JP4934369B2 - Peptide having blood pressure lowering effect - Google Patents

Description

  The present invention relates to a novel peptide having a blood pressure lowering action and usable for pharmaceuticals and foods, and a salt thereof, and a blood pressure lowering agent and a blood pressure lowering food comprising the peptide or a salt thereof as an active ingredient.

  In order to save patients suffering from hypertension, research on theoretical elucidation of hypertension has been actively conducted, and there have been many reports. The renin-angiotensin system is known as a representative in vivo factor that causes an increase in blood pressure, and the kallikrein-kinin system is known as a typical in vivo factor that acts to lower blood pressure, and angiotensin converting enzyme (hereinafter referred to as ACE) It is known to be greatly involved in both systems.

  ACE produces angiotensin II that increases blood pressure in the renin-angiotensin system, while inactivating bradykinin that decreases blood pressure in the kallikrein-kinin system. For this reason, ACE has a strong voltage boosting action. Therefore, the ACE inhibitor that suppresses the action of ACE suppresses the production of angiotensin II, which is a blood pressure increasing substance, and prevents inactivation of bradykinin, which lowers blood pressure, and thus can lower blood pressure. .

  From these points, research and development of ACE inhibitors have been actively conducted in recent years, and several compounds such as captopril have already been available as pharmaceuticals. On the other hand, it has been reported that certain types of peptides have an ACE inhibitory action, and several peptides of snake venom are derived from animals, and hydrolyzed peptides of wheat gluten as derived from plants (Patent Documents 1 and 2). ) And a peptide produced by treatment with soybean proteolytic enzyme (Patent Document 3) and the like have been proposed.

  However, most of the peptides having ACE inhibitory activity reported so far have weak blood pressure lowering effects. In particular, the peptides described in Patent Documents 2 and 3 require a large intake of 1 g / kg in order to actually reduce blood pressure. In other words, these peptides have a weak blood pressure lowering action, so that it is necessary to take a large amount in order to obtain a substantial effect, and it has been very difficult to put it into practical use as a blood pressure lowering agent.

  In addition, it has been pointed out that the peptides as described above have a kinin-enhancing effect as a result of fulfilling the ACE inhibitory function, which is likely to cause side effects such as dry cough, rash and flush.

JP-A-4-66594 JP-A-4-349893 JP-A-8-225593

  An object of the present invention is to provide a blood pressure lowering component that is safe without side effects, has a high blood pressure lowering effect, and can be used not only in pharmaceuticals but also in a form of food that can be easily obtained.

  In view of the above situation, the present inventors have made extensive studies and searched for a component that effectively lowers blood pressure. As a result, a novel pentapeptide having an amino acid sequence different from the known ACE inhibitory peptide, ie, a pentapeptide in which valine-proline-valine-proline-glutamine is arranged (Val-Pro-Val-Pro-Gln) It was successfully isolated from the hydrolyzate of wheat gluten. And it discovered that this pentapeptide had a high blood pressure lowering effect, and came to complete this invention.

In one embodiment, the present invention provides the following formula:
Val-Pro-Val-Pro-Gln
And a salt thereof.

  Furthermore, the present invention includes a blood pressure lowering agent and a food for lowering blood pressure, which comprise a peptide comprising the amino acid sequence represented by the above formula or a salt thereof as an active ingredient.

  According to the present invention, it is possible to provide a peptide, a blood pressure lowering agent, and a food for lowering blood pressure that are superior in safety and blood pressure lowering ability by a simpler method than the prior art.

Hereinafter, preferred embodiments of the present invention will be specifically described.
The peptide having a blood pressure-lowering action of the present invention was originally found after being separated and purified from the product of hydrolysis of wheat gluten with protease. In the case of the product separated and purified from wheat, all of the three amino acids Val, Pro and Gln contained in the peptide of the present invention are L-amino acids. However, the peptides of the present invention are not limited to peptides consisting only of L-amino acids, and may be amino acids that are optical isomers as long as they have the amino acid sequence described above. Even if the peptide is composed of all D-amino acids, even if any one of the five amino acids is a L-amino acid and the rest is a D-amino acid, chemical synthesis Any of these are included in the peptides of the present invention.

  The salt of the peptide of the present invention is not limited as long as it is a pharmaceutically acceptable salt, and examples thereof include acid addition salts and base addition salts. Examples of the acid addition salt include a salt with an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid, and a salt with an organic acid such as acetic acid, malic acid, succinic acid, tartaric acid or citric acid. Examples of the base addition salt include a salt with an alkali metal such as sodium or potassium, a salt with an alkaline earth metal such as calcium or magnesium, and a salt with an amine such as ammonium or triethylamine.

  Examples of the method for producing the peptide of the present invention include a method of chemically synthesizing or a method of separating and purifying from a natural product-derived protein such as a plant protein, preferably a hydrolyzate of wheat protein. The latter method uses wheat or the like taken on a daily basis as a raw material, and is preferable from the viewpoint of safety.

  When the peptide of the present invention is separated and purified from the hydrolyzate of wheat protein, the wheat protein used is mainly composed of gluten, but is also contained in wheat such as albumin, globulin, glutenin and gliadin. May contain other known proteins, and may inevitably contain impurities such as starch and fibers. In the present invention, wheat protein that has been subjected to chemical treatment or biological treatment with enzymes or the like in advance to reduce molecular weight or that has increased affinity with protease can be used as wheat protein. .

  As a protein hydrolysis method, a known hydrolysis method can be appropriately employed. Specifically, the method of hydrolyzing using protease etc. is mentioned. Examples of the method for hydrolyzing wheat protein using protease include the method described in JP-A-4-349893.

  Specifically, when a protein is hydrolyzed using a protease, a hydrolyzate can be produced by acting one or more types of protease in an aqueous medium. A method using an acidic protease alone or a method using an acidic protease and a neutral protease or an alkaline protease is preferable in that the target peptide can be obtained in a high yield.

  When acidic protease and neutral protease or alkaline protease are used, acidic protease and neutral protease or alkaline protease may be applied to hydrolysis treatment at the same time. However, after treatment with acidic protease, the acidic protease is deactivated first. Thereafter, treatment with a neutral protease or an alkaline protease is preferable because the dispersibility of the protein is improved in an acidic solution and the hydrolysis reaction efficiency is increased.

  When hydrolyzing a protein using a protease, the concentration of the protein in the aqueous medium needs to be appropriately adjusted depending on the type of protease to be used, but is usually adjusted to 1.0 to 20% by mass in the aqueous medium. Is good. As the aqueous medium, for example, water, physiological saline, citrate buffer, phosphate buffer or the like can be used.

  Examples of the acid protease include pepsin, a microorganism-derived acid protease, for example, an acid protease derived from Aspergillus, an aspartic protease derived from a Penicillium microorganism, an acid protease derived from an Aspergillus microorganism, an acid protease derived from a Bacillus microorganism, and an asphalt derived from Hilotake A tick protease or the like can be used. Acidic proteases are generally active in the acidic region at a pH of about 1.5 to 7.0. Among acid proteases, pepsin or koji mold-derived acid protease is preferably used because the target protein hydrolyzate can be obtained in high yield.

  One type of acidic protease may be used alone, or a plurality of types may be used in combination as long as acidic proteases do not adversely affect each other. The acidic protease may be used in a free state or may be used after being immobilized. In any case, it is preferable to use acidic protease at a rate of about 5,000 to 250,000 units per 100 g of dried protein.

  In the present specification, the protease activity (unit) was determined by using an 1% Hammerstein casein solution manufactured by Merck & Co., USA, as a substrate, and modified by Anson-Hagiwara (edited by Shiro Akahori, “Enzyme Research Method,” Volume 2, The reaction was performed at 30 ° C. for 30 minutes, and the amount of enzyme required to release 1 μg of tyrosine equivalent per minute was defined as 1 unit.

  When the treatment is performed using an acidic protease, it is usually carried out until the degree of hydrolysis of the protein reaches 30 to 60%.

  For example, when hydrolyzing wheat protein using pepsin or an acid protease derived from Aspergillus as the acidic protease, the pH of the aqueous medium containing the wheat protein is set to about 1.5 to 6.0, and the temperature is about 30 to 70 ° C. When the reaction is performed for about 2 to 25 hours, the degree of hydrolysis becomes 30 to 60%. At that time, the acidic protease is deactivated to stop the hydrolysis reaction. The inactivation of the acidic protease can be performed by increasing the pH of the solution to be higher than the active pH of the acidic protease or increasing the temperature (usually about 70 to 90 ° C.).

In the present specification, the degree of protein hydrolysis means the ratio of the protein mass dissolved in 0.75M trichloroacetic acid (TCA) to the total protein mass (dissolution rate in TCA), Desired.
Hydrolysis degree (%) = [(0.75M TCA dissolved protein mass) / (total protein mass)] × 100
In the case of further treatment with neutral protease or alkaline protease, thermosease, thermolysin, trypsin, chymotrypsin, pronase, subtilisin, esperase and the like can be used as neutral protease or alkaline protease. Among them, it is preferable to use thermolyse, thermolysin, or subtilisin because a protein hydrolyzate as a target product can be obtained in a high yield.

  Neutral proteases or alkaline proteases may be used alone or in combination as long as the proteases do not adversely affect each other. Further, the neutral protease or alkaline protease may be used in a free state or may be used after immobilization. In either case, about 100 to 10,000 neutral protease or alkaline protease per 100 g of dried protein. It is good to use at a rate of 500,000 units. The treatment with neutral protease or alkaline protease is preferably performed until the degree of hydrolysis of the protein reaches 60 to 90%.

  For example, when hydrolyzing wheat protein using thermolysin or thermosease as a neutral protease, the pH of the aqueous medium containing wheat protein is adjusted to about 7.0 to 8.0, and about 1 at a temperature of about 30 to 70 ° C. When the reaction is carried out for ˜25 hours, the hydrolysis degree becomes 60 to 90%, so that the protease is preferably deactivated at that time. The neutral protease or alkaline protease can be deactivated by a method in which the pH of the solution is lower than the active pH or the temperature is increased (usually about 80 to 100 ° C.).

  Next, an insoluble matter is separated and removed from the hydrolyzed solution to obtain a water-soluble component. Here, the insoluble matter means an insoluble matter present in the protease-treated product of the substrate defined in the present invention. A conventionally known method may be appropriately used to remove the insoluble matter. Specific methods include ordinary solid-liquid separation methods such as centrifugation, filtration, and decantation, and any one of them may be used alone, or a plurality may be used in combination.

  The water-soluble component after removing the insoluble matter may be used in the next step as it is, but after adsorbing and removing impurities contained therein using an adsorbent such as activated carbon or diatomaceous earth, the next step is performed. It is preferable to provide.

  The water-soluble component is mainly composed of a water-soluble peptide mixture obtained by protein hydrolysis, and further contains amino acids and other water-soluble substances. Examples of the method for purifying the protein hydrolyzate thus obtained include a method of fractionating (extracting) with water-containing alcohol and then purifying by gel filtration chromatography or high performance liquid chromatography (HPLC). The peptide of the present invention is soluble in water and hydrous alcohol. When hydrous alcohol is used, impurities insoluble in alcohol can be easily removed, and the purification step using HPLC can be simplified.

  When the water-soluble component is fractionated using a hydrous alcohol, it is preferable to form a concentrate from which the aqueous component has been removed in order to increase the alcohol concentration in the fraction and increase the efficiency of fractionation. Concentrates include both concentrated liquids and concentrated solids. As a method of forming a concentrated liquid, a vacuum concentration method such as a flash method or a centrifugal thin film method is used, and as a method of forming a concentrated solid, a spray drying method, a freeze drying method, a spray cool method, heat drying, or the like is appropriately used. You can choose. The water content of the concentrated liquid is preferably 40 to 70% by weight, and the water content of the concentrated solid is preferably 5 to 15% by weight. More preferably, a concentrated solid is used.

  When the protein hydrolyzate is fractionated using a hydrous alcohol, it is appropriate to use a linear or branched alcohol having 1 to 4 carbon atoms as the alcohol to be used, and examples thereof include methanol, ethanol, propanol and the like. It is done. Among these, it is more preferable to use ethanol in consideration of safety when taking the fractionated product and the purified product. Fractionation with hydrous alcohol is usually efficient when performed using an aqueous alcohol solution having a concentration of 70 to 100%.

  The fractionation method using the hydrous alcohol may be performed in one step or in multiple steps. For example, fractionation may be carried out in one step using a high concentration aqueous alcohol solution, or fractionation may be carried out by increasing the alcohol concentration in two steps: medium concentration alcohol aqueous solution-high concentration alcohol aqueous solution. When fractionated in two stages, impurities insoluble at each alcohol concentration can be efficiently removed, and the process of purification by subsequent chromatography can be further simplified.

  In the case of fractionation in one step, an alcohol aqueous solution having a concentration of 85 to 95% is added to the dried protein hydrolyzate and stirred to remove insoluble matters to obtain a sample for HPLC. In the case of fractionation in two stages, the dried protein hydrolyzate is first added with an aqueous alcohol solution having a concentration of 70 to 85% and stirred, and alcohol is further added to the supernatant from which insolubles have been removed to add 90 to 95% alcohol. Stir as an aqueous solution and remove insoluble matter again to make a sample for HPLC.

  When purifying by chromatography, it is preferable to once concentrate and dry the hydrous alcohol-soluble fraction obtained by the hydrous alcohol fraction. The method may be in accordance with a conventional method, and can be appropriately selected from evaporation, freeze drying, hot air drying, vacuum drying, spray drying and the like. When the peptide of the present invention is purified using HPLC, the concentrated alcohol-containing water-soluble alcohol-soluble fraction may be directly subjected to HPLC, or may be subjected to HPLC after rough purification by gel filtration column chromatography or the like.

  In the separation by HPLC, for example, a reverse phase column or an ion exchange column mainly used in peptide HPLC can be used, and these are silica-based (silica gel, etc.) and polymer-based (vinyl polymer) depending on the base of the filler. Etc.). In the present invention, a reverse phase column using a silica-based filler is preferably used.

  Examples of the substituent for the reverse phase filler include an octadecylsilyl group, an octyl group, a phenyl group, and a diphenyl group. These substituents are usually introduced into silanol groups on the surface of the silica gel, but those obtained by end-capping residual silanol groups by trimethylsilylation are preferably used.

  Specific examples of the separation column include Inertsil ODS-3 (GL Science) in which an octadecylsilyl group is bonded to a silica gel.

  A polar solvent is used as a mobile phase in HPLC using a reverse phase column. As the polar solvent, for example, a lower alcohol such as methanol, ethanol or isopropyl alcohol, or an aqueous solution such as acetonitrile or tetrahydrofuran can be used, and acetonitrile is preferably used. In addition, it is preferable to add trifluoroacetic acid to the polar solvent for the purpose of stabilizing the baseline and preventing peak tailing.

  The flow rate of the mobile phase in HPLC can be appropriately selected according to the use of the column, and is usually 0.1 to 100.0 mL / min, preferably 1 to 30 mL / min. The temperature of a separation column is 20-50 degreeC normally, Preferably it is 30-45 degreeC.

  As a detector in HPLC, it is usually preferable to use a UV detector, set the detection wavelength to 205 to 270 nm, preferably around 210 nm or 234 nm, and sort according to the obtained signal.

  The fraction collected in this manner is analyzed for amino acid sequence using a peptide sequencer, and the fraction containing a peptide having the amino acid sequence of Val-Pro-Val-Pro-Gln is collected. The peptide can be obtained.

Therefore, in one embodiment, the present invention is a method for producing a peptide consisting of an amino acid sequence represented by Val-Pro-Val-Pro-Gln,
(a) a step of hydrolyzing wheat protein with an acidic protease in an aqueous medium, or a step of hydrolyzing wheat protein with an acidic protease in an aqueous medium and further hydrolyzing with a neutral or alkaline protease;
(b) removing the insoluble matter from the hydrolysis product of step (a) and recovering the water-soluble component;
(c) removing the aqueous component from the water-soluble component recovered in step (b) and recovering the concentrate;
(d) fractionating the concentrate collected in step (c) with hydrous alcohol to fractionate the peptide mixture as hydrous alcohol-soluble fraction; and
(e) including a step of separating the hydrous alcohol-soluble fraction fractionated in step (d) by HPLC and collecting a fraction containing a peptide having an amino acid sequence represented by Val-Pro-Val-Pro-Gln. It relates to said method.

  Subsequently, a chemical synthesis method of the peptide of the present invention will be described. The peptide of the present invention is a pentapeptide in which five amino acids are bonded, and can be synthesized by either a liquid phase synthesis method or a solid phase synthesis method. When the solid phase synthesis method is employed, the peptide synthesizer (for example, Biolynx 4170 manufactured by Pharmacia) or the like can also be used for synthesis.

  As a specific synthesis method, a known method can be used. For example, an amino group such as a Boc (t-butyloxycarbonyl) group or an Fmoc (9-fluorenylmethoxycarbonyl) group is added to glutamine which is the C-terminal amino acid of the peptide Val-Pro-Val-Pro-Gln of the present invention. Proline protected with a protecting group and activated with a carboxyl group is condensed. Subsequently, after removing the protecting group of the proline amino group of the Pro-Gln dipeptide, the amino group is protected and the valine activated with the carboxyl group is condensed. In this way, by sequentially condensing amino acids from the C-terminal side to the N-terminal side, a peptide having the amino acid sequence of Val-Pro-Val-Pro-Gln can be synthesized. In this case, by using the L-form and D-form of each amino acid, it is possible to synthesize a peptide in which any one to four of the five amino acids are L-amino acids and the rest are D-amino acids.

  In the case of the solid phase synthesis method, the peptide synthesized in this way is dissociated from the carrier, and after removing all protecting groups, the peptide is purified by an ordinary method such as HPLC using a reversed phase column. be able to.

  Since the peptide of the present invention has a high blood pressure lowering action, it can be used as an active ingredient in blood pressure lowering agents and foods for lowering blood pressure. The peptide of the present invention, blood pressure lowering agent and food for lowering blood pressure (the blood pressure lowering agent of the present invention may include food for lowering blood pressure) can be administered to humans and various animals, Can lower blood pressure and suppress blood pressure increase in humans and animals. In addition, since the peptide of the present invention has a low angiotensin converting enzyme inhibitory action, it does not have actions such as dry cough, rash, and flush derived from the angiotensin converting enzyme inhibitory action. Therefore, the antihypertensive agent comprising the peptide of the present invention as an active ingredient is advantageous in that it has no side effects and is safe. Furthermore, since the peptide of the present invention has a high blood pressure lowering action as described above, a peptide having a conventional blood pressure lowering action requires a large amount of administration, whereas it can exert an effect at a low dose. It is practical as an active ingredient for blood pressure lowering agents.

  A suitable dose of the blood pressure lowering agent of the present invention varies depending on various conditions such as age, weight, sex, symptom, animal type and the like of the human or animal to be administered, and can be appropriately set. The antihypertensive agent of the present invention can be administered either orally or parenterally. In the case of oral administration, the dosage of the blood pressure lowering agent of the present invention is usually 1 to 1000 mg, preferably 10 to 500 mg, as a daily dose, based on the mass of the peptide which is an active ingredient.

  Furthermore, the blood pressure lowering agent of the present invention can be blended with various additives used in the manufacture of pharmaceuticals, foods, and feeds, and may coexist with various substances. Such substances and additives include various fats and oils, crude drugs, amino acids, polyhydric alcohols, natural polymers, vitamins, minerals, dietary fibers, surfactants, purified water, excipients, stabilizers, pH adjusters, Antioxidants, sweeteners, flavoring ingredients, acidulants, colorants, flavors and the like can be mentioned.

  Examples of the various fats and oils include vegetable oils such as soybean oil, safflower oil, and olive oil, and animal fats such as beef tallow and sardine oil.

  Examples of the herbal medicine include cow yellow, ground yellow, eggplant, royal jelly, carrot and deer.

  Examples of the amino acid include cysteine, leucine, arginine and the like.

  Examples of the polyhydric alcohol include ethylene glycol, polyethylene glycol, propylene glycol, glycerin, sugar alcohol and the like. Examples of the sugar alcohol include sorbitol, erythritol, xylitol, maltitol, mannitol and the like.

  Examples of the natural polymer include gum arabic, agar, water-soluble corn fiber, gelatin, xanthan gum, casein, gluten or gluten hydrolyzate, lecithin, and dextrin.

  Examples of the various vitamins include vitamin C (ascorbic acid), vitamin B group, vitamin E (tocopherol), vitamin A, D, K, riboflavin butyrate, and the like. The vitamin B group includes vitamin B1 derivatives, vitamin B2, vitamin B6, vitamin B12, and various vitamin B complexes such as biotin, pantothenic acid, nicotinic acid, and folic acid. Examples of vitamin B1 derivatives include thiamine or a salt thereof, thiamine disulfide, fursultiamine or a salt thereof, dicetiamine, bisbuthiamine, bisbenchamine, benfotiamine, thiamine monophosphate disulfide, chicotiamine, octothiamine, prosultiamine, etc. All the compounds having the physiological activity of vitamin B1 are included.

  Examples of the mineral include calcium, magnesium, zinc, iron and the like.

  Examples of dietary fiber include gums, mannan, pectin, hemicellulose, lignin, β-glucan, xylan, and arabinoxylan.

  Examples of the surfactant include glycerin fatty acid ester, sorbitan fatty acid ester, and sucrose fatty acid ester.

  Examples of the excipient include sucrose, glucose, corn starch, calcium phosphate, lactose, dextrin, starch, crystalline cellulose, and cyclodextrin.

  In addition to the above, various additives include, for example, taurine, glutathione, carnitine, creatine, coenzyme Q, glucuronic acid, glucuronolactone, theanine, γ-aminobutyric acid, capsaicin, various organic acids, flavonoids, polyphenols Catechins, xanthine derivatives, indigestible oligosaccharides such as fructooligosaccharides, polyvinylpyrrolidone and the like.

  The amount of these additives is appropriately determined according to the type of additive and the amount of intake to be desired, but is generally in the range of 0.01 to 30% by mass, preferably 0.1 to 10% by mass. % Range.

  The blood pressure lowering agent of the present invention may be administered alone or together with a solid carrier or a liquid carrier usually used in the pharmaceutical industry. Moreover, the blood pressure lowering agent of the present invention can be used in combination with or combined with other drugs. The administration form at that time can be any form such as tablets, pills, powders, capsules, granules, powders, syrups, aqueous solutions, injections, drinks, liquid foods and the like.

  Furthermore, the blood pressure lowering agent of the present invention can also be added to food and feed. Therefore, the present invention also relates to a food for lowering blood pressure containing the peptide of the present invention or a salt thereof. The food of the present invention includes health foods and functional foods such as various forms of foods such as powders, tablets, fine granules, tablets, granules, capsules, liquid foods and the like. Such a form of food can be produced using conventional means after adding an appropriate excipient (for example, starch, processed starch, lactose, glucose, water, etc.) to the peptide of the present invention. As specific examples of the food of the present invention, coffee beverage, tea beverage, fruit juice beverage, soft drink, milk beverage, butter, mayonnaise, shortening, margarine, various salad dressings, breads, noodles, cooked rice, pasta, Examples include confectionery, cookies, chocolate, candy, chewing gum, various seasonings, and various diet products.

  By ingesting the food containing the peptide having a blood pressure lowering action of the present invention or a salt thereof, effects such as a reduction in blood pressure or suppression of an increase in blood pressure can be expected.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples at all.

Example 1 Separation and purification of a new pentapeptide A reaction kettle was charged with 267 g of wheat gluten (active gluten, manufactured by Weston Foods Limited), 1700 mL of water, and 32 mL of 1N citric acid, and the pH was adjusted to 4.0 using citric acid. Then, 0.4 g (20000 units) of acidic protease (Protease M Amano, manufactured by Amano Enzyme) was added, heated to 45 ° C., and reacted for 5 hours. Sodium hydroxide was added to the reaction solution to adjust the solution to pH 7.5, and the temperature was maintained at 85 ° C. for 10 minutes to deactivate the acidic protease.

  Next, 2000 mL of water was added, the temperature was adjusted to 65 ° C., 4 g (320,000 units) of neutral protease (Thermoase, manufactured by Daiwa Kasei) was added and reacted for 4 hours, and then the temperature was maintained at 90 ° C. for 20 minutes. Sex protease was inactivated.

  The reaction solution was centrifuged (8000 G, 30 minutes) to remove insoluble matters, and the supernatant was recovered to obtain a water-soluble component. Then, 8 g of activated carbon and 60 g of diatomaceous earth were added and stirred at room temperature for 1 hour. . Next, filtration was performed, and the solid matter was separated and removed to recover the liquid, and lyophilized to obtain 150 g of a dry solid.

  To 100 g of the dried solid obtained above, 2000 mL of 80% (v / v) ethanol was added and stirred, and insolubles were removed by filtration. To the remaining supernatant, 2000 mL of 99.5% ethanol was added to further adjust the ethanol concentration to 90%. The generated insoluble material was removed by filtration. The supernatant was concentrated under reduced pressure and dried to obtain 40.78 g of a fraction.

This solid fraction was further fractionated by gel filtration chromatography using YMCgel ODS-AQ12S50 (2.5 × 40 cm), and the fraction eluted with 10% acetonitrile-0.1% aqueous trifluoroacetic acid was purified by HPLC. The peak indicated by the arrow in FIG. 1 was collected and purified. As a result, 180 mg of peptide was obtained.
HPLC separation conditions Column: Inertsil ODS-3 φ20 × 250 mm (manufactured by GL Science)
Mobile phase: 10% acetonitrile-0.1% trifluoroacetic acid aqueous solution Flow rate: 10 mL / min Detector: UV spectrophotometer 210 nm
Temperature: 40 ° C

  When the amino acid sequence of the purified peptide was analyzed using a peptide sequencer, it was confirmed to have the amino acid sequence Val-Pro-Val-Pro-Gln.

Comparative Example 1 Isolation of ACE Inhibitory Peptide Derived from Wheat Ile-Ala-Pro, an ACE inhibitory peptide derived from wheat, which was an ACE inhibitor, was produced with reference to JP-A-4-66594.

  1000 g of wheat gluten was dispersed and dissolved in 20 L of 0.03 mol / L hydrochloric acid, and distilled water was added to make 40 L. The pH was adjusted to 2.0 using hydrochloric acid, 600000 units of pepsin (manufactured by Roche) were added, and the mixture was reacted at 37 ° C. for 15 hours. After completion of the reaction, the pH was adjusted to 4.4 with 5 mol / L sodium hydroxide, and an acidic protease (Protease M Amano, manufactured by Amano Enzyme) 100000 unit was added and reacted at 45 ° C. for 5 hours. Subsequently, the pH was adjusted to 6.0, and the enzyme was inactivated by heating at 90 ° C. for 20 minutes. After removing insolubles by centrifugation, the supernatant was freeze-dried to obtain 674.1 g of a dry solid.

  The dried solid obtained above was fractionated by gel filtration chromatography using YMCgel ODS-AQ12S50 (2.5 × 40 cm), and a fraction having high ACE inhibitory activity was collected. Further purified using HPLC. As a result, 67 mg of peptide was obtained. When the amino acid sequence of the purified peptide was analyzed using a peptide sequencer, it was confirmed that it had an amino acid sequence of Ile-Ala-Pro.

Test Example 1: Blood pressure lowering effect test (single administration) and its evaluation The systolic blood pressure (SBP) of a 12-week-old spontaneously hypertensive rat (SHR rat) was measured using a non-animal blood pressure monitor (MK- 2000; manufactured by Muromachi Kikai Co., Ltd.), and animals having SBP of 200 mmHg or more were used for the test.

  The peptides of Example 1 and Comparative Example 1 and captopril (manufactured by Sigma-Aldrich) as a positive control were separately dissolved in water for injection, adjusted to the dose shown in Table 1, and given to the animals once orally Administered. Blood pressure measurements were taken before administration and 2, 4, 6, 8 and 24 hours after administration. The control group was orally administered with water for injection, and the number of animals in one group was 10.

  In addition, as for the rats used, 11 weeks old SHR / Izm male rats were purchased from Japan SLC Co., Ltd., and after the preliminary breeding including a quarantine period of 1 week or longer, the animals in which no abnormalities were observed in the general state were selected. And used.

  The measurement results are shown in Table 1 as the difference between the SBP before administration and the SBP at each time point after administration (Change of SBP), and the difference in Change of SBP at each time point from the control group.

  As is clear from Table 1, it is clear that the animals administered with the peptide of Example 1 of the present invention have a much greater decrease in blood pressure than the animals administered with the peptide showing the ACE inhibitory activity of Comparative Example 1. became. That is, it was shown that the peptide of the present invention has a very strong blood pressure lowering action as compared with conventionally known blood pressure lowering peptides.

  Moreover, the animal which administered the peptide of Example 1 of this invention showed the blood pressure fall equivalent to the animal which administered captopril approved as a blood pressure lowering agent. That is, it was shown that the blood pressure lowering action of the peptide of the present invention is sufficient for practical use as an active ingredient of a blood pressure lowering agent.

Example 2 Preparation of Tablets 20 g of the peptide obtained in the same manner as in Example 1, 73.8 g of crystalline cellulose (Asahi Kasei) and 5 g of polyvinylpyrrolidone (BASF) were mixed, and 30 mL of ethanol was added thereto, followed by a wet method. Granules were produced according to a conventional method. After the granules were dried, 1.2 g of magnesium stearate was added to obtain granules for tableting, and tableting was performed using a tableting machine to produce 100 tablets each having 1 g.

Example 3 Production of Granules 20 g of the peptide obtained in the same manner as in Example 1, 160 g of lactose (DMV) and 60 g of crystalline cellulose (Asahi Kasei) were mixed, and 130 mL of ethanol was added thereto, using a kneader. The kneading was carried out for 5 minutes by a usual method. After kneading, the mixture was sieved with 10 mesh and dried at 50 ° C. in a dryer. After drying, the particles were sized to obtain 240 g of granules.

Example 4 Manufacture of syrup preparation Purified water (400 g) was boiled, and while stirring this, 750 g of white sugar and 10 g of the peptide obtained in the same manner as in Example 1 were added and dissolved, and the resulting mixture was wiped with heat and purified. Water was added to make a total volume of 1000 mL to produce a syrup.

  Since the peptide of the present invention has a low angiotensin converting enzyme inhibitory action, it does not have side effects such as dry cough, rash, and flush resulting from the angiotensin converting enzyme inhibitory action. Moreover, the blood pressure lowering agent of the present invention is extremely practical because it can be easily produced using easily available and inexpensive raw materials and can be blended into various foods and drinks because of its high safety.

Wheat gluten is treated with protease, the reaction solution is centrifuged, the supernatant is filtered, ethanol is added to the dried solid obtained by lyophilizing the liquid, and the supernatant is concentrated under reduced pressure to dryness. An HPLC chart obtained as a result of fractionation by gel filtration chromatography and subjecting the elution part with 10% acetonitrile-0.1% trifluoroacetic acid aqueous solution to HPLC is shown.

Claims (3)

The following formula:
Val-Pro-Val-Pro-Gln
Or a salt thereof comprising the amino acid sequence represented by   A blood pressure lowering agent comprising the peptide according to claim 1 or a salt thereof.   A food additive comprising the peptide according to claim 1 or a salt thereof.

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