WO2017150548A1 - Peptide - Google Patents

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Publication number
WO2017150548A1
WO2017150548A1 PCT/JP2017/007870 JP2017007870W WO2017150548A1 WO 2017150548 A1 WO2017150548 A1 WO 2017150548A1 JP 2017007870 W JP2017007870 W JP 2017007870W WO 2017150548 A1 WO2017150548 A1 WO 2017150548A1
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WIPO (PCT)
Prior art keywords
peptide
ghrelin
secretion
promoting
food
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PCT/JP2017/007870
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French (fr)
Japanese (ja)
Inventor
耕作 大日向
絢也 中戸
隼人 青木
浩 岩倉
Original Assignee
国立大学法人京都大学
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Application filed by 国立大学法人京都大学 filed Critical 国立大学法人京都大学
Priority to JP2018503338A priority Critical patent/JP6957033B2/en
Publication of WO2017150548A1 publication Critical patent/WO2017150548A1/en

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    • 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
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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
    • 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
    • A23L33/185—Vegetable proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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 a novel peptide.
  • Ghrelin is a gastrointestinal hormone that has an appetite promoting action and a growth hormone secretion promoting action.
  • Japan which faces a super-aging society, development of a ghrelin secretion promoting substance that contributes to the improvement of anorexia associated with aging and the reduction of growth hormone secretion is desired.
  • Patent Document 1 describes a low molecular weight peptide that activates a ghrelin receptor.
  • Patent Document 2 describes a growth hormone secretion promoting composition derived from soybean protein. However, a food-derived peptide that promotes the secretion of ghrelin has not been known.
  • An object of the present invention is to provide a novel peptide having a ghrelin secretion-promoting action, and a medicine and food containing the peptide.
  • the present inventor has intensively studied to solve the above problems. As a result, a peptide having a ghrelin secretion-promoting action was found in the tryptic digest of beta-conglycinin protein.
  • the present invention has been completed based on such findings and further studies.
  • the present invention includes the following aspects.
  • Item 1 A peptide having the amino acid sequence NKNPFLFGSNR (SEQ ID NO: 1) at the N-terminus.
  • Item 2 The peptide according to Item 1, which is derived from a trypsin digest of soybean beta-conglycinin protein.
  • a pharmaceutical composition comprising the peptide according to Item 3, Item 1 or 2 as an active ingredient.
  • a ghrelin secretion promoter comprising the peptide according to Item 4, Item 1 or 2 as an active ingredient.
  • Item 6 A food characterized by adding the peptide according to Item 1, Item 2 or Item 2.
  • Item 7 The food according to Item 5 or 6 for promoting appetite and / or promoting secretion of growth hormone.
  • Item 8 A method for promoting appetite and / or promoting growth hormone secretion, comprising a step of administering the peptide of Item 8, Item 1 or 2 to a patient or a preliminary group in need.
  • Item 9 The peptide according to Item 1 or 2 for promoting appetite and / or promoting growth hormone secretion.
  • Item 10 Use of the peptide according to Item 1 or 2 for producing a medicament or food for promoting appetite and / or promoting secretion of growth hormone.
  • the pharmaceutical composition and food containing the peptide of the present invention as an active ingredient exerts an appetite promoting action and a growth hormone secreting promoting action based on the ghrelin secretion promoting action. Based on these actions, it can be suitably used for the prevention and treatment of anorexia, muscle mass loss and locomotive syndrome.
  • the peptide of the present invention has low side effects and is suitable for long-term use.
  • the pharmaceutical composition and food of the present invention are effective for oral administration.
  • the peptide of the present invention is an enzyme digest of soybean beta-conglycinin protein, side effects are not a problem.
  • soybean beta-conglycinin protein is contained in a large amount in soybean, so that it can be produced at low cost.
  • the peptide of the present invention is a peptide having an 11-residue amino acid sequence of NKNPFLGSNR (SEQ ID NO: 1) at the N-terminus.
  • the peptide of the present invention preferably has 11 to 50 amino acid residues, more preferably 11 to 25 amino acid residues, still more preferably 11 to 15 amino acid residues, and particularly preferably 11, 12, 13 or 14 amino acid residues. . 1 to 10th amino acid sequence, 1 to 9th amino acid sequence, 1 to 8th amino acid sequence, 1 to 7th amino acid sequence, 1 to 6th amino acid sequence in the amino acid sequence shown in SEQ ID NO: 1, A peptide comprising the 1st to 5th amino acid sequence, the 1st to 4th amino acid sequence, and the 1st to 3rd amino acid sequence is also exemplified as another embodiment of the present invention.
  • the peptide of the present invention includes a peptide having an amino acid sequence of NKNPFLFGSNR (SEQ ID NO: 1) and a peptide having an amino acid sequence of NKNPFLFGSNRFETLFKNQYGR (SEQ ID NO: 2).
  • the amino acids constituting the peptide are L-form amino acids, D-form amino acids, or DL-form amino acids (the racemate and any amino acid in which either enantiomer is excessive if the D-form and L-form amino acids are mixed). Any of the above can be used.
  • Preferable is a peptide consisting only of L-form amino acids or only D-form amino acids, particularly a peptide consisting only of L-form amino acids.
  • the peptide used by this invention may be any form of each enantiomer thru
  • the peptide can have modifications.
  • the amino terminus (N terminus) of the peptide may be a free amino group (NH 2 —), or may have a modification such as an acetyl group (CH 3 CO—).
  • the carboxy terminus (C terminus) of the peptide may be a free carboxyl group (—COOH) or may have a modification such as an amide group.
  • the amino acid residue of the peptide may be unmodified or may have a modification such as a phosphate group or a sugar chain.
  • the peptide of the present invention may be a salt (acid addition salt or base salt).
  • Acid addition salts include inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, perchloric acid, citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, p-toluenesulfonic acid And salts of organic acids such as benzenesulfonic acid, methanesulfonic acid and trifluoroacetic acid.
  • the base salt include alkali metal salts such as sodium, potassium and lithium, and alkaline earth metal salts such as calcium and magnesium.
  • the peptide of the present invention may be a solvate.
  • Solvates include solvates such as water (in the case of hydrates), methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, and dimethoxyethane. Things.
  • the peptide of the present invention can be obtained by hydrolysis of a natural protein or polypeptide, or can be obtained by chemical synthesis.
  • the peptide of the present invention can be obtained by hydrolysis of trypsin of a soybean main storage protein beta-conglycinin ( ⁇ -conglycinin, ⁇ -CG) protein.
  • Trypsin is an enzyme derived from the pancreas in the case of mammals such as humans, and is a known proteolytic enzyme (protease) (EC 3.4.21.4). Trypsin can be used as a food additive in Japan. As the trypsin, commercially available products such as reagent grade and food additive grade can be used.
  • the substrate to be hydrolyzed by trypsin is not particularly limited as long as it contains soybean ⁇ -CG protein.
  • soybean itself pomace obtained by extracting soybean oil from soybean (also referred to as meal or defatted soybean), purified beta-conglycinin protein and the like can be mentioned.
  • the reaction temperature can be appropriately selected from 30 to 70 ° C, 30 to 40 ° C, 40 to 70 ° C, 50 to 65 ° C, and the like.
  • the reaction time can be appropriately selected from about 30 minutes to 48 hours, about 1 to 10 hours, and about 2 to 8 hours.
  • the pH at which the reaction is carried out can be appropriately selected from about pH 6.5 to 8.5 and pH 7 to 8. In one preferred embodiment, the reaction can be performed for about 2 to 8 hours under conditions of a temperature of about 30 to 40 ° C. and a pH of 6.5 to 8.5 (particularly about pH 7.5).
  • the peptide of the present invention may not be obtained under conditions where hydrolysis is excessive.
  • trypsin is deactivated by heating to a temperature at which trypsin is deactivated (for example, heating at a temperature exceeding 80 ° C. for about 5 to 60 minutes).
  • reaction product of hydrolysis may be used as it is, or an active ingredient peptide may be separated and used by purification.
  • the peptide of the present invention can also be obtained by peptide synthesis. That is, in a liquid phase method or a solid phase method, which is a commonly used method for peptide synthesis, a method using an active ester such as HBTU, a raw material having a reactive carboxyl group and a raw material having a reactive amino group, or carbodiimide In a peptide synthesis such as a method using a coupling agent such as When the resulting condensate has a protecting group, it can also be produced by removing the protecting group.
  • the C-terminal carboxyl group is a chlorotrityl resin, chloromethyl resin, oxymethyl resin, p- It is bound to a carrier such as an alkoxybenzyl alcohol resin.
  • the condensation reaction is carried out in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.
  • the protecting group is removed, but in the case of the solid phase method, the bond between the C-terminus of the peptide and the resin is further cleaved.
  • the peptides of the present invention are purified according to conventional methods. Examples thereof include ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like. Synthesis of the synthesized peptide is analyzed by a protein sequencer that reads the amino acid sequence from the C-terminal by the Edman degradation method, GC-MS, or the like.
  • the peptide of the present invention can also be synthesized by an enzymatic method (see WO2003 / 010307).
  • the peptide of the present invention has an action of promoting the secretion of ghrelin.
  • Ghrelin is a biologically derived peptide having the following structure. Specifically, it consists of 28 amino acid residues, and the third serine residue is modified with n-octanoic acid. Ghrelin exhibits an appetite promoting action and a growth hormone secretion promoting action.
  • the peptide of the present invention that promotes the secretion of ghrelin also has an action based on the promotion of ghrelin secretion, such as an appetite promoting action (feeding promoting action) and a growth hormone secreting promoting action.
  • an appetite promoting action feeding promoting action
  • a growth hormone secreting promoting action a growth hormone secreting promoting action.
  • the peptide of the present invention can be used for the treatment of anorexia in the elderly and the like.
  • the growth promoting action of growth hormone it can be used for prevention of muscle mass loss, prevention or treatment of locomotive syndrome (locomo), and the like.
  • the fact that the peptide of the present invention promotes the secretion amount of ghrelin can be evaluated by methods known to those skilled in the art. For example, a test substance is added to the ghrelin-secreting cell MGN3-1 described in the literature: Iwakura ⁇ H et.al. Endocrinology. 2010 Jun; 151 (6): 2940-5, and ghrelin in the medium recovered after culturing for a predetermined time. The amount of ghrelin secreted can be measured by quantifying. Ghrelin can be quantified by an immunochemical technique such as ELISA (Enzyme-Linked ImmunoSorbent Assay).
  • ELISA Enzyme-Linked ImmunoSorbent Assay
  • the peptide of the present invention can be provided as a pharmaceutical composition or a food (food composition).
  • the route of administration of the peptide of the present invention or a product containing the peptide is not particularly limited, and any of oral administration, parenteral administration, and rectal administration can be adopted, orally or parenterally. Can be administered. Among these, oral administration is preferable from the viewpoint of high effect.
  • the dosage of this peptide varies depending on the administration method, the condition and age of the administered person, but is usually 0.01 mg / kg to 500 mg / kg, preferably 0.05 mg / kg to 100 mg / kg per adult day. More preferably, it is 0.1 to 30 mg / kg.
  • the peptide (active ingredient) of the present invention can be administered in the form of a pharmaceutical composition prepared by mixing with a pharmaceutical carrier.
  • a pharmaceutical carrier a substance that is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.
  • the peptide of the present invention can be used as a medicine or food per se, or alone or together with a suitable non-toxic carrier for ingestion, diluent or excipient (tablet, uncoated tablet, dragee, effervescent tablet, Film-coated tablets, chewable tablets, etc.), capsules, troches, powders, fine granules, granules, solutions, suspensions, emulsions, pastes, creams, injections (amino acid infusions, electrolyte infusions, etc.) Or a preparation for food or medicine such as sustained release preparations such as enteric tablets, capsules and granules.
  • the content of the peptide in the food can be appropriately selected, but is generally in the range of 0.01 to 100% by weight.
  • Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These preparations are prepared according to a conventional method.
  • the liquid preparation may be dissolved or suspended in water or other appropriate solvent at the time of use. Tablets and granules may be coated by a known method.
  • injection it is prepared by dissolving the peptide of the present invention in water, but it may be dissolved in physiological saline or glucose solution as necessary, and a buffer or preservative may be added. Good.
  • These preparations may contain the peptide of the present invention in a proportion of 0.01% to 100% by weight, preferably 1 to 90% by weight. These formulations may also contain other therapeutically valuable ingredients.
  • an active ingredient and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, anhydrous silicic acid and the like are mixed to form a powder, or if necessary, sucrose, Add a binder such as hydroxypropylcellulose and polyvinylpyrrolidone, a disintegrant such as carboxymethylcellulose and carboxymethylcellulose calcium, and wet or dry granulate to form granules.
  • these powders and granules may be tableted as they are or after adding a lubricant such as magnesium stearate or talc.
  • granules or tablets should be coated with an enteric solvent base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methyl methacrylate polymer and coated with an enteric solvent preparation, or with ethylcellulose, carnauba wax, hardened oil, etc. You can also.
  • an enteric solvent base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methyl methacrylate polymer and coated with an enteric solvent preparation, or with ethylcellulose, carnauba wax, hardened oil, etc. You can also.
  • powders or granules are filled into hard capsules, or active ingredients are dissolved as they are or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin film to form soft capsules. Can do.
  • an active ingredient and a sweetener such as sucrose, sorbitol, and glycerin are dissolved in water to add a transparent syrup, further essential oil, ethanol, etc. to make an elixir, Gum arabic, tragacanth, polysorbate 80, sodium carboxymethyl cellulose and the like may be added to form an emulsion or suspension.
  • a transparent syrup such as sucrose, sorbitol, and glycerin
  • Examples of foods that can be prepared by adding and blending the peptide of the present invention include so-called health foods, functional foods, functional labeling foods, dietary supplements, supplements, foods for specified health use, foods for the sick, and combinations for the sick It may be a food (Ministry of Health, Labor and Welfare, a special-purpose food) or a food for the elderly (Ministry of Health, Labor and Welfare, a special-purpose food), uncoated tablet, film-coated tablet, sugar-coated tablet, granule, powder, tablet, capsule (hard capsule and soft capsule And chewable type, syrup type, drink type, etc.
  • the preparation of food containing the peptide according to the present invention can be carried out by a method known per se.
  • Ghrelin-secreting cells MGN3-1 were seeded at 1 ⁇ 10 5 cells / well in a 96-well plate and cultured in a medium for 24 hours. After culturing, the cells were washed with DPBS, and 100 ⁇ L of a test substance (Buffer: 50 ⁇ M sodium octanoate / DMEM) was added. Only the buffer to which no test substance was added was used as a control. After further culturing for 4 hours, the medium was collected and a supernatant was obtained by centrifugation. 10 ⁇ L of 1N HCl was added to the supernatant and stored at ⁇ 80 ° C.
  • Buffer 50 ⁇ M sodium octanoate / DMEM
  • the concentration of ghrelin in the sample was evaluated by ELISA (Bertin Pharma, Ghrelin (Acylated) EIAKit A05117).
  • ⁇ Production example> Enzyme digest
  • ⁇ -CG 1: 100 (weight ratio, final concentration of ⁇ -CG: 20 mg / ml), and attached in the attached buffer. Reaction was performed.
  • the enzymes and reaction conditions used were as follows: (I) Trypsin (manufactured by SIGMA, T1426); reaction temperature: 37 ° C., reaction time: 5 hours, reaction pH: 7.5.
  • the sample was boiled (100 ° C., 10 minutes) to stop the enzyme reaction.
  • peptide According to a conventional method, peptide NKNPFLFGSNR (SEQ ID NO: 1; Example 1), peptide NKNPFLFGSNRFETLFKNQYGR (SEQ ID NO: 2; Example 2), peptide FETLFKNQYGR (SEQ ID NO: 3; Comparative Example 1), peptide HKNKNPFLFGSNR (SEQ ID NO: 4; Comparative Example 2) and NPFLFGSNR (SEQ ID NO: 5; Comparative Example 3) was synthesized.
  • Test Example 1 ⁇ -CG Trypsin Digest ⁇ -CG Trypsin Digest was evaluated for the effect on ghrelin secretion using ghrelin-secreting cells MGN3-1.
  • the horizontal axis “Ghrelin (% of control)” indicates the ratio (mass percentage) to the measured value of ghrelin secretion when MGN3-1 is added to the control (Control, Buffer without addition of test substance). .
  • ⁇ -CG trypsin digest ( ⁇ -CG Digest) was found to have an activity to increase ghrelin secretion and promote ghrelin secretion.
  • Test Example 2 Peptide (1) About the peptide NKNPFLFGSNRFETLFKNQYGR (sequence number 2) contained in the tryptic digest of ⁇ -CG, the effect on ghrelin secretion was evaluated in the same manner as in Test Example 1.
  • peptide NKNPFLFGSNR SEQ ID NO: 1
  • peptide FETLFKNQYGR SEQ ID NO: 3
  • the peptide NKNPFLFGSNRFETLFKNQYGR (SEQ ID NO: 2) was found to have an activity of promoting strong ghrelin secretion at 1000 ⁇ M.
  • the peptide NKNPFLFGSNR (SEQ ID NO: 1) was found to have an activity of promoting strong ghrelin secretion at 1 ⁇ M or more.
  • Test Example 3 Peptide (2) The effects of the peptides HKNKNPFLFGSNR (SEQ ID NO: 4) and NPFLFGSNR (SEQ ID NO: 5) on ghrelin secretion were also evaluated. Also tested was 100 ⁇ M Noradrenaline, which is known to promote ghrelin secretion as a subject.
  • Test Example 4 Mechanism of increase in ghrelin secretion amount It was verified whether the action of the peptide of the present invention promotes the synthesis of ghrelin or the secretion of already synthesized ghrelin. As test substances, trypsin digest of ⁇ -CG and peptide NKNPFLFGSNR (SEQ ID NO: 1) were used.
  • the ghrelin in the culture solution was evaluated in the same manner as in Test Examples 1 and 2.
  • ghrelin in cells after removing the medium, RIPA Buffer ⁇ (manufactured by Nacalai Tesque) was added to ghrelin-secreting cell MGN3-1 to dissolve it, and the supernatant was obtained by centrifuging the lysate. It was. 10 ⁇ L of 1N HCl was added to the supernatant and stored at ⁇ 80 ° C. The concentration of ghrelin in the sample was evaluated in the same manner as in Test Examples 1 and 2.
  • FIG. 4 shows the quantitative results of ghrelin in the culture solution.
  • FIG. 4B shows the quantitative results of ghrelin in the cells. Since ghrelin in the cells did not increase, it was revealed that the peptide of the present invention has an action of enhancing secretion rather than promoting ghrelin synthesis.
  • Test Example 5 Mechanism of action The mechanism of action leading to the promotion of ghrelin secretion was verified. The effect of peptide NKNPFLFGSNR (SEQ ID NO: 1) on intracellular cAMP concentration and intracellular calcium ion concentration ([Ca 2+ ]) was evaluated.
  • ghrelin-secreting cells MGN3-1 were seeded at 1 ⁇ 10 5 cells / well in a 96-well plate and cultured in a medium for 24 hours. After culturing, the cells were washed with DPBS, and 45 ⁇ L of a test substance (Buffer: Krebs-Ringer-HEPES buffer containing 0.5 mM IBMX) was added. After further incubation for 30 minutes, intracellular cAMP was quantified using HitHunter cAMP Assay for small molecules (DiscoveRx).
  • Buffer Krebs-Ringer-HEPES buffer containing 0.5 mM IBMX
  • the intracellular calcium ion concentration was measured according to the method described in the literature: Kagebayashi Tet.al. Mol. Nutr. Food Res. 2012 Sep; 56 (9): 1456-63.
  • FIG. 5A shows the quantitative results of intracellular cAMP.
  • FIG. 5B shows changes in intracellular calcium ion concentration ( ⁇ [Ca 2+ ] i).
  • Noradrenaline is known to promote ghrelin secretion through an increase in intracellular cAMP concentration and intracellular calcium ion concentration. Involvement of Gs-type trimeric G protein is known.
  • the peptide of the present invention promotes ghrelin secretion without changing the intracellular cAMP concentration and intracellular calcium ion concentration, it is considered that the peptide is via a different route from the known route. .
  • Test Example 6 Administration to a living body A trypsin digest of ⁇ -CG (30 mg / kg) and peptide NKNPFLFGSNR (SEQ ID NO: 1) (0.3 mg / kg) were administered to mice (ddY mice, male, 34 to 42 g). The effect on eating behavior was evaluated.
  • ⁇ -CG tryptic digest dissolved in physiological saline and peptide NKNPFLFGSNR (SEQ ID NO: 1) were orally administered to mice, and then pre-weighed feed (CE-2, manufactured by Claire Japan) The amount of food intake (Food intake (g / 6 hrs)) was calculated by measuring the feed weight after 6 hours.
  • peptide NKNPFLFGSNR SEQ ID NO: 1
  • CE-2 manufactured by CLEA Japan
  • the pre-weighed mixed feed was given to the mice, and the feed weight (Food intake (g / 24 hrs)) was calculated by measuring the weight of the feed after 24 hours.

Abstract

This peptide has an amino acid sequence NKNPFLFGSNR (SEQ ID NO: 1) at the N terminus thereof.

Description

ペプチドpeptide
　本発明は、新規なペプチドに関する。 The present invention relates to a novel peptide.
　グレリンは、食欲促進作用と成長ホルモン分泌促進作用を有する消化管ホルモンである。超高齢化社会を迎える我が国では、加齢に伴う食欲不振や成長ホルモン分泌減少の改善に寄与する、グレリン分泌促進物質の開発が望まれている。 Ghrelin is a gastrointestinal hormone that has an appetite promoting action and a growth hormone secretion promoting action. In Japan, which faces a super-aging society, development of a ghrelin secretion promoting substance that contributes to the improvement of anorexia associated with aging and the reduction of growth hormone secretion is desired.
　特許文献１には、グレリン受容体を活性化する低分子ペプチドが記載されている。特許文献２には、大豆タンパク質由来の成長ホルモン分泌促進組成物が記載されている。しかしながら、グレリンの分泌を促進する食品由来のペプチドは知られていなかった。 Patent Document 1 describes a low molecular weight peptide that activates a ghrelin receptor. Patent Document 2 describes a growth hormone secretion promoting composition derived from soybean protein. However, a food-derived peptide that promotes the secretion of ghrelin has not been known.
特開2005-082489JP2005-082489 特開2006-347946JP 2006-347946
　本発明は、グレリンの分泌促進作用を有する新規なペプチド、並びに、該ペプチドを含む医薬及び食品を提供することを目的とする。 An object of the present invention is to provide a novel peptide having a ghrelin secretion-promoting action, and a medicine and food containing the peptide.
　本発明者は、上記課題を解決すべく、鋭意研究を重ねてきた。その結果、ベータコングリシニンタンパク質のトリプシン消化物中に、グレリンの分泌促進作用を有するペプチドを見出した。本発明はかかる知見に基づいて、さらに検討を重ねて完成されたものである。 The present inventor has intensively studied to solve the above problems. As a result, a peptide having a ghrelin secretion-promoting action was found in the tryptic digest of beta-conglycinin protein. The present invention has been completed based on such findings and further studies.
　すなわち、本発明は以下の態様を包含する。 That is, the present invention includes the following aspects.
　項１、Ｎ末端にアミノ酸配列NKNPFLFGSNR（配列番号１）を有するペプチド。 Item 1. A peptide having the amino acid sequence NKNPFLFGSNR (SEQ ID NO: 1) at the N-terminus.
　項２、大豆のベータコングリシニンタンパク質のトリプシン消化物に由来する、項１に記載のペプチド。 Item 2. The peptide according to Item 1, which is derived from a trypsin digest of soybean beta-conglycinin protein.
　項３、項１または２に記載のペプチドを有効成分とする医薬組成物。 A pharmaceutical composition comprising the peptide according to Item 3, Item 1 or 2 as an active ingredient.
　項４、項１または２に記載のペプチドを有効成分とするグレリン分泌促進剤。 A ghrelin secretion promoter comprising the peptide according to Item 4, Item 1 or 2 as an active ingredient.
　項５、項１または２に記載のペプチドを含有する食品。 A food containing the peptide according to Item 5, Item 1 or 2.
　項６、項１または２に記載のペプチドを添加することを特徴とする食品。 Item 6. A food characterized by adding the peptide according to Item 1, Item 2 or Item 2.
　項７、食欲促進及び／または成長ホルモンの分泌促進のための、項５または６に記載の食品。 Item 7. The food according to Item 5 or 6 for promoting appetite and / or promoting secretion of growth hormone.
　項８、項１または２に記載のペプチドを必要とする患者または予備群に投与する工程を含む、食欲を促進及び／または成長ホルモンの分泌を促進する方法。 Item 8. A method for promoting appetite and / or promoting growth hormone secretion, comprising a step of administering the peptide of Item 8, Item 1 or 2 to a patient or a preliminary group in need.
　項９、食欲を促進及び／または成長ホルモンの分泌を促進するための、項１または２に記載のペプチド。  Item 9. The peptide according to Item 1 or 2 for promoting appetite and / or promoting growth hormone secretion.
　項１０、食欲を促進及び／または成長ホルモンの分泌を促進するための医薬または食品を製造するための、項１または２に記載のペプチドの使用。 Item 10. Use of the peptide according to Item 1 or 2 for producing a medicament or food for promoting appetite and / or promoting secretion of growth hormone.
　本発明のペプチドを有効成分とする医薬組成物、食品は、グレリンの分泌促進作用に基づき、食欲促進や成長ホルモンの分泌促進の作用を発揮する。これらの作用に基づき、食欲不振、筋肉量低下、ロコモティブシンドロームの予防や治療に好適に使用することができる。本発明のペプチドは、副作用が低く長期の服用に適したものである。また、本発明の医薬組成物、食品は、経口投与で有効である。 The pharmaceutical composition and food containing the peptide of the present invention as an active ingredient exerts an appetite promoting action and a growth hormone secreting promoting action based on the ghrelin secretion promoting action. Based on these actions, it can be suitably used for the prevention and treatment of anorexia, muscle mass loss and locomotive syndrome. The peptide of the present invention has low side effects and is suitable for long-term use. The pharmaceutical composition and food of the present invention are effective for oral administration.
　本発明のペプチドは、大豆ベータコングリシニンタンパク質の酵素消化物であるので、副作用は問題にならない。また、大豆ベータコングリシニンタンパク質は、大豆中に多量に含まれるので、低コストで製造できる。 Since the peptide of the present invention is an enzyme digest of soybean beta-conglycinin protein, side effects are not a problem. In addition, soybean beta-conglycinin protein is contained in a large amount in soybean, so that it can be produced at low cost.
酵素消化物がグレリン分泌に与える影響。Effects of enzyme digests on ghrelin secretion. ペプチドがグレリン分泌に与える影響。Effect of peptides on ghrelin secretion. ペプチドがグレリン分泌に与える影響。Effect of peptides on ghrelin secretion. グレリン分泌量増加の作用機構の検証結果。The verification result of the action mechanism of ghrelin secretion increase. グレリン分泌促進の作用機序の検証結果。The verification result of the action mechanism of ghrelin secretion promotion. 経口投与による摂食行動への影響。Effects on oral feeding behavior.
　本発明のペプチドは、Ｎ末端にＮＫＮＰＦＬＦＧＳＮＲ（配列番号１）の１１残基のアミノ酸配列を有するペプチドである。 The peptide of the present invention is a peptide having an 11-residue amino acid sequence of NKNPFLGSNR (SEQ ID NO: 1) at the N-terminus.
　本発明のペプチドは、好ましくは１１～５０アミノ酸残基、より好ましくは１１～２５アミノ酸残基、さらに好ましくは１１～１５アミノ酸残基、特に好ましくは１１、１２、１３若しくは１４アミノ酸残基を有する。配列番号１に示すアミノ酸配列のうち、１～１０番目のアミノ酸配列、１～９番目のアミノ酸配列、１～８番目のアミノ酸配列、１～７番目のアミノ酸配列、１～６番目のアミノ酸配列、１～５番目のアミノ酸配列、１～４番目のアミノ酸配列及び１～３番目のアミノ酸配列からなるペプチドも本発明の別の態様として挙げられる。 The peptide of the present invention preferably has 11 to 50 amino acid residues, more preferably 11 to 25 amino acid residues, still more preferably 11 to 15 amino acid residues, and particularly preferably 11, 12, 13 or 14 amino acid residues. . 1 to 10th amino acid sequence, 1 to 9th amino acid sequence, 1 to 8th amino acid sequence, 1 to 7th amino acid sequence, 1 to 6th amino acid sequence in the amino acid sequence shown in SEQ ID NO: 1, A peptide comprising the 1st to 5th amino acid sequence, the 1st to 4th amino acid sequence, and the 1st to 3rd amino acid sequence is also exemplified as another embodiment of the present invention.
　本発明のペプチドとしては、NKNPFLFGSNR（配列番号１）のアミノ酸配列を有するペプチド、NKNPFLFGSNRFETLFKNQYGR（配列番号２）のアミノ酸配列を有するペプチドが包含される。 The peptide of the present invention includes a peptide having an amino acid sequence of NKNPFLFGSNR (SEQ ID NO: 1) and a peptide having an amino acid sequence of NKNPFLFGSNRFETLFKNQYGR (SEQ ID NO: 2).
　ペプチドを構成するアミノ酸は、Ｌ体のアミノ酸、Ｄ体のアミノ酸或いはＤＬ体のアミノ酸（Ｄ体とＬ体が混合されたアミノ酸であればラセミ体といずれか一方のエナンチオマーが過剰なアミノ酸のいずれも含まれる）のいずれを使用することができる。好ましくはＬ体のアミノ酸のみ、或いはＤ体のアミノ酸のみからなるペプチド、特にＬ体のアミノ酸のみからなるペプチドが好ましい。 The amino acids constituting the peptide are L-form amino acids, D-form amino acids, or DL-form amino acids (the racemate and any amino acid in which either enantiomer is excessive if the D-form and L-form amino acids are mixed). Any of the above can be used. Preferable is a peptide consisting only of L-form amino acids or only D-form amino acids, particularly a peptide consisting only of L-form amino acids.
　また、本発明で使用するペプチドが２以上の不斉炭素を含む場合、各エナンチオマーないしジアステレオマー或いはこれらの任意の比率の混合物のいずれの形態でもあり得る。
エナンチオマーまたはジアステレオマーの分離は、通常のカラムで行う方法、光学活性カラムを使用したり、光学活性基を導入して誘導体の形態で光学分割した後、その光学活性基を除去する方法や、光学活性の酸または塩基との塩を形成して光学分割する方法などの公知のいずれの方法を用いることができる。 Moreover, when the peptide used by this invention contains two or more asymmetric carbons, it may be any form of each enantiomer thru | or a diastereomer, or the mixture of these arbitrary ratios.
Separation of enantiomers or diastereomers is carried out using a normal column, using an optically active column, introducing an optically active group and optically resolving it in the form of a derivative, and then removing the optically active group, Any known method such as a method of optical resolution by forming a salt with an optically active acid or base can be used.
　ペプチドは、修飾を有することができる。ペプチドのアミノ末端（Ｎ末端）は、遊離のアミノ基（ＮＨ２－）であっても、アセチル基（ＣＨ３ＣＯ－）などの修飾を有するものであってもよい。ペプチドのカルボキシ末端（Ｃ末端）は、遊離のカルボキシル基（－ＣＯＯＨ）であっても、アミド基などの修飾を有するものであってもよい。ペプチドのアミノ酸残基は、無修飾ものであっても、リン酸基、糖鎖などの修飾を有するものであってもよい。 The peptide can have modifications. The amino terminus (N terminus) of the peptide may be a free amino group (NH 2 —), or may have a modification such as an acetyl group (CH 3 CO—). The carboxy terminus (C terminus) of the peptide may be a free carboxyl group (—COOH) or may have a modification such as an amide group. The amino acid residue of the peptide may be unmodified or may have a modification such as a phosphate group or a sugar chain.
　本発明のペプチドは、塩（酸付加塩又は塩基塩）であってもよい。酸付加塩としては、塩酸、硫酸、硝酸、リン酸、臭化水素酸、過塩素酸などの無機塩、クエン酸、コハク酸、マレイン酸、フマル酸、リンゴ酸、酒石酸、ｐ－トルエンスルホン酸、ベンゼンスルホン酸、メタンスルホン酸、トリフルオロ酢酸などの有機酸の塩が挙げられる。塩基塩としては、ナトリウム、カリウム、リチウムなどのアルカリ金属塩、カルシウム、マグネシウムなどのアルカリ土類金属塩などが挙げられる。 The peptide of the present invention may be a salt (acid addition salt or base salt). Acid addition salts include inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, perchloric acid, citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, p-toluenesulfonic acid And salts of organic acids such as benzenesulfonic acid, methanesulfonic acid and trifluoroacetic acid. Examples of the base salt include alkali metal salts such as sodium, potassium and lithium, and alkaline earth metal salts such as calcium and magnesium.
　本発明のペプチドは、溶媒和物であってもよい。溶媒和物としては、水(水和物の場合)、メタノール、エタノール、イソプロパノール、酢酸、テトラヒドロフラン、アセトン、ジメチルホルムアミド、ジメチルスルホキシド、ジメチルアセトアミド、アセトアミド、エチレングリコール、プロピレングリコール、ジメトキシエタンなどの溶媒和物が挙げられる。 The peptide of the present invention may be a solvate. Solvates include solvates such as water (in the case of hydrates), methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, and dimethoxyethane. Things.
　本発明のペプチドは、天然のタンパク質ないしポリペプチドの加水分解により得ることもでき、化学合成により得ることもできる。 The peptide of the present invention can be obtained by hydrolysis of a natural protein or polypeptide, or can be obtained by chemical synthesis.
　例えば、本発明のペプチドは、大豆の主要貯蔵タンパク質ベータコングリシニン（β-conglycinin、β-CG）タンパク質の、トリプシンの加水分解により得ることができる。 For example, the peptide of the present invention can be obtained by hydrolysis of trypsin of a soybean main storage protein beta-conglycinin (β-conglycinin, β-CG) protein.
　トリプシンは、ヒトなどの哺乳類の場合は膵臓由来の酵素であり、公知のタンパク質分解酵素（プロテアーゼ）である（EC3.4.21.4）。トリプシンは、我が国において食品添加物として使用することができる。トリプシンは、試薬グレード、食品添加物グレードなどの市販されているものを使用することができる。 Trypsin is an enzyme derived from the pancreas in the case of mammals such as humans, and is a known proteolytic enzyme (protease) (EC 3.4.21.4). Trypsin can be used as a food additive in Japan. As the trypsin, commercially available products such as reagent grade and food additive grade can be used.
　トリプシンにより加水分解をする基質は、大豆β-CGタンパク質を含むものであればとくに限定されない。例えば、大豆それ自体、大豆から大豆油を抽出した搾りかす（ミール、脱脂大豆ともいう。）、精製したベータコングリシニンタンパク質などが挙げられる。 The substrate to be hydrolyzed by trypsin is not particularly limited as long as it contains soybean β-CG protein. For example, soybean itself, pomace obtained by extracting soybean oil from soybean (also referred to as meal or defatted soybean), purified beta-conglycinin protein and the like can be mentioned.
　トリプシンによる加水分解は、本発明のペプチドが得られる条件で行う。反応温度は３０～７０℃、３０～４０℃、４０～７０℃、５０～６５℃などから適宜選択することができる。反応時間は、３０分～４８時間程度、１～１０時間程度、２～８時間程度などから適宜選択することができる。反応を行うｐＨは、ｐH６．５～８．５程度、ｐH７～８程度から適宜選択することができる。一つの好適な態様においては、３０～４０℃程度の温度、ｐH６．５～８．５（特に、ｐH７．５程度）の条件下で、２～８時間程度反応させることができる。 Hydrolysis with trypsin is carried out under conditions that give the peptide of the present invention. The reaction temperature can be appropriately selected from 30 to 70 ° C, 30 to 40 ° C, 40 to 70 ° C, 50 to 65 ° C, and the like. The reaction time can be appropriately selected from about 30 minutes to 48 hours, about 1 to 10 hours, and about 2 to 8 hours. The pH at which the reaction is carried out can be appropriately selected from about pH 6.5 to 8.5 and pH 7 to 8. In one preferred embodiment, the reaction can be performed for about 2 to 8 hours under conditions of a temperature of about 30 to 40 ° C. and a pH of 6.5 to 8.5 (particularly about pH 7.5).
　加水分解が過度に行われる条件下では、本発明のペプチドが得られない場合がある。 The peptide of the present invention may not be obtained under conditions where hydrolysis is excessive.
　必要に応じて、トリプシンが失活する温度に加熱（例えば、８０℃を超える温度で５～６０分程度での加熱）することで、トリプシンを失活させる。 If necessary, trypsin is deactivated by heating to a temperature at which trypsin is deactivated (for example, heating at a temperature exceeding 80 ° C. for about 5 to 60 minutes).
　加水分解の反応生成物は、そのまま使用してもよく、精製により有効成分のペプチドを分離して使用してもよい。 The reaction product of hydrolysis may be used as it is, or an active ingredient peptide may be separated and used by purification.
　また本発明のペプチドは、ペプチド合成法で取得することもできる。即ち、ペプチド合成に通常用いられる方法である液相法または固相法で、反応性カルボキシル基を有する原料と、反応性アミノ基を有する原料とをＨＢＴＵ等の活性エステルを用いた方法や、カルボジイミドなどのカップリング剤を用いた方法等のペプチド合成において通常の方法により縮合させることができる。生成する縮合物が保護基を有する場合、その保護基を除去することによっても製造し得る。 The peptide of the present invention can also be obtained by peptide synthesis. That is, in a liquid phase method or a solid phase method, which is a commonly used method for peptide synthesis, a method using an active ester such as HBTU, a raw material having a reactive carboxyl group and a raw material having a reactive amino group, or carbodiimide In a peptide synthesis such as a method using a coupling agent such as When the resulting condensate has a protecting group, it can also be produced by removing the protecting group.
　この反応工程において反応に関与すべきでない官能基は、保護基により保護される。アミノ基の保護基としては、例えばベンジルオキシカルボニル（ＣＢＺ）、ｔ－ブチルオキシカルボニル（Ｂｏｃ），９－フルオレニルメチルオキシカルボニル（Ｆｍｏｃ）等が挙げられる。カルボキシル基の保護剤としては例えばアルキルエステル、ベンジルエステル等を形成し得る基が挙げられるが、固相法の場合は、Ｃ末端のカルボキシル基はクロロトリチル樹脂、クロルメチル樹脂、オキシメチル樹脂、ｐ－アルコキシベンジルアルコール樹脂等の担体に結合している。縮合反応は、カルボジイミド等の縮合剤の存在下にあるいはＮ－保護アミノ酸活性エステルまたはペプチド活性エステルを用いて実施する。 In this reaction step, functional groups that should not participate in the reaction are protected by protecting groups. Examples of amino-protecting groups include benzyloxycarbonyl (CBZ), t-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the carboxyl group-protecting agent include groups capable of forming alkyl esters, benzyl esters, and the like. In the solid phase method, the C-terminal carboxyl group is a chlorotrityl resin, chloromethyl resin, oxymethyl resin, p- It is bound to a carrier such as an alkoxybenzyl alcohol resin. The condensation reaction is carried out in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.
　縮合反応終了後、保護基は除去されるが、固相法の場合はさらにペプチドのＣ末端と樹脂との結合を切断する。更に、本発明のペプチドは通常の方法に従い精製される。例えばイオン交換クロマトグラフィー、逆相液体クロマトグラフィー、アフィニティークロマトグラフィー等が挙げられる。合成したペプチドの合成はエドマン分解法でＣ－末端からアミノ酸配列を読み取るプロテインシークエンサー、ＧＣ－ＭＳ等で分析される。 After completion of the condensation reaction, the protecting group is removed, but in the case of the solid phase method, the bond between the C-terminus of the peptide and the resin is further cleaved. Furthermore, the peptides of the present invention are purified according to conventional methods. Examples thereof include ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like. Synthesis of the synthesized peptide is analyzed by a protein sequencer that reads the amino acid sequence from the C-terminal by the Edman degradation method, GC-MS, or the like.
　本発明のペプチドは、酵素法によっても合成することが可能である（WO2003/010307参照）。 The peptide of the present invention can also be synthesized by an enzymatic method (see WO2003 / 010307).
　本発明のペプチドは、グレリンの分泌を促進する作用を有する。グレリンは、下記の構造を有する生体由来のペプチドである。具体的には、アミノ酸残基２８基からなり、３番目のセリン残基がｎ－オクタン酸で修飾されている。グレリンは、食欲促進作用と成長ホルモン分泌促進作用を示す。 The peptide of the present invention has an action of promoting the secretion of ghrelin. Ghrelin is a biologically derived peptide having the following structure. Specifically, it consists of 28 amino acid residues, and the third serine residue is modified with n-octanoic acid. Ghrelin exhibits an appetite promoting action and a growth hormone secretion promoting action.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　従って、グレリンの分泌を促進する本発明のペプチドは、食欲促進作用（摂食促進作用）、成長ホルモンの分泌促進作用等のグレリンの分泌促進に基づく作用をも有する。食欲促進作用（摂食促進作用）に基づいて、本発明のペプチドは、高齢者における食欲不振の治療のためなどに使用することができる。成長ホルモンの分泌促進作用に基づいて、筋肉量低下の予防、ロコモティブシンドローム（ロコモ）の予防若しくは治療のためなどに使用することができる。 Therefore, the peptide of the present invention that promotes the secretion of ghrelin also has an action based on the promotion of ghrelin secretion, such as an appetite promoting action (feeding promoting action) and a growth hormone secreting promoting action. Based on the appetite promoting action (feeding promoting action), the peptide of the present invention can be used for the treatment of anorexia in the elderly and the like. Based on the growth promoting action of growth hormone, it can be used for prevention of muscle mass loss, prevention or treatment of locomotive syndrome (locomo), and the like.
　本発明のペプチドがグレリンの分泌量を促進することは、当業者に公知の手法で評価をすることができる。例えば、文献：Iwakura H et.al. Endocrinology. 2010 Jun;151(6):2940-5に記載のグレリン分泌細胞MGN3-1に試験物質を添加して、所定時間培養後に回収した培地中のグレリンを定量することで、グレリンの分泌量を測定することができる。グレリンの定量は、ELISA（Enzyme-Linked ImmunoSorbentAssay）などの免疫化学的手法により行うことができる。 The fact that the peptide of the present invention promotes the secretion amount of ghrelin can be evaluated by methods known to those skilled in the art. For example, a test substance is added to the ghrelin-secreting cell MGN3-1 described in the literature: Iwakura 文献 H et.al. Endocrinology. 2010 Jun; 151 (6): 2940-5, and ghrelin in the medium recovered after culturing for a predetermined time. The amount of ghrelin secreted can be measured by quantifying. Ghrelin can be quantified by an immunochemical technique such as ELISA (Enzyme-Linked ImmunoSorbent Assay).
　本発明のペプチドは、医薬組成物または食品（食品組成物）として提供されうる。 The peptide of the present invention can be provided as a pharmaceutical composition or a food (food composition).
　本発明のペプチドまたはこれを含有する製品の投与経路は特に限定されるものではなく、経口投与、非経口投与、直腸内投与のいずれを採用することも可能であり、経口的あるいは非経口的に投与することができる。中でも、効果が高いとの観点から、経口投与が好ましい。 The route of administration of the peptide of the present invention or a product containing the peptide is not particularly limited, and any of oral administration, parenteral administration, and rectal administration can be adopted, orally or parenterally. Can be administered. Among these, oral administration is preferable from the viewpoint of high effect.
　本ペプチドの投与量は、投与方法、投与される者の状態や年齢等により異なるが、成人１日あたり通常は０．０１ｍｇ／ｋｇ～５００ｍｇ／ｋｇ、好ましくは０．０５ｍｇ／ｋｇ～１００ｍｇ／ｋｇ、より好ましくは０．１～３０ｍｇ／ｋｇである。本発明のペプチド(有効成分)は、製剤用担体と混合して調製した医薬組成物の形で投与することができる。製剤用担体としては、製剤分野において常用され、かつ本発明のペプチドと反応しない物質が用いられる。 The dosage of this peptide varies depending on the administration method, the condition and age of the administered person, but is usually 0.01 mg / kg to 500 mg / kg, preferably 0.05 mg / kg to 100 mg / kg per adult day. More preferably, it is 0.1 to 30 mg / kg. The peptide (active ingredient) of the present invention can be administered in the form of a pharmaceutical composition prepared by mixing with a pharmaceutical carrier. As a pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.
　本発明のペプチドはそれ自体医薬または食品として利用することができ、或いは単独で、もしくは適当な無毒性の経口摂取用担体、希釈剤または賦形剤とともに、タブレット（素錠、糖衣錠、発泡錠、フィルムコート錠、チュアブル錠など）、カプセル、トローチ、粉末、細粒剤、顆粒剤、液剤、懸濁液、乳濁液、ペースト、クリーム、注射剤（アミノ酸輸液、電解質輸液等の輸液に配合する場合を含む）、或いは腸溶性の錠剤、カプセル剤、顆粒剤などの徐放性製剤などの食品用もしくは医薬用の製剤にすることが可能である。食品中のペプチドの含有量は適宜選択が可能であるが一般に、０．０１～１００重量％の範囲である。 The peptide of the present invention can be used as a medicine or food per se, or alone or together with a suitable non-toxic carrier for ingestion, diluent or excipient (tablet, uncoated tablet, dragee, effervescent tablet, Film-coated tablets, chewable tablets, etc.), capsules, troches, powders, fine granules, granules, solutions, suspensions, emulsions, pastes, creams, injections (amino acid infusions, electrolyte infusions, etc.) Or a preparation for food or medicine such as sustained release preparations such as enteric tablets, capsules and granules. The content of the peptide in the food can be appropriately selected, but is generally in the range of 0.01 to 100% by weight.
　具体的には、医薬または食品に加えることができる製剤用担体ないし経口摂取用担体、希釈剤または賦形剤のような物質の例として乳糖、ブドウ糖、マンニット、デキストリン、シクロデキストリン、デンプン、蔗糖、メタケイ酸アルミン酸マグネシウム、合成ケイ酸アルミニウム、カルボキシメチルセルロースナトリウム、ヒドロキシプロピルデンプン、カルボキシメチルセルロースカルシウム、イオン交換樹脂、メチルセルロース、ゼラチン、アラビアゴム、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン、ポリビニルアルコール、軽質無水ケイ酸、ステアリン酸マグネシウム、タルク、トラガント、ベントナイト、ビーガム、酸化チタン、ソルビタン脂肪酸エステル、ラウリル硫酸ナトリウム、グリセリン、脂肪酸グリセリンエステル、精製ラノリン、グリセロゼラチン、ポリソルベート、マクロゴール、植物油、ロウ、流動パラフィン、白色ワセリン、フルオロカーボン、非イオン性界面活性剤、プロピレングルコール、水等が挙げられる。 Specifically, examples of substances such as pharmaceutical carriers or ingestible carriers, diluents or excipients that can be added to medicines or foods include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, and sucrose. , Magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light Silica, magnesium stearate, talc, tragacanth, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate Potassium, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, water and the like.
　剤型としては、錠剤、カプセル剤、顆粒剤、散剤、シロップ剤、懸濁剤、坐剤、軟膏、クリーム剤、ゲル剤、貼付剤、吸入剤、注射剤等が挙げられる。これらの製剤は常法に従って調製される。尚、液体製剤にあっては、用時、水又は他の適当な溶媒に溶解または懸濁する形であってもよい。また錠剤、顆粒剤は周知の方法でコーティングしてもよい。注射剤の場合には、本発明のペプチドを水に溶解させて調製されるが、必要に応じて生理食塩水あるいはブドウ糖溶液に溶解させてもよく、また緩衝剤や保存剤を添加してもよい。 Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These preparations are prepared according to a conventional method. The liquid preparation may be dissolved or suspended in water or other appropriate solvent at the time of use. Tablets and granules may be coated by a known method. In the case of injection, it is prepared by dissolving the peptide of the present invention in water, but it may be dissolved in physiological saline or glucose solution as necessary, and a buffer or preservative may be added. Good.
　これらの製剤は、本発明のペプチドを０．０１％～１００重量％、好ましくは１～９０重量％の割合で含有することができる。これらの製剤はまた、治療上価値のある他の成分を含有していてもよい。 These preparations may contain the peptide of the present invention in a proportion of 0.01% to 100% by weight, preferably 1 to 90% by weight. These formulations may also contain other therapeutically valuable ingredients.
　経口投与用の固形製剤を製造するには、有効成分と賦形剤成分例えば乳糖、澱粉、結晶セルロース、乳酸カルシウム、無水ケイ酸などと混合して散剤とするか、さらに必要に応じて白糖、ヒドロキシプロピルセルロース、ポリビニルピロリドンなどの結合剤、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウムなどの崩壊剤などを加えて湿式又は乾式造粒して顆粒剤とする。錠剤を製造するには、これらの散剤及び顆粒剤をそのまま或いはステアリン酸マグネシウム、タルクなどの滑沢剤を加えて打錠すればよい。これらの顆粒又は錠剤はヒドロキシプロピルメチルセルロースフタレート、メタクリル酸－メタクリル酸メチルポリマーなどの腸溶剤基剤で被覆して腸溶剤製剤、あるいはエチルセルロース、カルナウバロウ、硬化油などで被覆して持続性製剤とすることもできる。また、カプセル剤を製造するには、散剤又は顆粒剤を硬カプセルに充填するか、有効成分をそのまま或いはグリセリン、ポリエチレングリコール、ゴマ油、オリーブ油などに溶解した後ゼラチン膜で被覆し軟カプセルとすることができる。 In order to produce a solid preparation for oral administration, an active ingredient and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, anhydrous silicic acid and the like are mixed to form a powder, or if necessary, sucrose, Add a binder such as hydroxypropylcellulose and polyvinylpyrrolidone, a disintegrant such as carboxymethylcellulose and carboxymethylcellulose calcium, and wet or dry granulate to form granules. In order to produce tablets, these powders and granules may be tableted as they are or after adding a lubricant such as magnesium stearate or talc. These granules or tablets should be coated with an enteric solvent base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methyl methacrylate polymer and coated with an enteric solvent preparation, or with ethylcellulose, carnauba wax, hardened oil, etc. You can also. In order to produce capsules, powders or granules are filled into hard capsules, or active ingredients are dissolved as they are or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin film to form soft capsules. Can do.
　経口投与用の液状製剤を製造するには、有効成分と白糖、ソルビトール、グリセリンなどの甘味剤とを水に溶解して透明なシロップ剤、更に精油、エタノールなどを加えてエリキシル剤とするか、アラビアゴム、トラガント、ポリソルベート８０、カルボキシメチルセルロースナトリウムなどを加えて乳剤又は懸濁剤としてもよい。これらの液状製剤には所望により矯味剤、着色剤、保存剤などを加えてもよい。 In order to produce a liquid preparation for oral administration, an active ingredient and a sweetener such as sucrose, sorbitol, and glycerin are dissolved in water to add a transparent syrup, further essential oil, ethanol, etc. to make an elixir, Gum arabic, tragacanth, polysorbate 80, sodium carboxymethyl cellulose and the like may be added to form an emulsion or suspension. These liquid preparations may contain a flavoring agent, a coloring agent, a preservative and the like as desired.
　本発明に係るペプチドを添加、配合して調製しうる食品の具体的形態としては、例えば、飲料類（コーヒー、ココア、ジュース、清涼飲料、ミネラル飲料、茶飲料、緑茶、紅茶、烏龍茶、乳飲料、乳酸菌飲料、ヨーグルト飲料、炭酸飲料、その他ノンアルコール飲料、アルコール飲料など）、菓子類（ハードキャンディー、ガム、グミ、ゼリー、プディング、ムース、ケーキ、キャンデー、クッキー、クラッカー、ビスケット、チョコレート、氷菓（アイスクリーム、アイスキャンディ、シャーベット、かき氷など）など）、ふりかけ、ドレッシング、調味料、大豆加工食品（豆腐、味噌、醤油、湯葉、きな粉、納豆など）、食肉加工食品(ハンバーグ、ミートローフ、ミートボール、つくねなど)、魚肉加工食品（かまぼこ、ちくわなど）、レトルト食品、ゼリー状食品（ゼリー、寒天、ゼリー状飲料等）、等を挙げることができる。本発明のペプチドを添加・配合して調製しうる食品としては、いわゆる健康食品、機能性食品、機能性表示食品、栄養補助食品、サプリメント、特定保健用食品、病者用食品・病者用組合せ食品（厚生労働省、特別用途食品の一種）又は高齢者用食品（厚生労働省、特別用途食品の一種）としてもよく、素錠、フィルムコート錠、糖衣錠、顆粒、粉末、タブレット、カプセル（ハードカプセルとソフトカプセルとのいずれも含む。）、チュアブルタイプ、シロップタイプ、ドリンクタイプ等とすることもできる。本発明に係るペプチドを添加・配合した食品の調製は、それ自体公知の方法で行うことができる。 Specific examples of foods that can be prepared by adding and blending the peptide according to the present invention include, for example, beverages (coffee, cocoa, juice, soft drinks, mineral drinks, tea drinks, green tea, tea, oolong tea, and milk drinks. , Lactic acid bacteria beverages, yogurt beverages, carbonated beverages, other non-alcoholic beverages, alcoholic beverages, etc.), confectionery (hard candy, gum, gummi, jelly, pudding, mousse, cake, candy, cookies, crackers, biscuits, chocolate, ice confectionery ( Ice cream, ice candy, sherbet, shaved ice, etc.), sprinkles, dressing, seasoning, processed soybean food (tofu, miso, soy sauce, yuba, kinako, natto, etc.), processed meat food (hamburger, meatloaf, meatballs, etc.) Tsukune etc.), processed fish food (kamaboko, chikuwa etc.) Retort food, jelly-like food (jelly, agar-agar, a jelly-like beverages, and the like), and the like can be given. Examples of foods that can be prepared by adding and blending the peptide of the present invention include so-called health foods, functional foods, functional labeling foods, dietary supplements, supplements, foods for specified health use, foods for the sick, and combinations for the sick It may be a food (Ministry of Health, Labor and Welfare, a special-purpose food) or a food for the elderly (Ministry of Health, Labor and Welfare, a special-purpose food), uncoated tablet, film-coated tablet, sugar-coated tablet, granule, powder, tablet, capsule (hard capsule and soft capsule And chewable type, syrup type, drink type, etc. The preparation of food containing the peptide according to the present invention can be carried out by a method known per se.
　次に実施例により本発明を更に具体的に説明する。しかし下記の実施例は本発明の範囲を限定するものではない。 Next, the present invention will be described more specifically with reference to examples. However, the following examples do not limit the scope of the present invention.
　＜方法＞
　（グレリン分泌活性の評価）
　グレリン分泌細胞MGN3-1を96ウェルプレートに1 x 105 cells/well播種し、培地中24時間培養した。培養後、細胞をDPBSで洗浄し、試験物質100μL（Buffer：50 μM sodium octanoate/DMEM）を添加した。なお、試験物質を添加しないBufferのみをcontrolとした。さらに4時間培養をした後に、培地を回収し遠心分離処理により上清（supernatant）を得た。上清に1N HClを10μL添加し、-80℃で保存をした。 <Method>
(Evaluation of ghrelin secretion activity)
Ghrelin-secreting cells MGN3-1 were seeded at 1 × 10 5 cells / well in a 96-well plate and cultured in a medium for 24 hours. After culturing, the cells were washed with DPBS, and 100 μL of a test substance (Buffer: 50 μM sodium octanoate / DMEM) was added. Only the buffer to which no test substance was added was used as a control. After further culturing for 4 hours, the medium was collected and a supernatant was obtained by centrifugation. 10 μL of 1N HCl was added to the supernatant and stored at −80 ° C.
　試料中のグレリンの濃度をELISA法（Bertin Pharma社製、Ghrelin (Acylated) EIAKit A05117）により評価した。 The concentration of ghrelin in the sample was evaluated by ELISA (Bertin Pharma, Ghrelin (Acylated) EIAKit A05117).
　＜製造例＞
　（酵素消化物）
　精製したβコングリシニン（β-CG）タンパク質と消化酵素とを、酵素：β-CG＝１：１００（重量比、β-CGの終濃度：20 mg/ml）で混合し、添付のバッファー中で反応を行った。 <Production example>
(Enzyme digest)
Purified β-conglycinin (β-CG) protein and digestive enzyme are mixed with enzyme: β-CG = 1: 100 (weight ratio, final concentration of β-CG: 20 mg / ml), and attached in the attached buffer. Reaction was performed.
　使用した酵素と反応条件は以下の通りとした：
（i）トリプシン（SIGMA社製、T1426）；反応温度：37℃、反応時間：5時間、反応pH：7.5。 The enzymes and reaction conditions used were as follows:
(I) Trypsin (manufactured by SIGMA, T1426); reaction temperature: 37 ° C., reaction time: 5 hours, reaction pH: 7.5.
　上記の反応時間の経過後、試料をボイル（100℃、10分間）し、酵素反応を停止した。 After the above reaction time, the sample was boiled (100 ° C., 10 minutes) to stop the enzyme reaction.
　（ペプチド）
　定法により、ペプチドNKNPFLFGSNR（配列番号１；実施例１）、ペプチドNKNPFLFGSNRFETLFKNQYGR（配列番号２；実施例２）、ペプチドFETLFKNQYGR（配列番号３；比較例１）ペプチドHKNKNPFLFGSNR（配列番号４；比較例２）及びNPFLFGSNR（配列番号５；比較例３）を合成した。 (peptide)
According to a conventional method, peptide NKNPFLFGSNR (SEQ ID NO: 1; Example 1), peptide NKNPFLFGSNRFETLFKNQYGR (SEQ ID NO: 2; Example 2), peptide FETLFKNQYGR (SEQ ID NO: 3; Comparative Example 1), peptide HKNKNPFLFGSNR (SEQ ID NO: 4; Comparative Example 2) and NPFLFGSNR (SEQ ID NO: 5; Comparative Example 3) was synthesized.
　（統計解析）
　試験により得られたたデータを、試行数ｎの平均（Mean）と標準誤差（Standard error of the mean、SEM）との和（Mean±SEM）で表した。2群間の比較にはt検定を用いた。3群間以上の比較には、データを１方向ANOVAにより解析し、引き続いて多重比較のためのTukey-Kramer試験を行った。p<0.05の場合（図中、”*”）に、有意差ありと判定した。 (Statistical analysis)
Data obtained by the test was expressed as the sum (Mean ± SEM) of the mean (Mean) of the number of trials n and the standard error of the mean (SEM). A t-test was used for comparison between the two groups. For comparisons between 3 groups or more, data were analyzed by one-way ANOVA, followed by a Tukey-Kramer test for multiple comparisons. When p <0.05 (“*” in the figure), it was determined that there was a significant difference.
　＜試験例＞
　試験例１：β-CGのトリプシン消化物
　β-CGのトリプシン消化物（β-CG Trypsin Digest）について、グレリン分泌細胞MGN3-1を用いて、グレリン分泌に与える影響を評価した。 <Test example>
Test Example 1: β-CG Trypsin Digest β-CG Trypsin Digest was evaluated for the effect on ghrelin secretion using ghrelin-secreting cells MGN3-1.
　結果を図１に示す。図中、横軸「Ghrelin (% of control)」は、対照（Control, 試験物質を添加しないBufferのみ）をMGN3-1に添加した場合のグレリン分泌量の測定値に対する割合（質量百分率）を示す。 The results are shown in FIG. In the figure, the horizontal axis “Ghrelin (% of control)” indicates the ratio (mass percentage) to the measured value of ghrelin secretion when MGN3-1 is added to the control (Control, Buffer without addition of test substance). .
　β-CGのトリプシン消化物（β-CG Trypsin Digest）は、グレリン分泌量を増加させる、グレリン分泌を促進する活性を有することが明らかとなった。 Β-CG trypsin digest (β-CG Digest) was found to have an activity to increase ghrelin secretion and promote ghrelin secretion.
　試験例２：ペプチド（１）
　β-CGのトリプシン消化物に含まれるペプチドNKNPFLFGSNRFETLFKNQYGR（配列番号２）について、試験例１と同様にしてグレリン分泌に与える影響を評価した。 Test Example 2: Peptide (1)
About the peptide NKNPFLFGSNRFETLFKNQYGR (sequence number 2) contained in the tryptic digest of β-CG, the effect on ghrelin secretion was evaluated in the same manner as in Test Example 1.
　さらに、ペプチドNKNPFLFGSNRFETLFKNQYGRのN末端側及びC末端側のそれぞれに対応する、ペプチドNKNPFLFGSNR（配列番号１）及びペプチドFETLFKNQYGR（配列番号３）についても、グレリン分泌に与える影響を評価した。 Furthermore, the effect of peptide NKNPFLFGSNR (SEQ ID NO: 1) and peptide FETLFKNQYGR (SEQ ID NO: 3) corresponding to the N-terminal side and C-terminal side of peptide NKNPFLFGSNRFETLFKNQYGR, respectively, on ghrelin secretion was evaluated.
　結果を図２に示す（n=3-4）。ペプチドNKNPFLFGSNRFETLFKNQYGR（配列番号２）は、1000μMで強力なグレリン分泌を促進する活性有することが明らかとなった。また、ペプチドNKNPFLFGSNR（配列番号１）は、1μM以上で強力なグレリン分泌を促進する活性を有することが明らかとなった。 The results are shown in Fig. 2 (n = 3-4). The peptide NKNPFLFGSNRFETLFKNQYGR (SEQ ID NO: 2) was found to have an activity of promoting strong ghrelin secretion at 1000 μM. In addition, the peptide NKNPFLFGSNR (SEQ ID NO: 1) was found to have an activity of promoting strong ghrelin secretion at 1 μM or more.
　試験例３：ペプチド（２）
　ペプチドHKNKNPFLFGSNR（配列番号４）及びNPFLFGSNR（配列番号５）についても、グレリン分泌に与える影響を評価した。また、対象としてグレリンの分泌を促進することが知られている100μMノルアドレナリン（Noradrenaline）についても試験を行った。 Test Example 3: Peptide (2)
The effects of the peptides HKNKNPFLFGSNR (SEQ ID NO: 4) and NPFLFGSNR (SEQ ID NO: 5) on ghrelin secretion were also evaluated. Also tested was 100 μM Noradrenaline, which is known to promote ghrelin secretion as a subject.
　結果を図３に示す（n=3-4）。ペプチドNKNPFLFGSNR（配列番号１）の結果との対比から、NKNPFLFGSNRの11残基がグレリンの分泌促進において特に重要であることが明らかとなった。 The results are shown in Fig. 3 (n = 3-4). From the comparison with the result of peptide NKNPFLFGSNR (SEQ ID NO: 1), it was revealed that 11 residues of NKNPFLFGSNR are particularly important in promoting ghrelin secretion.
　参考試験例
　試験例２及び３で用いた各ペプチドが、β-CGのトリプシン消化物に含まれることを質量分析法により確認した。結果を表１に示す。表中、RTは保持時間（retention time）を示す。 Reference Test Example It was confirmed by mass spectrometry that each peptide used in Test Examples 2 and 3 was contained in a tryptic digest of β-CG. The results are shown in Table 1. In the table, RT indicates a retention time.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　試験例４：グレリン分泌量増加の作用機構
　本発明のペプチドによる作用が、グレリンの合成を促進するか、すでに合成されたグレリンの分泌を促進するのかを検証した。試験物質としては、β-CGのトリプシン消化物及びペプチドNKNPFLFGSNR（配列番号１）を用いた。 Test Example 4: Mechanism of increase in ghrelin secretion amount It was verified whether the action of the peptide of the present invention promotes the synthesis of ghrelin or the secretion of already synthesized ghrelin. As test substances, trypsin digest of β-CG and peptide NKNPFLFGSNR (SEQ ID NO: 1) were used.
　培養液中のグレリンについては、試験例１及び２と同様にして評価した。 The ghrelin in the culture solution was evaluated in the same manner as in Test Examples 1 and 2.
　細胞中のグレリンについては、培地を除去した後、グレリン分泌細胞MGN3-1にRIPA Buffer (ナカライテスク社製)を添加して溶解し、溶解液を遠心分離することにより上清（supernatant）を得た。上清に1N HClを10μL添加し、-80℃で保存をした。試料中のグレリンの濃度は試験例１及び２と同様にして評価した。 Regarding ghrelin in cells, after removing the medium, RIPA Buffer 分泌 (manufactured by Nacalai Tesque) was added to ghrelin-secreting cell MGN3-1 to dissolve it, and the supernatant was obtained by centrifuging the lysate. It was. 10 μL of 1N HCl was added to the supernatant and stored at −80 ° C. The concentration of ghrelin in the sample was evaluated in the same manner as in Test Examples 1 and 2.
　結果を図４に示す（n=4-5）。図４Ａには培養液中のグレリンの定量結果を示す。図４Ｂには細胞中のグレリンの定量結果を示す。細胞中のグレリンは増加していないため、本発明のペプチドはグレリン合成促進ではなく分泌を亢進する作用を有することが明らかとなった。  The results are shown in FIG. 4 (n = 4-5). FIG. 4A shows the quantitative results of ghrelin in the culture solution. FIG. 4B shows the quantitative results of ghrelin in the cells. Since ghrelin in the cells did not increase, it was revealed that the peptide of the present invention has an action of enhancing secretion rather than promoting ghrelin synthesis.
　試験例５：作用機構
　グレリンの分泌促進に至る作用機構を検証した。ペプチドNKNPFLFGSNR（配列番号１）が、細胞内のcAMP濃度及び細胞内カルシウムイオン濃度（[Ca2+]）に与える影響を評価した。 Test Example 5: Mechanism of action The mechanism of action leading to the promotion of ghrelin secretion was verified. The effect of peptide NKNPFLFGSNR (SEQ ID NO: 1) on intracellular cAMP concentration and intracellular calcium ion concentration ([Ca 2+ ]) was evaluated.
　細胞内cAMP濃度の測定については、グレリン分泌細胞MGN3-1を96ウェルプレートに1 x 105 cells/well播種し、培地中で24時間培養した。培養後、細胞をDPBSで洗浄し、試験物質45μL（Buffer：0.5 mM IBMXを含むKrebs-Ringer-HEPES buffer）を添加した。さらに30分間培養をした後に、細胞内のcAMPをHitHunter cAMP Assay for small molecules (DiscoveRx社製)を用いて定量した。 For measurement of intracellular cAMP concentration, ghrelin-secreting cells MGN3-1 were seeded at 1 × 10 5 cells / well in a 96-well plate and cultured in a medium for 24 hours. After culturing, the cells were washed with DPBS, and 45 μL of a test substance (Buffer: Krebs-Ringer-HEPES buffer containing 0.5 mM IBMX) was added. After further incubation for 30 minutes, intracellular cAMP was quantified using HitHunter cAMP Assay for small molecules (DiscoveRx).
　細胞内カルシウムイオン濃度については、文献：Kagebayashi T et.al. Mol. Nutr. Food Res. 2012 Sep;56(9):1456-63に記載の方法に従って測定した。 The intracellular calcium ion concentration was measured according to the method described in the literature: Kagebayashi Tet.al. Mol. Nutr. Food Res. 2012 Sep; 56 (9): 1456-63.
　結果を図５に示す。図５Ａは、細胞内のcAMPの定量結果を示す。図５Ｂは、細胞内カルシウムイオン濃度の変化（Δ[Ca2+]i）を示す。ノルアドレナリンは、細胞内のcAMP濃度及び細胞内カルシウムイオン濃度の増加を介して、グレリンの分泌を促進することが知られている。Gs型の三量体Gタンパク質の関与が知られている。これに対して、本発明のペプチドは、細胞内のcAMP濃度及び細胞内カルシウムイオン濃度の変化を介さずにグレリンの分泌を促進するため、既知の経路とは異なる経路を介していると考えられる。 The results are shown in FIG. FIG. 5A shows the quantitative results of intracellular cAMP. FIG. 5B shows changes in intracellular calcium ion concentration (Δ [Ca 2+ ] i). Noradrenaline is known to promote ghrelin secretion through an increase in intracellular cAMP concentration and intracellular calcium ion concentration. Involvement of Gs-type trimeric G protein is known. On the other hand, since the peptide of the present invention promotes ghrelin secretion without changing the intracellular cAMP concentration and intracellular calcium ion concentration, it is considered that the peptide is via a different route from the known route. .
　試験例６：生体への投与
　マウス（ddYマウス、雄、34～42g）にβ-CGのトリプシン消化物（30 mg/kg）及びペプチドNKNPFLFGSNR（配列番号１）（0.3 mg/kg）を投与して、摂食行動への影響を評価した。 Test Example 6: Administration to a living body A trypsin digest of β-CG (30 mg / kg) and peptide NKNPFLFGSNR (SEQ ID NO: 1) (0.3 mg / kg) were administered to mice (ddY mice, male, 34 to 42 g). The effect on eating behavior was evaluated.
　単回投与については、生理食塩水に溶解したβ-CGのトリプシン消化物及びペプチドNKNPFLFGSNR（配列番号１）をマウスに経口投与した後、予め秤量した飼料（CE-2、日本クレア社製）を与え、6時間後に飼料重量を測定することにより摂食量（Food intake (g/6 hrs)）を算出した。 For single administration, β-CG tryptic digest dissolved in physiological saline and peptide NKNPFLFGSNR (SEQ ID NO: 1) were orally administered to mice, and then pre-weighed feed (CE-2, manufactured by Claire Japan) The amount of food intake (Food intake (g / 6 hrs)) was calculated by measuring the feed weight after 6 hours.
　混餌投与については、粉末飼料（CE-2、日本クレア社製）にペプチドNKNPFLFGSNR（配列番号１）を混合した（飼料中0.0003%, 0.0003% of diet）。予め秤量しておいた混合飼料をマウスに与え、24時間後に飼料重量を測定することにより摂食量（Food intake (g/24 hrs)）を算出した。 For mixed feeding, peptide NKNPFLFGSNR (SEQ ID NO: 1) was mixed with powdered feed (CE-2, manufactured by CLEA Japan) (0.0003% in feed, 0.0003% of diet). The pre-weighed mixed feed was given to the mice, and the feed weight (Food intake (g / 24 hrs)) was calculated by measuring the weight of the feed after 24 hours.
　結果を図６に示す。図６Ａは単回投与の結果を示す（n=5-6又はn=10-12）。図６Ｂは混餌投与の結果を示す（n=5-6又はn=10-12）。β-CGのトリプシン消化物及びペプチドNKNPFLFGSNR（配列番号１）は、経口投与によりマウスの摂食を促進することが明らかとなった。  The results are shown in FIG. FIG. 6A shows the results of a single dose (n = 5-6 or n = 10-12). FIG. 6B shows the results of dietary administration (n = 5-6 or n = 10-12). It was revealed that trypsin digest of β-CG and peptide NKNPFLFGSNR (SEQ ID NO: 1) promoted feeding of mice by oral administration.

Claims (10)

  1. 　Ｎ末端にアミノ酸配列NKNPFLFGSNR（配列番号１）を有するペプチド。 A peptide having the amino acid sequence NKNPFLFGSNR (SEQ ID NO: 1) at the N-terminus.
  2. 　大豆のベータコングリシニンタンパク質のトリプシン消化物に由来する、請求項１に記
    載のペプチド。     The peptide according to claim 1, which is derived from a tryptic digest of soybean beta-conglycinin protein.
  3. 　請求項１または２に記載のペプチドを有効成分とする医薬組成物。 A pharmaceutical composition comprising the peptide according to claim 1 or 2 as an active ingredient.
  4. 　請求項１または２に記載のペプチドを有効成分とするグレリン分泌促進剤。 A ghrelin secretion promoter comprising the peptide according to claim 1 or 2 as an active ingredient.
  5. 　請求項１または２に記載のペプチドを含有する食品。 A food containing the peptide according to claim 1 or 2.
  6. 　請求項１または２に記載のペプチドを添加することを特徴とする食品。 A food comprising the peptide according to claim 1 or 2 added thereto.
  7. 　食欲促進及び／または成長ホルモンの分泌促進のための、請求項５または６に記載の食
    品。     The food according to claim 5 or 6 for promoting appetite and / or promoting secretion of growth hormone.
  8. 　請求項１または２に記載のペプチドを必要とする患者または予備群に投与する工程を含
    む、食欲を促進及び／または成長ホルモンの分泌を促進する方法。     A method for promoting appetite and / or promoting secretion of growth hormone, comprising the step of administering to the patient or preparatory group in need of the peptide according to claim 1 or 2.
  9. 　食欲を促進及び／または成長ホルモンの分泌を促進するための、請求項１または２に記
    載のペプチド。     The peptide according to claim 1 or 2 for promoting appetite and / or promoting secretion of growth hormone.
  10. 　食欲を促進及び／または成長ホルモンの分泌を促進するための医薬または食品を製造するための、請求項１または２に記載のペプチドの使用。  Use of the peptide according to claim 1 or 2 for producing a medicament or food for promoting appetite and / or promoting secretion of growth hormone.
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