eggs, grains (e.g., wheat, barley, corn), beans (e.g., soybeans), seeds, nuts, vegetables, fruits, tea, and malt. From the viewpoint of relatively high proline content, meat, gelatin, milk, and grains are preferred.

[0051] The acyclic polypeptide contained in the composition of this embodiment may be a protein or a degradation product (sometimes referred to as a fragment) thereof. Examples of proteins include elastin, collagen, gelatin, keratin, myosin, actin, albumin, hemoglobin, milk casein, whey protein, gluten, hordein, and zein. From the viewpoint of containing relatively large amounts of proline, elastin, collagen, gelatin, milk casein, soybean globulin, wheat gluten, barley hordein, and corn zein are preferred.

[0052] The cyclic peptide in this embodiment is composed of several amino acid residues and may be a cyclic tripeptide or a cyclic dipeptide. In one aspect, the cyclic peptide is a dipeptide.

[0053] The composition of this embodiment is particularly suitable for use in the production of cyclic peptides, such as dipeptides represented by cyclo(Pro-X), in the blood, where X is any one selected from Gly, Val, Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, and Tyr. In one aspect, the acyclic polypeptide contained in the composition is acyclic elastin or a fragment thereof, as described above in "1. Composition."

[0054] According to the inventors' research, various peptides were increased in the blood of subjects who orally ingested elastin. Among the increased peptides were cyclic peptides, cyclo(Gly-Ala), cyclo(Pro-Gly), cyclo(Pro-Val), and cyclo(Pro-Ala). Pro-Gly was previously known as a peptide whose blood concentration increased after oral ingestion of elastin peptides (Shigemura et al. A pilot study for the detection of cyclic prolyl-hydroxyproline (Pro-Hyp) in human blood after ingestion of collagen hydrolysate. Nutrients 2018, 10, 1356). [Example]

[0055] [1: Identification of blood peptides] Humans were given enzymatically hydrolyzed elastin derived from pigs, and elastin-derived peptides containing cyclic dipeptides (as well as desmosine and isodesmosine, compounds unique to elastin) that appeared in the blood were identified.

[0056] (subject) (1) Selection of subjects The purpose of the study and the possibility of discontinuing participation at will were explained to seven healthy adult males who provided written consent.

[0057] (Test Method) (1) Study design: Open-label, uncontrolled study

[0058] (2) The test meals are as follows: Pig elastin peptide (enzymatic hydrolysate)

[0059] (3) Intake method and blood sampling The test meal is prepared by measuring the powder based on the body weight measured in advance so that the elastin content is 0.4g / kg body weight, and dissolving it in commercially available drinking water (soft water) so that the content is 10mL / g elastin (240mL / 24g elastin for a body weight of 60kg). Subjects fasted for 12 hours from the previous night (drinking water was permitted), and blood samples were taken from around 9:00 AM, pre-ingestion (0 minutes). Subjects were then instructed to consume the entire test meal within one minute, and blood samples were taken sequentially from a forearm vein at 15, 30, 60, 90, 120, and 240 minutes later (a total of seven samples were taken using a butterfly needle). Each sample was 10 mL, with approximately 3 mL discarded from the second and subsequent samples to remove blood from the needle (13 mL total). A total of 90 mL was taken per day. Subjects are prohibited from eating until all blood samples are taken on the day, and are required to drink 1 liter of water at any time between before ingestion (0 minutes) and after blood sampling until the end of the test (addition will be made if the amount is insufficient). During this time, subjects are only allowed to do desk work and are required to remain at rest.

[0060] (4) Plasma isolation Blood samples are collected using heparin-containing blood collection tubes. After collection, plasma is obtained by centrifugation, aliquoted, and stored frozen (-80°C) until analysis. Between collection and centrifugation, the blood is temporarily stored on ice.

[0061] (5) Analysis method Blood pretreatment, analytical methods, analysis, and statistical analysis were performed in accordance with previously reported methods.

[0062] Plasma obtained in the blood collection test was added with a three-fold volume of ethanol and centrifuged (3,000 x g, 4 °C, 10 minutes) to remove proteins. The supernatant after protein removal was centrifuged and dried, dissolved in 0.1% formic acid, and analyzed using an LCMS2020 (Shimadzu Corporation). Stable isotope-labeled Pro( 13 C5, 15 N-Pro) or Val( 13 C5, 15Analysis was performed using a peptide of the same sequence incorporating N-Val as an internal standard (Shigemura et al. A pilot study for the detection of cyclic prolyl-hydroxyproline (Pro-Hyp) in human blood after ingestion of collagen hydrolysate. Nutrients 2018, 10, 1356.).

[0063] (6) Measurement items Based on the sequence information of porcine elastin (SEQ ID NO: 2), taking into consideration the content of each amino acid and the ease of cleavage of the sequence, linear peptides and cyclic peptides composed of the following Pro, Gly, Val, and Ala are selected as candidates for detection and quantification.

[0064] [Table 1]

[0065] (result) The results are shown in the table below.

[0066] [Table 2]

[0067] Cyclic prolylglycine, cyclic prolylvaline, and cyclic prolylalanine were each detected at ∼1.5 nmol / mL.

[0068] [2: Gene expression] The functionality of cyclic prolylglycine, cyclic prolylvaline, and cyclic prolylalanine was evaluated.

[0069] The cyclic dipeptides used were prepared as follows: 0.3 mg of the linear dipeptide was dissolved in 20 μL of 200 mM aqueous ammonia and heated at 120° C. for 24 hours. After heating, the solution was centrifuged and dried, and then redissolved in water. The linear dipeptide and cyclic dipeptide were separated using an HPLC Prominence series (Shimadzu Corporation).

[0070] The effect of cyclic dipeptides on the expression of longevity genes (NAMPT, SIRT1) was evaluated using keratinocytes (HaCaT cells) by RT-qPCR.

[0071] (1) Preparation of total RNA using the High Pure RNA Isolation Kit Total RNA was prepared using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland) according to the manufacturer's protocol.

[0072] HaCaT cells were cultured at a final concentration of 6.0 × 10 4 Cells were seeded into a 6-well plate dish at 100 cells / mL and cultured for 24 hours. After 24 hours, each sample was added to the medium to a final concentration of 10 μM. After 48 hours, the medium was completely removed and 2 mL of 1x PBS was added to wash the cells. The PBS was removed, and 200 μL of 1x PBS and 400 μL of the High Pure RNA Isolation Kit's lysis-binding buffer were added. The plate was tilted for 4 minutes to distribute the solution evenly across the dish until the solution no longer became viscous. The bottom of the dish was scraped with the back of a tip, and the cell lysate was collected into a 1.5 mL tube. The collected cell lysate was then thoroughly suspended using a vortex mixer for 50 seconds.

[0073] The High Pure filter tube and collection tube included with the kit were assembled, and the entire volume of the cell lysate was added to the filter tube. Centrifuged at 10,000 rpm for 15 seconds. The solution discharged into the collection tube was discarded, and the filter tube and collection tube were reassembled. 90 μL of DNase Incubation Buffer and 10 μL of DNase I (the number of samples plus one) were added to a 1.5 mL tube per sample and mixed. 100 μL of this mixture was added to each filter tube and allowed to stand at room temperature for 15 minutes. After 15 minutes, 500 μL of Wash Buffer I included with the kit was added to the filter tube, and the tube was centrifuged at 10,000 rpm for 15 seconds. The solution in the collection tube was discarded, and the filter tube and collection tube were reassembled. 500 μL of Wash Buffer II was added to the filter tube, and the tube was centrifuged at 10,000 rpm for 15 seconds. The solution in the collection tube was discarded, and the filter tube and collection tube were reassembled. 200 μL of Wash Buffer II was added to the filter tube and centrifuged at 14,000 rpm for 2 minutes. After centrifugation, the filter tube was placed in a new, sterile 1.5 mL tube, and 100 μL of Elution Buffer was added to the center of the filter tube. The tube was then left to stand at room temperature for 3 minutes. After standing, the tube was centrifuged at 10,000 rpm for 1 minute to elute the total RNA.

[0074] The total RNA concentration in the solution was measured using Nano Drop 2000 (Thermo Fisher Scientific, Waltham, USA).

[0075] (2) RT-qPCR Each RNA was diluted to 6.25 ng / μL in 0.2 mL tubes with nuclease-free water, and then further diluted 10-fold. Primers for NAMPT and SIRT1 and SIRT3 were also diluted to 2 μM in 0.2 mL tubes. Taking into account the number of samples and genes, a premix was prepared by dissolving Go Taq qPCR Master Mix, Go Script RTMix, and nuclease-free water in a 25:1:4 ratio in a 1.5 mL tube. A 96-well PCR plate was placed on an ice plate, and 12 μL of premix, 2 μL each of primers F and R, and 4 μL each of diluted RNA were added to each well. The plate was then centrifuged for 10 seconds in a plate centrifuge and then loaded into the PCR system for analysis. The primer sequences are listed in Table 1.

[0076] RT-qPCR was performed using a Thermal Cycle Dicer Real Time System (Takara Bio Inc., Shiga, Japan). The reaction conditions were 37°C for 15 minutes (1 cycle), 95°C for 10 minutes (1 cycle), 95°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds (45 cycles), and 95°C for 15 seconds, 60°C for 30 seconds, and 95°C for 15 seconds (1 cycle). Detection was performed using FAM. Expression levels of the target genes were quantified using the ΔΔCt method.

[0077] [Table 3]

[0078] The results are shown in Figure 1. Resveratrol was used as a positive control, and the expression level of the target gene was expressed as a relative expression level to β-actin. The elastin-derived cyclic peptide significantly promoted NAMPT gene expression compared to the control.

[0079] The results are shown in Figure 2. Cyclic prolylalanine and cyclic prolylglycine significantly promoted the expression of the SIRT1 gene compared to the control.

[0080] [3: Analysis of porcine elastin peptides] The same porcine elastin peptides as those administered in the above experiment were analyzed.

[0081] 3-1: Amino acid composition analysis Porcine elastin peptides were hydrolyzed with hydrochloric acid and then quantified using an automatic amino acid analyzer (L-8900 Hitachi High-Tech Science Corporation).

[0082] The results are shown in the table below.

[0083] [Table 4]

[0084] 3-2: Molecular weight analysis Porcine elastin peptides were subjected to gel filtration HPLC and calculated from the elution time of the sample. Column used: Superdex 30 Increase 10 / 300GL Analysis software: Calculated from chromatographic waveforms using the GPC analysis software of the Shimadzu HPLC system

[0085] The average molecular weight (weight average molecular weight) obtained by GPC was 3,627.

[0086] The analysis results of the molecular weight distribution are shown in the table below.

[0087] [Table 5]

[0088] Nutritional Components The nutritional components of pork elastin peptides are shown in the table below.

[0089] [Table 6]

[0090] [Sequence listed in the sequence listing] SEQ ID NO:1 Bovine elastin amino acid sequence SEQ ID NO:2 Porcine elastin amino acid sequence SEQ ID NO:3 Chicken elastin amino acid sequence SEQ ID NO:4 PCR primer SEQ ID NO:5 PCR primer SEQ ID NO:6 PCR primer SEQ ID NO:7 PCR primer SEQ ID NO:8 PCR primer SEQ ID NO:9 PCR primer

Claims

1. A composition for promoting nicotinamide phosphoribosyltransferase (NAMPT) gene expression, comprising any one selected from cyclo(Pro-X), its salts, and its precursors.

2. The composition of claim 1, further having the function of promoting SIRT1 gene expression.

3. 2. The composition of claim 1, wherein X is any one selected from Gly, Val, Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, and Tyr.

4. 2. The composition of claim 1, wherein X is any one selected from Gly, Val, and Ala.

5. The composition according to claim 1 , wherein the composition comprises a precursor of cyclo(Pro-X), and the precursor of cyclo(Pro-X) is comprised of elastin or a fragment thereof.

6. An oral composition for producing cyclic peptides in the blood, comprising a non-cyclic polypeptide.

7. 7. The composition of claim 6, wherein the polypeptide is elastin, and the elastin is a porcine-derived elastin peptide.

8. The composition according to claim 7, wherein the elastin is a peptide consisting of the sequence set forth in SEQ ID NO:

1.

9. The composition of claim 6 , wherein the cyclic peptide is cyclo(Pro-X).

10. 7. The composition of claim 6, wherein X is any one selected from Gly, Val, Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, and Tyr.

11. 11. The composition of claim 6, wherein X is any one selected from Gly, Val, and Ala.

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