JP2010037261A - Hypotensive peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new peptide having ACE inhibitory action and its use. <P>SOLUTION: The peptide is composed of any of the amino acid sequences represented by (1): Asn-Asn-Tyr-Pro-Ser-Asp-Ile-Asp-Gln-Trp-His-Asp-Lys (Sequence No.1) and (2): Gly-Glu-Gly-Leu-Ile-Val-Tyr-His (Sequence No.2). The peptide is useful as an angiotensin-converting enzyme inhibitor, an antihypertensive agent, and an active ingredient in a food, particularly a food for preventing hypertension. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to novel peptides and uses thereof. More specifically, the present invention relates to a novel peptide having an angiotensin converting enzyme inhibitory action (hereinafter referred to as “ACE inhibitory action”) and exhibiting a hypotensive action based on the action, and uses thereof. Since the peptide of the present invention exhibits an antihypertensive action based on the ACE inhibitory action as described above, it is effective in preventing or treating hypertension.

  Hypertension itself is a disease with no subjective symptoms. However, if left untreated, secondary causes of serious diseases such as heart disease and cerebrovascular disorder, preventing and treating hypertension has been a major challenge.

For hypertension, Angiotensin Converting Enzyme (A)
CE ”) is known to be deeply involved, and it is possible to prevent or treat hypertension by inhibiting ACE. For this reason, many developments of antihypertensive agents containing a compound having an ACE inhibitory action as an active ingredient have been made.

  However, in order to prevent or treat hypertension, it is necessary to take a compound having an ACE inhibitory action for a long period of time. Therefore, it is important that the compound has an ACE inhibitory action but has low side effects. Requirements. In addition, as the concept of preventive medical health management has become widespread, there is a need for the development of functional foods that have the effect of preventing hypertension rather than pharmaceuticals.

The following Patent Documents 1 to 5 and Non-Patent Documents 1 to 6 disclose various peptides having an ACE inhibitory action. However, these peptides are all different from the peptides of the present invention, and peptides of the same sequence are not found in text search of the JPO electronic library and search by Google scalar and PubMed. It was found to be a novel peptide having an amino acid sequence different from that of the existing peptide.
Japanese Patent No. 30686656 JP 2002-145899 A JP 2002-338594 A JP 2005-255670 A JP 2007-261999 A Journal of Nutritional Biochemistry 13 (2002) pp.80-86 Diagnosis and New Drug, Vol. 39, No. 2, 2002, pp. 85-90 Journal of Japanese Society for Food Science and Technology, Vol. 50, No. 10, pages 457-462 (2003). Journal of Japan Society for Food Science and Technology, Vol. 50, No. 6, pp. 286-288 (2003). Journal of Japanese Society for Food Science and Technology, Vol. 50, No. 7, pp. 310-315 (2003) Journal of Japan Society for Food Science and Technology, Vol. 51, No. 1, pp. 34-37 (2004).

  An object of the present invention is to provide a novel peptide, more specifically, to provide a novel peptide that has an ACE inhibitory action and can be effectively used for the prevention or treatment of hypertension based on the action. Another object of the present invention is to provide uses of the above peptides, specifically, an angiotensin converting enzyme inhibitor (hereinafter referred to as “ACE inhibitor”), an antihypertensive agent and a food.

  As a result of repeated studies in view of the above problems, the present inventors have found that a peptide comprising a specific amino acid sequence has an ACE inhibitory action, and based on this finding, according to the peptide, the peptide has an ACE inhibitory action. Based on that, I was convinced that hypertension can be prevented or ameliorated.

The present invention has been completed based on such findings and includes the following embodiments:
Item 1. A peptide consisting of any of the amino acid sequences described in (1) or (2) below:
(1) Asn-Asn-Tyr-Pro-Ser-Asp-Ile-Asp-Gln-Trp-His-Asp-Lys (SEQ ID NO: 1)
(2) Gly-Glu-Gly-Leu-Ile-Val-Tyr-His (SEQ ID NO: 2).
Item 2. A composition containing at least one of the peptides according to Item 1.
Item 3. An angiotensin converting enzyme inhibitor (ACE inhibitor) comprising at least one of the peptides according to Item 1 as an active ingredient.
Item 4. An antihypertensive agent comprising at least one of the peptides according to item 1 as an active ingredient.
Item 5. A food comprising at least one of the peptides according to Item 1 as an active ingredient.
Item 6. Item 6. The food according to Item 5, which is a food for preventing hypertension.
Item 7. Item 7. The food according to Item 5 or Item 6, which is a food product other than a proteolysate obtained by trypsinizing royal jelly or an alcohol-modified protein thereof and a food containing the same.

  Since all of the peptides of the present invention have an ACE inhibitory action, they can exert an antihypertensive action based on the ACE inhibitory action. Therefore, the peptide of the present invention is effective for prevention or treatment of hypertension. That is, by ingesting the peptide of the present invention, blood pressure can be reduced, and hypertension and various diseases caused by hypertension can be prevented.

The peptide targeted by the present invention is characterized by comprising the amino acid sequence of either the following (1) or (2):
(1) Asn-Asn-Tyr-Pro-Ser-Asp-Ile-Asp-Gln-Trp-His-Asp-Lys (SEQ ID NO: 1)
(2) Gly-Glu-Gly-Leu-Ile-Val-Tyr-His (SEQ ID NO: 2).

  The peptide of the present invention is not particularly limited by the origin or acquisition method of the acquisition, and based on the amino acid sequence and the ACE inhibitory action, peptide synthesis methods, genetic engineering methods, isolation and purification methods, and fermentation By using a conventionally known method such as a method, it can be produced or prepared.

  The peptides (1) and (2) are characterized by having an ACE inhibitory action as shown in the Examples.

  As long as the peptides (1) and (2) have an ACE inhibitory action, they may be used as free (free) peptides after adjusting to the pH of the isoelectric point, After adjusting the pH to the basic side, the solvent is removed by freeze-drying, spray-drying, etc., or by adding a solvent such as ethanol to cause precipitation, an acid addition salt (an inorganic acid salt such as hydrochloride or fumarate). It can also be prepared in the form of an organic acid salt such as an acid salt) or a base salt (for example, an alkali metal salt such as Na or K). The base salt preferably suppresses the content of sodium as a pressurizing substance as much as possible. Such a peptide may be used in the form of a solution, but it is usually desirable to isolate and use it in the form of a solid, for example, a crystal, amorphous or powder.

  Peptides (1) and (2) of the present invention are used as ACE inhibitors, either alone or in combination with a non-toxic carrier, diluent or excipient acceptable as a food, pharmaceutical or reagent. can do. In this case, the ratio of the peptide (1) or (2) blended with the ACE inhibitor is not particularly limited as long as the ACE inhibitor has ACE inhibitory activity, and generally from 0.01 to 100% by weight. It is possible to select appropriately. The ACE inhibitory action of the ACE inhibitor can be evaluated by the method described in Example 3 described later.

  In addition, the peptides (1) and (2) of the present invention can be used alone as foods, pharmaceuticals or reagents. Therefore, the composition containing either peptide (1) or (2) targeted by the present invention, or both peptides (1) and (2) is based on the ACE inhibitory action or the action of the peptide. A composition utilizing an antihypertensive effect is preferable, and specifically includes a food composition, a pharmaceutical composition, and a reagent composition.

  When the peptides (1) and (2) of the present invention are prepared as a food composition or a pharmaceutical composition, both of them are used together with a nontoxic carrier, diluent or excipient acceptable in food or a pharmaceutical, and a tablet (elementary). Tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (powder), fine granules, granules, liquids, suspensions, emulsions, Prepare syrups, pastes, injections (including those prepared by mixing with distilled water, amino acid infusions, electrolyte infusions, etc. at the time of use) to make food or pharmaceutical preparations It is possible. The content of peptide (1) and (2) in the preparation is not particularly limited as long as the preparation has an ACE inhibitory action, but in general, it should be appropriately selected from the range of 0.01 to 100% by weight, respectively. Is possible.

  Specific examples of foods that can be prepared by blending the peptides of the present invention include, for example, beverages [soft drinks (coffee, cocoa, juice, mineral drinks, tea drinks, green tea, tea, oolong tea, etc.), milk Beverages, lactic acid bacteria beverages, yogurt beverages, carbonated beverages, alcoholic beverages (Japanese liquor, western liquor, fruit liquor, honey liquor, etc.), spreads (butter cream, chocolate cream, cheese cream, etc.), pastes (fruit paste, vegetable paste, sesame paste) , Seaweed paste, etc.), Western confectionery (chocolate, donut, pie, cream puff, muffin, waffle, gum, gummy, jelly, candy, cookies, crackers, biscuits, snack confectionery, cake, pudding, etc.), Japanese confectionery (rice cake, rice cracker, karinto, Hail, dumpling, ohagi, daifuku, castella, anmitsu, sheepkan etc. , Ice confectionery (ice cream, ice candy, sherbet), retort food (curry, beef bowl, miscellaneous cooking, porridge, soup, meat sauce, demiglace sauce, hamburger, oden seeds, steamed rice bowl etc.), instant food (instant ramen, instant udon, instant soba) , Instant Yakisoba, Instant Spaghetti, Instant Miso Soup, Powdered Soup, Powdered Juice, Hot Cake Mix, etc.), Bottled / Canned, Jelly Food (Jelly, Jelly Drink, etc.), Seasoning (Soy Sauce, Mirin, Vinegar, Sugar , Honey, miso, dressing, umami seasoning, compound seasoning, sauce, mayonnaise, ketchup, sprinkle, soup, dashi stock, bouillon, sauce, roux, etc.), beekeeping products (honey, royal jelly, propolis, pollen load (pollen) Dango), bees, etc.), dairy products (milk, cheese, yogurt, fresh cream) ), Processed fruits (jam, marmalade, syrup pickled, dried fruits, etc.), processed vegetables (vegetable jam, etc.), processed cereals (noodles, pasta, bread, rice noodles, etc.), pickles (pickled plums, takuan, kimchi pickles, shallow pickles) , Pickles, etc.), pickles (immediate pickles, kimchi pickles, etc.), fish products (kamaboko, chikuwa, hanpen, etc.), livestock products (ham, sausage, salami, bacon, etc.), delicacies , Salted, mirinboshi, smoked, etc.), dried food (seasoned seaweed, etc.), side dishes (green, fried, fried, grilled, boiled, vinegared, etc.), frozen food (fried shrimp, croquette, spring roll, tonkatsu, shrimp, Examples include dumplings, hamburger, takoyaki, Imagawa-yaki (rotary-baked), meat buns, buns, etc.) and fat foods (salad oil, margarine, butter).

  As foods that can be prepared by adding and blending the peptide of the present invention, so-called health foods, functional foods, dietary supplements, supplements, foods for specified health use, foods for sick people, combined foods for sick people (Ministry of Health, Labor and Welfare, It may be a food for special use) or a food for the elderly (Ministry of Health, Labor and Welfare, a kind of special use food). In that case, tablets as described above (plain tablets, dragees, effervescent tablets, film-coated tablets, chewables) Tablets, troches, etc.), pills, powders (powder), fine granules, granules, capsules (including both hard capsules and soft capsules), syrups, and drinks (liquids). Moreover, this invention can also be added and mix | blended with dog food, cat food, etc. as pet food. The preparation of food containing the peptide according to the present invention can be carried out by a method known per se.

  The peptide of the present invention can be continuously taken for the purpose of preventing hypertension, alleviating the tendency to hypertension, or regulating blood pressure. Therefore, functional peptides for preventing hypertension, particularly foods for specified health use. Useful as such. In particular, foods such as food for specified health use containing the peptide of the present invention preferably have normal high blood pressure (systolic blood pressure is 130-139 mmHg, diastolic blood pressure is 85-89 mmHg) and mild hypertension (systolic blood pressure 140-159 mmHg). To diastolic blood pressure 90-99 mmHg), it can be suitably used to prevent hypertension.

  When the peptide of the present invention or a composition containing the same is used for the purpose of preventing hypertension, the daily amount of peptide taken by an adult weighing 60 kg is generally 10 mg to 10 g, preferably 0.1 to 5 g. The range is preferably 0.5 to 3 g, particularly 0.5 to 1 g.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

Example 1 Preparation of Peptide (1) (1) Preparation of Royal Jelly Protein Hydrolyzate Add 1200 ml of purified water to 100 g of royal jelly ethanol-denatured protein obtained by adding ethanol to royal jelly, and stir in a 37 ° C water bath. After adjusting the pH to 8.0 by adding 20% by weight sodium hydroxide solution, add 250 mg of trypsin (derived from bovine pancreas, manufactured by Roche) and maintain the pH at 8.0 with a 1 mol / L sodium hydroxide test solution at 37 ° C. After time treatment, the enzyme was decomposed. Next, the obtained reaction solution was heated in a boiling water bath for 30 minutes to deactivate the enzyme, and then adjusted to pH 4.5 with 50% dilute hydrochloric acid. This was centrifuged (10000 rpm, 20 minutes), and the obtained supernatant was filtered through a membrane filter (0.8 μm) and freeze-dried to obtain 70.2 g of a royal jelly protein hydrolyzate.

(2) Fractionation with synthetic adsorbent 50 g of the royal jelly protein hydrolyzate prepared above was dissolved in 800 mL of water, and this was applied to a column packed with a synthetic adsorbent (Diaion HP20 (Mitsubishi Chemical)) under the following conditions. did.

<Column chromatography conditions>
Column: 11cmφ × 22cm (capacity: 2100mL)
Stationary phase: Synthetic adsorbent (Diaion HP20)
Mobile phase: water, methanol or a mixture thereof (methanol concentration: 0, 20, 40, 60, 80, 100%)
Column temperature: room temperature detector: UV detection (detection wavelength: 275 nm).

  The fraction eluted with a mobile phase having a methanol concentration of 60% was collected as fraction 5 (Fr. 5) (yield: 14.46 g).

(3) Sorting with large-diameter column 1 g of Fr. 5 collected above was dissolved in 4.5 mL of 15% strength acetonitrile aqueous solution containing trifluoroacetic acid at a ratio of 0.05%. This was subjected to a large-diameter column packed with TSK-gel (ODS) under the following conditions.

<Reverse phase column chromatography conditions>
Column: 55mmφ × 300nm
Stationary phase: TSK-gel ODS 120T (Tosoh)
Mobile phase: 15% acetonitrile / 0.05% trifluoroacetic acid Column temperature: Room temperature Flow rate: 42 mL / min
Detector: UV detection (detection wavelength: 220 nm).

Peak fractions (fractions 1 to 9) were collected by UV detection at a wavelength of 220 nm. This operation was repeated 5 times, and fraction 5 (Fr. 5-5) having a retention time of about 73 to 85 minutes was collected (total yield of 5 collections: 0.24 g).

(4) Fractionation with medium-diameter column (20 mmφ) Fr. 5-5 (0.24 g) collected above was dissolved in 3 mL of 0.2% trifluoroacetic acid. Of this, 1 mL was applied to a medium-diameter column packed with COSMOSIL-5C18-ARII (manufactured by Nacalai Tesque) under the following conditions.

<Column chromatography conditions>
Column: 20mmφ × 250nm
Stationary phase: COSMOSIL-5C18-ARII (Nacalai Tesque)
Mobile phase A: 0.05% trifluoroacetic acid mobile phase B: 50% methanol / 0.05% trifluoroacetic acid gradient Conditions: mobile phase B concentration 35% → 100% (0 minutes → 240 minutes)
Column temperature: 40 ° C
Flow rate: 8mL / min
Detector: UV detection (detection wavelength: 220 nm).

Peak fractions (fractions 1 to 11) were collected by UV detection at a wavelength of 220 nm. This operation was repeated three times, and fraction 4 (Fr. 5-5-4) having a retention time of about 67 to 77 minutes was collected (total yield of three collections: 51 mg).

(5) Fractionation with medium-diameter column (20 mmφ) Fr. 5-5-4 (51 mg) collected above was dissolved in 3 mL of 0.2% trifluoroacetic acid. Of this, 1 mL was applied to a medium-diameter column packed with COSMOSIL-5C18-ARII (manufactured by Nacalai Tesque) under the following conditions.

<Column chromatography conditions>
Column: 20mmφ × 250nm
Stationary phase: COSMOSIL-5C18-ARII (Nacalai Tesque)
Mobile phase: 22.5% methanol / 0.05% trifluoroacetic acid Flow rate: 8 mL / min
Column temperature: 40 ° C
Detector: UV detection (detection wavelength: 220 nm).

Peak fractions (fractions 1 to 8) were collected by UV detection at a wavelength of 220 nm. This operation was repeated three times, and fraction 6 (Fr. 5-5-4-6) having a retention time of about 91 to 97 minutes was collected (total yield of three collections: 6.0 mg).

(6) Identification of peptide (determination of amino acid sequence)
Toray Research Center was requested to identify the peptide contained in fraction 6 (Fr.5-5-4-6) obtained in (5) above, and the amino acid sequence was measured by N-terminal amino acid sequence analysis by Edman degradation. . As a result, it was confirmed that Fr. 5-5-4-6 contains a peptide having the following amino acid sequence.
・ Amino acid sequence of peptide (1)
Asn-Asn-Tyr-Pro-Ser-Asp-Ile-Asp-Gln-Trp-His-Asp-Lys (SEQ ID NO: 1).

Example 2 Preparation of peptide (2)
(1) Preparation of royal jelly protein hydrolyzate Add 100 ml of ethanol-denatured protein obtained by adding ethanol to royal jelly, add 1200 ml of purified water, and add 20 wt% sodium hydroxide solution while stirring in a 37 ° C water bath. After adjusting the pH to 8.0, 250 mg of trypsin (derived from bovine pancreas, manufactured by Roche) was added, and the mixture was treated at 37 ° C. for 24 hours while maintaining the pH at 8.0 with a 1 mol / L sodium hydroxide test solution to perform enzymatic degradation. Next, the obtained reaction solution was heated in a boiling water bath for 30 minutes to deactivate the enzyme, and then adjusted to pH 4.5 with 50% dilute hydrochloric acid. This was centrifuged (10000 rpm, 20 minutes), and the obtained supernatant was filtered through a membrane filter (0.8 μm) and freeze-dried to obtain 70.2 g of a royal jelly protein hydrolyzate.

(2) Preparative royal jelly 50 g by synthetic adsorbent was dissolved in 800 mL of water, and this was applied to a column packed with a synthetic adsorbent (Diaion HP20 (manufactured by Mitsubishi Chemical)) under the following conditions.

<Column chromatography conditions>
Column: 11cmφ × 22cm (capacity: 2100mL)
Stationary phase: Synthetic adsorbent (Diaion HP20)
Mobile phase: water, methanol or a mixture thereof (methanol concentration: 0, 20, 40, 60, 80, 100%)
Column temperature: room temperature detector: UV detection (detection wavelength: 275 nm).

  The fraction eluted with a mobile phase having a methanol concentration of 60% was collected as fraction 5 (Fr. 5) (yield: 14.46 g).

(3) Sorting with large-diameter column 1 g of Fr. 5 collected above was dissolved in 4.5 mL of 15% strength acetonitrile aqueous solution containing trifluoroacetic acid at a ratio of 0.05%. This was subjected to a large-diameter column packed with TSK-gel (ODS) under the following conditions.

<Reverse phase column chromatography conditions>
Column: 55mmφ × 300nm
Stationary phase: TSK-gel ODS 120T (Tosoh)
Mobile phase: 15% acetonitrile / 0.05% trifluoroacetic acid Column temperature: Room temperature Flow rate: 42 mL / min
Detector: UV detection (detection wavelength: 220 nm).

Peak fractions (fractions 1 to 9) were collected by UV detection at a wavelength of 220 nm. This operation was repeated 5 times, and fraction 6 (Fr. 5-6) having a retention time of about 85 to 93 minutes was collected (total yield of 5 collections: 0.38 g).

(4) Fractionation with medium-diameter column (20 mmφ) Fr. 5-6 (0.38 g) collected above was dissolved in 5 mL of 0.2% trifluoroacetic acid. Of this, 1 mL was applied to a medium-diameter column packed with COSMOSIL-5C18-ARII (manufactured by Nacalai Tesque) under the following conditions.

<Column chromatography conditions>
Column: 20mmφ × 250nm
Stationary phase: COSMOSIL-5C18-ARII (Nacalai Tesque)
Mobile phase A: 0.05% trifluoroacetic acid mobile phase B: 50% methanol / 0.05% trifluoroacetic acid gradient Conditions: mobile phase B concentration 45% → 100% (0 minutes → 240 minutes)
Column temperature: 40 ° C
Flow rate: 8mL / min
Detector: UV detection (detection wavelength: 220 nm).

Peak fractions (fractions 1 to 13) were collected by UV detection at a wavelength of 220 nm. This operation was repeated 5 times, and fraction 5 (Fr. 5-6-5) having a retention time of about 56 to 61 minutes was collected (total yield of 5 collections: 31 mg).

(5) Fractionation with medium-diameter column (10 mmφ) Fr. 5-6-5 (31 mg) collected above was dissolved in 3 mL of 0.2% trifluoroacetic acid. Of this, 1 mL was applied to a medium-diameter column packed with COSMOSIL-5C18-ARII (manufactured by Nacalai Tesque) under the following conditions.

<Column chromatography conditions>
Column: 20mmφ × 250nm
Stationary phase: COSMOSIL-5C18-ARII (Nacalai Tesque)
Mobile phase: 25% methanol / 0.05% trifluoroacetic acid Flow rate: 2 mL / min
Column temperature: 40 ° C
Detector: UV detection (detection wavelength: 220 nm).

Peak fractions (fractions 1 to 6) were collected by UV detection at a wavelength of 220 nm. This operation was repeated three times, and fraction 3 (Fr. 5-6-5-3) having a retention time of about 58 to 63 minutes was collected (total yield of three collections: 4.8 mg).

(6) Identification of peptide (determination of amino acid sequence)
The Toray Research Center was asked to identify the peptide contained in fraction 3 (Fr.5-6-5-3) obtained in (5) above, and the amino acid sequence was measured by N-terminal amino acid sequence analysis by Edman degradation. . As a result, it was confirmed that Fr. 5-6-5-3 contains a peptide having the following amino acid sequence.
・ Amino acid sequence of peptide (2)
Gly-Glu-Gly-Leu-Ile-Val-Tyr-His (SEQ ID NO: 2).

Example 3 Evaluation of angiotensin converting enzyme inhibitory action (ACE inhibitory action)
(1) Preparation of each solution
(a) 25mU / mL ACE solution / borate buffer (pH 8.3)
Prepare by dissolving 1mL of pH8.3 borate buffer in 0.25U or 1U / vial of angiotensin I converting enzyme (Sigma) from rabbit lung (0.25U or 1U / mL, frozen). This solution is appropriately diluted with a pH 8.3 borate buffer solution to prepare a 25 mU / mL solution (prepared at the time of use).

(b) 12.5 mM hippuryl-L-histidyl-L-leucine solution / borate buffer (pH 8.3)
Add pH8.3 borate buffer to Hippuryl-L-Histidyl-L-Leucine (HHL / H ₂ O, molecular weight 447.8, manufactured by Peptide Laboratories), and dissolve by heating in a boiling water bath for 3 minutes. Prepare {HHL · H ₂ O 5.5935 g + pH 8.3 borate buffer 1 mL: 12.5 mM (prepared when used)}.

(c) Internal standard solution
Dissolve 12 mg of m-methylhippuric acid (molecular weight: 193.20) in a water / acetonitrile mixture (9: 1) to make 100 mL, and use this as the internal standard solution (store in the refrigerator).

(d) Borate buffer (pH 8.3)
Liquid A: 0.2M boric acid solution (including 0.3M sodium chloride)
Dissolve 12.4 g of boric acid and 17.5 g of sodium chloride in water and adjust to 1000 mL.
Solution B: 0.05M sodium tetraborate solution (containing 0.3M sodium chloride) is prepared.
The solution B is added to the solution A to adjust the pH to 8.3 (stored in a refrigerator).

(2) Experimental method Each of the peptides (1) and (2) obtained in Examples 1 and 2 was dissolved in purified water, and this was taken up in a 25 μl test tube (reaction solution concentration 0.1 mg / mL). The rabbit lung-derived 25 mU / mL ACE solution / borate buffer (pH 8.3) 50 μl was added and incubated at 37 ° C. for 5 minutes. As a substrate, 50 μl of 12.5 mM hippuryl-L-histidyl-L-leucine (HHL, produced by Peptide Research) solution / borate buffer (pH 8.3) was added and incubated at 37 ° C. for 60 minutes. Next, to stop the reaction, add 125 μL of 0.5 M hydrochloric acid solution, add 100 μL of 0.625 mM m-methyl hippuric acid acetonitrile solution as an internal standard solution, and add 750 μL of ethyl acetate to extract hippuric acid as the reaction product. The mixture was stirred and centrifuged with a vortex mixer (2000 rpm, 5 min). 0.5 mL of the organic layer was taken and evaporated to dryness under a stream of nitrogen gas at 60 ° C., and the residue was dissolved in 0.5 mL of a mobile phase for HPLC to obtain a sample solution.

Separately, 25 μl of borate buffer solution (pH 8.3) is taken into a test tube, to which 50 μl of 25 mU / mL ACE solution / borate buffer solution (pH 8.3) derived from rabbit lung is added and incubated at 37 ° C. for 5 minutes. Hereinafter, the same operation as the above sample solution was performed to obtain a standard solution. In addition, 75 μl of borate buffer (pH 8.3) was placed in a test tube, incubated at 37 ° C. for 5 minutes, and thereafter operated in the same manner as the above sample solution to obtain a blank test solution. The HPLC method was performed on 20 μl each of the sample solution, standard solution and blank test solution under the following conditions, and the ratio of the peak area of hippuric acid to the peak area of m-methylhippuric acid was determined for each sample.

<HPLC conditions>
Column: L-column ODS 4.6 × 150mm (Chemicals Evaluation and Research Institute)
Mobile phase: 10 mM KH ₂ PO ₄ (pH 3.0) / acetonitrile = 17/3
Column temperature: 40 ° C
Flow rate: 1.0mL / min
Inflow volume: 20μL
Detector: UV (measurement wavelength: 228 nm).

Based on the ratio of the peak area of hippuric acid to the peak area of m-methylhippuric acid for each sample obtained by the HPLC, the ACE inhibition rate (IC (%)) was calculated by the following formula.

  The results are shown in the table.

From the above, peptides (1) and (2) consisting of the amino acid sequences shown in SEQ ID NOs: 1 and 2, respectively, have an ACE inhibitory action, and can exert a hypotensive action based on the ACE inhibitory action. It became clear. From this, it is considered that peptides (1) and (2) are both useful as active ingredients of antihypertensive agents, that is, therapeutic or preventive agents for hypertension.

[Prescription example]
Hereinafter, formulation examples including the peptide (1) or (2) of the present invention are shown.
Formulation Example 1 Granular food The following raw materials were mixed by a conventional method and molded into a tablet by a tableting machine. This tablet was film-coated for food by a conventional method to obtain a granular health food.
(Composition per grain)
Peptide (1) or (2) 250 mg
Starch 90 mg
Sucrose fatty acid ester 10 mg
350 mg total

Formulation Example 2 Drink After thoroughly stirring the following materials in water, the total amount was 100 mL.
(Composition for one bottle)
Peptide (1) or (2) 1 g
Lemon juice 20 mL
Propolis 0.3 g
Vitamin C 0.2 g
Honey 13 g
Appropriate amount of water
Total 100 mL

Formulation Example 3 Granules The following materials were granulated according to a conventional method to obtain sticky aluminum sachets.
(Composition for one bottle)
Peptide (1) or (2) 300 mg
Lactose 1000 mg
Dietary fiber 100 mg
Honey powder 20 mg
Vitamin C 5 mg
Total 1425 mg

SEQ ID NOs: 1 and 2 show the amino acid sequences of peptides having an ACE inhibitory action.

Claims (6)

A peptide consisting of any of the amino acid sequences shown in (1) or (2) below:
(1) Asn-Asn-Tyr-Pro-Ser-Asp-Ile-Asp-Gln-Trp-His-Asp-Lys (SEQ ID NO: 1)
(2) Gly-Glu-Gly-Leu-Ile-Val-Tyr-His (SEQ ID NO: 2).   A composition comprising at least one of the peptides according to claim 1.   An angiotensin converting enzyme inhibitor comprising at least one of the peptides according to claim 1 as an active ingredient.   An antihypertensive agent comprising at least one of the peptides according to claim 1 as an active ingredient.   A food comprising at least one of the peptides according to claim 1 as an active ingredient.   The food according to claim 5, which is a food for preventing hypertension.