WO2019107057A1 - Peptide and use thereof - Google Patents

Peptide and use thereof Download PDF

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Publication number
WO2019107057A1
WO2019107057A1 PCT/JP2018/040632 JP2018040632W WO2019107057A1 WO 2019107057 A1 WO2019107057 A1 WO 2019107057A1 JP 2018040632 W JP2018040632 W JP 2018040632W WO 2019107057 A1 WO2019107057 A1 WO 2019107057A1
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Prior art keywords
peptide
muscle
present
composition
seq
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PCT/JP2018/040632
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French (fr)
Japanese (ja)
Inventor
裕孝 富
三保子 河崎
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小林製薬株式会社
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Publication of WO2019107057A1 publication Critical patent/WO2019107057A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention relates to peptides and their use.
  • Muscle is one of the important tissues in the living body, and adult muscle mass is said to account for about 40% of body weight.
  • muscle mass tends to decrease due to various factors such as aging, lack of exercise, fixation of the body due to casts, etc.
  • the decrease in muscle mass causes a decrease in exercise capacity due to muscle atrophy and thus causes inconvenience in daily life.
  • muscle protein synthesis and degradation always occur, and enhanced muscle protein degradation leads to a decrease in muscle mass. Therefore, suppressing the degradation of muscle proteins is important in suppressing muscle atrophy.
  • An object of the present invention is to provide a peptide and the like capable of suppressing muscle atrophy.
  • the present inventors conducted intensive studies in view of the above problems, and found that a specific peptide can suppress muscle atrophy. As a result of further studies based on the findings, the present inventors have completed the present invention.
  • the present invention includes the following.
  • Item 1 A peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
  • Item 2. A composition comprising, as an active ingredient, a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
  • Item 3. A composition for suppressing muscle atrophy comprising, as an active ingredient, a peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
  • muscle atrophy can be suppressed.
  • muscle atrophy can be suppressed.
  • the muscle tissue maintenance rate in each group is shown.
  • the muscle tissue maintenance rate in each group is shown.
  • the expression rate of MuRF1 in each group is shown.
  • Peptide The present invention provides a peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
  • the peptide has an action of suppressing muscle atrophy.
  • the means for obtaining the peptide of the present invention is not limited, and can be prepared, for example, by conventionally known genetic engineering methods, chemical synthesis methods, and the like.
  • a polynucleotide encoding a peptide represented by any one of SEQ ID NOs: 1 to 13 is inserted into a vector or the like, and a transformant into which the vector or the like is incorporated is cultured It can be produced by obtaining a peptide.
  • the peptide of the present invention can be prepared by subjecting an object having an amino acid sequence encoding the peptide to an enzyme treatment or the like to obtain a desired fragment.
  • the peptide of the present invention can be synthesized and produced by a conventionally known chemical synthesis method according to the information of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13 and the nucleotide sequence encoding it.
  • Chemical synthesis methods include peptide synthesis methods by liquid phase method and solid phase method. Those skilled in the art can easily obtain and understand information such as the nucleotide sequence of a polynucleotide encoding a peptide of the present invention based on the sequence of the peptide of the present invention and the like.
  • the peptide of the present invention may be appropriately modified at its N-terminus, C-terminus, or internal as long as it has a muscle atrophy suppressive action, and may be in the form of a salt.
  • muscle atrophy can be suppressed.
  • Muscles can be classified roughly into skeletal muscle, smooth muscle and cardiac muscle.
  • skeletal muscle tends to decrease due to various factors such as aging, lack of exercise, fixation of body by cast, weightlessness in space, bed rest, inactivity at rest due to disease or injury, etc., malnutrition
  • the decrease causes a decrease in exercise capacity due to muscle atrophy, and in turn causes inconvenience in daily life.
  • muscle loss can be suppressed, that is, muscle atrophy can be suppressed.
  • suppression of muscle atrophy is evaluated by at least one of skeletal muscle weight, the expression level of Atrogin-1 in skeletal muscle, and the expression level of MuRF1 (muscle RING finger 1) in skeletal muscle.
  • Atrogin-1 and MuRF1 are genes associated with muscle atrophy, and it has been conventionally known that their expression levels increase in accelerated muscle protein degradation and muscle atrophy. Therefore, if at least one of suppression of decrease in skeletal muscle weight, suppression of expression of Atrogin-1 in skeletal muscle and suppression of expression of MuRF1 in skeletal muscle is observed using the peptide of the present invention, The peptide is determined to have a muscle atrophy inhibitory action, and the higher the degree of inhibition, the higher the muscle atrophy inhibitory action.
  • the skeletal muscle weight, the expression levels of Atrogin-1 and MuRF1, the degree of increase and decrease of these, and the like may be determined according to a conventionally known method in the present field, and can be determined with reference to, for example, the methods described in the following examples.
  • composition Containing Peptide The present invention provides a composition comprising, as an active ingredient, a peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
  • a peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13 (hereinafter sometimes referred to as the peptide of the present invention) is described in the same manner as described above.
  • composition of the present invention contains the peptide of the present invention as an active ingredient, and may contain the peptide of the present invention singly or in combination of two or more.
  • composition of the present invention comprises, in addition to the peptide of the present invention, an optionally pharmaceutically acceptable ingredient, It may further contain any component such as an edible component and a component usable for feed.
  • an excipient as an optional ingredient, an excipient, a binder, a disintegrant, a disintegration inhibitor, a lubricant, a stabilizer, an antiseptic, a pH adjuster, a filler, a solvent (water, Lower alcohols such as methanol, ethanol, isopropanol etc.
  • Alcohols such as polyhydric alcohols such as propylene glycol and 1,3-butylene glycol (anhydrous, water containing or not), hydrocarbons such as isoparaffin etc., diluent Flavor, coloring agent, flavoring agent, suspending agent, wetting agent, emulsifying agent, solubilizing agent, dispersing agent, buffer, bulking agent, thickening agent, surfactant, absorption promoter, adsorbent, coating agent, film Examples are forming agents, cooling agents, gelling agents, sweeteners, anti-inflammatory agents, various nutritional components and the like.
  • the optional components may be suitably determined in accordance with the form of the composition, the application subject, the route of application, the degree of symptoms, the degree of expected effects, etc.
  • One type may be used alone, or two or more types may be used. It may be used in combination, and the compounding amount may be determined appropriately.
  • composition of the present invention is not limited either, and may be appropriately set according to the application subject, application route and the like.
  • the composition of the present invention may be in the form of solution, emulsion, suspension, syrup, elixir, liquid form such as injection, powder, granule, fine granule, tablet (including troche, chewable etc.), round Examples are various forms such as semisolid or solid forms such as freeze-dried products of agents, capsules (including hard capsules and soft capsules), gels, creams, pastes, mousses, sheets, and liquid forms.
  • the composition of the present invention when it is in solid form, it may be used by mixing with a liquid such as water, and the composition of the present invention may be a sustained release dosage form .
  • the tablet may be, if necessary, a tablet having a conventional coating, such as sugar coated tablets, gelatin coated tablets, enteric coated tablets, film coated tablets, double tablets and multilayer tablets. it can.
  • the usage mode of the composition of the present invention is also not limited, and may be appropriately set according to the purpose.
  • Food compositions including beverages, functional health foods (including foods for specified health, nutritionally functional foods, functional indication foods, supplements, etc.), foods for sick persons) as usage modes of the composition of the present invention, Examples thereof include pharmaceutical compositions, feed compositions, food compositions, pharmaceutical compositions, additives to feeds and the like.
  • composition of the present invention may be produced according to a conventional procedure conventionally known in the form, mode of use, etc., and may be produced by mixing the peptide of the present invention and the above-mentioned optional components as required. .
  • the application target of the composition is also not limited, but human, non-human mammals and the like are exemplified.
  • the application amount of the composition of the present invention is not limited as long as the effects of the present invention can be obtained, and the physical size, age, symptoms, application route, application form, degree of symptoms, expected effects of subjects (target animals). It may be set appropriately according to the degree or the like.
  • the dose (intake) may be a daily dose (intake), based on an adult weighing 60 kg. It is exemplified that the peptide is preferably administered (ingested) to be 0.1 to 5000 mg, more preferably 0.5 to 5000 mg.
  • the composition of the present invention may be single dose (intake) or multiple dose (ingestion) per day.
  • the content of the peptide of the present invention is not limited as long as the effects of the present invention can be obtained, and the daily dose (intake) preferably falls within the above range. It may be set appropriately for reference.
  • the content of the peptide of the present invention to be incorporated into the composition of the present invention is preferably 0.001 to 99.9% by mass, more preferably 0.095 to 95%, as the content of the peptide of the present invention incorporated into the composition of the present invention. %, More preferably 0.1 to 95% by mass.
  • the decrease in muscle mass can be suppressed.
  • muscle atrophy can be suppressed.
  • the suppression of muscle atrophy is evaluated by at least one of skeletal muscle weight, the expression level of Atrogin-1 in skeletal muscle, and the expression level of MuRF1 in skeletal muscle, which is explained in the same manner as described above.
  • the present invention provides a composition for suppressing muscle atrophy, which comprises a peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13 as an active ingredient.
  • the peptide of the present invention described above and the composition of the present invention containing the peptide are aging, lack of exercise, fixation of the body by casting etc., weightlessness in space etc etc., bedridden, It is useful for suppressing muscle atrophy due to various factors such as inactivity at rest due to diseases or injuries, and malnutrition. Therefore, for the peptide of the present invention and a composition containing the peptide, for example, sarcopenia (primary or secondary), muscles, bones, joints, etc. involved in physical exercise are deteriorated, and it is necessary for bedriding or nursing care.
  • sarcopenia primary or secondary
  • the peptide of the present invention and a composition containing the peptide are, for example, for the purpose of maintaining the opportunity for employment of the senior generation, for the purpose of suppressing the aging of movement due to muscle loss due to aging and the like. It can be used preferably for the purpose of maintaining the enjoyment of a hobby requiring muscle strength such as tennis and dance, and also for the purpose of maintaining the muscle and muscle strength of a senior athlete.
  • the peptide of the present invention of the present invention and the composition containing the peptide are, for example, muscle weakness, aging, lack of exercise, inactivity due to diseases or injuries, etc., malnutrition, physical ability Decrease, activity in weightlessness, bedriding and needing care, muscle building, daily health maintenance, maintenance of youthfulness of movement, maintenance of fun of the required hobby of golf, tennis, dance etc. muscular strength, senior athletes It can be preferably used for a subject (target animal) concerned with muscles, muscle weakness and the like.
  • Test Example 1 Preparation of peptide A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 was synthesized. A peptide consisting of the amino acid sequence represented by each of SEQ ID NOs: 2 to 13 was similarly synthesized.
  • Test Example 2 Muscle atrophy suppression test 2-1. Test and evaluation procedure The muscle atrophy inhibitory effect of each peptide was evaluated according to the following procedure.
  • the mouse myoblast cell line C2C12 (manufactured by ATCC) is inoculated into Dulbecco's modified Eagle's medium (DMEM, manufactured by Wako) containing 10% FBS (fetal bovine serum) and then cultured in a carbon dioxide incubator (37 ° C.) Cultured. The resulting culture was seeded in a 24-well plate and cultured until it became semi-confluent. Then, the obtained culture solution was replaced with DMEM containing 2% horse serum, and cultured for 5 days to differentiate C2C12 cells into myotubes.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • DMEM medium without fetal calf serum
  • the culture medium was added such that the amount of peptide in each well was 0.01 mg / ml and 0.001 mg / ml.
  • cDNA was synthesized according to the procedure using PrimeScriptTM RT reagent Kit (manufactured by Takara Bio Inc.). Procedure using the obtained cDNA as a template and SYBR (registered trademark) Premix EX Taq (registered trademark) II (manufactured by Takara Bio Inc.) and Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by Takara Bio Inc.) According to, real-time PCR was performed to quantify the expression amount of Atrogin-1 gene. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control gene.
  • Atrogin-1 gene was analyzed using Thermal Cycler Dice (registered trademark) Real Time System III dedicated software (manufactured by Takara Bio Inc.). The analysis results are shown as relative values to the expression level of Atrogin-1 gene in the control group.
  • the Atrogin-1 gene is a gene specifically expressed in muscle tissue, and it is judged that the degradation of muscle protein is enhanced when the expression of the gene is high. As apparent from the results shown in Table 1, according to the peptide used in this test, the expression level of Atrogin-1 gene could be reduced. From this, it was found that the peptide used in this test is useful for suppressing the degradation of muscle proteins, particularly for suppressing the degradation of skeletal muscle proteins, that is, useful for suppressing muscle atrophy.
  • Test Example 3 Muscle atrophy suppression evaluation 3-1. Test procedure The muscle atrophy inhibitory effect of the peptide was evaluated according to the following procedure. In this test example, a peptide consisting of the amino acid sequences represented by SEQ ID NOS: 4 and 5 was used.
  • each rat was anesthetized by isoflurane inhalation anesthesia (2.5%). After that, the left leg was left untreated (normal foot), and the sciatic nerve in the right leg was excised (neurectomy leg). Then, each rat was continuously administered a test sample for 7 days, and was exsanguinated under inhalation deep anesthesia of isoflurane on the day after the end of administration, and was euthanized. Then, the muscles (legitimate muscle) of both sides (normal foot and nerve excision foot) were immediately separated and excised, the weight of each was measured, about 50 mg cut and immersed in RNA-Later (0.75 mL) and cryopreserved.
  • test group 1 As the sample administration, the peptide represented by SEQ ID NO: 4 in consideration of feed intake per rat (about 18 g / day), body weight (sample administration day), etc. on 2-3 days immediately before the test.
  • test sample 2 in which the peptide represented by SEQ ID NO: 5 was added to 1 ml of water was orally taken once a day to rats (peptide 6.90 mg / kg / day), and this was used as test group 2 did.
  • test samples 1 and 2 instead of the test samples 1 and 2, 1 ml of water was orally taken by the rat once a day, and this was used as a control group.
  • test samples 1 and 2 intended for people who want to maintain muscle in place of test samples 1 and 2 (leucine, lysine, valine, isoleucine, threonine, phenylalanine, methionine, citric acid, sucrose fatty acid ester, histidine, sweetener (Containing tryptophan, shellfish Ca, lecithin, flavoring agent, coloring agent, vitamin D / reduced maltose) according to the instruction of the commercial product, taking into account the equivalent of administration to rats, 1 The rats were orally ingested once a day (3000 mg / kg / day), and this was designated test group 3.
  • test samples 1 and 2 During the 7 days of administration of test samples 1 and 2, control, and commercial products, normal rat feed and water were used ad libitum except these were given.
  • the muscle tissue retention ratio (neural excise foot muscle weight ⁇ 100 / normal foot muscle weight) of the nerve excision foot muscle weight (right side) to the normal foot muscle weight (left side) of each group was determined. The calculation results are shown in FIG.
  • the amount of beta actin mRNA was quantified.
  • the primer used for real-time PCR used what was synthesize
  • Primer for MuRF1 aggactcctgccgagtgac (SEQ ID NO: 16, Forward primer) ttgtggctcagttcctcctttt (SEQ ID NO: 17, ReversePrimer) The results are shown in FIG.
  • FIG. 1 shows the muscle tissue retention rate of the nerve-resection foot muscle weight (right side) relative to the normal foot muscle weight (left side) of each group.
  • the muscle tissue maintenance rate was improved compared to the control group.
  • the test group 3 group given the commercially available product
  • FIG. 2 shows the muscle tissue maintenance rates in test groups 1 to 3 when the muscle tissue maintenance rate of the nerve tissue for foot muscle weight (right side) with respect to the normal foot muscle weight for the control group (left side) is 100%. Similar to FIG. 1, in FIG. 2 also, in the test groups 1 and 2, the muscle tissue maintenance rate was improved as compared with the control group. On the other hand, in the test group 3, the muscle tissue maintenance rate was lower than in the control group. This also shows that the peptide of the present invention was able to suppress the decrease in muscle weight.
  • FIG. 3 shows the expression rate of MuRF1.
  • the expression level of MuRF1 was reduced in test groups 1 and 2 as compared to the control group.
  • the expression level of the mRNA level of MuRF1 was increased compared to the control group.
  • the expression level of the MuRF1 gene is high, it is judged that the degradation of muscle protein is accelerated. From this, it was found that the peptide of the present invention is useful for suppressing the degradation of muscle protein.
  • the amount of the peptide of the present invention is, for example, preferably 0.1 to 5000 mg, more preferably 0.5 to 50 mg, as a daily dose (intake) based on an adult weighing 60 kg.
  • the administration (intake) of 5000 mg is exemplified.
  • the peptide represented by SEQ ID NO: 4 is, for example, more preferably 1 to 2000 mg, particularly preferably as a daily dose (intake) based on an adult of 60 kg body weight. Is administered (ingested) at 2 to 120 mg.
  • the peptide represented by SEQ ID NO: 5 is, for example, more preferably 1 to 3000 mg, particularly preferably, as a daily dose (intake) based on an adult of 60 kg body weight. Is administered (ingested) at 6 to 360 mg.
  • the administration (intake) amount is an example in the case of oral administration, and in parenteral administration, it may be appropriately determined with reference to the value.
  • a peptide consisting of an amino acid represented by any one of SEQ ID NOs: 1 to 13 and a composition containing the same are useful for inhibiting the degradation of muscle proteins, particularly for inhibiting the degradation of skeletal muscle proteins.
  • the peptide and a composition containing the same are useful for suppressing muscle atrophy, particularly for suppressing muscle atrophy of skeletal muscle.
  • the peptide and a composition containing the same are useful for sarcopenia, locomotive syndrome and the like, particularly muscle atrophy such as age-related muscle atrophy, disuse muscle atrophy and the like.

Abstract

[Problem] The purpose of the present invention is to provide a peptide, etc., that is capable of suppressing muscle atrophy. [Solution] A peptide comprising an amino acid sequence represented by any of SEQ ID NOS: 1-13. A composition having as an active ingredient a peptide comprising an amino acid sequence represented by any of SEQ ID NOS: 1-13. A composition for suppressing muscle atrophy having as an active ingredient a peptide comprising an amino acid sequence represented by any of SEQ ID NOS: 1-13.

Description

ペプチド及びその利用Peptide and use thereof
　本発明は、ペプチド及びその利用に関する。 The present invention relates to peptides and their use.
　筋肉は、生体において重要な組織の一つであり、成人の筋肉量は体重の約４０％を占めるといわれている。しかし、加齢、運動不足、ギプス等による身体の固定といった様々な要因により筋肉量は減少しやすく、筋肉量の減少は、筋萎縮による運動能力の低下、ひいては日常生活における不都合を引き起こす。 Muscle is one of the important tissues in the living body, and adult muscle mass is said to account for about 40% of body weight. However, muscle mass tends to decrease due to various factors such as aging, lack of exercise, fixation of the body due to casts, etc. The decrease in muscle mass causes a decrease in exercise capacity due to muscle atrophy and thus causes inconvenience in daily life.
　生体内では、常に、筋タンパク質の合成と分解が生じており、筋タンパク質分解の亢進は、筋肉量の減少につながる。このため、筋タンパク質の分解を抑制することは、筋萎縮を抑制する上で重要である。 In vivo, muscle protein synthesis and degradation always occur, and enhanced muscle protein degradation leads to a decrease in muscle mass. Therefore, suppressing the degradation of muscle proteins is important in suppressing muscle atrophy.
　これまでに、例えば、アキノワスレグサ（Hemerocallis fulva var. sempervirens）が筋萎縮を改善する作用を有することが報告されている（特許文献１）。 So far, it has been reported that, for example, Aquinogus rega (Hemerocallis fulva var. Sempervirens) has an action to improve muscle atrophy (Patent Document 1).
特開２０１５－１８９６８３号公報JP, 2015-189683, A
　本発明は、筋萎縮を抑制できるペプチド等を提供することを目的とする。 An object of the present invention is to provide a peptide and the like capable of suppressing muscle atrophy.
　本発明者らは、前記課題に鑑み鋭意検討を行ったところ、特定のペプチドが筋萎縮を抑制できることを見いだした。本発明者らは、該知見に基づき更に検討を重ねた結果、本発明を完成させるに至った。 The present inventors conducted intensive studies in view of the above problems, and found that a specific peptide can suppress muscle atrophy. As a result of further studies based on the findings, the present inventors have completed the present invention.
　本発明は、次に掲げるものを包含する。
項１．配列番号１～１３のいずれかで表されるアミノ酸配列からなるペプチド。
項２．配列番号１～１３のいずれかで表されるアミノ酸配列からなるペプチドを有効成分とする組成物。
項３．配列番号１～１３のいずれかで表されるアミノ酸配列からなるペプチドを有効成分とする筋萎縮抑制用組成物。 The present invention includes the following.
Item 1. A peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
Item 2. A composition comprising, as an active ingredient, a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
Item 3. A composition for suppressing muscle atrophy comprising, as an active ingredient, a peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
　本発明のペプチドによれば、筋萎縮を抑制することができる。また、本発明のペプチドを含有する組成物によれば、筋萎縮を抑制することができる。 According to the peptide of the present invention, muscle atrophy can be suppressed. Moreover, according to the composition containing the peptide of the present invention, muscle atrophy can be suppressed.
各群における筋組織維持率を示す。The muscle tissue maintenance rate in each group is shown. 各群における筋組織維持率を示す。The muscle tissue maintenance rate in each group is shown. 各群におけるMuRF1の発現率を示す。The expression rate of MuRF1 in each group is shown.
　以下、本発明について、より詳細に説明する。
１．ペプチド
　本発明は、配列番号１～１３のいずれかで表されるアミノ酸配列からなるペプチドを提供する。該ペプチドは、筋萎縮を抑制する作用を有する。 Hereinafter, the present invention will be described in more detail.
1. Peptide The present invention provides a peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13. The peptide has an action of suppressing muscle atrophy.
　本発明のペプチドの入手手段は制限されず、例えば、従来公知の遺伝子工学的手法や化学合成法等によって作製することができる。一例として、本発明のペプチドは、配列番号１～１３のいずれかで表されるペプチドをコードするポリヌクレオチドをベクター等に挿入し、該ベクター等が組み込まれた形質転換体を培養して所望のペプチドを取得することにより、作製することができる。また、本発明のペプチドは、該ペプチドをコードするアミノ酸配列を有する対象物を酵素処理等して、所望の断片を取得することにより作製することができる。また、本発明のペプチドは、配列番号１～１３のいずれかで表されるアミノ酸配列やこれをコードするヌクレオチド配列の情報に従って従来公知の化学合成法により合成して、作製することができる。化学合成法として、液相法や固相法によるペプチド合成法が包含される。当業者であれば、本発明のペプチドをコードするポリヌクレオチドの塩基配列等の情報も、本発明のペプチドの配列等に基づいて、容易に入手、理解することができる。 The means for obtaining the peptide of the present invention is not limited, and can be prepared, for example, by conventionally known genetic engineering methods, chemical synthesis methods, and the like. As an example, in the peptide of the present invention, a polynucleotide encoding a peptide represented by any one of SEQ ID NOs: 1 to 13 is inserted into a vector or the like, and a transformant into which the vector or the like is incorporated is cultured It can be produced by obtaining a peptide. In addition, the peptide of the present invention can be prepared by subjecting an object having an amino acid sequence encoding the peptide to an enzyme treatment or the like to obtain a desired fragment. In addition, the peptide of the present invention can be synthesized and produced by a conventionally known chemical synthesis method according to the information of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13 and the nucleotide sequence encoding it. Chemical synthesis methods include peptide synthesis methods by liquid phase method and solid phase method. Those skilled in the art can easily obtain and understand information such as the nucleotide sequence of a polynucleotide encoding a peptide of the present invention based on the sequence of the peptide of the present invention and the like.
　本発明のペプチドは、筋萎縮抑制作用を有する限り、そのＮ末端、Ｃ末端、内部において適宜修飾されていてもよく、また、塩の形態であってもよい。 The peptide of the present invention may be appropriately modified at its N-terminus, C-terminus, or internal as long as it has a muscle atrophy suppressive action, and may be in the form of a salt.
　本発明のペプチドによれば、筋萎縮を抑制することができる。 According to the peptide of the present invention, muscle atrophy can be suppressed.
　筋肉は、大きく分けると骨格筋、平滑筋、心筋に分類できる。例えば、骨格筋は、加齢、運動不足、ギプス等による身体の固定、宇宙での無重力状態、寝たきり、疾患や怪我等による安静時の不活動、低栄養といった様々な要因により減少する傾向にあり、その減少は、筋萎縮による運動能力の低下、ひいては日常生活における不都合を引き起こす。本発明のペプチドによれば、筋肉の減少を抑制することができ、すなわち、筋萎縮を抑制することができる。 Muscles can be classified roughly into skeletal muscle, smooth muscle and cardiac muscle. For example, skeletal muscle tends to decrease due to various factors such as aging, lack of exercise, fixation of body by cast, weightlessness in space, bed rest, inactivity at rest due to disease or injury, etc., malnutrition The decrease causes a decrease in exercise capacity due to muscle atrophy, and in turn causes inconvenience in daily life. According to the peptide of the present invention, muscle loss can be suppressed, that is, muscle atrophy can be suppressed.
　本発明において筋萎縮の抑制は、骨格筋重量、骨格筋中のAtrogin-1の発現量、骨格筋中のMuRF1(muscle RING finger 1)の発現量の少なくともいずれかにより評価される。Atrogin-1やMuRF1は、筋萎縮に関連する遺伝子であり、筋タンパク質の分解亢進や筋萎縮において、その発現量が増加することが従来知られている。このため、本発明のペプチドを用いて骨格筋重量の減少抑制、骨格筋中のAtrogin-1の発現量抑制、骨格筋中のMuRF1の発現量抑制の少なくともいずれかが認められれば、本発明のペプチドは筋萎縮抑制作用を有すると判断し、抑制の程度が高いほど筋萎縮抑制作用が高いと判断する。骨格筋重量、Atrogin-1やMuRF1の発現量、これらの増減の程度等は、本分野で従来公知の手法に従い決定すればよく、例えば後述の実施例に記載する手法を参考にして決定できる。 In the present invention, suppression of muscle atrophy is evaluated by at least one of skeletal muscle weight, the expression level of Atrogin-1 in skeletal muscle, and the expression level of MuRF1 (muscle RING finger 1) in skeletal muscle. Atrogin-1 and MuRF1 are genes associated with muscle atrophy, and it has been conventionally known that their expression levels increase in accelerated muscle protein degradation and muscle atrophy. Therefore, if at least one of suppression of decrease in skeletal muscle weight, suppression of expression of Atrogin-1 in skeletal muscle and suppression of expression of MuRF1 in skeletal muscle is observed using the peptide of the present invention, The peptide is determined to have a muscle atrophy inhibitory action, and the higher the degree of inhibition, the higher the muscle atrophy inhibitory action. The skeletal muscle weight, the expression levels of Atrogin-1 and MuRF1, the degree of increase and decrease of these, and the like may be determined according to a conventionally known method in the present field, and can be determined with reference to, for example, the methods described in the following examples.
２．ペプチドを含有する組成物
　本発明は、配列番号１～１３のいずれかで表されるアミノ酸配列からなるペプチドを有効成分とする組成物を提供する。 2. Composition Containing Peptide The present invention provides a composition comprising, as an active ingredient, a peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
　配列番号１～１３のいずれかで表されるアミノ酸配列からなるペプチド（以下、本発明のペプチドと称する場合がある）は、前述と同様に説明される。 A peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13 (hereinafter sometimes referred to as the peptide of the present invention) is described in the same manner as described above.
　本発明の組成物は、本発明のペプチドを有効成分として含有し、本発明のペプチドを１種単独で含有していてもよく、２種以上で含有していてもよい。 The composition of the present invention contains the peptide of the present invention as an active ingredient, and may contain the peptide of the present invention singly or in combination of two or more.
　本発明の組成物は、本発明のペプチドのほか、必要に応じて薬学的に許容可能な成分、
可食性の成分、飼料に使用可能な成分といった任意の成分を更に含有してもよい。　　 The composition of the present invention comprises, in addition to the peptide of the present invention, an optionally pharmaceutically acceptable ingredient,
It may further contain any component such as an edible component and a component usable for feed.
　本発明を制限するものではないが、任意の成分として、賦形剤、結合剤、崩壊剤、崩壊抑制剤、滑沢剤、安定剤、防腐剤、ｐＨ調整剤、充填剤、溶剤（水、メタノール、エタノール、イソプロパノール等の低級アルコール、プロピレングリコール、１，３－ブチレングリコール等の多価アルコール等のアルコール類（無水、含水の別を問わない）、イソパラフィン等の炭化水素類等）、希釈剤、香料、着色料、矯味剤、懸濁剤、湿潤剤、乳化剤、可溶化剤、分散剤、緩衝剤、増量剤、増粘剤、界面活性剤、吸収促進剤、吸着剤、コーティング剤、皮膜形成剤、清涼剤、ゲル化剤、甘味料、抗炎症剤、各種栄養成分等が例示される。 Although not limiting the present invention, as an optional ingredient, an excipient, a binder, a disintegrant, a disintegration inhibitor, a lubricant, a stabilizer, an antiseptic, a pH adjuster, a filler, a solvent (water, Lower alcohols such as methanol, ethanol, isopropanol etc. Alcohols such as polyhydric alcohols such as propylene glycol and 1,3-butylene glycol (anhydrous, water containing or not), hydrocarbons such as isoparaffin etc., diluent Flavor, coloring agent, flavoring agent, suspending agent, wetting agent, emulsifying agent, solubilizing agent, dispersing agent, buffer, bulking agent, thickening agent, surfactant, absorption promoter, adsorbent, coating agent, film Examples are forming agents, cooling agents, gelling agents, sweeteners, anti-inflammatory agents, various nutritional components and the like.
　任意の成分は、組成物の形態、適用対象、適用経路、症状の程度、期待される効果の程度等に応じて適宜決定すればよく、１種単独で使用してもよく、２種以上を組み合わせて使用してもよく、その配合量も適宜決定すればよい。 The optional components may be suitably determined in accordance with the form of the composition, the application subject, the route of application, the degree of symptoms, the degree of expected effects, etc. One type may be used alone, or two or more types may be used. It may be used in combination, and the compounding amount may be determined appropriately.
　本発明の組成物は、経口用、非経口用を問わず、その形態も制限されず、適用対象、適用経路等に応じて適宜設定すればよい。本発明の組成物の形態として、液剤、乳剤、懸濁剤、シロップ剤、エリキシル剤、注射剤等の液状形態、散剤、顆粒剤、細粒剤、錠剤（トローチ、チュアブル等を含む）、丸剤、カプセル剤（ハードカプセル、ソフトカプセルを含む）、ゲル状、クリーム状、ペースト状、ムース状、シート状、液状形態の凍結乾燥物等の半固形または固形形態等の各種形態が例示される。 The form of the composition of the present invention, whether for oral or parenteral use, is not limited either, and may be appropriately set according to the application subject, application route and the like. The composition of the present invention may be in the form of solution, emulsion, suspension, syrup, elixir, liquid form such as injection, powder, granule, fine granule, tablet (including troche, chewable etc.), round Examples are various forms such as semisolid or solid forms such as freeze-dried products of agents, capsules (including hard capsules and soft capsules), gels, creams, pastes, mousses, sheets, and liquid forms.
　また、例えば本発明の組成物が固形形態である場合、これは水等の液体と混合して使用してもよく、また、本発明の組成物は徐放性の剤形であってもよい。また、例えば錠剤は、必要に応じて、従来公知の剤皮を施した錠剤、例えば糖衣錠、ゼラチン被包錠、腸溶被錠、フィルムコーティング錠、また、二重錠、多層錠とすることができる。 Also, for example, when the composition of the present invention is in solid form, it may be used by mixing with a liquid such as water, and the composition of the present invention may be a sustained release dosage form . Also, for example, the tablet may be, if necessary, a tablet having a conventional coating, such as sugar coated tablets, gelatin coated tablets, enteric coated tablets, film coated tablets, double tablets and multilayer tablets. it can.
　本発明の組成物の使用態様も制限されず、目的に応じて適宜設定すればよい。本発明の組成物の使用態様として、食品組成物（飲料を含む、保健機能食品（特定保健用食品、栄養機能食品、機能性表示食品、サプリメント等を含む）、病者用食品を含む）、医薬組成物、飼料組成物、また、食品組成物、医薬組成物、飼料等への添加剤等が例示される。　 The usage mode of the composition of the present invention is also not limited, and may be appropriately set according to the purpose. Food compositions (including beverages, functional health foods (including foods for specified health, nutritionally functional foods, functional indication foods, supplements, etc.), foods for sick persons) as usage modes of the composition of the present invention, Examples thereof include pharmaceutical compositions, feed compositions, food compositions, pharmaceutical compositions, additives to feeds and the like.
　本発明の組成物は、前記形態、使用態様等における従来公知の通常の手順に従い製造すればよく、本発明のペプチドと、必要に応じて前記任意の成分とを混合等して製造すればよい。 The composition of the present invention may be produced according to a conventional procedure conventionally known in the form, mode of use, etc., and may be produced by mixing the peptide of the present invention and the above-mentioned optional components as required. .
　本発明において、組成物の適用対象も制限されないが、ヒト、ヒト以外の哺乳動物等が例示される。 In the present invention, the application target of the composition is also not limited, but human, non-human mammals and the like are exemplified.
　本発明の組成物の適用量は、本発明の効果が得られる限り制限されず、対象者（対象動物）の体格、年齢、症状、適用経路、適用形態、症状の程度、期待される効果の程度等に応じて適宜設定すればよい。本発明を制限するものではないが、本発明の組成物が経口組成物である場合、投与（摂取）量は、１日投与（摂取）量として、体重６０ｋｇの成人を基準として、本発明のペプチドを、好ましくは０．１～５０００ｍｇ、より好ましくは０．５～５０００ｍｇとなるように投与（摂取）することが例示される。本発明の組成物は、１日あたり単回投与（摂取）であってもよく複数回投与（摂取）であってもよい。 The application amount of the composition of the present invention is not limited as long as the effects of the present invention can be obtained, and the physical size, age, symptoms, application route, application form, degree of symptoms, expected effects of subjects (target animals). It may be set appropriately according to the degree or the like. Without limiting the present invention, when the composition of the present invention is an oral composition, the dose (intake) may be a daily dose (intake), based on an adult weighing 60 kg. It is exemplified that the peptide is preferably administered (ingested) to be 0.1 to 5000 mg, more preferably 0.5 to 5000 mg. The composition of the present invention may be single dose (intake) or multiple dose (ingestion) per day.
　本発明の組成物中、本発明のペプチドの含有量は、本発明の効果が得られる限り制限されず、１日投与（摂取）量が好ましくは前記範囲となるように、また、前記範囲を参考にして、適宜設定すればよい。この限りにおいて制限されないが、本発明の組成物に配合される本発明のペプチドの含有量として、組成物中、好ましくは０．００１～９９．９質量％、より好ましくは０．０９５～９５質量％、更に好ましくは０．１～９５質量％が例示される。 In the composition of the present invention, the content of the peptide of the present invention is not limited as long as the effects of the present invention can be obtained, and the daily dose (intake) preferably falls within the above range. It may be set appropriately for reference. The content of the peptide of the present invention to be incorporated into the composition of the present invention is preferably 0.001 to 99.9% by mass, more preferably 0.095 to 95%, as the content of the peptide of the present invention incorporated into the composition of the present invention. %, More preferably 0.1 to 95% by mass.
　本発明の組成物によれば、筋肉量の減少を抑制することができる。このため、本発明の組成物によれば、筋萎縮を抑制することができる。本発明において筋萎縮の抑制は、骨格筋重量、骨格筋中のAtrogin-1の発現量、骨格筋中のMuRF1の発現量の少なくともいずれかにより評価され、これは前述と同様に説明される。 According to the composition of the present invention, the decrease in muscle mass can be suppressed. For this reason, according to the composition of the present invention, muscle atrophy can be suppressed. In the present invention, the suppression of muscle atrophy is evaluated by at least one of skeletal muscle weight, the expression level of Atrogin-1 in skeletal muscle, and the expression level of MuRF1 in skeletal muscle, which is explained in the same manner as described above.
　このことから、本発明は、配列番号１～１３のいずれかで表されるアミノ酸配列からなるペプチドを有効成分とする筋萎縮抑制用組成物を提供するともいえる。 From this, it can be said that the present invention provides a composition for suppressing muscle atrophy, which comprises a peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13 as an active ingredient.
　また、これらのことから、前述の本発明のペプチド、また、該ペプチドを含有する本発明の組成物は、加齢、運動不足、ギプス等による身体の固定、宇宙等での無重力状態、寝たきり、疾患や怪我等による安静時の不活動、低栄養といった様々な要因による筋萎縮の抑制に有用である。従って、本発明のペプチド、また、該ペプチドを含有する組成物は、例えば、サルコペニア（一次性、二次性）、身体運動にかかわる筋肉、骨、関節等の機能が衰えて寝たきりや要介護になったりその危険が高いロコモティブシンドローム（運動器症候群）等の抑制を目的として好ましく使用でき、特に、加齢性筋萎縮、廃用性筋萎縮等の抑制を目的として好ましく使用できる。また、本発明のペプチド、また、該ペプチドを含有する組成物は、例えば、加齢等による筋肉減少による動きの老化の抑制を目的として、シニア世代の就業の機会の維持を目的として、ゴルフ、テニス、ダンス等の筋力が必要な趣味の楽しみの維持を目的として、また、シニアアスリートの筋肉や筋力の維持を目的として好ましく使用できる。 Further, from these facts, the peptide of the present invention described above and the composition of the present invention containing the peptide are aging, lack of exercise, fixation of the body by casting etc., weightlessness in space etc etc., bedridden, It is useful for suppressing muscle atrophy due to various factors such as inactivity at rest due to diseases or injuries, and malnutrition. Therefore, for the peptide of the present invention and a composition containing the peptide, for example, sarcopenia (primary or secondary), muscles, bones, joints, etc. involved in physical exercise are deteriorated, and it is necessary for bedriding or nursing care. It can be preferably used for the purpose of suppressing locomotive syndrome (motor organ syndrome) or the like, which is at high risk, and particularly for the purpose of suppressing age-related muscle atrophy, disuse muscle atrophy, and the like. In addition, the peptide of the present invention and a composition containing the peptide are, for example, for the purpose of maintaining the opportunity for employment of the senior generation, for the purpose of suppressing the aging of movement due to muscle loss due to aging and the like. It can be used preferably for the purpose of maintaining the enjoyment of a hobby requiring muscle strength such as tennis and dance, and also for the purpose of maintaining the muscle and muscle strength of a senior athlete.
　また、このことから、本発明の本発明のペプチド、また、該ペプチドを含有する組成物は、例えば、筋肉の衰え、加齢、運動不足、疾患や怪我等による不活動、低栄養、身体能力の低下、無重力状態での活動、寝たきりや要介護、筋肉づくり、日々の健康維持、動きの若々しさの維持、ゴルフ、テニス、ダンス等の筋力の必要な趣味の楽しみの維持、シニアアスリートの筋肉、筋力低下等が気になる対象者（対象動物）に好ましく使用できる。 In addition, from this, the peptide of the present invention of the present invention and the composition containing the peptide are, for example, muscle weakness, aging, lack of exercise, inactivity due to diseases or injuries, etc., malnutrition, physical ability Decrease, activity in weightlessness, bedriding and needing care, muscle building, daily health maintenance, maintenance of youthfulness of movement, maintenance of fun of the required hobby of golf, tennis, dance etc. muscular strength, senior athletes It can be preferably used for a subject (target animal) concerned with muscles, muscle weakness and the like.
　以下、実施例を挙げて本発明を説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.
試験例１．ペプチドの作製
　配列番号１で表されるアミノ酸配列からなるペプチドを合成した。配列番号２～１３のそれぞれで表されるアミノ酸配列からなるペプチドも同様に合成した。 Test Example 1 Preparation of peptide A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 was synthesized. A peptide consisting of the amino acid sequence represented by each of SEQ ID NOs: 2 to 13 was similarly synthesized.
試験例２．筋萎縮抑制試験
２－１．試験及び評価手順
　次の手順に従い、各ペプチドの筋萎縮抑制効果を評価した。 Test Example 2 Muscle atrophy suppression test
2-1. Test and evaluation procedure The muscle atrophy inhibitory effect of each peptide was evaluated according to the following procedure.
　マウス筋芽細胞株C2C12（ATCC社製）を、10% FBS（ウシ胎児血清；fetal bovine serum）含有ダルベッコ改変イーグル培地（DMEM、Wako社製）に接種し、炭酸ガスインキュベーター内（37℃）で培養した。得られた培養液を24ウェルプレートに播種し、セミコンフルエントになるまで培養した。次いで、得られた培養液を、2%ウマ血清含有DMEMに交換して、5日間培養し、C2C12細胞を筋管細胞へ分化させた。 The mouse myoblast cell line C2C12 (manufactured by ATCC) is inoculated into Dulbecco's modified Eagle's medium (DMEM, manufactured by Wako) containing 10% FBS (fetal bovine serum) and then cultured in a carbon dioxide incubator (37 ° C.) Cultured. The resulting culture was seeded in a 24-well plate and cultured until it became semi-confluent. Then, the obtained culture solution was replaced with DMEM containing 2% horse serum, and cultured for 5 days to differentiate C2C12 cells into myotubes.
　筋管細胞の形成を確認後、前記ペプチドを添加したDMEM培地（ウシ胎児血清不含有）を筋管細胞に添加した。この際、各ウェルのペプチド量を0.01mg/ml、0.001mg/mlとなるように、培地を添加した。DMEM培地（前記ペプチド不含有、ウシ胎児血清不含有）を筋管細胞に添加したものを、コントロールとした。試験は各群n＝3で実施した。 After confirming the formation of myotubes, DMEM medium (without fetal calf serum) containing the above peptide was added to myotubes. At this time, the culture medium was added such that the amount of peptide in each well was 0.01 mg / ml and 0.001 mg / ml. A control was performed by adding DMEM medium (the above-mentioned peptide-free and fetal bovine serum-free) to myotubes. The test was performed in each group n = 3.
　各ペプチドと筋管細胞とを、炭酸ガスインキュベーター内（37℃）で6時間反応させたのち、ISOGEN II（株式会社ニッポンジーン製）を用いてtotal RNAを回収した。 Each peptide was reacted with myotube cells in a carbon dioxide gas incubator (37 ° C.) for 6 hours, and then total RNA was recovered using ISOGEN II (manufactured by Nippon Gene Co., Ltd.).
　回収したRNAを鋳型とし、PrimeScript（商標）RT reagent Kit（タカラバイオ株式会社製）を用いて手順に従い、cDNAを合成した。得られたcDNAを鋳型とし、SYBR（登録商標）Premix EX Taq（商標）II（タカラバイオ株式会社製）及びThermal Cycler Dice（登録商標）Real Time System III（タカラバイオ株式会社製）を用いて手順に従い、リアルタイムPCRを行い、Atrogin-1遺伝子の発現量を定量した。内部標準遺伝子にはGAPDH（glyceraldehyde-3-phosphate dehydrogenase）を用いた。また、Atrogin-1遺伝子の発現量を、Thermal Cycler Dice（登録商標） Real Time System III専用ソフトウェア（タカラバイオ株式会社製）を用いて解析した。解析結果を、コントロール群のAtrogin-1遺伝子の発現量に対する相対値で示した。 Using the collected RNA as a template, cDNA was synthesized according to the procedure using PrimeScriptTM RT reagent Kit (manufactured by Takara Bio Inc.). Procedure using the obtained cDNA as a template and SYBR (registered trademark) Premix EX Taq (registered trademark) II (manufactured by Takara Bio Inc.) and Thermal Cycler Dice (registered trademark) Real Time System III (manufactured by Takara Bio Inc.) According to, real-time PCR was performed to quantify the expression amount of Atrogin-1 gene. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control gene. In addition, the expression level of Atrogin-1 gene was analyzed using Thermal Cycler Dice (registered trademark) Real Time System III dedicated software (manufactured by Takara Bio Inc.). The analysis results are shown as relative values to the expression level of Atrogin-1 gene in the control group.
２－２．結果
　結果を表１に示す。 2-2. Results The results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表中、例えば「２０％」は、コントロール群でのAtrogin-1遺伝子の発現量を１とした場合、ペプチド添加群でのAtrogin-1遺伝子の発現量が０．８であり、ペプチドの添加によって、Atrogin-1遺伝子の発現量が２０％低減されたことを意味する。 In the table, for example, “20%” indicates that when the expression amount of Atrogin-1 gene in the control group is 1, the expression amount of Atrogin-1 gene in the peptide addition group is 0.8, and the addition of the peptide , Means that the expression level of Atrogin-1 gene was reduced by 20%.
　表１から明らかなように、前述のペプチドを添加した群ではいずれも、コントロール群より、Atrogin-1遺伝子の発現量が減少した。 As apparent from Table 1, in all the groups to which the above-mentioned peptides were added, the expression level of the Atrogin-1 gene was reduced compared to the control group.
　Atrogin-1遺伝子は、筋組織で特異的に発現する遺伝子であり、該遺伝子の発現が高いと筋タンパク質の分解が亢進していると判断される。表１に示す結果から明らかなように、本試験で使用したペプチドによれば、Atrogin-1遺伝子の発現量を低減することができた。このことから、本試験で使用したペプチドは、筋タンパク質の分解抑制、特に骨格筋タンパク質の分解抑制に有用であり、すなわち筋萎縮抑制に有用であることが分かった。 The Atrogin-1 gene is a gene specifically expressed in muscle tissue, and it is judged that the degradation of muscle protein is enhanced when the expression of the gene is high. As apparent from the results shown in Table 1, according to the peptide used in this test, the expression level of Atrogin-1 gene could be reduced. From this, it was found that the peptide used in this test is useful for suppressing the degradation of muscle proteins, particularly for suppressing the degradation of skeletal muscle proteins, that is, useful for suppressing muscle atrophy.
試験例３．筋萎縮抑制評価
３－１．試験手順
　次の手順に従い、ペプチドの筋萎縮抑制効果を評価した。本試験例では、配列番号４、５で表されるアミノ酸配列からなるペプチドを用いた。 Test Example 3 Muscle atrophy suppression evaluation
3-1. Test procedure The muscle atrophy inhibitory effect of the peptide was evaluated according to the following procedure. In this test example, a peptide consisting of the amino acid sequences represented by SEQ ID NOS: 4 and 5 was used.
　被験動物としてSD系ラット（雄、6週齢、日本エスエルシー株式会社より入手）を用いた。ラット1匹／飼育ケージ（136mm W×208mm D×115mm H）、室温約23℃、照明時間12時間／日（7～19時）、通常のラット用飼料及び水は自由摂取にて、ラットを個別飼育した。各ラットの体重を測定し、体重をもとに乱塊法で群分けした。試験は各群n＝６で実施した。 As test animals, SD rats (male, 6 weeks old, obtained from Japan SLC Co., Ltd.) were used. 1 rat / rearing cage (136 mm W x 208 mm D x 115 mm H), room temperature about 23 ° C, lighting time 12 hours / day (7 to 19 o'clock), normal rat food and water are freely available I reared individually. The weight of each rat was measured and divided into groups based on the weight based on the lumped mass method. The test was performed in each group n = 6.
　7日間の馴化飼育後、イソフルラン吸入麻酔（2.5%）により各ラットに麻酔を施した。
その後、左側下肢を非処置（正常足）とし、右側下肢の坐骨神経を切除した（神経切除足）。次いで、各ラットに被験試料を7日間連続投与し、投与終了日の翌日にイソフルランの吸入深麻酔下に放血し、安楽死させた。その後、直ちに両側（正常足及び神経切除足）の筋肉（ヒラメ筋）を分離し摘出し、それぞれの重量を測定し、約50mg切り分け、RNA-Later （0.75mL）に浸漬後、凍結保存した。 After 7 days of acclimatization, each rat was anesthetized by isoflurane inhalation anesthesia (2.5%).
After that, the left leg was left untreated (normal foot), and the sciatic nerve in the right leg was excised (neurectomy leg). Then, each rat was continuously administered a test sample for 7 days, and was exsanguinated under inhalation deep anesthesia of isoflurane on the day after the end of administration, and was euthanized. Then, the muscles (legitimate muscle) of both sides (normal foot and nerve excision foot) were immediately separated and excised, the weight of each was measured, about 50 mg cut and immersed in RNA-Later (0.75 mL) and cryopreserved.
　なお、試料投与として、試験直前2～3日のラット1匹あたりの飼料摂取量（約18ｇ／1日）、体重（試料投与日）等を考慮して、配列番号４で表されるペプチドを水1mlに添加した分散液（被験試料１）を1日1回ラットに経口摂取させた（ペプチド量2.22mg／kg／day）。これを試験群１とした。 As the sample administration, the peptide represented by SEQ ID NO: 4 in consideration of feed intake per rat (about 18 g / day), body weight (sample administration day), etc. on 2-3 days immediately before the test. The dispersion (Test sample 1) added to 1 ml of water was orally taken to rats once a day (peptide amount: 2.22 mg / kg / day). This was designated test group 1.
　同様に、配列番号５で表されるペプチドを水1mlに添加した分散液（被験試料２）を1日1回ラットに経口摂取させ（ペプチド6.90mg／kg／day）、これを試験群２とした。 Similarly, a dispersion (test sample 2) in which the peptide represented by SEQ ID NO: 5 was added to 1 ml of water was orally taken once a day to rats (peptide 6.90 mg / kg / day), and this was used as test group 2 did.
　被験試料１、２に代えて、水1mlを1日1回ラットに経口摂取させ、これをコントロール群とした。 Instead of the test samples 1 and 2, 1 ml of water was orally taken by the rat once a day, and this was used as a control group.
　また、被験試料１、２に代えて、筋肉の維持を求める人を対象とする市販品（ロイシン、リジン、バリン、イソロイシン、スレオニン、フェニルアラニン、メチオニン、クエン酸、ショ糖脂肪酸エステル、ヒスチジン、甘味料、トリプトファン、貝Ca、レシチン、香料、着色料、ビタミンD／還元麦芽糖を含有）を、該市販品の説明書に従って、ラットへの投与換算を考慮して、水1mlに添加した分散液を1日1回ラットに経口摂取させ（3000 mg／kg／day）、これを試験群３とした。 In addition, commercial products intended for people who want to maintain muscle in place of test samples 1 and 2 (leucine, lysine, valine, isoleucine, threonine, phenylalanine, methionine, citric acid, sucrose fatty acid ester, histidine, sweetener (Containing tryptophan, shellfish Ca, lecithin, flavoring agent, coloring agent, vitamin D / reduced maltose) according to the instruction of the commercial product, taking into account the equivalent of administration to rats, 1 The rats were orally ingested once a day (3000 mg / kg / day), and this was designated test group 3.
　被験試料１、２、コントロール、市販品の投与7日間中、これらを与える以外は、通常のラット用飼料及び水を自由摂取とした。 During the 7 days of administration of test samples 1 and 2, control, and commercial products, normal rat feed and water were used ad libitum except these were given.
３－２．評価手順
　前述の通り、筋肉の重量を測定した。 3-2. Evaluation Procedure Muscle weights were measured as described above.
　測定した重量に基づいて、各群の正常足筋重量（左側）に対する神経切除足筋重量（右側）の筋組織維持率（神経切除足筋重量×100／正常足筋重量）を求めた。算出結果を図１に示す。 Based on the measured weights, the muscle tissue retention ratio (neural excise foot muscle weight × 100 / normal foot muscle weight) of the nerve excision foot muscle weight (right side) to the normal foot muscle weight (left side) of each group was determined. The calculation results are shown in FIG.
　また、測定した重量に基づいて、コントロール群の正常足筋重量（左側）に対する神経切除足筋重量（右側）の筋組織維持率を100%とした時の、試験群１～３における筋組織維持率を求めた。算出結果を図２に示す。 In addition, based on the measured weight, the muscle tissue maintenance in the test groups 1 to 3 when the muscle tissue maintenance rate of the nerve resection foot muscle weight (right side) to the normal foot muscle weight (left side) of the control group is 100%. The rate was determined. The calculation results are shown in FIG.
　また、前述の通りRNA-Later （RNA Stabilization Reagent（Qiagen社製））にて保存した筋肉から、Pure Link（商標）RNA mini kit（invitrogen社製）を用いてtotal RNAを抽出した。抽出したRNAは、SuperScript（商標）VILO（商標）MasterMix（invitrogen社製）を用いてcDNAに逆転写し、リアルタイムPCR法に従いMuRF1（muscle RING finger 1）のmRNA量を定量した。内部標準として、ベータアクチンのmRNA量を定量した。なお、リアルタイムPCRに用いたプライマーはSIGMA-ALDLICH(http://www.genosys.jp/news/news_ssl.html)で合成したものを使用した。具体的には、次の通りである。
ベータアクチン(Actb)用プライマー：cccgcgagtacaaccttct（配列番号１４、Forward primer）、cgtcatccatggcgaact（配列番号１５、ReversePrimer）
MuRF1用プライマー：aggactcctgccgagtgac（配列番号１６、Forward primer）ttgtggctcagttcctcctt（配列番号１７、ReversePrimer）
　結果を図３に示す。 In addition, total RNA was extracted from the muscle preserved with RNA-Later (RNA Stabilization Reagent (manufactured by Qiagen)) as described above, using Pure LinkTM RNA mini kit (manufactured by invitrogen). The extracted RNA was reversely transcribed to cDNA using SuperScript (trademark) VILO (trademark) MasterMix (manufactured by Invitrogen), and the mRNA amount of MuRF1 (muscle RING finger 1) was quantified according to a real-time PCR method. As an internal standard, the amount of beta actin mRNA was quantified. In addition, the primer used for real-time PCR used what was synthesize | combined by SIGMA-ALDICH (http://www.genosys.jp/news/news_ssl.html). Specifically, it is as follows.
Primer for beta actin (Actb): cccgcgagtacaaccttct (SEQ ID NO: 14, Forward primer), cgtcatccatggcgaact (SEQ ID NO: 15, ReversePrimer)
Primer for MuRF1: aggactcctgccgagtgac (SEQ ID NO: 16, Forward primer) ttgtggctcagttcctcctt (SEQ ID NO: 17, ReversePrimer)
The results are shown in FIG.
３－３．結果
　前述の通り、図１には、各群の正常足筋重量（左側）に対する神経切除足筋重量（右側）の筋組織維持率を示す。図１に示す通り、コントロール群と比較して、試験群１及び２（本発明のペプチドを含有する被験試料１、２を与えた群）では筋組織維持率が向上した。一方、試験群３（市販品を与えた群）ではコントロール群と同等であり、筋組織維持率の向上は認められなかった。このことから、本発明のペプチドによれば、筋重量の減少を抑制できたことが分かった。 3-3. Results As described above, FIG. 1 shows the muscle tissue retention rate of the nerve-resection foot muscle weight (right side) relative to the normal foot muscle weight (left side) of each group. As shown in FIG. 1, in the test groups 1 and 2 (groups receiving test samples 1 and 2 containing the peptide of the present invention), the muscle tissue maintenance rate was improved compared to the control group. On the other hand, in the test group 3 (group given the commercially available product), it was equal to the control group, and no improvement in muscle tissue maintenance rate was observed. From this, it was found that the peptide of the present invention was able to suppress the decrease in muscle weight.
　図２には、コントロール群の正常足筋重量（左側）に対する神経切除足筋重量（右側）の筋組織維持率を100%とした時の、試験群１～３における筋組織維持率を示す。図１と同様に、図２においても、コントロール群と比較して、試験群１及び２は筋組織維持率が向上した。一方、試験群３ではコントロール群よりも筋組織維持率が低くなった。このことからも、本発明のペプチドによれば、筋重量の減少を抑制できたことが分かった。 FIG. 2 shows the muscle tissue maintenance rates in test groups 1 to 3 when the muscle tissue maintenance rate of the nerve tissue for foot muscle weight (right side) with respect to the normal foot muscle weight for the control group (left side) is 100%. Similar to FIG. 1, in FIG. 2 also, in the test groups 1 and 2, the muscle tissue maintenance rate was improved as compared with the control group. On the other hand, in the test group 3, the muscle tissue maintenance rate was lower than in the control group. This also shows that the peptide of the present invention was able to suppress the decrease in muscle weight.
　図３には、MuRF1の発現率を示す。図３から理解できる通り、コントロール群と比較して、試験群１及び２では、MuRF1の発現量が低下した。これに対して、試験群３ではコントロール群よりもMuRF1のmRNA量の発現量が増加した。MuRF1遺伝子の発現量が多いと筋タンパク質の分解が亢進していると判断される。このことから、本発明のペプチドは、筋タンパク質の分解抑制に有用であることが分かった。 FIG. 3 shows the expression rate of MuRF1. As can be understood from FIG. 3, the expression level of MuRF1 was reduced in test groups 1 and 2 as compared to the control group. On the other hand, in the test group 3, the expression level of the mRNA level of MuRF1 was increased compared to the control group. When the expression level of the MuRF1 gene is high, it is judged that the degradation of muscle protein is accelerated. From this, it was found that the peptide of the present invention is useful for suppressing the degradation of muscle protein.
　なお、本試験の結果に基づけば、体重６０ｋｇの成人を基準とした１日投与（摂取）量として、本発明のペプチドを、例えば、好ましくは０．１～５０００ｍｇ、より好ましくは０．５～５０００ｍｇを投与（摂取）することが例示される。更に、本発明を制限するものではないが、体重６０ｋｇの成人を基準とした１日投与（摂取）量として、配列番号４で表されるペプチドは、例えば、更に好ましくは１～２０００ｍｇ、特に好ましくは２～１２０ｍｇを投与（摂取）することが例示される。また、本発明を制限するものではないが、体重６０ｋｇの成人を基準とした１日投与（摂取）量として、配列番号５で表されるペプチドは、例えば、更に好ましくは１～３０００ｍｇ、特に好ましくは６～３６０ｍｇを投与（摂取）することが例示される。該投与（摂取）量は経口投与の場合の一例であり、非経口投与においては、該値を参考にして適宜決定すればよい。 In addition, based on the results of this test, the amount of the peptide of the present invention is, for example, preferably 0.1 to 5000 mg, more preferably 0.5 to 50 mg, as a daily dose (intake) based on an adult weighing 60 kg. The administration (intake) of 5000 mg is exemplified. Furthermore, although the present invention is not limited thereto, the peptide represented by SEQ ID NO: 4 is, for example, more preferably 1 to 2000 mg, particularly preferably as a daily dose (intake) based on an adult of 60 kg body weight. Is administered (ingested) at 2 to 120 mg. Although the present invention is not limited, the peptide represented by SEQ ID NO: 5 is, for example, more preferably 1 to 3000 mg, particularly preferably, as a daily dose (intake) based on an adult of 60 kg body weight. Is administered (ingested) at 6 to 360 mg. The administration (intake) amount is an example in the case of oral administration, and in parenteral administration, it may be appropriately determined with reference to the value.
　これらの結果から、配列番号１～１３のいずれかで表されるアミノ酸からなるペプチド、これを含有する組成物は、筋タンパク質の分解抑制、特に骨格筋タンパク質の分解抑制に有用であることが分かった。また、該ペプチド、これを含有する組成物は、筋萎縮抑制、特に骨格筋の筋萎縮抑制に有用であることが分かった。また、該ペプチド、これを含有する組成物は、サルコペニア、ロコモティブシンドローム等、特に、加齢性筋萎縮、廃用性筋萎縮等といった筋萎縮に有用であることが分かった。 From these results, it has been found that a peptide consisting of an amino acid represented by any one of SEQ ID NOs: 1 to 13 and a composition containing the same are useful for inhibiting the degradation of muscle proteins, particularly for inhibiting the degradation of skeletal muscle proteins The In addition, it has been found that the peptide and a composition containing the same are useful for suppressing muscle atrophy, particularly for suppressing muscle atrophy of skeletal muscle. In addition, it has been found that the peptide and a composition containing the same are useful for sarcopenia, locomotive syndrome and the like, particularly muscle atrophy such as age-related muscle atrophy, disuse muscle atrophy and the like.

Claims (3)

  1. 配列番号１～１３のいずれかで表されるアミノ酸配列からなるペプチド。 A peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
  2. 配列番号１～１３のいずれかで表されるアミノ酸配列からなるペプチドを有効成分とする組成物。 A composition comprising, as an active ingredient, a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
  3. 配列番号１～１３のいずれかで表されるアミノ酸配列からなるペプチドを有効成分とする筋萎縮抑制用組成物。 A composition for suppressing muscle atrophy comprising, as an active ingredient, a peptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004277329A (en) * 2003-03-14 2004-10-07 Nipro Corp Porphyrin compound, its albumin inclusion compound and artificial oxygen carrier
WO2009145363A1 (en) * 2008-05-30 2009-12-03 ソムノクエスト株式会社 Composition comprising a hot water extract of hemerocallis plants, said composition being effective in treating fatigue by having antidepressant properties or by improving sleep
WO2011106717A1 (en) * 2010-02-26 2011-09-01 University Of Florida Research Foundation, Inc. Mitochondrial-targeted antioxidants protect against mechanical ventilation-induced diaphragm dysfunction and skeletal muscle atrophy
JP2015189683A (en) * 2014-03-27 2015-11-02 小林製薬株式会社 Muscle mass increasing agent and muscle atrophy improver comprising hemerocallis fulva var
WO2017001515A1 (en) * 2015-06-30 2017-01-05 Firmenich Sa Marine peptides and muscle health

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004277329A (en) * 2003-03-14 2004-10-07 Nipro Corp Porphyrin compound, its albumin inclusion compound and artificial oxygen carrier
WO2009145363A1 (en) * 2008-05-30 2009-12-03 ソムノクエスト株式会社 Composition comprising a hot water extract of hemerocallis plants, said composition being effective in treating fatigue by having antidepressant properties or by improving sleep
WO2011106717A1 (en) * 2010-02-26 2011-09-01 University Of Florida Research Foundation, Inc. Mitochondrial-targeted antioxidants protect against mechanical ventilation-induced diaphragm dysfunction and skeletal muscle atrophy
JP2015189683A (en) * 2014-03-27 2015-11-02 小林製薬株式会社 Muscle mass increasing agent and muscle atrophy improver comprising hemerocallis fulva var
WO2017001515A1 (en) * 2015-06-30 2017-01-05 Firmenich Sa Marine peptides and muscle health

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YAMADA SHIGERU ET AL.: "Effects of soybean peptide AM intake on muscle weakness in the Elderly", SOY PROTEIN RESEARCH, JAPAN, vol. 16, no. 34, 2013, pages 112 - 118 *

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