WO2013047128A1 - Peptide and angiotensin-converting enzyme inhibitor - Google Patents

Peptide and angiotensin-converting enzyme inhibitor Download PDF

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Publication number
WO2013047128A1
WO2013047128A1 PCT/JP2012/072609 JP2012072609W WO2013047128A1 WO 2013047128 A1 WO2013047128 A1 WO 2013047128A1 JP 2012072609 W JP2012072609 W JP 2012072609W WO 2013047128 A1 WO2013047128 A1 WO 2013047128A1
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peptide
converting enzyme
present
blood pressure
lys
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PCT/JP2012/072609
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French (fr)
Japanese (ja)
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明男 山田
越智　大介
琢磨 桜井
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森永乳業株式会社
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Priority to JP2013536124A priority Critical patent/JP5430803B2/en
Publication of WO2013047128A1 publication Critical patent/WO2013047128A1/en

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    • 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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel peptide and an angiotensin converting enzyme inhibitor containing the peptide.
  • An angiotensin converting enzyme inhibitor can be used as food, feed, and medicine.
  • Angiotensin converting enzyme is an enzyme that acts on angiotensin I generated from angiotensinogen by cleaving with renin, liberates two amino acids at the C-terminal, and converts it into angiotensin II.
  • Angiotensin converting enzyme generates angiotensin II having a strong pressor action and also has an action of inactivating bradykinin having a hypotensive action. Because of these actions, angiotensin converting enzyme inhibitors are used as therapeutic agents for hypertension, and captopril, renibase, lisinopril and the like are known as commercially available drugs.
  • angiotensin converting enzyme inhibitors are recommended to be actively used as first-line drugs for many pathologies such as heart disease, kidney disease, cerebrovascular disorder, arteriosclerotic disease, diabetes, metabolic syndrome, and elderly hypertension. (High Blood Pressure Treatment Guidelines 2009).
  • Non-patent Document 1 peptides having an angiotensin converting enzyme inhibitory action have been found in natural products. Since these are composed of natural amino acids, they can be taken orally as nutritional sources, as well as exhibiting blood pressure lowering effects. It is used as a health food.
  • peptides obtained by degrading casein with an enzyme Patent Documents 1 to 11, Non-Patent Document 1 are examples.
  • many other natural peptides having an angiotensin converting enzyme inhibitory action are known (Non-patent Document 2).
  • An object of the present invention is to provide a novel peptide having an angiotensin converting enzyme inhibitory action and an angiotensin converting enzyme inhibitor containing the same.
  • the present inventors have intensively studied to solve the above problems. That is, by synthesizing a hexapeptide in which lysine is fixed on the C-terminal side and 5 types of amino acids (arginine, leucine, isoleucine, proline, serine) are randomly bound to the peptide, high angiotensin converting enzyme inhibition is observed in the mixture. It has been found that there is a novel peptide having activity, and that the peptide has a specific sequence, and the present invention has been completed.
  • the present invention provides a peptide consisting of the following amino acid sequence.
  • Xaa-Arg-Xaa-Pro-Ser-Lys SEQ ID NO: 1 (However, each amino acid is L-form, and Xaa is independently Ile or Leu.)
  • the peptide of the present invention preferably has any of the following amino acid sequences.
  • the present invention also provides an angiotensin converting enzyme inhibitor containing the peptide as an active ingredient.
  • the present invention also provides a blood pressure lowering agent containing the peptide as an active ingredient.
  • the present invention also provides a method for lowering blood pressure in a subject comprising administering the peptide to a subject in need of blood pressure reduction.
  • the present invention also provides use of the peptide for lowering blood pressure in a subject in need of blood pressure reduction.
  • the separation chromatogram by the reverse phase HPLC of a synthetic peptide mixture The horizontal axis represents time (minutes), and the vertical axis represents absorbance at 215 nm.
  • the peptide of the present invention has a sequence represented by Xaa-Arg-Xaa-Pro-Ser-Lys (SEQ ID NO: 1).
  • the peptide of the present invention may be a salt of this peptide.
  • Leu is an L-leucine residue
  • Arg is an L-arginine residue
  • Ile is an L-isoleucine residue
  • Pro is an L-proline residue
  • Ser is an L-serine residue
  • Lys is an L-lysine residue. Indicates a group.
  • Xaa is independently Ile or Leu.
  • the peptide of the present invention has any of the following amino acid sequences. a) Ile-Arg-Ile-Pro-Ser-Lys (SEQ ID NO: 2) b) Ile-Arg-Leu-Pro-Ser-Lys (SEQ ID NO: 3) c) Leu-Arg-Leu-Pro-Ser-Lys (SEQ ID NO: 4) d) Leu-Arg-Ile-Pro-Ser-Lys (SEQ ID NO: 5)
  • the peptide of the present invention can be produced by chemical synthesis.
  • the chemical synthesis of the peptide of the present invention can be carried out by a liquid phase method or a solid phase method usually used for the synthesis of oligopeptides.
  • the synthesized peptide is deprotected as necessary to remove unreacted reagents, by-products and the like. Such peptide synthesis can be performed using a commercially available peptide synthesizer.
  • the peptide of the present invention is preferably isolated and purified from the above synthesized (mixed) product.
  • Peptide purification is usually performed in the same manner as that used for oligopeptide purification, such as ion exchange chromatography, adsorption chromatography, reverse phase chromatography, partition chromatography, gel filtration chromatography, and other various chromatography methods. , Solvent precipitation, salting out, and partitioning between the two liquid phases, and the like.
  • the fraction containing the target substance can be determined using the angiotensin converting enzyme inhibitory action described below as an index, and the active component of those fractions can be determined by mass spectrometry or / and a protein sequencer. Can be identified.
  • the peptide of the present invention can also be produced by expressing a recombinant DNA encoding the peptide in an appropriate host cell.
  • vectors and hosts necessary for the production of recombinant DNA those usually used for the production of proteins and peptides can be used.
  • the peptide of the present invention can be used as an active ingredient of an angiotensin converting enzyme inhibitor.
  • the peptide of the present invention has an angiotensin converting enzyme inhibitory action.
  • an angiotensin converting enzyme is inhibited, the production of angiotensin II and the inactivation of bradykinin by the enzyme are suppressed, resulting in a blood pressure lowering effect. Therefore, the peptide or angiotensin converting enzyme inhibitor of the present invention is a preventive or therapeutic agent for various diseases derived from hypertension such as cerebral hemorrhage, cerebral infarction, angina pectoris, myocardial infarction, renal failure, etc., specifically blood pressure. It can be used as a depressant.
  • one form of the present invention is a blood pressure lowering agent containing the peptide of the present invention as an active ingredient.
  • an angiotensin converting enzyme inhibitor is known to have an effect on essential hypertension of unknown cause, and the peptide of the present invention is expected to show a therapeutic or preventive effect on essential hypertension.
  • it can also be used as a therapeutic or prophylactic agent for diseases such as cardiac hypertrophy and angina which are known to be effective for angiotensin converting enzyme inhibitors.
  • Another aspect of the present invention is a method for lowering blood pressure in a subject comprising administering a peptide of the present invention to a subject in need of blood pressure reduction.
  • Another aspect of the present invention is the use of a peptide of the present invention for lowering blood pressure in a subject in need of blood pressure reduction.
  • the subject requiring blood pressure lowering is not particularly limited as long as it is effective to inhibit angiotensin converting enzyme, but patients having various diseases derived from the above-mentioned hypertension, patients with essential hypertension, and cardiac hypertrophy And patients having diseases such as angina. Also, in these methods and uses, “decrease in blood pressure” can be “treatment of disease”.
  • the peptide of this invention can be mix
  • One type of peptide may be blended, or a mixture of any two or more types may be used.
  • additives such as carriers, excipients, binders, disintegrants, lubricants, colorant stabilizers, flavoring agents, diluents, and solvents for injections can be used for pharmaceutical formulation.
  • Specific preparations include tablets (including sugar-coated tablets, enteric-coated tablets, buccal tablets), powders, capsules (including enteric capsules and soft capsules), granules (including those coated), pills,
  • Illustrative examples include troches, encapsulated liposomes, solutions, and pharmaceutically acceptable sustained release formulations.
  • Carriers and excipients used in these formulations include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder and the like as binders
  • Examples thereof include starch, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose and the like.
  • the disintegrant include starch, agar, gelatin powder, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate.
  • lubricant magnesium stearate, hydrogenated vegetable oil, macrogol, etc.
  • colorant red No. 2, yellow No. 4, and blue No. 1, etc., which are allowed to be added to pharmaceuticals, etc.
  • components that are usually used in the manufacture of pharmaceuticals can be used for stabilizers, flavoring agents, diluents, solvents for injections, and the like.
  • Tablets and granules are sucrose, hydroxypropylcellulose, purified shellac, gelatin, sorbitol, glycerin, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate as necessary And a methacrylic acid polymer.
  • the angiotensin converting enzyme inhibitor or blood pressure lowering agent of the present invention may be administered either orally or parenterally, but oral administration is preferred.
  • Parenteral administration includes intravenous injection, rectal administration, inhalation and the like.
  • Examples of the dosage form for oral administration include tablets, capsules, troches, syrups, granules, powders, ointments and the like.
  • the medicine and pharmaceutical composition which have the angiotensin converting enzyme inhibitory action known or discovered in the future can also be used together with the peptide of this invention.
  • the drug or pharmaceutical composition to be used in combination may be contained as one of the active ingredients in the drug of the present invention, or it is not contained in the drug of the present invention and is combined with the drug of the present invention as a separate drug. May be commercialized.
  • the peptide of the present invention can be contained in food as an active ingredient, and processed as a food having an angiotensin converting enzyme inhibitory action or blood pressure lowering action as one embodiment of an angiotensin converting enzyme inhibitor or blood pressure lowering agent. .
  • Such foods may be in the form of liquids, pastes, solids, powders, etc., in addition to tablet confections, liquid foods, feeds (including for pets), etc., for example, bread, macaroni, spaghetti, noodles, cakes Flour products such as mix, fried flour, bread crumbs, instant noodles, cup noodles, retort / cooked food, cooked canned food, microwave food, instant soup / stew, instant miso soup / soup, canned soup, freeze-dried food, other instant foods
  • Instant foods such as foods; canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, processed cereals (cereal processed products); canned seafood, fish ham and sausages, fish paste products, Processed marine products such as marine delicacy, tsukudani, etc .; Livestock canned and pasted products, livestock processed products such as livestock ham and sausage; processed milk, milk drinks, yogurts Milk and dairy products such as lactic acid bacteria beverages, cheese, ice cream, formula milk powder, cream and other
  • the peptide of the present invention can be contained in the feed as an active ingredient, and processed as a feed having an angiotensin converting enzyme inhibitory action or blood pressure lowering action.
  • the form of the feed is not particularly limited.
  • the peptide powder of the present invention or an aqueous solution thereof is used for cereals such as corn, wheat, barley, rye, and milo; soybean oil cake, rapeseed oil cake, coconut oil cake Vegetable oils such as flaxseed, flaxseed oil, rice bran, rice bran, defatted rice bran, etc .; production corn such as corn gluten meal, corn jam meal; fish meal, skim milk powder, whey, yellow grease, tallow, etc. Animal feeds; yeasts such as torula yeast and beer yeast; mineral feeds such as tricalcium phosphate and calcium carbonate; fats and oils; simple amino acids; sugars and the like. Examples of the form of the feed include pet food, livestock feed, and fish feed.
  • the amount of the peptide of the present invention (the total amount in the case of multiple peptides) ) Is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and 0.1% by mass with respect to the final composition of the angiotensin converting enzyme inhibitor or blood pressure lowering agent.
  • the above is particularly preferable.
  • the dose of the angiotensin converting enzyme inhibitor or blood pressure lowering agent of the present invention varies depending on age, symptoms, etc., but is usually 0.001 to 3000 mg / day, preferably 0.01 to 30 mg / day as the amount of the peptide of the present invention. It may be administered once a day or divided into 2 to 3 times a day.
  • the angiotensin converting enzyme inhibitor or blood pressure lowering agent of the present invention is ingested or taken, the effects of the present invention are sufficiently exhibited at any timing before, between, and after a meal.
  • the eluent (A / B) is maintained at 98/2 for 5 minutes, 30 minutes to the eluent (75/25), 10 minutes to the eluent (50/50), and further eluent ( Gradient elution was performed in 3 minutes until 20/80), and then the synthetic peptide mixture was separated under a gradient condition of maintaining the eluent (20/80) for 5 minutes (chromatogram: FIG. 1).
  • the eluate was fractionated for each peak, and the angiotensin converting enzyme inhibitory ability was measured by the method described later, and the fractions having strong angiotensin converting enzyme inhibitory ability were retention times of 34.0 minutes, 34.4 minutes, 34.7 minutes, and 35.1. Eluted in minutes.
  • the peptides contained in these fractions were analyzed with a protein sequencer (PPSQ-23A) manufactured by Shimadzu Corporation, and found to be Ile-Arg-Ile-Pro-Ser-Lys (hereinafter referred to as HP1), Ile-Arg-Leu-Pro-Ser-Lys (hereinafter referred to as HP2), Leu-Arg-Leu-Pro-Ser-Lys (hereinafter referred to as HP3), and Leu-Arg-Ile-Pro- Ser-Lys (hereinafter referred to as HP4).
  • HP1 Ile-Arg-Ile-Pro-Ser-Lys
  • HP3 Ile-Arg-Leu-Pro-Ser-Lys
  • HP4 Leu-Arg-Ile-Pro- Ser-Lys
  • Example 2 [Angiotensin-converting enzyme inhibitory action of peptides]
  • Test method Angiotensin converting enzyme inhibition was measured according to the method of Kashman et al. [Biochemical pharmacology, Vol. 20, pages 1637 to 1648 (1971)].
  • the peptide of the present invention HP1, HP2, HP3, HP4 obtained in Example 1, the peptide described in Agricultural and Biological Chemistry 49 (5), 1405-1409, 1985 (Phe-Phe-Val-Ala- Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys: SEQ ID NO: 7) and the peptide (Met-Lys-Pro) described in Japanese Patent No. 38169921 (WO2003 / 044044)) were used. All of these peptides were chemically synthesized in the same manner as in Example 1.
  • the sample was dissolved in 0.1 M borate buffer (containing 0.3 M NaCl, pH 8.3), 0.08 ml was placed in a test tube, and then 0.1 M borate buffer (containing 0.3 M NaCl) 0.2 ml of an enzyme substrate (Hiprilhistidylleucine, Sigma) adjusted to 5 mM at pH 8.3) was added, and the mixture was incubated at 37 ° C. for 3 minutes. Subsequently, 0.02 ml of rabbit lung angiotensin converting enzyme (manufactured by Sigma) prepared to be 0.1 U / ml by adding distilled water was added and reacted at 37 ° C. for 30 minutes.
  • an enzyme substrate Hiprilhistidylleucine, Sigma
  • Inhibitory activity was determined from the following formula, and IC50 [sample concentration ( ⁇ M) required to inhibit angiotensin converting enzyme activity by 50%] was determined.
  • Inhibition rate (AB) / (AC) ⁇ 100%
  • Test results are shown in Table 1. As is clear from Table 1, all of the peptides of the present invention (HP1, HP2, HP3, HP4) were found to have potent angiotensin converting enzyme inhibitory activity.
  • Example 3 [Antihypertensive action of peptides in animals]
  • Test method Fourteen 12-week-old SHR / Hos male rats (purchased from Japan SLC Co., Ltd.) were preliminarily raised for 1 week, and the blood pressure of the rats was measured using a small animal non-invasive automatic sphygmomanometer (MK-2000, Muromachi Kikai Co., Ltd.).
  • the group was divided into two groups so that the average systolic blood pressure before administration for each group was almost the same value, and then the animals were fasted for about 14 hours after being divided into two groups.
  • the peptide obtained in Example 1 (Leu-Arg-Ile-Pro-Ser-Lys: HP4) was dissolved in water for injection and orally administered to rats at a rate of 5 mL / kg body weight (3 mg / kg body weight as HP4).
  • the blood pressure of the rats was measured immediately before sample administration, 1, 3, 6 and 8 hours after sample administration.
  • a novel peptide having an angiotensin converting enzyme inhibitory action is provided.
  • An angiotensin converting enzyme inhibitor containing the peptide can be used as a medicine. Since the peptides of the present invention are all composed of natural (L) amino acids, they are highly safe and can be used in foods.

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Abstract

A peptide comprising the amino acid sequence shown below can be used as an angiotensin enzyme inhibitor or a hypotensive agent. Xaa-Arg-Xaa-Pro-Ser-Lys (In the formula, each amino acid residue is in an L-form; and Xaa's independently represent Ile or Leu.)

Description

ペプチドおよびアンジオテンシン変換酵素阻害剤Peptides and angiotensin converting enzyme inhibitors
　本発明は、新規なペプチド及び同ペプチドを含有するアンジオテンシン変換酵素阻害剤に関する。アンジオテンシン変換酵素阻害剤は、食品、飼料、及び医薬として利用することができる。 The present invention relates to a novel peptide and an angiotensin converting enzyme inhibitor containing the peptide. An angiotensin converting enzyme inhibitor can be used as food, feed, and medicine.
　アンジオテンシン変換酵素（ＡＣＥ）は、レニンによる切断によりアンジオテンシノーゲンから生じるアンジオテンシンＩに働き、Ｃ末端の２個のアミノ酸を遊離させて、アンジオテンシンIIに変換する酵素である。アンジオテンシン変換酵素は強い昇圧作用を有するアンジオテンシンIIを生成させるとともに、降圧作用を有するブラジキニンを不活性化する作用も有している。このような作用から、アンジオテンシン変換酵素阻害剤は、高血圧の治療薬として使用されており、カプトプリル、レニベース、リシノプリルなどが市販薬として知られている。また、アンジオテンシン変換酵素阻害剤は、心疾患、腎疾患、脳血管障害、動脈硬化性疾患、糖尿病、メタボリックシンドローム、高齢者高血圧など多くの病態に対して第一選択薬として積極的な使用が勧められている（高血圧治療ガイドライン２００９）。 Angiotensin converting enzyme (ACE) is an enzyme that acts on angiotensin I generated from angiotensinogen by cleaving with renin, liberates two amino acids at the C-terminal, and converts it into angiotensin II. Angiotensin converting enzyme generates angiotensin II having a strong pressor action and also has an action of inactivating bradykinin having a hypotensive action. Because of these actions, angiotensin converting enzyme inhibitors are used as therapeutic agents for hypertension, and captopril, renibase, lisinopril and the like are known as commercially available drugs. In addition, angiotensin converting enzyme inhibitors are recommended to be actively used as first-line drugs for many pathologies such as heart disease, kidney disease, cerebrovascular disorder, arteriosclerotic disease, diabetes, metabolic syndrome, and elderly hypertension. (High Blood Pressure Treatment Guidelines 2009).
　一方、アンジオテンシン変換酵素阻害作用を有するペプチドが天然物中から見出されている。これらは天然型のアミノ酸から構成されているため栄養源として経口摂取は勿論のこと、血圧低下作用を示す以外には、重い副作用を示すことはなく安全性が高いため、血圧降下作用を有する特定保健用食品として利用されている。例えば、カゼインを酵素により分解して得たペプチド類（特許文献１～１１、非特許文献１）がその例である。また、その他にもアンジオテンシン変換酵素阻害作用を有する天然型のペプチドが数多く知られている（非特許文献２）。 On the other hand, peptides having an angiotensin converting enzyme inhibitory action have been found in natural products. Since these are composed of natural amino acids, they can be taken orally as nutritional sources, as well as exhibiting blood pressure lowering effects. It is used as a health food. For example, peptides obtained by degrading casein with an enzyme (Patent Documents 1 to 11, Non-Patent Document 1) are examples. In addition, many other natural peptides having an angiotensin converting enzyme inhibitory action are known (Non-patent Document 2).
特開昭５８－１０９４２５号JP 58-109425 A 特開昭５９－４４３２３号JP 59-44323 特開昭５９－４４３２４号JP 59-44324 A 特開昭６１－３６２２６号JP 61-36226 特開昭６１－３６２２７号JP 61-36227 A 特開平６－２７７０９０号JP-A-6-277090 特開平６－２７７０９１号JP-A-6-277091 特開平６－２７９４９１号JP-A-6-279491 特開平７－１０１９８２号JP 7-101982 特開平７－１０１９８５号JP 7-101985 A 特許第３８１６９２１号Japanese Patent No. 3816921
　上記のように、アンジオテンシン変換酵素阻害作用を有する種々のペプチドが知られているが、これらのペプチド類では、未だ食品中の機能として、アンジオテンシン変換酵素阻害活性は不十分である。よって、天然物型で、一層高いアンジオテンシン変換酵素阻害活性を有するペプチドの取得と、その食品又は医薬等への応用が望まれているところである。
　本発明は、アンジオテンシン変換酵素阻害作用を有する新規ペプチド、及びそれを含むアンジオテンシン変換酵素阻害剤を提供することを課題とする。 As described above, various peptides having an angiotensin converting enzyme inhibitory action are known, but these peptides still have insufficient angiotensin converting enzyme inhibitory activity as a function in food. Therefore, it is desired to obtain a natural product-type peptide having higher angiotensin converting enzyme inhibitory activity and to apply it to food or medicine.
An object of the present invention is to provide a novel peptide having an angiotensin converting enzyme inhibitory action and an angiotensin converting enzyme inhibitor containing the same.
　本発明者らは、前記課題を解決するために鋭意検討を行った。すなわち、リジンをＣ端側に固定して５種類のアミノ酸（アルギニン、ロイシン、イソロイシン、プロリン、セリン）をランダムにペプチド結合させたヘキサペプチドを合成することにより、その混合物中に高いアンジオテンシン変換酵素阻害活性を有する新規なペプチドが存在し、さらに同ペプチドが特定の配列を有することを見出し、本発明を完成するに至った。 The present inventors have intensively studied to solve the above problems. That is, by synthesizing a hexapeptide in which lysine is fixed on the C-terminal side and 5 types of amino acids (arginine, leucine, isoleucine, proline, serine) are randomly bound to the peptide, high angiotensin converting enzyme inhibition is observed in the mixture. It has been found that there is a novel peptide having activity, and that the peptide has a specific sequence, and the present invention has been completed.
　本発明は、下記のアミノ酸配列からなるペプチドを提供する。
Ｘａａ－Ａｒｇ－Ｘａａ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（配列番号１）
（但し、各アミノ酸はＬ－体であり、Ｘａａは、独立してＩｌｅ又はＬｅｕである。）
　本発明のペプチドは、以下のアミノ酸配列のいずれかを有することを好ましい形態としている。
ａ）Ｉｌｅ－Ａｒｇ－Ｉｌｅ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（配列番号２）
ｂ）Ｉｌｅ－Ａｒｇ－Ｌｅｕ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（配列番号３）
ｃ）Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（配列番号４）
ｄ）Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（配列番号５）
　本発明はまた、前記ペプチドを有効成分として含有するアンジオテンシン変換酵素阻害剤を提供する。
　また本発明は、前記ペプチドを有効成分として含有する血圧降下剤を提供する。
　また本発明は、血圧降下を必要とする対象に、前記ペプチドを投与することを含む、前記対象の血圧を降下させる方法を提供する。
　また本発明は、血圧降下を必要とする対象の血圧を降下させるための、前記ペプチドの使用を提供する。 The present invention provides a peptide consisting of the following amino acid sequence.
Xaa-Arg-Xaa-Pro-Ser-Lys (SEQ ID NO: 1)
(However, each amino acid is L-form, and Xaa is independently Ile or Leu.)
The peptide of the present invention preferably has any of the following amino acid sequences.
a) Ile-Arg-Ile-Pro-Ser-Lys (SEQ ID NO: 2)
b) Ile-Arg-Leu-Pro-Ser-Lys (SEQ ID NO: 3)
c) Leu-Arg-Leu-Pro-Ser-Lys (SEQ ID NO: 4)
d) Leu-Arg-Ile-Pro-Ser-Lys (SEQ ID NO: 5)
The present invention also provides an angiotensin converting enzyme inhibitor containing the peptide as an active ingredient.
The present invention also provides a blood pressure lowering agent containing the peptide as an active ingredient.
The present invention also provides a method for lowering blood pressure in a subject comprising administering the peptide to a subject in need of blood pressure reduction.
The present invention also provides use of the peptide for lowering blood pressure in a subject in need of blood pressure reduction.
合成ペプチド混合物の逆相ＨＰＬＣによる分離クロマトグラム。横軸は時間（分）を、縦軸は215nmでの吸光度を、それぞれ示す。The separation chromatogram by the reverse phase HPLC of a synthetic peptide mixture. The horizontal axis represents time (minutes), and the vertical axis represents absorbance at 215 nm.
　次に、本発明の好ましい実施形態について詳細に説明する。ただし、本発明は以下の好ましい実施形態に限定されず、本発明の範囲内で自由に変更することができるものである。尚、本明細書において百分率は特に断りのない限り質量による表示である。
　本発明のペプチドは、Ｘａａ－Ａｒｇ－Ｘａａ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（配列番号１）で表される配列を有する。また、本発明のペプチドは、このペプチドの塩類であってもよい。本発明において、ＬｅｕはＬ－ロイシン残基、ＡｒｇはＬ－アルギニン残基、ＩｌｅはＬ－イソロイシン残基、ＰｒｏはＬ－プロリン残基、ＳｅｒはＬ－セリン残基、ＬｙｓはＬ－リジン残基を示す。また、Ｘａａは、独立してＩｌｅ又はＬｅｕである。 Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention. In the present specification, percentages are expressed by mass unless otherwise specified.
The peptide of the present invention has a sequence represented by Xaa-Arg-Xaa-Pro-Ser-Lys (SEQ ID NO: 1). The peptide of the present invention may be a salt of this peptide. In the present invention, Leu is an L-leucine residue, Arg is an L-arginine residue, Ile is an L-isoleucine residue, Pro is an L-proline residue, Ser is an L-serine residue, and Lys is an L-lysine residue. Indicates a group. Xaa is independently Ile or Leu.
　すなわち、本発明のペプチドは以下のアミノ酸配列のいずれかを有する。
ａ）Ｉｌｅ－Ａｒｇ－Ｉｌｅ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（配列番号２）
ｂ）Ｉｌｅ－Ａｒｇ－Ｌｅｕ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（配列番号３）
ｃ）Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（配列番号４）
ｄ）Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（配列番号５） That is, the peptide of the present invention has any of the following amino acid sequences.
a) Ile-Arg-Ile-Pro-Ser-Lys (SEQ ID NO: 2)
b) Ile-Arg-Leu-Pro-Ser-Lys (SEQ ID NO: 3)
c) Leu-Arg-Leu-Pro-Ser-Lys (SEQ ID NO: 4)
d) Leu-Arg-Ile-Pro-Ser-Lys (SEQ ID NO: 5)
　本発明のペプチドは、化学合成によって製造することができる。本発明のペプチドの化学合成は、オリゴペプチドの合成に通常用いられている液相法または固相法によって行うことができる。合成されたペプチドは、必要に応じて脱保護され、未反応試薬、副生物等を除去する。このようなペプチドの合成は、市販のペプチド合成装置を用いて行うことができる。上記合成（混合）物から、好ましくは本発明のペプチドを単離、精製する。ペプチドの精製は、通常、オリゴペプチドの精製に用いられているのと同様の手法、例えばイオン交換クロマトグラフィー、吸着クロマトグラフィー、逆相クロマトグラフィー、分配クロマトグラフィー、ゲル濾過クロマトグラフィー等の各種クロマトグラフィー、溶媒沈殿、塩析、２種の液相間での分配等の方法を適宜組み合わせることによって、行うことができる。本発明のペプチドの精製に際しては、目的物質を含む画分は、後述するアンジオテンシン変換酵素阻害作用を指標として決定することができ、それらの画分の活性成分は質量分析法又は／およびプロテインシーケンサーにより同定することができる。 The peptide of the present invention can be produced by chemical synthesis. The chemical synthesis of the peptide of the present invention can be carried out by a liquid phase method or a solid phase method usually used for the synthesis of oligopeptides. The synthesized peptide is deprotected as necessary to remove unreacted reagents, by-products and the like. Such peptide synthesis can be performed using a commercially available peptide synthesizer. The peptide of the present invention is preferably isolated and purified from the above synthesized (mixed) product. Peptide purification is usually performed in the same manner as that used for oligopeptide purification, such as ion exchange chromatography, adsorption chromatography, reverse phase chromatography, partition chromatography, gel filtration chromatography, and other various chromatography methods. , Solvent precipitation, salting out, and partitioning between the two liquid phases, and the like. In the purification of the peptide of the present invention, the fraction containing the target substance can be determined using the angiotensin converting enzyme inhibitory action described below as an index, and the active component of those fractions can be determined by mass spectrometry or / and a protein sequencer. Can be identified.
　また、本発明のペプチドは、同ペプチドをコードする組換えＤＮＡを適当な宿主細胞で発現させることによっても、製造することができる。組換えＤＮＡの作成に必要なベクター、及び宿主は、通常タンパク質やペプチドの製造に用いられているものを使用することができる。 The peptide of the present invention can also be produced by expressing a recombinant DNA encoding the peptide in an appropriate host cell. As vectors and hosts necessary for the production of recombinant DNA, those usually used for the production of proteins and peptides can be used.
　本発明のペプチドは、アンジオテンシン変換酵素阻害剤の有効成分として使用することができる。本発明のペプチドは、アンジオテンシン変換酵素阻害作用を有している。アンジオテンシン変換酵素が阻害されると、同酵素によるアンジオテンシンIIの生成、及び、ブラジキニンの不活性化が抑制されるため、結果として血圧降下作用を示す。したがって、本発明のペプチド又はアンジオテンシン変換酵素阻害剤は、高血圧に由来する種々の疾患、例えば脳出血、脳梗塞、狭心症、心筋梗塞、腎不全等に対する予防剤又は治療剤、具体的には血圧降下剤等として使用することができる。すなわち、本発明の一形態は、本発明のペプチドを有効成分として含有する血圧降下剤である。また、アンジオテンシン変換酵素阻害剤は、原因不明の本態性高血圧にも効果があることが知られており、本発明のペプチドも本態性高血圧に対する治療又は予防効果を示すことが期待される。その他、アンジオテンシン変換酵素阻害剤が有効であることが知られている心肥大、狭心病等の疾患に対しても、治療、予防薬として使用することができる。
　本発明の他の形態は、血圧降下を必要とする対象に本発明のペプチドを投与することを含む、前記対象の血圧を降下させる方法である。
　また、本発明の他の形態は、血圧降下を必要とする対象の血圧を降下させるための本発明のペプチドの使用である。
　血圧降下を必要とする対象としては、アンジオテンシン変換酵素を阻害することが有効である限り特に制限されないが、上記の高血圧に由来する種々の疾患を有する患者、本態性高血圧の患者、及び、心肥大、狭心病等の疾患を有する患者が挙げられる。また、これらの方法及び使用において、「血圧の降下」は「疾患の治療」であり得る。 The peptide of the present invention can be used as an active ingredient of an angiotensin converting enzyme inhibitor. The peptide of the present invention has an angiotensin converting enzyme inhibitory action. When an angiotensin converting enzyme is inhibited, the production of angiotensin II and the inactivation of bradykinin by the enzyme are suppressed, resulting in a blood pressure lowering effect. Therefore, the peptide or angiotensin converting enzyme inhibitor of the present invention is a preventive or therapeutic agent for various diseases derived from hypertension such as cerebral hemorrhage, cerebral infarction, angina pectoris, myocardial infarction, renal failure, etc., specifically blood pressure. It can be used as a depressant. That is, one form of the present invention is a blood pressure lowering agent containing the peptide of the present invention as an active ingredient. Moreover, an angiotensin converting enzyme inhibitor is known to have an effect on essential hypertension of unknown cause, and the peptide of the present invention is expected to show a therapeutic or preventive effect on essential hypertension. In addition, it can also be used as a therapeutic or prophylactic agent for diseases such as cardiac hypertrophy and angina which are known to be effective for angiotensin converting enzyme inhibitors.
Another aspect of the present invention is a method for lowering blood pressure in a subject comprising administering a peptide of the present invention to a subject in need of blood pressure reduction.
Another aspect of the present invention is the use of a peptide of the present invention for lowering blood pressure in a subject in need of blood pressure reduction.
The subject requiring blood pressure lowering is not particularly limited as long as it is effective to inhibit angiotensin converting enzyme, but patients having various diseases derived from the above-mentioned hypertension, patients with essential hypertension, and cardiac hypertrophy And patients having diseases such as angina. Also, in these methods and uses, “decrease in blood pressure” can be “treatment of disease”.
　本発明のペプチドは、医薬、飲食品、飼料、化粧品、医薬部外品等に配合することができる。配合されるペプチドは、１種類でもよく、任意の２種以上の混合物であってもよい。
　例えば、医薬の製剤化にあたっては、担体、賦形剤、結合剤、崩壊剤、滑沢剤、着色剤安定剤、矯味矯臭剤、希釈剤、注射剤用溶剤等の添加剤を使用できる。具体的製剤として、錠剤（糖衣錠、腸溶性コーティング錠、バッカル錠を含む。）、散剤、カプセル剤（腸溶性カプセル、ソフトカプセルを含む。）、顆粒剤（コーティングしたものを含む。）、丸剤、トローチ剤、封入リポソーム剤、液剤、又はこれらの製剤学的に許容され得る徐放製剤等を例示することができる。 The peptide of this invention can be mix | blended with a pharmaceutical, food-drinks, feed, cosmetics, a quasi-drug, etc. One type of peptide may be blended, or a mixture of any two or more types may be used.
For example, additives such as carriers, excipients, binders, disintegrants, lubricants, colorant stabilizers, flavoring agents, diluents, and solvents for injections can be used for pharmaceutical formulation. Specific preparations include tablets (including sugar-coated tablets, enteric-coated tablets, buccal tablets), powders, capsules (including enteric capsules and soft capsules), granules (including those coated), pills, Illustrative examples include troches, encapsulated liposomes, solutions, and pharmaceutically acceptable sustained release formulations.
　これらの製剤に用いる担体及び賦形剤としては、乳糖、ブドウ糖、白糖、マンニトール、馬鈴薯澱粉、トウモロコシ澱粉、炭酸カルシウム、リン酸カルシウム、硫酸カルシウム、結晶セルロース、カンゾウ末、ゲンチアナ末等を、結合剤としては澱粉、ゼラチン、シロップ、ポリビニルアルコール、ポリビニルエーテル、ポリビニルピロリドン、ヒドロキシプロピルセルロース、エチルセルロース、メチルセルロース、カルボキシメチルセルロース等を例示することができる。
　また、崩壊剤としては、澱粉、寒天、ゼラチン末、カルボキシメチルセルロースナトリウム、カルボキシメチルセルロースカルシウム、結晶セルロース、炭酸カルシウム、炭酸水素ナトリウム、及びアルギン酸ナトリウム等を例示することができる。
　更に、滑沢剤としては、ステアリン酸マグネシウム、水素添加植物油、及びマクロゴール等を、着色剤としては医薬品に添加することが許容されている赤色２号、黄色４号、及び青色１号等を、それぞれ例示することができる。
　その他、安定剤、矯味矯臭剤、希釈剤、注射剤用溶剤等についても、通常、医薬の製造に用いられる成分を用いることができる。 Carriers and excipients used in these formulations include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder and the like as binders Examples thereof include starch, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose and the like.
Examples of the disintegrant include starch, agar, gelatin powder, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate.
Furthermore, as the lubricant, magnesium stearate, hydrogenated vegetable oil, macrogol, etc., and as the colorant, red No. 2, yellow No. 4, and blue No. 1, etc., which are allowed to be added to pharmaceuticals, etc. , Respectively.
In addition, components that are usually used in the manufacture of pharmaceuticals can be used for stabilizers, flavoring agents, diluents, solvents for injections, and the like.
　錠剤及び顆粒剤は、必要に応じ白糖、ヒドロキシプロピルセルロース、精製セラック、ゼラチン、ソルビトール、グリセリン、エチルセルロース、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、フタル酸セルロースアセテート、フタル酸ヒドロキシプロピルメチルセルロース、メチルメタクリレート、及びメタアクリル酸重合体等により被膜することもできる。 Tablets and granules are sucrose, hydroxypropylcellulose, purified shellac, gelatin, sorbitol, glycerin, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate as necessary And a methacrylic acid polymer.
　本発明のアンジオテンシン変換酵素阻害剤又は血圧降下剤は、経口投与、非経口投与のいずれによって投与されてもよいが、経口投与が好ましい。非経口投与としては、静注、直腸投与、吸入等が挙げられる。経口投与の剤型としては、錠剤、カプセル剤、トローチ剤、シロップ剤、顆粒剤、散剤、軟膏等が挙げられる。また、公知の、もしくは将来的に見出されるアンジオテンシン変換酵素阻害作用を有する薬剤、医薬組成物を本発明のペプチドと併用することもできる。併用する薬剤又は医薬組成物は、本発明の薬剤中に有効成分の一つとして含有させてもよいし、本発明の薬剤中には含有させずに、別個の薬剤として本発明の薬剤と組み合わせて商品化してもよい。 The angiotensin converting enzyme inhibitor or blood pressure lowering agent of the present invention may be administered either orally or parenterally, but oral administration is preferred. Parenteral administration includes intravenous injection, rectal administration, inhalation and the like. Examples of the dosage form for oral administration include tablets, capsules, troches, syrups, granules, powders, ointments and the like. Moreover, the medicine and pharmaceutical composition which have the angiotensin converting enzyme inhibitory action known or discovered in the future can also be used together with the peptide of this invention. The drug or pharmaceutical composition to be used in combination may be contained as one of the active ingredients in the drug of the present invention, or it is not contained in the drug of the present invention and is combined with the drug of the present invention as a separate drug. May be commercialized.
　また、本発明のペプチドを有効成分として食品中に含有させ、アンジオテンシン変換酵素阻害剤又は血圧降下剤の一態様として、アンジオテンシン変換酵素阻害作用又は血圧降下作用を有する食品として加工することも可能である。
　このような食品としては、液状、ペースト状、固体、粉末等の形態を問わず、錠菓、流動食、飼料（ペット用を含む）等のほか、例えば、パン、マカロニ、スパゲッティ、めん類、ケーキミックス、から揚げ粉、パン粉等の小麦粉製品；即席めん、カップめん、レトルト・調理食品、調理缶詰、電子レンジ食品、即席スープ・シチュー、即席みそ汁・吸い物、スープ缶詰、フリーズ・ドライ食品、その他の即席食品等の即席食品類；農産缶詰、果実缶詰、ジャム・マーマレード類、漬物、煮豆類、農産乾物類、シリアル（穀物加工品）等の農産加工品； 水産缶詰、魚肉ハム・ソーセージ、水産練り製品、水産珍味類、つくだ煮類等の水産加工品；畜産缶詰・ペースト類、畜肉ハム・ソーセージ等の畜産加工品；加工乳、乳飲料、ヨーグルト類、乳酸菌飲料類、チーズ、アイスクリーム類、調製粉乳類、クリーム、その他の乳製品等の乳・乳製品；バター、マーガリン類、植物油等の油脂類；しょうゆ、みそ、ソース類、トマト加工調味料、みりん類、食酢類等の基礎調味料；調理ミックス、カレーの素類、たれ類、ドレッシング類、めんつゆ類、スパイス類、その他の複合調味料等の複合調味料・食品類；素材冷凍食品、半調理冷凍食品、調理済冷凍食品等の冷凍食品；キャラメル、キャンディー、チューインガム、チョコレート、クッキー、ビスケット、ケーキ、パイ、スナック、クラッカー、和菓子、米菓子、豆菓子、デザート菓子、その他の菓子などの菓子類；炭酸飲料、天然果汁、果汁飲料、果汁入り清涼飲料、果肉飲料、果粒入り果実飲料、野菜系飲料、豆乳、豆乳飲料、コーヒー飲料、お茶飲料、粉末飲料、濃縮飲料、スポーツ飲料、栄養飲料、アルコール飲料、その他の嗜好飲料等の嗜好飲料類、ベビーフード、ふりかけ、お茶潰けのり等のその他の市販食品等；育児用調製粉乳；経腸栄養食；機能性食品（特定保健用食品、栄養機能食品）等が挙げられる。 Further, the peptide of the present invention can be contained in food as an active ingredient, and processed as a food having an angiotensin converting enzyme inhibitory action or blood pressure lowering action as one embodiment of an angiotensin converting enzyme inhibitor or blood pressure lowering agent. .
Such foods may be in the form of liquids, pastes, solids, powders, etc., in addition to tablet confections, liquid foods, feeds (including for pets), etc., for example, bread, macaroni, spaghetti, noodles, cakes Flour products such as mix, fried flour, bread crumbs, instant noodles, cup noodles, retort / cooked food, cooked canned food, microwave food, instant soup / stew, instant miso soup / soup, canned soup, freeze-dried food, other instant foods Instant foods such as foods; canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, processed cereals (cereal processed products); canned seafood, fish ham and sausages, fish paste products, Processed marine products such as marine delicacy, tsukudani, etc .; Livestock canned and pasted products, livestock processed products such as livestock ham and sausage; processed milk, milk drinks, yogurts Milk and dairy products such as lactic acid bacteria beverages, cheese, ice cream, formula milk powder, cream and other dairy products; fats and oils such as butter, margarine, vegetable oil; soy sauce, miso, sauces, tomato processed seasonings , Mirins, basic seasonings such as vinegar; cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices, and other complex seasonings and foods; frozen foods, Semi-cooked frozen foods, frozen foods such as cooked frozen foods; caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pie, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, other confectionery Sweets: carbonated drink, natural fruit juice, fruit juice drink, soft drink with fruit juice, fruit drink, fruit drink with fruit granules, vegetable drink, soy milk, soy milk drink Coffee beverages, tea beverages, powdered beverages, concentrated beverages, sports beverages, nutritional beverages, alcoholic beverages, other beverages such as other favorite beverages, baby foods, sprinkles, other commercially available foods such as tea crush paste, etc. for childcare Examples include formula milk powder; enteral nutrition; functional food (food for specified health use, functional food for nutrition).
　さらに、アンジオテンシン変換酵素阻害剤又は血圧降下剤の一態様として、本発明のペプチドを有効成分として飼料中に含有させ、アンジオテンシン変換酵素阻害作用又は血圧降下作用を有する飼料として加工することも可能である。
　飼料の形態としては特に制限されず、例えば、本発明のペプチドの粉末やこれらの水溶液（シロップ等）等を、トウモロコシ、小麦、大麦、ライ麦、マイロ等の穀類；大豆油粕、ナタネ油粕、ヤシ油粕、アマニ油粕等の植物性油粕類；フスマ、麦糠、米糠、脱脂米糠等の糠類；コーングルテンミール、コーンジャムミール等の製造粕類；魚粉、脱脂粉乳、ホエイ、イエローグリース、タロー等の動物性飼料類；トルラ酵母、ビール酵母等の酵母類；第三リン酸カルシウム、炭酸カルシウム等の鉱物質飼料；油脂類；単体アミノ酸；糖類等に配合することにより製造できる。飼料の形態としては、例えば、ペットフード、家畜飼料、養魚飼料等が挙げられる。 Furthermore, as an embodiment of an angiotensin converting enzyme inhibitor or blood pressure lowering agent, the peptide of the present invention can be contained in the feed as an active ingredient, and processed as a feed having an angiotensin converting enzyme inhibitory action or blood pressure lowering action. .
The form of the feed is not particularly limited. For example, the peptide powder of the present invention or an aqueous solution thereof (syrup or the like) is used for cereals such as corn, wheat, barley, rye, and milo; soybean oil cake, rapeseed oil cake, coconut oil cake Vegetable oils such as flaxseed, flaxseed oil, rice bran, rice bran, defatted rice bran, etc .; production corn such as corn gluten meal, corn jam meal; fish meal, skim milk powder, whey, yellow grease, tallow, etc. Animal feeds; yeasts such as torula yeast and beer yeast; mineral feeds such as tricalcium phosphate and calcium carbonate; fats and oils; simple amino acids; sugars and the like. Examples of the form of the feed include pet food, livestock feed, and fish feed.
　本発明のアンジオテンシン変換酵素阻害剤又は血圧降下剤（医薬品、飲食品、飼料、化粧品、医薬部外品等の各態様）において、本発明のペプチドの配合量（ペプチドが複数種の場合は合計量）は、アンジオテンシン変換酵素阻害剤又は血圧降下剤の最終組成物に対して、０．００１質量％以上であることが好ましく、０．０１質量％以上であることがより好ましく、０．１質量％以上であることが特に好ましい。 In the angiotensin converting enzyme inhibitor or antihypertensive agent of the present invention (each aspect of pharmaceuticals, foods and drinks, feeds, cosmetics, quasi drugs, etc.), the amount of the peptide of the present invention (the total amount in the case of multiple peptides) ) Is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and 0.1% by mass with respect to the final composition of the angiotensin converting enzyme inhibitor or blood pressure lowering agent. The above is particularly preferable.
　本発明のアンジオテンシン変換酵素阻害剤又は血圧降下剤の投与量は、年齢、症状等により異なるが、通常、本発明のペプチドの量として０．００１～３０００ｍｇ／日、好ましくは０．０１～３０ｍｇ／日であり、１日１回、又は２回から３回に分けて投与してもよい。また、本発明のアンジオテンシン変換酵素阻害剤又は血圧降下剤を摂取又は服用する場合は、食前、食間、食後のいずれのタイミングであっても、本発明の効果は十分に発揮されるものである。 The dose of the angiotensin converting enzyme inhibitor or blood pressure lowering agent of the present invention varies depending on age, symptoms, etc., but is usually 0.001 to 3000 mg / day, preferably 0.01 to 30 mg / day as the amount of the peptide of the present invention. It may be administered once a day or divided into 2 to 3 times a day. In addition, when the angiotensin converting enzyme inhibitor or blood pressure lowering agent of the present invention is ingested or taken, the effects of the present invention are sufficiently exhibited at any timing before, between, and after a meal.
　以下に実施例を用いて本発明をさらに詳しく説明するが、本発明はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
＜実施例１＞
（１）ペプチドの化学合成
　ペプチドシンセサイザー（Ｍｏｄｅｌ ４３３Ａ型、アプライドバイオシステムズ社）を使用し、５種類のアミノ酸誘導体混合物〔Ｆｍｏｃ－Ｌ－Ｌｅｕ、Ｆｍｏｃ－Ｌ－Ａｒｇ（Ｍｔｒ）、Ｆｍｏｃ－Ｌ－Ｉｌｅ、Ｆｍｏｃ－Ｌ－Ｓｅｒ（ｔＢｕ）、Ｆｍｏｃ－Ｌ－Ｐｒｏ〕、Ｆｍｏｃ－Ｌ－Ｌｙｓ（Ｂｏｃ）－Ｗａｎｇ Ｒｅｓｉｎ（いずれも国産化学）を原料に用いて、固相合成法によりＡＡ(6)－ＡＡ(5)－ＡＡ(4)－ＡＡ(3)－ＡＡ(2)－Ｌｙｓ〔ＡＡ(n)は、５種類のアミノ酸のいずれかであり、合成されるペプチドは５5＝３１２５種類〕（配列番号６）の混合物の合成を行った。操作はアプライドバイオシステムズ社のマニュアルに従って行った。合成反応後、脱保護した。得られたペプチドは、下記ＨＰＬＣ条件で精製した。 <Example 1>
(1) Peptide Chemical Synthesis Using a peptide synthesizer (Model 433A type, Applied Biosystems), a mixture of five amino acid derivatives [Fmoc-L-Leu, Fmoc-L-Arg (Mtr), Fmoc-L-Ile , Fmoc-L-Ser (tBu), Fmoc-L-Pro], Fmoc-L-Lys (Boc) -Wang Resin (all from domestic chemistry) and AA (6)- AA (5) -AA (4) -AA (3) -AA (2) -Lys [AA (n) is one of five amino acids, and 5 5 = 3125 peptides are synthesized] ( A mixture of SEQ ID NO: 6) was synthesized. The operation was performed according to the manual of Applied Biosystems. After the synthesis reaction, it was deprotected. The obtained peptide was purified under the following HPLC conditions.
（２）ＨＰＬＣによるペプチドの分離
　逆相ＨＰＬＣで上記合成混合物の分離を行った。このＨＰＬＣ条件は下記ＨＰＬＣ条件１に示した。 (2) Separation of peptides by HPLC The synthesis mixture was separated by reverse phase HPLC. The HPLC conditions are shown in the following HPLC condition 1.
〔ＨＰＬＣ条件１〕
　カラム ：ＣａｄｅｎｚａＣＤ－１８　１０ｍｍＩ．Ｄ．×２５０ｍｍ（インタクト社製）
　検出：ＵＶ ２１５ｎｍ
　流速：３ｍｌ／分
　溶離液Ａ：０．１％ ＴＦＡを含む水溶液
　溶離液Ｂ：０．１％ ＴＦＡを含むアセトニトリル溶液 [HPLC condition 1]
Column: Cadenza CD-18 10 mmI. D. × 250mm (manufactured by Intact)
Detection: UV 215nm
Flow rate: 3 ml / min Eluent A: Aqueous solution containing 0.1% TFA Eluent B: Acetonitrile solution containing 0.1% TFA
　サンプルをカラムに導入後、溶離液（Ａ／Ｂ）を９８／２で５分間維持し、溶離液（７５／２５）まで３０分間、溶離液（５０／５０）まで１０分間、更に溶離液（２０／８０）まで３分間でグラジエント溶出を行い、その後５分間溶離液（２０／８０）を維持するグラジエント条件で、合成ペプチド混合物を分離した（クロマトグラム：図１）。
　各ピーク毎に溶出液を分画し、後述する方法でアンジオテンシン変換酵素阻害能を測定したところ、強いアンジオテンシン変換酵素阻害能を持つ画分が、リテンションタイム34.0分、34.4分、34.7分、及び35.1分に溶出された。 After the sample is introduced into the column, the eluent (A / B) is maintained at 98/2 for 5 minutes, 30 minutes to the eluent (75/25), 10 minutes to the eluent (50/50), and further eluent ( Gradient elution was performed in 3 minutes until 20/80), and then the synthetic peptide mixture was separated under a gradient condition of maintaining the eluent (20/80) for 5 minutes (chromatogram: FIG. 1).
The eluate was fractionated for each peak, and the angiotensin converting enzyme inhibitory ability was measured by the method described later, and the fractions having strong angiotensin converting enzyme inhibitory ability were retention times of 34.0 minutes, 34.4 minutes, 34.7 minutes, and 35.1. Eluted in minutes.
　これらの画分に含まれるペプチドについて、島津製作所製のプロテイン・シーケンサー（ＰＰＳＱ－２３Ａ）で分析を行ったところ、それぞれＩｌｅ－Ａｒｇ－Ｉｌｅ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（以下、ＨＰ１と記載する）、Ｉｌｅ－Ａｒｇ－Ｌｅｕ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（以下、ＨＰ２と記載する）、Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（以下、ＨＰ３と記載する）、及び、Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ（以下、ＨＰ４と記載する）であった。 The peptides contained in these fractions were analyzed with a protein sequencer (PPSQ-23A) manufactured by Shimadzu Corporation, and found to be Ile-Arg-Ile-Pro-Ser-Lys (hereinafter referred to as HP1), Ile-Arg-Leu-Pro-Ser-Lys (hereinafter referred to as HP2), Leu-Arg-Leu-Pro-Ser-Lys (hereinafter referred to as HP3), and Leu-Arg-Ile-Pro- Ser-Lys (hereinafter referred to as HP4).
＜実施例２＞
［ペプチドのアンジオテンシン変換酵素阻害作用］
（１）試験方法
　アンジオテンシン変換酵素阻害の測定は、カッシュマンらの方法〔バイオケミカル・ファーマコロジー２０巻、１６３７～１６４８頁（１９７１）〕に準じて行った。
　試料として、実施例１で得られた本発明ペプチド（HP1、HP2、HP3、HP4）、Agricultural and Biological Chemistry 49(5), 1405-1409, 1985に記載のペプチド（Phe－Phe－Val－Ala－Pro－Phe－Pro－Glu－Val－Phe－Gly－Lys：配列番号７）、及び特許第３８１６９２１号（WO2003/044044））に記載のペプチド（Ｍｅｔ－Ｌｙｓ－Ｐｒｏ）を用いた。これらのペプチドは、いずれも実施例１と同様にして化学合成したものである。 <Example 2>
[Angiotensin-converting enzyme inhibitory action of peptides]
(1) Test method Angiotensin converting enzyme inhibition was measured according to the method of Kashman et al. [Biochemical pharmacology, Vol. 20, pages 1637 to 1648 (1971)].
As a sample, the peptide of the present invention (HP1, HP2, HP3, HP4) obtained in Example 1, the peptide described in Agricultural and Biological Chemistry 49 (5), 1405-1409, 1985 (Phe-Phe-Val-Ala- Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys: SEQ ID NO: 7) and the peptide (Met-Lys-Pro) described in Japanese Patent No. 38169921 (WO2003 / 044044)) were used. All of these peptides were chemically synthesized in the same manner as in Example 1.
　試料を０．１Ｍホウ酸緩衝液（０．３Ｍ ＮａＣｌを含む、ｐＨ８．３）に溶解し、試験管に０．０８ｍｌ入れた後、０．１Ｍホウ酸緩衝液（０．３Ｍ ＮａＣｌを含む、ｐＨ８．３）で５ｍＭに調製した酵素基質（ヒプリルヒスチジルロイシン、シグマ社製）０．２ｍｌを添加し、３７℃で３分間保温した。次いで、蒸留水を添加して０．１Ｕ／ｍｌになるように調製したウサギ肺のアンジオテンシン変換酵素（シグマ社製）０．０２ｍｌを添加し、３７℃で３０分間反応させた。 The sample was dissolved in 0.1 M borate buffer (containing 0.3 M NaCl, pH 8.3), 0.08 ml was placed in a test tube, and then 0.1 M borate buffer (containing 0.3 M NaCl) 0.2 ml of an enzyme substrate (Hiprilhistidylleucine, Sigma) adjusted to 5 mM at pH 8.3) was added, and the mixture was incubated at 37 ° C. for 3 minutes. Subsequently, 0.02 ml of rabbit lung angiotensin converting enzyme (manufactured by Sigma) prepared to be 0.1 U / ml by adding distilled water was added and reacted at 37 ° C. for 30 minutes.
　その後、１Ｎ塩酸０．２５ｍｌを添加して反応を終了した後、１．７ｍｌの酢酸エチルを加え、２０秒間激しく攪件し、３０００ｒｐｍで１０分間遠心分離して、酢酸エチル層を１．４ｍｌ採取した。得られた酢酸エチル層を加熱して溶媒を除去した後、蒸留水を１．０ｍｌ添加し、抽出したヒプリル酸の吸収（２２８ｎｍの吸光度）を測定して、これを酵素活性とした。 Thereafter, 0.25 ml of 1N hydrochloric acid was added to complete the reaction, 1.7 ml of ethyl acetate was added, and the mixture was vigorously stirred for 20 seconds, centrifuged at 3000 rpm for 10 minutes, and 1.4 ml of the ethyl acetate layer was collected. did. The obtained ethyl acetate layer was heated to remove the solvent, and then 1.0 ml of distilled water was added, and the absorption of the extracted hyprilic acid (absorbance at 228 nm) was measured to determine the enzyme activity.
　次の式から、阻害活性を求め、ＩＣ５０［アンジオテンシン変換酵素の活性を５０％阻害するために必要な試料濃度（μＭ）］を決定した。 Inhibitory activity was determined from the following formula, and IC50 [sample concentration (μM) required to inhibit angiotensin converting enzyme activity by 50%] was determined.
阻害率＝（Ａ－Ｂ）／（Ａ－Ｃ）×１００％
　Ａ：試料（ペプチド）を含まない場合の酵素活性（２２８ｎｍの吸光度）
　Ｂ：試料添加の場合の酵素活性（２２８ｎｍの吸光度）
　Ｃ：酵素および試料を添加しない場合の酵素活性（２２８ｎｍの吸光度） Inhibition rate = (AB) / (AC) × 100%
A: Enzyme activity when sample (peptide) is not included (absorbance at 228 nm)
B: Enzyme activity when adding sample (absorbance at 228 nm)
C: Enzyme activity when no enzyme and sample are added (absorbance at 228 nm)
（２）試験結果
　本試験結果を表１に示す。表１から明らかなとおり、本発明のペプチド（HP1、HP2、HP3、HP4）は、いずれも強力なアンジオテンシン変換酵素阻害活性を有することが判明した。 (2) Test results The test results are shown in Table 1. As is clear from Table 1, all of the peptides of the present invention (HP1, HP2, HP3, HP4) were found to have potent angiotensin converting enzyme inhibitory activity.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
＜実施例３＞
［ペプチドの動物における血圧降下作用］
（１）試験方法
　１２週齢のＳＨＲ／Ｈｏｓ雄性ラット１４匹（日本エスエルシー株式会社より購入）を、１週間予備飼育し、ラットの血圧を小動物非観血式自動血圧計（ＭＫ－２０００，室町機械（株）社製）を用いて測定した。 <Example 3>
[Antihypertensive action of peptides in animals]
(1) Test method Fourteen 12-week-old SHR / Hos male rats (purchased from Japan SLC Co., Ltd.) were preliminarily raised for 1 week, and the blood pressure of the rats was measured using a small animal non-invasive automatic sphygmomanometer (MK-2000, Muromachi Kikai Co., Ltd.).
　収縮期血圧を指標とし、群毎の投与前の平均収縮期血圧がほぼ同じ値になるように、１群あたり７匹となるように２群に分けた後、約１４時間絶食し、試験群には実施例１で得られたペプチド（Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ：HP4）を注射用水に溶解し、ラットに５ｍＬ／体重ｋｇ（HP4として３ｍｇ／体重ｋｇ）の割合で経口投与し、試料投与直前、試料投与の１、３、６及び８時間後に、ラットの血圧を測定した。 Using the systolic blood pressure as an index, the group was divided into two groups so that the average systolic blood pressure before administration for each group was almost the same value, and then the animals were fasted for about 14 hours after being divided into two groups. For example, the peptide obtained in Example 1 (Leu-Arg-Ile-Pro-Ser-Lys: HP4) was dissolved in water for injection and orally administered to rats at a rate of 5 mL / kg body weight (3 mg / kg body weight as HP4). The blood pressure of the rats was measured immediately before sample administration, 1, 3, 6 and 8 hours after sample administration.
　対照群には、前記HP4水溶液の代わりに注射用水を同容量経口投与し、試料投与直前、試料投与の１、３、６及び８時間後に、ラットの血圧を測定した。
（２）試験結果 In the control group, the same volume of water for injection was orally administered instead of the HP4 aqueous solution, and the blood pressure of the rats was measured immediately before sample administration, 1, 3, 6 and 8 hours after sample administration.
(2) Test results
　本試験結果を表２に示す。表２から明らかなとおり、試験群（HP4投与群）において持続的に血圧降下が認められたのに対し、対照群では認められなかった。従って、本発明のペプチド（Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ：HP4）に、動物に対する血圧降下作用があることが判明した。
　なお、実施例１で得られた他のペプチド（HP1、HP2、HP3）についても同様の試験を行った結果、HP4と同じように血圧降下作用を有することが判明した。 The test results are shown in Table 2. As is clear from Table 2, blood pressure was continuously decreased in the test group (HP4 administration group), but not in the control group. Therefore, it was found that the peptide of the present invention (Leu-Arg-Ile-Pro-Ser-Lys: HP4) has a blood pressure lowering effect on animals.
In addition, as a result of conducting the same test also about the other peptides (HP1, HP2, HP3) obtained in Example 1, it was found to have a blood pressure lowering effect like HP4.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　本発明によれば、アンジオテンシン変換酵素阻害作用を有する新規ペプチドが提供される。同ペプチドを含有するアンジオテンシン変換酵素阻害剤は、医薬として利用することができる。本発明のペプチドは、すべて天然型（Ｌ体）のアミノ酸から構成されるため、安全性が高く食品にも用いることができる。 According to the present invention, a novel peptide having an angiotensin converting enzyme inhibitory action is provided. An angiotensin converting enzyme inhibitor containing the peptide can be used as a medicine. Since the peptides of the present invention are all composed of natural (L) amino acids, they are highly safe and can be used in foods.

Claims (9)

  1. 　下記のアミノ酸配列からなるペプチド。
    Ｘａａ－Ａｒｇ－Ｘａａ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓ
    （但し、各アミノ酸はＬ－体であり、Ｘａａは、独立してＩｌｅ又はＬｅｕである。）     A peptide consisting of the following amino acid sequence.
    Xaa-Arg-Xaa-Pro-Ser-Lys
    (However, each amino acid is L-form, and Xaa is independently Ile or Leu.)
  2. 　Ｉｌｅ－Ａｒｇ－Ｉｌｅ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓからなるペプチドである、請求項１に記載のペプチド。 The peptide according to claim 1, which is a peptide consisting of Ile-Arg-Ile-Pro-Ser-Lys.
  3. 　Ｉｌｅ－Ａｒｇ－Ｌｅｕ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓからなるペプチドである、請求項１に記載のペプチド。 The peptide according to claim 1, which is a peptide consisting of Ile-Arg-Leu-Pro-Ser-Lys.
  4. 　Ｌｅｕ－Ａｒｇ－Ｌｅｕ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓからなるペプチドである、請求項１に記載のペプチド。 The peptide according to claim 1, which is a peptide comprising Leu-Arg-Leu-Pro-Ser-Lys.
  5. 　Ｌｅｕ－Ａｒｇ－Ｉｌｅ－Ｐｒｏ－Ｓｅｒ－Ｌｙｓからなるペプチドである、請求項１に記載のペプチド。 The peptide according to claim 1, which is a peptide comprising Leu-Arg-Ile-Pro-Ser-Lys.
  6. 　請求項１～５のいずれか一項に記載のペプチドを有効成分として含有するアンジオテンシン変換酵素阻害剤。 An angiotensin converting enzyme inhibitor comprising the peptide according to any one of claims 1 to 5 as an active ingredient.
  7. 　請求項１～５のいずれか一項に記載のペプチドを有効成分として含有する血圧降下剤。 An antihypertensive agent comprising the peptide according to any one of claims 1 to 5 as an active ingredient.
  8. 　血圧降下を必要とする対象に、請求項１～５のいずれか一項に記載のペプチドを投与することを含む、前記対象の血圧を降下させる方法。 A method for lowering blood pressure of a subject, comprising administering the peptide according to any one of claims 1 to 5 to a subject in need of blood pressure reduction.
  9. 　血圧降下を必要とする対象の血圧を降下させるための、請求項１～５のいずれか一項に記載のペプチドの使用。 Use of the peptide according to any one of claims 1 to 5 for lowering blood pressure of a subject in need of blood pressure reduction.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001106699A (en) * 1999-10-05 2001-04-17 Suetsuna Yoko New hexapeptide and angiotensin-converting enzyme inhibitor
JP2007261999A (en) * 2006-03-28 2007-10-11 Yamada Bee Farm Corp Hypotensive peptide derived from royal jelly
JP2007326790A (en) * 2006-06-06 2007-12-20 Aomori Prefecture Method for producing acetic acid-enzyme treated product of apios americana, peptide derived from apios americana and method for producing the same
JP2008156307A (en) * 2006-12-26 2008-07-10 Asahi Breweries Ltd Angiotensin converting enzyme inhibitor, angiotensin converting enzyme inhibiting composition and food and drink containing the same
JP2009500404A (en) * 2005-06-30 2009-01-08 キャンピナ・ネダーランド・ホールディング・ビー．ブイ． Peptides that inhibit angiotensin converting enzyme
JP2010520274A (en) * 2007-03-06 2010-06-10 フリーズランド ブランズ ビー．ブイ． Methods for providing ACE inhibitory peptides from whey and the like

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001106699A (en) * 1999-10-05 2001-04-17 Suetsuna Yoko New hexapeptide and angiotensin-converting enzyme inhibitor
JP2009500404A (en) * 2005-06-30 2009-01-08 キャンピナ・ネダーランド・ホールディング・ビー．ブイ． Peptides that inhibit angiotensin converting enzyme
JP2007261999A (en) * 2006-03-28 2007-10-11 Yamada Bee Farm Corp Hypotensive peptide derived from royal jelly
JP2007326790A (en) * 2006-06-06 2007-12-20 Aomori Prefecture Method for producing acetic acid-enzyme treated product of apios americana, peptide derived from apios americana and method for producing the same
JP2008156307A (en) * 2006-12-26 2008-07-10 Asahi Breweries Ltd Angiotensin converting enzyme inhibitor, angiotensin converting enzyme inhibiting composition and food and drink containing the same
JP2010520274A (en) * 2007-03-06 2010-06-10 フリーズランド ブランズ ビー．ブイ． Methods for providing ACE inhibitory peptides from whey and the like

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AKIO YAMADA ET AL.: "Koketsuatsu Shizen Hassho Rat ni Okeru MKP o Gan'yu suru Casein Bunkaibutsu no Koatsu Sayo", THE PHARMACEUTICAL SOCIETY OF JAPAN DAI 130 NENKAI YOSHISHU 3, vol. 130TH, 5 March 2010 (2010-03-05), pages 216, 30P-PM099 *

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