JP2018184367A - peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide having e.g. a potent hypotensive action.SOLUTION: A peptide has an amino acid sequence selected from (i) FWGK (sequence number 1) and (ii) FW (sequence number 2).SELECTED DRAWING: None

Description

  The present invention relates to peptides. The present invention also relates to a pharmaceutical composition, an antihypertensive agent, an antifeedant and a food containing the peptide.

  Hypertension is called a silent killer because it causes diseases directly related to death such as stroke and myocardial infarction, and it is required to prevent and treat it.

  Many physiologically active peptides having various physiological actions have been found from enzyme digests of food proteins. The onset of arteriosclerotic diseases is known to be triggered by a decrease in the function of blood vessels, particularly vascular endothelium, and attention is paid to the function of physiologically active peptides on blood vessels.

  Patent Literature 1 and Non-Patent Literature 1 disclose peptides having actions such as feeding suppression, anti-obesity, arterial relaxation, blood pressure lowering, and prevention of metabolic syndrome.

International Publication WO2012 / 070554

Kagebayashi T et al. Mol Nutr Food Res. 2012 Sep; 56 (9): 1456-63.

  An object of this invention is to provide the peptide which has actions, such as a blood pressure fall stronger than a well-known peptide.

  The present inventor has conducted extensive studies to solve the above problems, and as a result, (i) the peptide having FWKGK (SEQ ID NO: 1) and (ii) FW (SEQ ID NO: 2) has a high blood pressure lowering effect. I found out. Based on such knowledge, the present invention has been completed through further studies.

  That is, the present invention includes the following aspects.

Item 1. A peptide having an amino acid sequence from any of the following:
(I) FWGK (SEQ ID NO: 1)
(Ii) FW (SEQ ID NO: 2)

  Item 2. The peptide of claim 1, derived from a subtilisin digest of bovine serum albumin protein.

  Item 3. A pharmaceutical composition comprising the peptide according to Item 3, Item 1 or 2 as an active ingredient.

  Item 4. An antihypertensive agent comprising the peptide according to item 4, item 1 or 2 as an active ingredient.

  Item 5. An antifeedant containing the peptide according to Item 5, Item 1 or 2 as an active ingredient.

  Claim | item 6, the foodstuff containing the peptide of claim | item 1 or 2.

  Item 7. A food comprising the peptide according to item 7, item 1 or 2.

  Item 8. The food according to claim 6 or 7 for lowering blood pressure.

  Item 9. The food according to claim 6 or 7 for suppressing feeding.

  Item 10. A method for lowering blood pressure and / or suppressing feeding behavior, comprising the step of administering the peptide according to Item 10, Item 1 or 2, to a patient or a preparatory group in need.

  Item 11. The peptide according to Item 1 or 2, for lowering blood pressure and / or suppressing feeding behavior.

  Item 12. Use of the peptide according to Item 1 or 2 for producing a medicament or food for lowering blood pressure and / or suppressing feeding behavior.

  A pharmaceutical composition or food containing the peptide of the present invention as an active ingredient has a high blood pressure lowering action, has low side effects, and is suitable for long-term use. Furthermore, it also has an antifeedant action. The pharmaceutical composition and food of the present invention are particularly effective for oral administration.

  Furthermore, natural short-chain peptides can be ingested as foods, and it can be expected that hypertensive individuals ingest them as foods to prevent diseases such as arteriosclerosis. It is also effective in preventing metabolic syndrome.

  Since the peptide of the present invention can be produced as an enzyme digest of bovine serum albumin protein, side effects are not a problem. Moreover, since the bovine serum albumin protein can be easily obtained, the peptide of the present invention can be produced at low cost.

Evaluation result of blood pressure lowering action (enzyme digest) Evaluation result of blood pressure lowering action (peptide) Evaluation result of blood pressure lowering action (peptide) Results of STC-1 cell calcium responsiveness test (peptide) Results of arterial relaxation experiment (peptide) Effects of inhibitors on arterial relaxation.

  The peptide of the present invention is a peptide (dipeptide) having an amino acid sequence of (i) 4 residues of FWGK (SEQ ID NO: 1) or (ii) 2 residues of FW (SEQ ID NO: 2).

  In another aspect of the present invention, the peptide of the present invention is a peptide comprising the amino acid sequence of (i) 4 residues of FWGK (SEQ ID NO: 1) or (ii) 2 residues of FW (SEQ ID NO: 2). In this embodiment, as long as the amino acid sequence is included, amino acid residues can be added to the N-terminal side and / or the C-terminal side (preferably, the C-terminal side). 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, still more preferably 4, 3, 2, or 1 amino acid. be able to.

  In addition, one or more (for example, 2 or 3), preferably 1 amino acid residue in the above amino acid sequence can be substituted within the range not impairing the effects of the present invention. In addition, one or more (for example, 2 or 3), preferably 1 amino acid residue in the above amino acid sequence can be inserted within a range not impairing the effects of the present invention. In addition, one or more (for example, 2 or 3), preferably 1 amino acid residue in the above amino acid sequence can be deleted within a range not impairing the effects of the present invention.

  The added amino acid residue may be a natural amino acid or a non-natural amino acid. Natural amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Constituent amino acid residues and other amino acid residues such as selenocysteine, N-formylmethionine, pyrrolysine, pyroglutamine and the like are included.

  The amino acids constituting the peptide are L-form amino acids, D-form amino acids, or DL-form amino acids (the racemate and any amino acid in which either enantiomer is excessive if the D-form and L-form amino acids are mixed). Any of the above can be used. Preferable is a peptide consisting only of L-form amino acids or only D-form amino acids, particularly a peptide consisting only of L-form amino acids.

  Moreover, when the peptide used by this invention contains two or more asymmetric carbons, it may be any form of each enantiomer thru | or a diastereomer, or the mixture of these arbitrary ratios. Separation of enantiomers or diastereomers is performed using a normal column, a method using an optically active column, a method of introducing an optically active group and optically resolving it in the form of a derivative, and then removing the optically active group, Any known method such as a method of optical resolution by forming a salt with an optically active acid or base can be used.

The peptide can have modifications. The amino terminus (N terminus) of the peptide may be a free amino group (NH ₂ —), or may have a modification such as an acetyl group (CH ₃ CO—). The carboxy terminus (C terminus) of the peptide may be a free carboxyl group (—COOH) or may have a modification such as an amide group. The amino acid residue of the peptide may be unmodified or may have a modification such as a phosphate group or a sugar chain.
The peptide of the present invention may be a salt (acid addition salt or base salt). Acid addition salts include inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, perchloric acid, citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, p-toluenesulfonic acid And salts of organic acids such as benzenesulfonic acid, methanesulfonic acid and trifluoroacetic acid. 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.

The peptide of the present invention may be a solvate. Solvates include solvates such as water (in the case of hydrates), methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, and dimethoxyethane. Things.
The peptide of the present invention can be obtained by hydrolysis of a natural protein or polypeptide, or can be obtained by chemical synthesis.

  For example, the peptides of the present invention can be obtained by hydrolysis of the bovine serum albumin protein subtilisin.

  Accordingly, the present invention includes a composition obtained by treating bovine serum albumin protein with subtilisin.

  Subtilisin (also called subtilisin or subtilisin) is an enzyme derived from Bacillus subtilis and is a known proteolytic enzyme (protease) (EC 3.4.21.62). Subtilisin is a kind of serine protease and has endopeptidase activity. Subtilisin can be used as a food additive in Japan. As subtilisin, commercially available products such as reagent grade and food additive grade can be used.

  The substrate to be hydrolyzed by subtilisin is not particularly limited as long as it contains bovine serum albumin protein. Examples include milk, whey (whey), blood (whole blood), plasma, serum, purified bovine serum albumin protein, and the like.

  Hydrolysis with subtilisin is carried out under conditions that give the peptide of the present invention. The reaction temperature can be appropriately selected from 30 to 70 ° C, 30 to 40 ° C, 40 to 70 ° C, 50 to 65 ° C, and the like. The reaction time can be appropriately selected from about 30 minutes to 48 hours, about 1 to 10 hours, and about 2 to 8 hours. The pH at which the reaction is carried out can be appropriately selected from about pH 6.5 to 8.5 and pH 7 to 8. In one suitable aspect, it can be made to react on the conditions of the temperature of about 30-40 degreeC, and pH 6.5-8.5 (especially about pH 7.5). The reaction time can be, for example, about 2 to 8 hours.

  The peptide of the present invention may not be obtained under conditions where hydrolysis is excessively performed.

  If necessary, subtilisin is inactivated by heating to a temperature at which subtilisin is inactivated (for example, heating at a temperature exceeding 80 ° C. for about 5 to 60 minutes).

  The reaction product of hydrolysis may be used as it is, or the active ingredient peptide may be separated and used by purification.

  The peptide of the present invention can also be obtained by peptide synthesis. That is, in a liquid phase method or a solid phase method, which is a commonly used method for peptide synthesis, a method using an active ester such as HBTU, a raw material having a reactive carboxyl group and a raw material having a reactive amino group, or carbodiimide In a peptide synthesis such as a method using a coupling agent such as When the resulting condensate has a protecting group, it can also be produced by removing the protecting group.

  Functional groups that should not participate in the reaction in this reaction step are protected by protecting groups. Examples of the protecting group for the amino group include benzyloxycarbonyl (CBZ), t-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the carboxyl group-protecting agent include groups capable of forming alkyl esters, benzyl esters and the like. In the solid phase method, the C-terminal carboxyl group is a chlorotrityl resin, chloromethyl resin, oxymethyl resin, p- It is bound to a carrier such as an alkoxybenzyl alcohol resin. The condensation reaction is carried out in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.

  After completion of the condensation reaction, the protecting group is removed, but in the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved. Furthermore, the peptides of the present invention are purified according to conventional methods. Examples thereof include ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like. Synthesis of the synthesized peptide is analyzed by a protein sequencer that reads the amino acid sequence from the C-terminal by the Edman degradation method, GC-MS, or the like.

  The peptide of the present invention can also be synthesized by an enzymatic method (see International Publication WO2003 / 010307).

The peptide of the present invention has a strong blood pressure lowering action and arterial relaxation action. As shown in the Examples described later, these actions are suppressed by lorglumide, which is an antagonist of CCK ₁ receptor, and thus these actions are considered to be mediated by activation of CCK ₁ receptor.

  Moreover, since the peptide of this invention has the effect | action which accelerates | stimulates release | release of endogenous feeding suppression peptide CCK, it also has a feeding suppression effect. The peptide of the present invention can be expected to exhibit a natural feeling of fullness as it is an action via the endogenous feeding inhibitory peptide CCK that enhances satisfaction after eating.

  Therefore, the peptide of the present invention has a blood pressure lowering action, an arterial relaxation action, an antifeeding action and an antiobesity action through an antifeeding action, and is further effective in preventing arteriosclerosis. Effective for prevention. In patients with metabolic syndrome, it has been reported that CCK responsiveness is lowered, and from this viewpoint, it is more suitable as a preventive agent for metabolic syndrome.

  The peptide of the present invention can be provided as a pharmaceutical composition or a food (food composition).

  The administration route of the peptide of the present invention or a product containing the peptide is not particularly limited, and oral administration, parenteral administration (for example, intramuscular or intravenous), rectal administration, and the like can be employed. Of these, oral administration or rectal administration, particularly oral administration is preferred from the viewpoint of high effect.

  The dose of the peptide varies depending on the administration method, the condition and age of the administered person. The dosage is usually 0.01 mg / kg to 500 mg / kg, preferably 0.05 mg / kg to 100 mg / kg, more preferably 0.1 to 30 mg / kg per day for adults of patients or preliminary groups in need. It is. The peptide (active ingredient) of the present invention can be administered in the form of a pharmaceutical composition prepared by mixing with a pharmaceutical carrier. As a pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.

  The peptide of the present invention can itself be used as a medicine or food. Tablets (plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, etc.), capsule hard capsules and soft capsules, alone or in combination with a suitable non-toxic carrier, diluent or excipient for oral intake Including both. ), Lozenges, powders, fine granules, granules, solutions, suspensions, emulsions, pastes, creams, injections (including when mixed in infusions such as amino acid infusions and electrolyte infusions), or enteric It is possible to prepare a preparation for food or pharmaceutical use such as a sustained release preparation such as a tablet, capsule or granule.

  One aspect of the present invention includes a pharmaceutical composition comprising the peptide of the present invention and a diluent, carrier, or excipient recognized as a pharmaceutical. As another embodiment, a food containing the peptide of the present invention (for example, it can be contained by adding) can be mentioned.

  The content of the peptide in the medicine or food can be appropriately selected, but is generally in the range of 0.01 to 100% by weight.

  Specifically, examples of substances such as pharmaceutical carriers or ingestible carriers, diluents or excipients that can be added to medicines or foods include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, and sucrose. , Magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light Silica, magnesium stearate, talc, tragacanth, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate Um, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, water and the like.

  Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These preparations are prepared according to a conventional method. The liquid preparation may be dissolved or suspended in water or other appropriate solvent at the time of use. Tablets and granules may be coated by a known method. In the case of injection, it is prepared by dissolving the peptide of the present invention in water, but it may be dissolved in physiological saline or glucose solution as necessary, and a buffer or preservative may be added. Good.

  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 therapeutically valuable ingredients.

  In order to produce a solid preparation for oral administration, an active ingredient and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, anhydrous silicic acid and the like are mixed to form a powder, or if necessary, sucrose, Add a binder such as hydroxypropylcellulose or polyvinylpyrrolidone, a disintegrant such as carboxymethylcellulose or carboxymethylcellulose calcium, and wet or dry granulate to form granules. In order to produce tablets, these powders and granules may be tableted as they are or after adding a lubricant such as magnesium stearate or talc. These granules or tablets should be coated with an enteric solvent base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methyl methacrylate polymer and coated with an enteric solvent preparation, or with ethylcellulose, carnauba wax, hardened oil, etc. You can also. In order to produce capsules, powders or granules are filled into hard capsules, or active ingredients are dissolved as they are or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin film to form soft capsules. Can do.

  In order to produce a liquid preparation for oral administration, an active ingredient and a sweetener such as sucrose, sorbitol, and glycerin are dissolved in water to add a transparent syrup, further essential oil, ethanol, etc. to make an elixir, Gum arabic, tragacanth, polysorbate 80, sodium carboxymethyl cellulose and the like may be added to form an emulsion or suspension. These liquid preparations may contain a flavoring agent, a coloring agent, a preservative and the like as desired.

  Specific examples of foods that can be prepared by adding and blending the peptide according to the present invention include, for example, beverages (coffee, cocoa, juice, soft drinks, mineral drinks, tea drinks, green tea, tea, oolong tea, and milk drinks. , Lactic acid bacteria beverages, yogurt beverages, carbonated beverages, other non-alcoholic beverages, alcoholic beverages, etc.), confectionery (hard candy, gum, gummi, jelly, pudding, mousse, cake, candy, cookies, crackers, biscuits, chocolate, ice confectionery ( Ice cream, ice candy, sherbet, shaved ice, etc.), sprinkles, dressing, seasoning, processed soybean food (tofu, miso, soy sauce, yuba, kinako, natto, etc.), processed meat food (hamburger, meatloaf, meatballs, etc.) Tsukune), processed fish food (kamaboko, chikuwa, etc.), Toruto food, jelly-like food (jelly, agar-agar, a jelly-like beverages, and the like), and the like can be given. Examples of foods that can be prepared by adding and blending the peptide of the present invention include so-called health foods, functional foods, functional labeling foods, dietary supplements, supplements, foods for specified health use, foods for the sick, and combinations for the sick May be food (Ministry of Health, Labor and Welfare, special-purpose food) or food for the elderly (Ministry of Health, Labor and Welfare, special-purpose food), uncoated tablets, film-coated tablets, dragees, granules, powders, tablets, capsules (hard capsules and soft capsules) And chewable type, syrup type, drink type, etc. The preparation of food containing the peptide according to the present invention can be carried out by a method known per se.

  The food containing the peptide of the present invention can be produced, for example, by adding the peptide of the present invention to a known food. Specific examples of foods that can be produced by adding the peptides according to the invention include, for example, beverages (coffee, cocoa, juice, soft drinks, mineral drinks, tea drinks, green tea, tea, oolong tea, milk drinks, lactic acid bacteria drinks , Yogurt beverages, carbonated beverages, other non-alcoholic beverages, alcoholic beverages, etc.), confectionery (hard candy, gum, gummi, jelly, pudding, mousse, cake, candy, cookies, crackers, biscuits, chocolate, ice confectionery (ice cream, Iced candy, sherbet, shaved ice, etc.), sprinkles, dressings, seasonings, dairy products (milk, processed milk, cream, butter, cheese, whey, ice creams, milk, milk powder, fermented milk (Yogurt, etc.), lactic acid bacteria beverages, milk beverages, etc., processed soybean foods (tofu, Salmon, soy sauce, yuba, kinako, natto, etc.), processed meat foods (hamburg, meatloaf, meatballs, tsukuyane, etc.), processed fish foods (kamaboko, chikuwa, etc.), retort foods, jelly-like foods (jelly, agar, jelly-like) Beverages, etc.). Preparation of the food to which the peptide according to the present invention is added can be performed by a method known per se.

  In addition, when the food containing the peptide of the present invention uses a product containing bovine serum albumin such as milk or whey as a raw material, the method for producing the food includes the step of hydrolysis with subtilisin. A food containing the peptide of the invention can be produced. In another aspect, a food containing the peptide of the present invention can also be produced by including a step of producing the peptide of the present invention such as lyophilization, acid and / or alkali treatment in the method for producing the food.

  The food containing the peptide of the present invention is a so-called health food, functional indication food, food for specified health use, dietary supplement, supplement, food for the sick, combination food for the sick (Ministry of Health, Labor and Welfare, a kind of special-purpose food) It can be a food for the elderly (Ministry of Health, Labor and Welfare, a kind of special-purpose food).

  Next, the present invention will be described more specifically with reference to examples. However, the following examples do not limit the scope of the present invention.

<Method>
The evaluation of the blood pressure lowering effect, the calcium responsiveness test of enteroendocrine cells, and the arterial relaxation experiment were performed according to the methods described in Patent Document 1 and Non-Patent Document 1.
(Evaluation of blood pressure lowering effect)
(i) Animal used Male rats with spontaneous hypertension (SHRs / Izm) (manufactured by SLC, Japan) were used. SHR rats were housed in a room controlled at 23 ° C., 12 hours / 12 hours light / dark cycle. The food was given as solid SP feed (Funabashi Farm) and CE-2 (Clea Japan), and was freely ingested with water.

(ii) Non-invasive blood pressure measurement experiment (Tail-cuff method)
Measure systolic blood pressure by Tail-cuff method for unanesthetized SHR rats. For blood pressure measurement, MK-2000 (Muromachi Kikai Co., Ltd.) was used. Animals trained on the tail-cuff apparatus for about 3 weeks were used in this experiment. Each sample was dissolved in physiological saline and forcibly administered orally using a metal sonde. Blood pressure measurement was performed immediately before administration and after the elapsed time from administration shown in each table (Time after administration (h); 2, 4, 6, 24, 48 hours).

  Evaluation was performed by calculating a change in systolic blood pressure (ΔSystolic blood pressure) immediately before administration.

(Calcium responsiveness test of enteroendocrine cells (STC-1))
Each sample was added using mouse intestinal endocrine cells STC-1, and cell responsiveness was evaluated. In STC-1 cells known as intestinal endocrine cells having CCK secretion ability, it is known that the intracellular calcium concentration increases during CCK secretion. CCK is an endogenous antifeedant hormone and is known to cause a feeling of fullness when its secretion is stimulated.

STC-1 cells were seeded in a 96-well plate at approximately 50,000 cells per well and incubated at 37 ° C. for 48 hours. Thereafter, 100 μl of fluorescent reagent Fluo4 was added to each well, and further incubated at 37 ° C. for 1 hour. Thereafter, the sample was administered, the fluorescence intensity was measured, and the change in intracellular calcium concentration (Δ [Ca ²⁺ ]) was calculated.

(Arterial relaxation experiment)
The mesenteric artery of an SHR rat (15-34 weeks old) was removed, and a specimen was prepared by incising in a spiral. Krebs-Henseleit nutrient solution ((120 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO ₄ , 1.2 mM KH ₂ PO 4, 2.5 mM CaCl ₂ , 25 mM NaHCO ₃ , 10 mM glucose), 37 ° C. The sample was suspended in a Magnus tube filled with 5% CO ₂ and 95% O ₂ mixed gas saturation), and the change in tension (tension) was recorded on a polygraph via a strain transducer (manufactured by Saneisha).

  The artery was contracted in advance with phenylephrine, and a sample was added at a stable position to measure the degree of relaxation. The relaxation rate was calculated with the degree of contraction when the artery was completely relaxed with papaverine as the ratio of the contraction level to 100%.

<Production example>
(Enzyme digest)
Purified bovine serum albumin (BSA) protein (manufactured by SIGMA) and digestive enzyme are mixed with enzyme: BSA = 1: 100 (weight ratio, final concentration of BSA: 20 mg / ml), and attached in the attached buffer. Reaction was performed.

The enzymes and reaction conditions used were as follows:
(I) Subtilisin (manufactured by SIGMA); reaction temperature: 37 ° C., reaction time: 5 hours, reaction pH: 7.5.

After elapse of the above reaction time, the sample was boiled (100 ° C., 10 minutes) to stop the enzyme reaction.
Further, the mixture was centrifuged at 3500 rpm × 10 minutes (4 ° C.), and the resulting supernatant was lyophilized.

(peptide)
Peptide FWGK (SEQ ID NO: 1), peptide FW (SEQ ID NO: 2) and peptide RF (SEQ ID NO: 3) were synthesized by a conventional method.
(Statistical analysis)
Data obtained by the test was expressed as the sum of the mean (Mean) of the number of trials n and the standard error of the mean (SEM). A t-test was used for comparison between the two groups. For comparisons between 3 groups or more, data were analyzed by one-way ANOVA, followed by a Tukey-Kramer test for multiple comparisons. When p <0.05 (“*” in the figure) and p <0.01 (“**” in the figure) with respect to the control (Control), it was determined that there was a significant difference. When p <0.10 (“#” in the figure) with respect to the control (Control), it was determined that there was a significant tendency.

<Experiment and results>
(Example 1: Blood pressure lowering action (enzyme digest))
The blood pressure lowering effect was evaluated using SHR rats orally administered with 15 mg / kg or 50 mg / kg of bovine serum albumin protein digested with subtilisin as a test substance (n = 10). Administration of saline alone as the solvent was used as a control (hereinafter the same).

  The results are shown in FIG. When BSA subtilisin digest is orally administered at 15 mg / kg and 50 mg / kg, it exhibits a blood pressure lowering effect.

(Example 2: Blood pressure lowering action (peptide))
Peptide FWGK and peptide FW were used as test substances, peptide FWGK was 5.0 μg / kg or 50 μg / kg, and peptide FW was 0.05 mg / kg. SHR rats administered orally at 0.5 mg / kg or 1.5 mg / kg were used to evaluate blood pressure lowering effects (n = 5-10 for peptide FWGK, n = 10-20 for peptide FW).

  The results are shown in FIGS. Peptide FWGK exhibits a blood pressure lowering effect significantly when administered at a low dose of 50 μg / kg. Furthermore, the blood pressure lowering effect persists until 6 hours after administration.

  Peptide FW exhibits a blood pressure lowering effect when 1.5 mg / kg is orally administered. The hypotensive effect persists until 6 hours after administration.

(Example 3: Calcium responsiveness test of intestinal endocrine cells (STC-1) (peptide))
STC-1 cells were tested for calcium responsiveness using peptide FWGK, peptide FW and peptide RF as test substances. Each peptide was used at 1 mM.

  The results are shown in FIG. Peptide FWGK, peptide FW and peptide RF are significantly more calcium responsive than control (control), and it is considered that CCK release is promoted. In particular, peptide FWGK and peptide FW are considered to have significantly greater changes in intracellular calcium and promote CCK release more strongly than peptide RF, which has been known to promote CCK release. Be

  The reactivity was dose-dependent (data not shown).

(Example 4: Arterial relaxation experiment (peptide))
Using the peptide FWGK, peptide FW and peptide RF as test substances, the arterial relaxation action was evaluated by an arterial relaxation experiment (n = 2).

The results are shown in FIG. For all of peptide FWGK, peptide FW, and peptide RF, arterial relaxation was observed in a dose-dependent manner. In particular, peptide FWGK showed a high arterial relaxation action even at a low concentration of 10 ⁻⁷ M (FIG. 5A).

(Example 5: Arterial relaxation experiment (peptide))
Using peptide FWGK as a test substance, the arterial relaxation action was evaluated by an arterial relaxation experiment in the presence or absence of various inhibitors (n = 3-4).

Inhibitors include CCK ₁ receptor antagonist (CCK ₁ R Antagonist) lorgumide (30 μM), cyclooxygenase inhibitor (COX Inhibitor) indometachin (3 μM), and nitric oxide L-NAME (100 μM) which is a synthetic enzyme inhibitor (NOS Inhibitor) was used.

  The peptide FWGK was administered after pretreatment with the inhibitor.

The results are shown in FIG. When lorglumide, a CCK ₁ receptor antagonist, was added, arterial relaxation was significantly inhibited. The relaxation rate did not decrease even when other inhibitors were added. Therefore, it is considered that the arterial relaxation action of peptide FWGK is mediated by activation of CCK ₁ receptor.

  A summary of the results of Examples 1-5 above is shown in Table 1.

(Reference Example 1)
The ratio of peptide FWGK and peptide FW contained in the subtilisin digest of bovine serum albumin protein was quantified by LC-MS.

LC-MS was performed under the following conditions (manufactured by Waters).
LC: Acquity UPLC system
Column: Acquity BEH-C18
MS: Xevo Q-TOF.

  The results are shown in Table 2. A numerical value is shown by the production rate (Yield mol%) by mol%.

Claims (12)

A peptide having an amino acid sequence from any of the following:
(I) FWGK (SEQ ID NO: 1)
(Ii) FW (SEQ ID NO: 2)   The peptide according to claim 1, which is derived from a subtilisin digest of bovine serum albumin protein.   A pharmaceutical composition comprising the peptide according to claim 1 or 2 as an active ingredient.   An antihypertensive agent comprising the peptide according to claim 1 or 2 as an active ingredient.   The antifeedant which uses the peptide of Claim 1 or 2 as an active ingredient.   A food containing the peptide according to claim 1 or 2.   A food comprising the peptide according to claim 1 or 2 added thereto.   The food according to claim 6 or 7, for lowering blood pressure.   The food according to claim 6 or 7, which is used for suppressing eating.   A method for lowering blood pressure and / or suppressing feeding behavior, comprising a step of administering the peptide according to claim 1 or 2 to a patient or a preliminary group in need.   The peptide according to claim 1 or 2, for lowering blood pressure and / or suppressing feeding behavior.   Use of the peptide according to claim 1 or 2 for the manufacture of a medicament or food for lowering blood pressure and / or suppressing feeding behavior.

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