WO2021107080A1 - Peptide - Google Patents

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Publication number
WO2021107080A1
WO2021107080A1 PCT/JP2020/044180 JP2020044180W WO2021107080A1 WO 2021107080 A1 WO2021107080 A1 WO 2021107080A1 JP 2020044180 W JP2020044180 W JP 2020044180W WO 2021107080 A1 WO2021107080 A1 WO 2021107080A1
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WIPO (PCT)
Prior art keywords
peptide
amino acid
seq
ghrelin
promoting
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PCT/JP2020/044180
Other languages
French (fr)
Japanese (ja)
Inventor
耕作 大日向
加奈 谷川
志門 阿部
浩 岩倉
大 佐藤
鈴木　秀幸
Original Assignee
国立大学法人京都大学
公益財団法人かずさＤｎａ研究所
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Publication of WO2021107080A1 publication Critical patent/WO2021107080A1/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
    • A23L33/17—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
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • 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
    • 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
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to a novel peptide.
  • Ghrelin is a gastrointestinal hormone that promotes appetite and growth hormone secretion.
  • Japan which is facing a super-aging society, the development of ghrelin secretion-promoting substances that contribute to the improvement of anorexia and growth hormone secretion decrease associated with aging is desired, and the development of food-derived ghrelin-promoting substances is particularly desired. ing.
  • Patent Document 1 describes a peptide having a ghrelin secretagogue action.
  • An object of the present invention is to provide a novel peptide having a ghrelin secretagogue action, and pharmaceuticals and foods containing the peptide.
  • the present inventor has been diligently researching to solve the above problems. As a result, a peptide having a ghrelin secretagogue action was found in the digested product of gluten chymotrypsin. The present invention has been completed by further studying based on such findings.
  • Item 1 A peptide comprising the amino acid sequence YPTSL (SEQ ID NO: 1), the amino acid sequence LSVTSPQQVSY (SEQ ID NO: 2) or the amino acid sequence SQQQQPVLPQQPSF (SEQ ID NO: 3).
  • Item 2. The peptide according to Item 1, which is derived from a chymotrypsin digest of gluten.
  • Item 3. A pharmaceutical composition containing the peptide according to Item 1 or 2 as an active ingredient.
  • Item 4. A ghrelin secretagogue containing the peptide according to Item 1 or 2 as an active ingredient.
  • Item 5. A food containing the peptide according to Item 1 or 2.
  • Item 7. Item 5.
  • Item 8. A method for promoting appetite and / or promoting growth hormone secretion, which comprises the step of administering the peptide according to item 1 or 2 to a patient or a preliminary group requiring the peptide.
  • 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 secretagogue-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 loss, and locomotive syndrome.
  • the peptide of the present invention has low side effects and is suitable for long-term administration.
  • the pharmaceutical composition and food of the present invention are effective by oral administration.
  • the peptide of the present invention is an enzymatic digest of gluten, side effects are not a problem.
  • gluten is abundant in wheat, it can be produced at low cost.
  • the effect of enzyme digests on ghrelin secretion The effect of peptides on ghrelin secretion.
  • the effect of peptides on ghrelin secretion The effect of peptides on ghrelin secretion.
  • the effect of peptides on ghrelin secretion Effect of oral administration on feeding behavior.
  • the peptide of the present invention is a peptide containing a 5-residue amino acid sequence of YPTSL (SEQ ID NO: 1), a peptide containing an 11-residue amino acid sequence of LSVTSPQQVSY (SEQ ID NO: 2), or 14 residues of SQQQQPVLPQQPSF (SEQ ID NO: 3).
  • SEQ ID NO: 1 a 5-residue amino acid sequence of YPTSL
  • SEQ ID NO: 2 a peptide containing an 11-residue amino acid sequence of LSVTSPQQVSY
  • SEQ ID NO: 3 14 residues of SQQQPVLPQQPSF
  • the peptide of the present invention is a peptide consisting of a 5-residue amino acid sequence of YPTSL (SEQ ID NO: 1), a peptide consisting of an 11-residue amino acid sequence of LSVTSPQQVSY (SEQ ID NO: 2), or SQQQQPVLPQQPSF (SEQ ID NO: 3) 14 It also includes peptides consisting of the amino acid sequence of residues.
  • amino acid sequences shown in SEQ ID NO: 2 the 1st to 10th amino acid sequences, the 1st to 9th amino acid sequences, the 1st to 8th amino acid sequences, and the 1st to 7th amino acid sequences counting from the N-terminal or C-terminal.
  • Another aspect of the present invention is a peptide consisting of the 1st to 6th amino acid sequences, the 1st to 5th amino acid sequences, the 1st to 4th amino acid sequences, and the 1st to 3rd amino acid sequences.
  • amino acid sequences shown in SEQ ID NO: 3 the 1st to 13th amino acid sequences, the 1st to 12th amino acid sequences, the 1st to 11th amino acid sequences, and the 1st to 10th amino acid sequences counted from the N-terminal or the C-terminal.
  • a peptide consisting of the 1st to 3rd amino acid sequences is also mentioned as another aspect of the present invention.
  • an amino acid residue can be added to the N-terminal side and / or the C-terminal side (preferably the N-terminal side) of the above amino acid sequence.
  • the number of amino acid residues to be added is not limited, and is about 20 amino acid residues, preferably about 10 amino acid residues, more preferably about 5 amino acid residues, and further preferably 4, 3, 2, or 1 amino acid. be able to.
  • the amino acids that make up the peptide are either L-form amino acids, D-form amino acids, or DL-form amino acids (if the amino acids are a mixture of D-form and L-form, racemic and one of the amino acids in excess of enantiomer). Included) can be used.
  • a peptide consisting of only L-form amino acids or only D-form amino acids, particularly a peptide consisting of only L-form amino acids is preferable.
  • the peptide used in the present invention may be in any form of each enantiomer or diastereomer or a mixture thereof in any ratio. Separation of enantiomers or diastereomers can be performed by using a normal column, using an optically active column, or by introducing an optically active group and performing optical resolution in the form of a derivative, and then removing the optically active group. Any known method can be used, such as a method of forming a salt with an optically active acid or base and performing optical resolution.
  • Peptides can have modifications.
  • the amino terminus (N-terminus) of the peptide may be a free amino group (NH 2- ) or one having 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 one having 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, nitrate, phosphoric acid, hydrobromic acid and perchloric acid, citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid and p-toluenesulfonic acid.
  • Salts of organic acids such as benzenesulfonic acid, methanesulfonic acid, trifluoroacetic acid and the like.
  • 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 water (in the case of hydrates), methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, dimethoxyethane and the like. Things can be mentioned.
  • the peptide of the present invention can be obtained by hydrolysis of chymotrypsin of gluten.
  • Chymotrypsin is a pancreas-derived enzyme in the case of mammals such as humans, and is a known proteolytic enzyme (protease) (EC.3.4.21.1, EC.3.4.21.2). Chymotrypsin can be used as a food additive in Japan. As the chymotrypsin, commercially available products such as reagent grade and food additive grade can be used.
  • the substrate hydrolyzed by chymotrypsin is not particularly limited as long as it contains gluten.
  • gluten for example, wheat itself, wheat protein, refined gluten and the like.
  • 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, about 2 to 8 hours, and the like.
  • the pH at which the reaction is carried out can be appropriately selected from pH 6.5 to 8.5 and pH 7 to 8. In one preferred embodiment, the reaction can be carried out under the conditions of a temperature of about 30 to 40 ° C. and a pH of 6.5 to 8.5 (particularly, a pH of about 7.5) for about 2 to 8 hours.
  • the peptide of the present invention may not be obtained.
  • chymotrypsin is inactivated by heating to a temperature at which chymotrypsin is inactivated (for example, heating at a temperature exceeding 80 ° C. for about 5 to 60 minutes).
  • the hydrolysis reaction product may be used as it is, or the peptide of the active ingredient may be separated and used by purification.
  • the peptide of the present invention can also be obtained by a peptide synthesis method. That is, in the liquid phase method or the solid phase method, which is a method usually used for peptide synthesis, a method using an active ester such as HBTU for a raw material having a reactive carboxyl group and a raw material having a reactive amino group, or a carbodiimide It can be condensed by a usual method in peptide synthesis such as a method using a coupling agent such as. If the resulting condensate has a protecting group, it can also be produced by removing the protecting group.
  • a peptide synthesis method That is, in the liquid phase method or the solid phase method, which is a method usually used for peptide synthesis, a method using an active ester such as HBTU for a raw material having a reactive carboxyl group and a raw material having a reactive amino group, or a carbodiimide It can be condensed by a usual method in peptide synthesis
  • the amino-protecting group include benzyloxycarbonyl (CBZ), t-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) and the like.
  • the carboxyl group protective agent include groups capable of forming an alkyl ester, a benzyl ester, etc.
  • the C-terminal carboxyl group is a chlorotrityl resin, a chloromethyl resin, an 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 with 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-terminal of the peptide and the resin is further cleaved.
  • the peptides of the invention are purified according to conventional methods. Examples thereof include ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like. The synthesis of the synthesized peptide is analyzed by a protein sequencer, GC-MS, etc., which reads the amino acid sequence from the C-terminal by the Edman degradation method.
  • 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 biological 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 has an appetite-promoting effect and a growth hormone secretagogue-promoting effect.
  • 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 secretion promoting action.
  • an appetite promoting action feeding promoting action
  • a growth hormone secretion promoting action a growth hormone secretion promoting action.
  • the peptides of the present invention can be used for the treatment of anorexia in the elderly and the like.
  • the secretagogue action of growth hormone it can be used for prevention of muscle loss, prevention or treatment of locomotive syndrome (locomo), and the like.
  • the fact that the peptide of the present invention promotes the amount of ghrelin secreted can be evaluated by a method known to those skilled in the art.
  • the amount of ghrelin secreted can be measured by quantifying.
  • Ghrelin can be quantified by an immunochemical method such as ELISA (Enzyme-Linked ImmunoSorbentAssay).
  • 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 same is not particularly limited, and any of oral administration, parenteral administration, and rectal administration can be adopted, orally or parenterally. Can be administered. Of these, oral administration is preferable from the viewpoint of high effect.
  • the dose of this peptide varies depending on the administration method, the condition and age of the person to be administered, but is usually 0.01 mg / kg to 500 mg / kg, preferably 0.05 mg / kg to 100 mg / kg per day for an adult. , More preferably 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 carrier for preparation.
  • a carrier for preparation a substance that is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.
  • the peptides of the invention can be used as pharmaceuticals or foods themselves, or tablets (uncoated tablets, sugar-coated tablets, effervescent tablets, alone or with suitable non-toxic carriers, diluents or excipients).
  • tablets uncoated tablets, sugar-coated tablets, effervescent tablets, alone or with suitable non-toxic carriers, diluents or excipients.
  • suitable non-toxic carriers diluents or excipients.
  • It can be a food- or pharmaceutical preparation such as a sustained-release preparation such as an enteric-coated tablet, capsule, or granule.
  • 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.
  • Dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections and the like. These formulations are prepared according to a conventional method. The liquid preparation may be dissolved or suspended in water or another suitable solvent at the time of use. Further, tablets and granules may be coated by a well-known method. In the case of an injection, the peptide of the present invention is prepared by dissolving it in water, but it may be dissolved in physiological saline or glucose solution if necessary, or a buffer or preservative may be added. Good.
  • compositions can 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 ingredients of therapeutic value.
  • the active ingredient and excipient ingredients such as lactose, starch, crystalline cellulose, calcium lactate, silicic anhydride, etc. are mixed to form a powder, or sucrose, if necessary.
  • a binder such as hydroxypropyl cellulose or polyvinylpyrrolidone, a disintegrant such as carboxymethyl cellulose or carboxymethyl cellulose calcium is added, and wet or dry granulation is performed to obtain granules.
  • these powders and granules may be tableted as they are or by 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 methyl methacrylate-methylmethacrylate polymer to form an enteric solvent preparation, or to be coated with ethyl cellulose, carnauba wax, cured oil, etc. to form a long-acting preparation. You can also.
  • enteric solvent base such as hydroxypropylmethylcellulose phthalate or methyl methacrylate-methylmethacrylate polymer to form an enteric solvent preparation
  • ethyl cellulose, carnauba wax, cured oil, etc. to form a long-acting preparation.
  • To manufacture capsules hard capsules are filled with powders or granules, or the active ingredient is used as it is or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc. and then coated with a gelatin film to form soft capsules. Can be done.
  • the active ingredient and sweeteners such as sucrose, sorbitol, and glycerin are dissolved in water to make a transparent syrup, and essential oil, ethanol, etc. are added to make an elixir.
  • Arabia gum, tragant, polysorbate 80, sodium carboxymethyl cellulose and the like may be added as an emulsion or a suspending agent.
  • a flavoring agent, a coloring agent, a preservative, or the like may be added to these liquid preparations.
  • Specific forms of foods that can be prepared by adding or blending the peptides according to the present invention include, for example, beverages (coffee, cocoa, juice, soft beverages, mineral beverages, tea beverages, green tea, tea, crow dragon tea, dairy beverages). , Lactic acid drinks, yogurt drinks, carbonated drinks, other non-alcoholic drinks, alcoholic drinks, etc.), confectionery (hard candy, gum, gummy, jelly, pudding, mousse, cake, candy, cookies, crackers, biscuits, chocolate, ice cream ( Ice cream, ice candy, sherbet, shaved ice, etc.), sprinkles, dressings, seasonings, processed soybean foods (tofu, miso, soy sauce, yuba, kina flour, natto, etc.), processed meat foods (hamburger, meat loaf, meat balls, etc.) Tsukune, etc.), processed fish meat foods (kamaboko, chikuwa, etc.), retort foods, jelly-like foods (jelly, agar, jelly-
  • Foods that can be prepared by adding or blending the peptides of the present invention include so-called health foods, functional foods, foods with functional claims, nutritional 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 type of special-purpose food) or a food for the elderly (Ministry of Health, Labor and Welfare, a type of special-purpose food), uncoated tablets, film-coated tablets, sugar-coated tablets, granules, powders, tablets, capsules (hard capsules and soft capsules). ), Chewable type, syrup type, drink type, etc. can also be used.
  • the food containing and blending the peptide according to the present invention can be prepared by a method known per se.
  • Ghrelin-secreting cells MGN3-1 were seeded in 96-well plates at 1 x 10 5 cells / well and cultured in medium for 24 hours. After culturing, the cells were washed with DPBS, and 100 ⁇ L (Buffer: 50 ⁇ M sodium octanoate / DMEM) of the test substance was added. Only the Buffer to which the test substance was not added was used as the control. After further culturing for 4 hours, the medium was collected and centrifuged to obtain a supernatant. 10 ⁇ L of 1N HCl was added to the supernatant and stored at -80 ° C.
  • the concentration of ghrelin in the sample was evaluated by the ELISA method (Ghrelin (Acylated) EIA Kit A05117 manufactured by Bertin Pharma).
  • the enzymes used and the reaction conditions were as follows: (I) Trypsin (manufactured by Sigma-Aldrich, T8003); reaction temperature: 37 ° C., reaction time: 5 hours, reaction pH: 7.5. (Ii) Chymotrypsin (manufactured by Sigma-Aldrich, C4129); reaction temperature: 37 ° C., reaction time: 5 hours, reaction pH: 7.5.
  • the sample was boiled (100 ° C., 10 minutes) to stop the enzymatic reaction.
  • peptide YPTSL SEQ ID NO: 1; Example 1
  • peptide LSVTSPQQVSY SEQ ID NO: 2; Example 2
  • peptide SQQQQPVLPQQPSF SEQ ID NO: 3; Example 3
  • peptide HVSAEHQAASL SEQ ID NO: 4; Comparative Example 1
  • Peptide YPTS SEQ ID NO: 5; Comparative Example 2
  • Peptide PTSL SEQ ID NO: 6; Comparative Example 3
  • Peptide QQPQQQYPLGQGSF SEQ ID NO: 7; Comparative Example 4
  • Peptide YLSVTSPQQVSY SEQ ID NO: 8; Comparative Example 5
  • Peptide QQPQQYPSGQGSF SEQ ID NO: 9; Comparative Example 6
  • Test Example 1 Enzyme digestion of gluten The effect of gluten trypsin or chymotrypsin digest on ghrelin secretion was evaluated using ghrelin-secreting cell MGN3-1.
  • Fig. 1 The results are shown in Fig. 1.
  • the vertical axis "Ghrelin (% of control)" indicates the ratio (percentage) of the ghrelin secretion amount to the measured value when the control (only Buffer to which the test substance is not added) is added to MGN3-1.
  • Test Example 2 Peptide (1) The effects of the peptide YPTSL (SEQ ID NO: 1; Example 1) and the peptide HVSAEHQAASL (SEQ ID NO: 4; Comparative Example 1) contained in the digested product of gluten chymotrypsin on ghrelin secretion were evaluated in the same manner as in Test Example 1.
  • the results are shown in Fig. 2.
  • the peptide YPTSL (SEQ ID NO: 1; Example 1) was found to have an activity of promoting ghrelin secretion.
  • the peptide HVSAEHQAASL (SEQ ID NO: 4; Comparative Example 1) was not confirmed to have an activity of promoting ghrelin secretion.
  • Test Example 3 Peptide (2) The effect of 1 mM or 10 mM peptide YPTSL (SEQ ID NO: 1; Example 1) on ghrelin secretion was evaluated in the same manner as in Test Example 1.
  • Test Example 4 Peptide (3) Effects of peptide YPTSL (SEQ ID NO: 1; Example 1), peptide YPTS (SEQ ID NO: 5; Comparative Example 2), and peptide PTSL (SEQ ID NO: 6; Comparative Example 3) on ghrelin secretion in the same manner as in Test Example 1. Was evaluated.
  • Test Example 5 Peptide (4) Peptide QQPQQQYPLGQGSF (SEQ ID NO: 7; Comparative Example 4), peptide LSVTSPQQVSY (SEQ ID NO: 2; Example 2), peptide SQQQQPVLPQQPSF (SEQ ID NO: 3; Example 3), peptide YLSVTSPQQVSY (SEQ ID NO: 8) contained in the digested product of gluten chymotrypsin. Comparative Example 5) and peptide QQPQQQYPSGQGSF (SEQ ID NO: 9; Comparative Example 6) were evaluated for their effect on ghrelin secretion in the same manner as in Test Example 1.
  • Fig. 5 The results are shown in Fig. 5. It was revealed that the peptide LSVTSPQQVSY (SEQ ID NO: 2; Example 2) and the peptide SQQQQPVLPQQPSF (SEQ ID NO: 3; Example 3) have an activity of promoting ghrelin secretion. In addition, it was revealed that the activity of promoting ghrelin secretion increases in a peptide concentration-dependent manner.
  • the peptide QQPQQQQYPLGQGSF (SEQ ID NO: 7; Comparative Example 4), the peptide YLSVTSPQQVSY (SEQ ID NO: 8; Comparative Example 5), and the peptide QQPQQQYPSGQGSF (SEQ ID NO: 9; Comparative Example 6) were confirmed to have an activity of promoting ghrelin secretion. There wasn't.
  • Test Example 6 In-vivo administration Mice (ddY mice, males, 42-56 g) were administered a digested product of gluten chymotrypsin to evaluate the effect on feeding behavior.
  • a pre-weighed feed (CE-2, manufactured by Japan Claire) is given, and the feed weight is measured after 20, 60, and 120 minutes. The amount of food intake was calculated accordingly.

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Abstract

Provided are a novel peptide which has a ghrelin secretion-promoting effect, and a medicine and food product which contain said peptide. Specifically provided is a peptide which contains the amino acid sequence YPTSL (sequence number 1), amino acid sequence LSVTSPQQVSY (sequence number 2) or amino acid sequence SQQQQPVLPQQPSF (sequence number 3).

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おり、特に食品由来のグレリン促進物質の開発が望まれている。 Ghrelin is a gastrointestinal hormone that promotes appetite and growth hormone secretion. In Japan, which is facing a super-aging society, the development of ghrelin secretion-promoting substances that contribute to the improvement of anorexia and growth hormone secretion decrease associated with aging is desired, and the development of food-derived ghrelin-promoting substances is particularly desired. ing.
　特許文献１には、グレリン分泌促進作用を有するペプチドについて記載されている。 Patent Document 1 describes a peptide having a ghrelin secretagogue action.
国際公開第2017/150548号International Publication No. 2017/150548
　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 secretagogue action, and pharmaceuticals and foods 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、グルテンのキモ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 been diligently researching to solve the above problems. As a result, a peptide having a ghrelin secretagogue action was found in the digested product of gluten chymotrypsin. The present invention has been completed by further studying based on such findings.
　すなわち、本発明は以下の態様を包含する。
項１．
アミノ酸配列YPTSL（配列番号１）、アミノ酸配列LSVTSPQQVSY（配列番号２）又はアミノ酸配列SQQQQPVLPQQPSF（配列番号３）を含むペプチド。
項２．
グルテンのキモトリプシン消化物に由来する、項１に記載のペプチド。
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。
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。
項５．
項１または２に記載のペプチドを含有する食品。
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。
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。
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。
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に記載のペプチドの使用。 That is, the present invention includes the following aspects.
Item 1.
A peptide comprising the amino acid sequence YPTSL (SEQ ID NO: 1), the amino acid sequence LSVTSPQQVSY (SEQ ID NO: 2) or the amino acid sequence SQQQQPVLPQQPSF (SEQ ID NO: 3).
Item 2.
Item 2. The peptide according to Item 1, which is derived from a chymotrypsin digest of gluten.
Item 3.
A pharmaceutical composition containing the peptide according to Item 1 or 2 as an active ingredient.
Item 4.
A ghrelin secretagogue containing the peptide according to Item 1 or 2 as an active ingredient.
Item 5.
A food containing the peptide according to Item 1 or 2.
Item 6.
A food product to which the peptide according to Item 1 or 2 is added.
Item 7.
Item 5. The food according to Item 5 or 6 for promoting appetite and / or promoting the secretion of growth hormone.
Item 8.
A method for promoting appetite and / or promoting growth hormone secretion, which comprises the step of administering the peptide according to item 1 or 2 to a patient or a preliminary group requiring the peptide.
Item 9.
Item 2. Use of the peptide according to Item 1 or 2 for promoting appetite and / or promoting the 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 secretagogue-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 loss, and locomotive syndrome. The peptide of the present invention has low side effects and is suitable for long-term administration. Moreover, the pharmaceutical composition and food of the present invention are effective by oral administration.
　本発明のペプチドは、グルテンの酵素消化物であるので、副作用は問題にならない。また、グルテンは、小麦中に多く含まれるので、低コストで製造できる。 Since the peptide of the present invention is an enzymatic digest of gluten, side effects are not a problem. In addition, since gluten is abundant in wheat, 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。The effect of enzyme digests on ghrelin secretion. ペプチドがグレリン分泌に与える影響。The effect of peptides on ghrelin secretion. ペプチドがグレリン分泌に与える影響。The effect of peptides on ghrelin secretion. ペプチドがグレリン分泌に与える影響。The effect of peptides on ghrelin secretion. ペプチドがグレリン分泌に与える影響。The effect of peptides on ghrelin secretion. 経口投与による摂食行動への影響。Effect of oral administration on feeding behavior.
　本発明のペプチドは、YPTSL（配列番号１）の５残基のアミノ酸配列を含むペプチド、LSVTSPQQVSY（配列番号２）の１１残基のアミノ酸配列を含むペプチド、又はSQQQQPVLPQQPSF（配列番号３）の１４残基のアミノ酸配列を含むペプチドである。また本発明のペプチドは、YPTSL（配列番号１）の５残基のアミノ酸配列からなるペプチド、LSVTSPQQVSY（配列番号２）の１１残基のアミノ酸配列からなるペプチド、又はSQQQQPVLPQQPSF（配列番号３）の１４残基のアミノ酸配列からなるペプチドをも包含する。 The peptide of the present invention is a peptide containing a 5-residue amino acid sequence of YPTSL (SEQ ID NO: 1), a peptide containing an 11-residue amino acid sequence of LSVTSPQQVSY (SEQ ID NO: 2), or 14 residues of SQQQQPVLPQQPSF (SEQ ID NO: 3). A peptide containing the amino acid sequence of the group. The peptide of the present invention is a peptide consisting of a 5-residue amino acid sequence of YPTSL (SEQ ID NO: 1), a peptide consisting of an 11-residue amino acid sequence of LSVTSPQQVSY (SEQ ID NO: 2), or SQQQQPVLPQQPSF (SEQ ID NO: 3) 14 It also includes peptides consisting of the amino acid sequence of residues.
　配列番号２に示すアミノ酸配列のうち、Ｎ末端またはＣ末端から数えて、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。また、配列番号３に示すアミノ酸配列のうち、Ｎ末端またはＣ末端から数えて、１～１３番目のアミノ酸配列、１～１２番目のアミノ酸配列、１～１１番目のアミノ酸配列、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。 Of the amino acid sequences shown in SEQ ID NO: 2, the 1st to 10th amino acid sequences, the 1st to 9th amino acid sequences, the 1st to 8th amino acid sequences, and the 1st to 7th amino acid sequences counting from the N-terminal or C-terminal. Another aspect of the present invention is a peptide consisting of the 1st to 6th amino acid sequences, the 1st to 5th amino acid sequences, the 1st to 4th amino acid sequences, and the 1st to 3rd amino acid sequences. Further, among the amino acid sequences shown in SEQ ID NO: 3, the 1st to 13th amino acid sequences, the 1st to 12th amino acid sequences, the 1st to 11th amino acid sequences, and the 1st to 10th amino acid sequences counted from the N-terminal or the C-terminal. Amino acid sequence 1st to 9th amino acid sequence, 1st to 8th amino acid sequence, 1st to 7th amino acid sequence, 1st to 6th amino acid sequence, 1st to 5th amino acid sequence, 1st to 4th amino acid sequence And a peptide consisting of the 1st to 3rd amino acid sequences is also mentioned as another aspect of the present invention.
　本発明のペプチドの別の態様として、上記アミノ酸配列に対して、Ｎ末端側及び／又はＣ末端側（好ましくは、Ｎ末端側）にアミノ酸残基を付加することができる。付加されるアミノ酸残基の個数は限定されず、２０アミノ酸残基程度、好ましくは１０アミノ酸残基程度、より好ましくは５アミノ酸残基程度、さらに好ましくは４、３、２、若しくは１アミノ酸とすることができる。 As another aspect of the peptide of the present invention, an amino acid residue can be added to the N-terminal side and / or the C-terminal side (preferably the N-terminal side) of the above amino acid sequence. The number of amino acid residues to be added is not limited, and is about 20 amino acid residues, preferably about 10 amino acid residues, more preferably about 5 amino acid residues, and further preferably 4, 3, 2, or 1 amino acid. be able to.
　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 that make up the peptide are either L-form amino acids, D-form amino acids, or DL-form amino acids (if the amino acids are a mixture of D-form and L-form, racemic and one of the amino acids in excess of enantiomer). Included) can be used. Preferably, a peptide consisting of only L-form amino acids or only D-form amino acids, particularly a peptide consisting of only L-form amino acids is preferable.
　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。 Further, when the peptide used in the present invention contains two or more asymmetric carbons, it may be in any form of each enantiomer or diastereomer or a mixture thereof in any ratio. Separation of enantiomers or diastereomers can be performed by using a normal column, using an optically active column, or by introducing an optically active group and performing optical resolution in the form of a derivative, and then removing the optically active group. Any known method can be used, such as a method of forming a salt with an optically active acid or base and performing optical resolution.
　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。 Peptides can have modifications. The amino terminus (N-terminus) of the peptide may be a free amino group (NH 2- ) or one having 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 one having 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, nitrate, phosphoric acid, hydrobromic acid and perchloric acid, citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid and p-toluenesulfonic acid. , Salts of organic acids such as benzenesulfonic acid, methanesulfonic acid, trifluoroacetic acid and the like. 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 water (in the case of hydrates), methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, dimethoxyethane and the like. Things can be mentioned.
　本発明のペプチドは、グルテンの、キモトリプシンの加水分解により得ることができる。 The peptide of the present invention can be obtained by hydrolysis of chymotrypsin of gluten.
　キモトリプシンは、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（EC.3.4.21.1、EC.3.4.21.2）。キモトリプシンは、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。キモトリプシンは、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。 Chymotrypsin is a pancreas-derived enzyme in the case of mammals such as humans, and is a known proteolytic enzyme (protease) (EC.3.4.21.1, EC.3.4.21.2). Chymotrypsin can be used as a food additive in Japan. As the chymotrypsin, commercially available products such as reagent grade and food additive grade can be used.
　キモトリプシンにより加水分解をする基質は、グルテンを含むものであればとくに限定されない。例えば、小麦それ自体、小麦タンパク質、精製したグルテンなどが挙げられる。 The substrate hydrolyzed by chymotrypsin is not particularly limited as long as it contains gluten. For example, wheat itself, wheat protein, refined gluten and the like.
　キモ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 chymotrypsin is performed under the conditions under which the peptide of the present invention can be obtained. 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, about 2 to 8 hours, and the like. The pH at which the reaction is carried out can be appropriately selected from pH 6.5 to 8.5 and pH 7 to 8. In one preferred embodiment, the reaction can be carried out under the conditions of a temperature of about 30 to 40 ° C. and a pH of 6.5 to 8.5 (particularly, a pH of about 7.5) for about 2 to 8 hours.
　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。 Under conditions where hydrolysis is excessive, the peptide of the present invention may not be obtained.
　必要に応じて、キモ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、キモトリプシンを失活させる。 If necessary, chymotrypsin is inactivated by heating to a temperature at which chymotrypsin is inactivated (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 hydrolysis reaction product may be used as it is, or the peptide of the active ingredient 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 a peptide synthesis method. That is, in the liquid phase method or the solid phase method, which is a method usually used for peptide synthesis, a method using an active ester such as HBTU for a raw material having a reactive carboxyl group and a raw material having a reactive amino group, or a carbodiimide It can be condensed by a usual method in peptide synthesis such as a method using a coupling agent such as. If 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。 Functional groups that should not be involved in the reaction in this reaction step are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl (CBZ), t-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the carboxyl group protective agent include groups capable of forming an alkyl ester, a benzyl ester, etc. In the case of the solid phase method, the C-terminal carboxyl group is a chlorotrityl resin, a chloromethyl resin, an 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 with 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 the condensation reaction is completed, the protecting group is removed, but in the case of the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved. In addition, the peptides of the invention are purified according to conventional methods. Examples thereof include ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like. The synthesis of the synthesized peptide is analyzed by a protein sequencer, GC-MS, etc., which reads the amino acid sequence from the C-terminal by the Edman degradation method.
　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 biological 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 has an appetite-promoting effect and a growth hormone secretagogue-promoting effect.
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 secretion promoting action. Based on the appetite-promoting action (feeding-promoting action), the peptides of the present invention can be used for the treatment of anorexia in the elderly and the like. Based on the secretagogue action of growth hormone, it can be used for prevention of muscle 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 amount of ghrelin secreted can be evaluated by a method known to those skilled in the art. For example, reference: Iwakura Het.al. Endocrinology. 2010 Jun; 151 (6): Ghrelin in a medium collected by adding a test substance to the ghrelin-secreting cell MGN3-1 described in 2940-5 and culturing for a predetermined time. The amount of ghrelin secreted can be measured by quantifying. Ghrelin can be quantified by an immunochemical method such as ELISA (Enzyme-Linked ImmunoSorbentAssay).
　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 same is not particularly limited, and any of oral administration, parenteral administration, and rectal administration can be adopted, orally or parenterally. Can be administered. Of 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 dose of this peptide varies depending on the administration method, the condition and age of the person to be administered, but is usually 0.01 mg / kg to 500 mg / kg, preferably 0.05 mg / kg to 100 mg / kg per day for an adult. , More preferably 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 carrier for preparation. As the 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 peptides of the invention can be used as pharmaceuticals or foods themselves, or tablets (uncoated tablets, sugar-coated tablets, effervescent tablets, alone or with suitable non-toxic carriers, diluents or excipients). (Film-coated tablets, chewable tablets, etc.), capsules, troches, powders, fine granules, granules, liquids, suspensions, emulsions, pastes, creams, injections (amino acid infusions, electrolyte infusions, etc.) It can be a food- or pharmaceutical preparation such as a sustained-release preparation such as an enteric-coated tablet, capsule, or granule. 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 or oral carriers, diluents or excipients that can be added to pharmaceuticals or foods include lactose, glucose, mannites, dextrin, cyclodextrin, starch, crustacea. , Magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethyl cellulose, hydroxypropyl starch, carboxymethyl cellulose calcium, ion exchange resin, methyl cellulose, gelatin, arabic rubber, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, light Silica anhydride, magnesium stearate, talc, tragant, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin , White vaseline, fluorocarbon, nonionic surfactant, 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。 Dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections and the like. These formulations are prepared according to a conventional method. The liquid preparation may be dissolved or suspended in water or another suitable solvent at the time of use. Further, tablets and granules may be coated by a well-known method. In the case of an injection, the peptide of the present invention is prepared by dissolving it in water, but it may be dissolved in physiological saline or glucose solution if necessary, or 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 can 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 ingredients of therapeutic value.
　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。 To produce a solid preparation for oral administration, the active ingredient and excipient ingredients such as lactose, starch, crystalline cellulose, calcium lactate, silicic anhydride, etc. are mixed to form a powder, or sucrose, if necessary. A binder such as hydroxypropyl cellulose or polyvinylpyrrolidone, a disintegrant such as carboxymethyl cellulose or carboxymethyl cellulose calcium is added, and wet or dry granulation is performed to obtain granules. In order to produce tablets, these powders and granules may be tableted as they are or by 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 methyl methacrylate-methylmethacrylate polymer to form an enteric solvent preparation, or to be coated with ethyl cellulose, carnauba wax, cured oil, etc. to form a long-acting preparation. You can also. To manufacture capsules, hard capsules are filled with powders or granules, or the active ingredient is used as it is or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc. and then coated with a gelatin film to form soft capsules. Can be done.
　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。 To produce a liquid preparation for oral administration, the active ingredient and sweeteners such as sucrose, sorbitol, and glycerin are dissolved in water to make a transparent syrup, and essential oil, ethanol, etc. are added to make an elixir. Arabia gum, tragant, polysorbate 80, sodium carboxymethyl cellulose and the like may be added as an emulsion or a suspending agent. If desired, a flavoring agent, a coloring agent, a preservative, or the like may be added to these liquid preparations.
　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 forms of foods that can be prepared by adding or blending the peptides according to the present invention include, for example, beverages (coffee, cocoa, juice, soft beverages, mineral beverages, tea beverages, green tea, tea, crow dragon tea, dairy beverages). , Lactic acid drinks, yogurt drinks, carbonated drinks, other non-alcoholic drinks, alcoholic drinks, etc.), confectionery (hard candy, gum, gummy, jelly, pudding, mousse, cake, candy, cookies, crackers, biscuits, chocolate, ice cream ( Ice cream, ice candy, sherbet, shaved ice, etc.), sprinkles, dressings, seasonings, processed soybean foods (tofu, miso, soy sauce, yuba, kina flour, natto, etc.), processed meat foods (hamburger, meat loaf, meat balls, etc.) Tsukune, etc.), processed fish meat foods (kamaboko, chikuwa, etc.), retort foods, jelly-like foods (jelly, agar, jelly-like beverages, etc.), etc. can be mentioned. Foods that can be prepared by adding or blending the peptides of the present invention include so-called health foods, functional foods, foods with functional claims, nutritional 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 type of special-purpose food) or a food for the elderly (Ministry of Health, Labor and Welfare, a type of special-purpose food), uncoated tablets, film-coated tablets, sugar-coated tablets, granules, powders, tablets, capsules (hard capsules and soft capsules). ), Chewable type, syrup type, drink type, etc. can also be used. The food containing and blending the peptide according to the present invention can be prepared 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 in more detail with reference to Examples. However, the examples below do not limit the scope of the 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、培地中で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 secretory activity)
Ghrelin-secreting cells MGN3-1 were seeded in 96-well plates at 1 x 10 5 cells / well and cultured in medium for 24 hours. After culturing, the cells were washed with DPBS, and 100 μL (Buffer: 50 μM sodium octanoate / DMEM) of the test substance was added. Only the Buffer to which the test substance was not added was used as the control. After further culturing for 4 hours, the medium was collected and centrifuged to obtain a supernatant. 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) EIA Kit A05117）により評価した。 The concentration of ghrelin in the sample was evaluated by the ELISA method (Ghrelin (Acylated) EIA Kit A05117 manufactured by Bertin Pharma).
　＜製造例＞
　（酵素消化物）
　精製した小麦グルテンと消化酵素とを、酵素：グルテン＝１：１００（重量比、グルテンの終濃度：10 mg/ml）で混合し、添付の溶液中で反応を行った。 <Manufacturing example>
(Enzyme digestive product)
The purified wheat gluten and the digestive enzyme were mixed at an enzyme: gluten = 1: 100 (weight ratio, final concentration of gluten: 10 mg / ml), and the reaction was carried out in the attached solution.
　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-Aldrich社製、T8003）；反応温度：37℃、反応時間：5時間、反応pH： 7.5。
（ii）キモトリプシン（Sigma-Aldrich社製、C4129）；反応温度：37℃、反応時間：5時間、反応pH：7.5。 The enzymes used and the reaction conditions were as follows:
(I) Trypsin (manufactured by Sigma-Aldrich, T8003); reaction temperature: 37 ° C., reaction time: 5 hours, reaction pH: 7.5.
(Ii) Chymotrypsin (manufactured by Sigma-Aldrich, C4129); 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 had elapsed, the sample was boiled (100 ° C., 10 minutes) to stop the enzymatic reaction.
　（ペプチド）
　定法により、ペプチドYPTSL（配列番号１；実施例１）、ペプチドLSVTSPQQVSY（配列番号２；実施例２）、ペプチドSQQQQPVLPQQPSF（配列番号３；実施例３）ペプチドHVSAEHQAASL（配列番号４；比較例１）、ペプチドYPTS（配列番号５；比較例２）、ペプチドPTSL（配列番号６；比較例３）、ペプチドQQPQQQYPLGQGSF（配列番号７；比較例４）、ペプチドYLSVTSPQQVSY（配列番号８；比較例５）、及びペプチドQQPQQQYPSGQGSF（配列番号９；比較例６）を合成した。 (peptide)
By routine, peptide YPTSL (SEQ ID NO: 1; Example 1), peptide LSVTSPQQVSY (SEQ ID NO: 2; Example 2), peptide SQQQQPVLPQQPSF (SEQ ID NO: 3; Example 3) peptide HVSAEHQAASL (SEQ ID NO: 4; Comparative Example 1), Peptide YPTS (SEQ ID NO: 5; Comparative Example 2), Peptide PTSL (SEQ ID NO: 6; Comparative Example 3), Peptide QQPQQQYPLGQGSF (SEQ ID NO: 7; Comparative Example 4), Peptide YLSVTSPQQVSY (SEQ ID NO: 8; Comparative Example 5), and Peptide QQPQQQYPSGQGSF (SEQ ID NO: 9; Comparative Example 6) was synthesized.
　（統計解析）
　試験により得られたデータを、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。図２はDunnetの検定を行った。 (Statistical analysis)
The data obtained by the test was expressed as the sum of the mean (Mean) of the number of trials n and the standard error (SEM). The t-test was used for comparison between the two groups. For comparisons between 3 or more groups, data were analyzed by one-way ANOVA, followed by Tukey-Kramer test for multiple comparisons. When p <0.05 (“*” in the figure), it was judged that there was a significant difference. Figure 2 shows Dunnet's test.
　＜試験例＞
　試験例１：グルテンの酵素消化物
　グルテンのトリプシン又はキモトリプシン消化物について、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: Enzyme digestion of gluten The effect of gluten trypsin or chymotrypsin digest on ghrelin secretion was evaluated using ghrelin-secreting cell MGN3-1.
　結果を図１に示す。図中、縦軸「Ghrelin (% of control)」は、対照（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（百分率）を示す。 The results are shown in Fig. 1. In the figure, the vertical axis "Ghrelin (% of control)" indicates the ratio (percentage) of the ghrelin secretion amount to the measured value when the control (only Buffer to which the test substance is not added) is added to MGN3-1.
　グルテンのキモトリプシン消化物は、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。また、グレリン分泌を促進する活性は、キモトリプシンの濃度依存的に増加することが明らかとなった。一方、グルテンのトリプシン消化物においては、同様の効果は確認されなかった。 It was revealed that the digested product of gluten chymotrypsin has an activity of increasing ghrelin secretion and promoting ghrelin secretion. In addition, it was revealed that the activity of promoting ghrelin secretion increases in a concentration-dependent manner of chymotrypsin. On the other hand, no similar effect was confirmed in the tryptic digest of gluten.
　試験例２：ペプチド（１）
　グルテンのキモトリプシン消化物に含まれるペプチドYPTSL（配列番号１；実施例１）、ペプチドHVSAEHQAASL（配列番号４；比較例１）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)
The effects of the peptide YPTSL (SEQ ID NO: 1; Example 1) and the peptide HVSAEHQAASL (SEQ ID NO: 4; Comparative Example 1) contained in the digested product of gluten chymotrypsin on ghrelin secretion were evaluated in the same manner as in Test Example 1.
　結果を図２に示す。ペプチドYPTSL（配列番号１；実施例１）は、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。一方、ペプチドHVSAEHQAASL（配列番号４；比較例１）については、グレリン分泌を促進する活性が確認されなかった。 The results are shown in Fig. 2. The peptide YPTSL (SEQ ID NO: 1; Example 1) was found to have an activity of promoting ghrelin secretion. On the other hand, the peptide HVSAEHQAASL (SEQ ID NO: 4; Comparative Example 1) was not confirmed to have an activity of promoting ghrelin secretion.
　試験例３：ペプチド（２）
　１ｍＭ又は１０ｍＭのペプチドYPTSL（配列番号１；実施例１）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 3: Peptide (2)
The effect of 1 mM or 10 mM peptide YPTSL (SEQ ID NO: 1; Example 1) on ghrelin secretion was evaluated in the same manner as in Test Example 1.
　結果を図３に示す。ペプチドYPTSL（配列番号１；実施例１）によるグレリン分泌を促進する活性は、ペプチドの濃度依存的に増加することが明らかとなった。 The results are shown in Fig. 3. It was revealed that the activity of the peptide YPTSL (SEQ ID NO: 1; Example 1) to promote ghrelin secretion increases in a concentration-dependent manner of the peptide.
　試験例４：ペプチド（３）
　ペプチドYPTSL（配列番号１；実施例１）、ペプチドYPTS（配列番号５；比較例２）、及びペプチドPTSL（配列番号６；比較例３）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 4: Peptide (3)
Effects of peptide YPTSL (SEQ ID NO: 1; Example 1), peptide YPTS (SEQ ID NO: 5; Comparative Example 2), and peptide PTSL (SEQ ID NO: 6; Comparative Example 3) on ghrelin secretion in the same manner as in Test Example 1. Was evaluated.
　結果を図４に示す。ペプチドYPTSL（配列番号１；実施例１）において確認されたグレリン分泌を促進する活性は、ペプチドYPTS（配列番号５；比較例２）、及びペプチドPTSL（配列番号６；比較例３）においては、確認されなかった。これらの結果から、ペプチドYPTSLの５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. 4. The activity of promoting ghrelin secretion confirmed in peptide YPTSL (SEQ ID NO: 1; Example 1) was found in peptide YPTS (SEQ ID NO: 5; Comparative Example 2) and peptide PTSL (SEQ ID NO: 6; Comparative Example 3). Not confirmed. From these results, it was clarified that 5 residues of the peptide YPTSL are particularly important in promoting ghrelin secretion.
　試験例５：ペプチド（４）
　グルテンのキモトリプシン消化物に含まれるペプチドQQPQQQYPLGQGSF（配列番号７；比較例４）、ペプチドLSVTSPQQVSY（配列番号２；実施例２）、ペプチドSQQQQPVLPQQPSF（配列番号３；実施例３）、ペプチドYLSVTSPQQVSY（配列番号８；比較例５）、及びペプチドQQPQQQYPSGQGSF（配列番号９；比較例６）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 5: Peptide (4)
Peptide QQPQQQYPLGQGSF (SEQ ID NO: 7; Comparative Example 4), peptide LSVTSPQQVSY (SEQ ID NO: 2; Example 2), peptide SQQQQPVLPQQPSF (SEQ ID NO: 3; Example 3), peptide YLSVTSPQQVSY (SEQ ID NO: 8) contained in the digested product of gluten chymotrypsin. Comparative Example 5) and peptide QQPQQQYPSGQGSF (SEQ ID NO: 9; Comparative Example 6) were evaluated for their effect on ghrelin secretion in the same manner as in Test Example 1.
　結果を図５に示す。ペプチドLSVTSPQQVSY（配列番号２；実施例２）、及びペプチドSQQQQPVLPQQPSF（配列番号３；実施例３）については、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。また、グレリン分泌を促進する活性は、ペプチドの濃度依存的に増加することが明らかとなった。一方、ペプチドQQPQQQYPLGQGSF（配列番号７；比較例４）、ペプチドYLSVTSPQQVSY（配列番号８；比較例５）、及びペプチドQQPQQQYPSGQGSF（配列番号９；比較例６）については、グレリン分泌を促進する活性が確認されなかった。 The results are shown in Fig. 5. It was revealed that the peptide LSVTSPQQVSY (SEQ ID NO: 2; Example 2) and the peptide SQQQQPVLPQQPSF (SEQ ID NO: 3; Example 3) have an activity of promoting ghrelin secretion. In addition, it was revealed that the activity of promoting ghrelin secretion increases in a peptide concentration-dependent manner. On the other hand, the peptide QQPQQQQYPLGQGSF (SEQ ID NO: 7; Comparative Example 4), the peptide YLSVTSPQQVSY (SEQ ID NO: 8; Comparative Example 5), and the peptide QQPQQQYPSGQGSF (SEQ ID NO: 9; Comparative Example 6) were confirmed to have an activity of promoting ghrelin secretion. There wasn't.
　試験例６：生体への投与
　マウス（ddYマウス、雄、42～56g）にグルテンのキモトリプシン消化物を投与して、摂食行動への影響を評価した。 Test Example 6: In-vivo administration Mice (ddY mice, males, 42-56 g) were administered a digested product of gluten chymotrypsin to evaluate the effect on feeding behavior.
　生理食塩水に溶解した小麦グルテンのキモトリプシン消化物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、20、60、及び120分後に飼料重量を測定することにより摂食量を算出した。 After oral administration of a digested product of wheat gluten chymotrypsin dissolved in physiological saline to mice, a pre-weighed feed (CE-2, manufactured by Japan Claire) is given, and the feed weight is measured after 20, 60, and 120 minutes. The amount of food intake was calculated accordingly.
　結果を図６に示す。グルテンのキモトリプシン消化物は、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。また摂食促進作用は、キモトリプシン消化物の濃度依存的に増加することが明らかとなった。 The results are shown in Fig. 6. Gluten chymotrypsin digests were found to promote mouse feeding by oral administration. It was also clarified that the feeding promoting effect increases depending on the concentration of the digested chymotrypsin.

Claims (9)

  1. 　アミノ酸配列YPTSL（配列番号１）、アミノ酸配列LSVTSPQQVSY（配列番号２）又はアミノ酸配列SQQQQPVLPQQPSF（配列番号３）を含むペプチド。 Peptide containing amino acid sequence YPTSL (SEQ ID NO: 1), amino acid sequence LSVTSPQQVSY (SEQ ID NO: 2) or amino acid sequence SQQQQPVLPQQPSF (SEQ ID NO: 3).
  2. 　グルテンのキモ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 digested product of gluten chymotrypsin.
  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 containing 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 secretagogue containing 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 product to which the peptide according to claim 1 or 2 is added.
  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 the 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 growth hormone secretion, which comprises the step of administering the peptide according to claim 1 or 2 to a patient or a preliminary group requiring the peptide.
  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に記載のペプチドの使用。 Use of the peptide according to claim 1 or 2 to promote appetite and / or promote growth hormone secretion.
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