KR101106303B1 - A antihypertensive peptide - Google Patents

Abstract

The present invention relates to a peptide prepared from oyster through enzyme treatment, and more particularly, to a peptide capable of inhibiting angiotensin converting enzyme (ACE).

In particular, the peptide of the present invention is excellent in inhibiting ability of angiotensin converting enzyme to treat or improve cardiovascular diseases including hypertension, as well as food-derived oysters, which ensures safety and does not cause side effects. It can be widely used in the related industry in that it can replace artificial materials for the treatment or amelioration of chemically manufactured cardiovascular diseases.

Angiotensin Converting Enzyme, Hypertension, Food-Derived Peptides

Description

Peptide with hypotension {A ANTIHYPERTENSIVE PEPTIDE}

The present invention relates to a peptide prepared from oyster through enzyme treatment, and more particularly, to a peptide capable of inhibiting angiotensin converting enzyme (ACE).

Peptides derived from food proteins in food components have been reported to exhibit a wide range of physiological activities, such as anticancer, lowering blood pressure, lowering serum cholesterol, enhancing immunity and promoting calcium absorption. Specifically, opioid peptides, which are involved in the contraction of smooth muscles such as analgesic, anesthesia, intestine, and appetite control in vivo, phagocytosis peptides for immunopotentiation, and angiotensin-I converting enzyme (ACE) ) Peptides that belong to antithrombotic peptides that inhibit platelet aggregation.

Angiotensin converting enzymes are dicarboxy peptides found in the proximal tubules of the blood vessels and kidneys, endothelial, heart, lung, activated macrophages, and brain tissues. They are produced by renin in the renin-angiotensin system, a mammalian blood pressure and water balance regulator. Activated angiotensin I from an angiotensinogen is converted to angiotensin II. Angiotensin II acts on four types of AT receptors in the adrenal glands, vascular smooth muscle cells, kidneys, heart, etc., which cause vasoconstriction, aldosterone and vasopressin release, tubular sodium absorption, and blood flow to the kidneys. It is reported that this can lead to various lesions in the cardiovascular, renal and central nervous areas.

Therefore, angiotensin converting enzyme inhibitors or angiotensin converting enzyme inhibitors (ACE inhibitors) are reported to be able to treat or prevent cardiovascular diseases such as hypertension, heart disease, arteriosclerosis, or cerebral hemorrhage or kidney disease, and many studies have been conducted. It's going on. Specifically, the results of various clinical studies and clinical trials confirming that it can effectively reduce chronic kidney disease, arteriosclerosis, heart attack and death thereof are reported.

Based on these results now, chemically synthesized such as ramipril, captopril, enalapil, lisinopril, fosinoril or spirapril, etc. Many angiotensin converting enzyme inhibitors are used to treat hypertension. However, these compounds are easily degraded in pharmaceutical dosage forms, which means they are not stable and act on other cells, causing problems with side effects such as weakness throughout the body, vomiting, coughing, headache, anorexia, and taste disorders. It is reported to be.

In order to solve this problem, there is an urgent need to develop an angiotensin converting enzyme inhibitor derived from a natural substance which does not cause side effects and ensures safety. In particular, in the case of angiotensin converting enzyme inhibitors derived from natural substances used as foods, those obtained from food protein hydrolysates or food protein hydrolysates have been reported.

In this regard, according to recent studies, angiotensin converting enzyme inhibitory activity was generally found to have a high effect on low molecular peptides of 3,000 Da or less. In particular, peptides having excellent angiotensin converting enzyme inhibitory activity derived from food proteins have a high molecular weight. It was confirmed that it was 1,000 Da or less. In addition, in the case of peptides having 5 or more amino acid residues, it is reported that the number of amino acid residues is required to be less than 5 because they may undergo hydrolysis by enteric enzymes without being absorbed into a circular structure upon oral administration. However, food-derived angiotensin converting enzyme inhibitors satisfying all of the above conditions have not been reported.

In order to solve the problems of the prior art as described above, the present invention can inhibit angiotensin converting enzyme (ACE), the side effect is not a problem from food origin, and to provide a peptide that ensures safety It is done.

In order to achieve the above object, the present invention provides a novel peptide derived from oysters that can inhibit angiotensin converting enzyme (ACE). Specifically, the peptide may be a peptide including the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or acid addition salts thereof.

In addition, the present invention provides a composition for the treatment or improvement of cardiovascular disease or hyperlipidemia comprising the peptide as an active ingredient in order to achieve the above object.

The present invention also provides a nucleotide encoding the peptide to achieve the above object.

In addition, the present invention provides a vector comprising a nucleotide encoding the peptide to achieve the above object.

The present invention also provides a method for preparing a composition for treating or preventing cardiovascular disease.

The inventors of the present invention, while studying a food-derived angiotensin converting enzyme inhibitor, confirmed that the peptide of the present invention obtained by enzymatic treatment of oysters is very excellent angiotensin converting enzyme inhibitory effect, and completed the present invention based on this.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a novel peptide derived from oysters that can inhibit angiotensin converting enzyme (ACE).

The angiotensin converting enzyme is mainly present in the pulmonary capillaries and induces vasoconstriction by decomposing two terminal amino acids of angiotensin I in an inactive state in the bloodstream, and induces aldosterone secretion to increase body fluids to increase blood pressure. Angiotensin II is an enzyme that converts into angiotensin II, which is capable of raising blood pressure, and the angiotensin converting enzyme inhibitor inhibits the angiotensin converting enzyme and exhibits a vasodilation effect.

The peptide is preferably a peptide comprising an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or acid addition salts thereof, for example, a peptide having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or acid addition salts thereof Can be. The peptide may be present in isolated or substantially pure form. In addition, the peptide may be a substance derived from a peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The acid addition salt may be a pharmaceutically acceptable acid addition salt, and the pharmaceutically acceptable acid addition salt refers to a salt that maintains the biological activity of the peptide and minimizes side effects such as unwanted toxin effects.

The acid addition salt may be a pharmaceutically acceptable inorganic acid addition salt or organic acid addition salt, and for example, hydrochloride, hydrobromide, sulfate, nitrate, acetate, benzoate, maleate, fumarate, succinate, tartarate, citrate, Oxalate, methanesulfonate, toluenesulfonate, aspartate or glutamate.

The present invention may also be a nucleotide encoding a peptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The nucleotide may be an isolated nucleotide.

The present invention also provides a vector comprising the nucleotide. The type of the vector may be changed depending on the host and the expression condition to be transformed using the vector. If the vector contains the nucleotide, other conditions are not limited.

The present invention also provides a composition for treating or improving cardiovascular disease comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

The peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 may preferably be a peptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The cardiovascular disease may be any one selected from the group consisting of hypertension, heart disease, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, atherosclerosis, and arteriosclerosis, preferably hypertension.

The hypertension is a blood vessel-related disease, and means arterial hypertension with high arterial pressure. The hypertension may cause arteriosclerosis or hypertensive heart disease.

The atherosclerosis refers to a disease in which the walls of the arteries are thickened and the elasticity of the arteries is reduced. When the atherosclerosis develops, the blood supply to the organs that received the blood from the hardened arteries is reduced, so that additional diseases may develop. An example of a typical disease related to this is coronary artery disease in which atherosclerosis changes in the coronary arteries that supply blood to the muscles of the heart.

The composition comprising the peptide as an active ingredient may include one or more of a pharmaceutical diluent selected from saline, buffered saline, dextrose, water, glycerol and ethanol, diluent is not limited thereto.

The composition may be applied differently depending on the purpose of administration and the disease. The amount of active ingredient administered substantially depends on a variety of related factors, such as the disease to be treated, the extent of the patient's condition, whether it is co-administered with other drugs (eg chemotactic drugs), the patient's age, gender, weight, food, time of administration, It should be determined in consideration of the route of administration and the ratio of the composition. The composition may be adjusted according to the type and severity of the disease and the route of administration, and may be administered once or divided into 1 to 3 times a day, but is not limited thereto.

The compositions of the present invention can be administered orally or parenterally. Parenteral administration refers to administration of medications other than oral, ie rectal, intravenous, peritoneal and muscular, arterial, transdermal, nasal, inhaled, ocular, and subcutaneous.

The composition may be formulated in any form such as oral dosage form, injectable solution or topical formulation. The formulation is preferably prepared for oral and injectable administration (true solution, suspension or emulsion), most preferably in oral form such as tablets, capsules, soft capsules, aqueous pharmaceuticals, pills, granules and the like. desirable.

In the above formulation, the peptide of the present invention may be filled into a soft capsule without an excipient, and may be made into a suitable formulation after being mixed or diluted with the carrier. Examples of suitable carriers include starch, water, saline, Ringer's solution, dextrose and the like.

The composition may be applied as a pharmaceutical composition or a food composition.

The pharmaceutical composition may be directly applied to animals including humans. The animal is a biological group corresponding to a plant, and mainly eats organic matter as nutrients, and means that digestion, excretion, and respiratory organs are differentiated, and preferably, vertebrates, more preferably mammals. The mammal may be human, pig, cow or goat, etc., preferably human.

The pharmaceutical composition may include a peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 alone as an active ingredient, and additional ingredients, pharmaceutically acceptable or nutritious according to the formulation, method of use and purpose of use. It may further comprise a pharmaceutically acceptable carrier, excipient, diluent or accessory.

More specifically, the pharmaceutical composition may be further added to the nutrients, vitamins, electrolytes, flavoring agents, coloring agents, neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, and stabilizers. It may further contain an agent, a preservative, glycerin, alcohol, a carbonation agent used in the carbonated beverage, and the like. In addition, the carrier, excipient or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose Selected from the group consisting of loose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, saline It may be one or more, but is not limited to any conventional carrier, excipient or diluent can be used. The components may be added independently or in combination to the peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 which is the active ingredient.

The pharmaceutical composition comprises 0.001% to 99.9% by weight, preferably 0.1% to 99% by weight, more preferably 1, of the peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 relative to the total weight of the composition It may include 50% by weight.

In addition, the pharmaceutical composition may further include conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives when formulated, and oral or parenteral. Can be used

Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, water, or the like in the active ingredient. It may be prepared by mixing cross or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients may include, for example, wetting agents, sweeteners, fragrances, and preservatives. have.

In addition, the formulation of the pharmaceutical composition of the present invention may be in a preferred form depending on the method of use, in particular by adopting methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated. Examples of specific formulations include warnings, granules, lotions, linings, limonades, powders, syrups, ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, liquid extracts, emulsions, Suspensions, premises, acupuncture, eye drops, tablets, suppositories, injections, spirits, capsules, creams, pills, soft or hard gelatin capsules.

Furthermore, the pharmaceutical compositions of the present invention are preferably formulated using appropriate methods known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. Can be converted.

The dosage of the pharmaceutical composition according to the present invention may be appropriately selected by those skilled in the art in consideration of the method of administration, the age, sex and weight of the recipient, the severity of the disease, and the like. For example, the pharmaceutical composition of the present invention may be administered at 0.000001 mg / kg / day to 1000 mg / kg / day, based on the peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

In addition, the pharmaceutical composition of the present invention, in addition to the peptide containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient, further comprising a compound having a known cardiovascular disease therapeutic effect, in particular, a natural-derived material used in foods It may be included in 5 parts by weight to 100 parts by weight with respect to 100 parts by weight of the active ingredient.

The present invention also provides a food composition comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. Examples of the food composition of the present invention include food, food additives, beverages or beverage additives.

The food composition comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient may further comprise suitable carriers, excipients and diluents commonly used in the preparation thereof.

In the present specification, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process. It is intended to include all foods, food additives, health functional foods and beverages, preferably gum or candy.

Examples of the food to which the peptide containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention can be added include various foods, beverages, gums, candy, tea, vitamin complexes, functional foods, and the like. In addition, the food in the present invention includes special nutritional products (e.g., prepared oils, infants, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, ice cream, etc., natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages, and other health supplements. The food, beverage or food additive may be prepared by a conventional production method.

Functional food in the present invention is the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food that is designed and processed to fully express the gymnastic function related to the living body, preferably the functional food of the present invention is sufficient to express the gymnastic function related to the prevention or improvement of cardiovascular disease or hyperlipidemia to the living body. It means food that can. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In the present invention, the drink is a generic term for drinking to quench thirst or to enjoy the taste and is intended to include a functional drink. The beverage contains, as an essential ingredient, a peptide containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient, and there are no particular restrictions on other ingredients, and various flavoring agents such as conventional drinks. Or natural carbohydrate or the like as an additional component. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and Sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate may generally be about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention. It may further contain a pulp for.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components can be used independently or in combination. The additive may be 0 to 100,000 parts by weight, preferably 0.00001 to 10,000 parts by weight, per 100 parts by weight of the peptide including the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention, but is not limited thereto.

Functional beverage in the present invention is a biological defense rhythm control, disease prevention and the like having a beverage group or a beverage composition that has added value to the beverage by using physical, biochemical, biotechnological techniques, etc. to function and express the function of the beverage to a specific purpose Means a beverage that is designed and processed to fully express the gymnastics function related to recovery.

The functional beverage is not particularly limited in addition to the peptide containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention as an essential ingredient in the indicated ratio, and various flavors or Natural carbohydrates and the like may be included as additional components. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and xylitol Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The natural carbohydrate may be included in an amount of 0 to 20 parts by weight, preferably 1 to 18 parts by weight, more preferably 5 to 12 parts by weight, per 100 parts by weight of the composition of the present invention.

In addition, in a food composition aimed at preventing or improving cardiovascular disease or hyperlipidemia, the amount of the peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is 0.00001% to 50% by weight of the total food weight It may be included as, but is not limited thereto.

The present invention also relates to a method for preparing a composition for treating or preventing cardiovascular disease.

The method may include treating transglutaminase in the oyster and treating proteolytic enzymes in the oyster treated with the transglutaminase.

More specifically, grinding the oysters; And treating the ground glutamic oyster with transglutaminase and treating the transglutaminase oyster with proteolytic enzymes.

The oyster may be poached in water, the temperature of the water may be from 80 to 150 ℃, the duration of the blanch may be 10 seconds to 10 minutes or 30 seconds to 2 minutes.

The grinding of the oysters is performed by adding water corresponding to about 1 to 4 times or 1.5 times to 3 times the weight of the boiled oysters, and using a grinder for 30 seconds to 5 minutes or 1 minute to 3 minutes to 500 rpm to It may be carried out under the conditions of 5,000 rpm or 1,000 rpm to 3,000 rpm.

The transglutaminase may be added 1 to 5% or 1.5 to 2.5% of the concentration of the oyster protein. The reaction conditions and reaction time may be appropriately controlled by the type of enzyme, for example, the reaction may be carried out for 10 minutes to 120 minutes or 30 minutes to 90 minutes at 20 to 40 ℃ or 25 to 35 ℃. By treatment of the transglutaminase, new covalent bonds between proteins can be formed.

The proteolytic enzyme treatment may be performed after inactivation of the transglutaminase by performing a heat treatment on the oyster ground treated with the transglutaminase. The heat treatment may be performed at 50 to 150 ° C. or 80 to 120 ° C. for 10 minutes to 120 minutes or 30 minutes to 90 minutes.

The protease may be a conventional protease, for example Protamex or Neutrase. The throughput of the protease may be 0.5 to 5% or 1 to 2% of the concentration of oyster protein, respectively. Specifically, the treatment of the protease may be performed by treating Neutrase after treating Protamex, and the reaction conditions, reaction time, and enzyme inactivation may be appropriately controlled by each enzyme type. For example, the reaction conditions may be carried out for 10 minutes to 120 minutes or 30 minutes to 90 minutes at 20 to 60 ℃ or 35 to 45 ℃. Heat treatment for inactivation of the protease may be carried out at 50 to 150 ℃ or 80 to 120 ℃ for 10 minutes to 120 minutes or 30 minutes to 90 minutes.

In addition, the production method is the step of adding ethanol such that the final concentration is 60% (v / v) to the enzyme treatment and left for 30 to 120 minutes or 45 minutes to 90 minutes in a low temperature room of 2 to 6 ℃ After precipitation of the ethanol insoluble material and residual protein, the step of removing by centrifugation may be further performed. In addition, the step of filtering the supernatant obtained by removing the precipitate may be further performed.

In addition, the present invention relates to a composition for treating or preventing cardiovascular diseases made by the method. The cardiovascular disease treatment or prevention composition may include an oyster enzyme treatment.

In the present invention, peptides or acid addition salts can inhibit angiotensin converting enzyme (ACE), which is useful for the treatment, prevention, or improvement of cardiovascular diseases, and is derived from oysters, which are foods, which ensure safety. Side effects are not a problem. In addition, since the three sides can be obtained by the enzyme treatment from the oyster harvested in abundance in the sea of Korea, the production of the peptide has the advantage of excellent economical efficiency compared to the conventional chemical artificial synthesis. Therefore, the peptide of the present invention can be widely used in the related industry in that it can replace an artificial chemical for treating or ameliorating existing chemically produced cardiovascular disease.

Hereinafter, the preparation examples and examples are presented in order to explain the present invention in more detail. However, the following Preparation Examples and Examples are only illustrated to aid the understanding of the present invention, and may be modified in various other forms, and the scope of the present invention is not limited to the following Examples.

Example 1 Preparation of Oyster Protein Enzyme Treated Products

1-1. Preparation of Oyster Protein Enzyme Treated Products

3 kg of aquaculture oysters purchased from the water haul association in Tongyeong was boiled in boiling water for 1 minute, and then drained using a pole. The water corresponding to twice the weight of the oyster was added to the oysters boiled in boiling water, and then crushed at 3000 rpm for 2 minutes using a grinder. Protein concentration of the oyster grinding was measured using the Lowry method. Hydrolysates were prepared by adding and reacting each transglutaminase and / or protease to a concentration of 2% relative to the measured protein concentration. Specifically, the hydrolyzate is Experimental Example 1 and proteolytic enzymes treated with transglutaminase (transglutaminase, TGase, Ajinomoto Co, Japan), Protamex (Novozyme-Korea, Korea) and Neutrase (Novozyme-Korea, Korea) Experimental Example 2 which was treated only with Protamex and Neutrase.

Preparation of the hydrolyzate of Experimental Example 1 was carried out in more detail as follows. First, a transglutaminase is added to the oyster pulverized product so as to have a concentration of 1%, and a reaction is performed at 30 ° C. for 1 hour to form a covalent bond, and then at 100 ° C. for 1 hour. Heat treatment was used to inactivate the enzyme. Protamex was added to the enzyme treatment treated with transglutaminase so as to have a concentration of 1% based on the measured protein concentration, and the reaction was performed at 40 ° C. for 1 hour, followed by heat treatment at 100 ° C. for 1 hour. The enzyme was inactivated. Neutrase was added to the Protamex-treated enzyme treatment so as to have a concentration of 1% relative to the measured protein concentration, and the reaction was performed at 50 ° C. for 1 hour, followed by heat treatment at 100 ° C. for 1 hour to burn the enzyme. Activated. Experimental Example 1 was obtained by obtaining the supernatant of the enzyme treatment.

In addition, Experimental Example 2 was added to Protamex so as to have a concentration of 1% of the measured protein concentration in the oyster pulverization, the reaction was carried out for 1 hour at 40 ℃, heat treatment for 1 hour at 100 ℃ to burn the enzyme After activation, Protrax-treated enzyme treated Neutrase was added at a concentration of 1% relative to the measured protein concentration, and the reaction was performed at 50 ° C. for 1 hour, followed by heat treatment at 100 ° C. for 1 hour. Was inactivated. Experimental Example 2 was obtained by obtaining a supernatant of the enzyme treatment.

Ethanol was added to the obtained Experimental Example 1 and Experimental Example 2 to a final concentration of 60% (v / v), and left for 1 hour in a low temperature room at 4 ℃ to precipitate the ethanol insoluble matter and residual protein, then centrifuged Removal was carried out using separation (8000 × g, 25 min). The supernatant obtained by removing the precipitate was filtered through a membrane of 0.45 μm, dried by a method of evaporating ethanol at a temperature of 40 ℃ or less using a rotary vacuum evaporator (N-type, EYELA, Japan) of Experimental Example 1 The hydrolyzate and the hydrolyzate of Experimental Example 2 were prepared. Each of the hydrolyzates was found to be about 29% (28.5 to 30.1%) of the oysters used at the beginning of the experiment, based on dry weight.

1-2. Confirmation of hypotension

The method for confirming the blood pressure lowering ability of the hydrolyzate and the following active ingredients of Experimental Example 1 and Experimental Example 2 obtained in Example 1-1 was performed by measuring the ACE inhibitory ability.

The hydrolyzate of Experimental Example 1 and Experimental Example 2 obtained in Example 1-1 was diluted with distilled water and confirmed by adjusting the final concentration to 0.5 mg / ml. The hypotension was measured using ACE inhibitory ability.

More specifically, ACE inhibition was performed based on the method of Wu et al. (2002).

Specifically, 5 mM hippuryl-His-Leu (HHL) and 0.25 mUnit ACE was prepared with 0.1 M borate buffer (pH 8.3) containing 0.3 M NaCl. 40 uL of sample and 150 uL of ACE were added to the Eppendorf tube. The reaction was stirred for 10 minutes in a 37 ° C. constant temperature water bath, and then 100 uL HHL was added and reacted for 30 minutes in a 37 ° C. temperature water bath. After completion of the reaction, 150 uL of 1 M HCl was added to stop the enzymatic reaction, and the supernatant was aliquoted by centrifugation at 10000 × g for 10 minutes. The separated supernatant was measured by HPLC (Hitachi, Japan) to determine the content of the advantageous hippuric acid (HA). For quantification of HA, 40 uL of reaction stop solution was injected into a reversed phase column (Watchers 120 ODS-AP, 4.6 x 250 mm, 5 μm, Daiso, Japan). As solvent A, H ₂ O containing 0.1% TFA was used, and as solvent B, 100% CH ₃ CN containing 0.1% TFA was used. Gradient was detected at 228 nm absorbance under linear alignment at 5 → 60% B solvent / 20 min. As a reference solution, 40 uL of solvent A was used instead of the sample, and the flow rate was eluted under a condition of 1 mL / min. Enzyme inhibitory activity was calculated by the following formula 1, by measuring the ACE inhibitory activity for each sample concentration was linear regression analysis of the inhibitory activity by concentration. In addition, the sample concentration required to inhibit 50% of the ACE inhibitory activity was defined as IC ₅₀ .

[Calculation 1]

ACE inhibition (%) = (HA of Reference-HA of sample) / HA x 100 of Reference

The results of confirming the inhibitory ability of the angiotensin converting enzyme before the sample treatment of blood pressure lowering ability according to the purification steps of the hydrolyzates of Experimental Example 1 and Experimental Example 2 are shown in Tables 1 and 2 below.

[Table 1]

Sample Name Hydrolyzate Cation-exchange chromatography Exclusion Chromatography Experimental Example 2 38.1% 86.7% 80.0% Experimental Example 1 48.9% 87.0% 79.8%

As shown in Table 1, as the hydrolyzate of Experimental Example 2 was purified, it was confirmed that the angiotensin converting enzyme inhibitory ability was excellent, and it was determined that it is preferable to extract a peptide having angiotensin converting enzyme inhibitory ability from Experimental Example 2.

1-3. Active ingredient recovery

After dissolving the hydrolyzate of Experimental Example 1 and Experimental Example 2 obtained in Example 1-1 in deionized water, the ultrafiltration unit (8200, Amicon Co., USA) equipped with a 5 kDa membrane (5 kDa membrane) Filtered. After performing the ultrafiltration, the obtained supernatant and residue were vacuum-dried to prepare an active ingredient dry powder of each oyster hydrolyzate.

1-4. Preparation of Fractions

The dry powder (hereinafter referred to as TGPN) obtained in Example 1-2 was dissolved in 20% CH ₃ CN and ultrafiltration with an ultrafilter (8200, Amicon Co., USA) equipped with a 1kDa membrane, followed by ultrafiltration (1 kDa). TGPN) and the filtrate (TGPN below 1 kDa) were separated, and the angiotensin converting enzyme inhibitory ability was measured using 10 ml of 10% CH ₃ CN. As a result, it was confirmed that IC ₅₀ of TGPN greater than 1 kDa was 4.5157 mg / ml, and IC ₅₀ of TGPN less than 1 kDa was 0.6142 mg / ml, and TGPN of 1 kDa or less was excellent in inhibiting angiotensin converting enzyme. Purifying the TGPN of less than 1 kDa excellent in the inhibition, the step of separating the active ingredient was performed. The purification method performed to separate the active ingredient is as shown in Figure 1, in detail as follows.

In the first step, a size exclusion column (Hiroad 16/60 superdex 30 pg, 16 × 600 mm, Pharmacia, Sweden) was used to separate the low-breaking TGPN (30.1298 mg / ml, 20 ml) by molecular weight. It was detected by using a 226 nm absorbance, the solvent was used 20% CH ₃ CN, the flow rate was carried out under 1 ml / min conditions, and each 2 ml aliquot. The molecular weight of each 2 ml aliquot sample was calculated by preparing a standard curve through the elution volume of each standard protein under the same conditions. Angiotensin converting enzyme inhibitory ability was measured for each fraction divided by each molecular weight.

The second step was detected by 254 nm absorbance using a reverse phase HPLC column (218TP510 Protein & peptide C ₁₈ , 10 × 250 mm, 5 μm, Grace Vydac, USA). As solvent A, H ₂ O containing 0.1% TFA was used, and as solvent B, CH ₃ CN containing 0.1% TFA was used, and the concentration gradient was 0 to 50% B solvent / 50min. The flow rate was partially purified under the condition of 3 mL / min. Angiotensin inhibitory capacity of 1/65 of each fraction was measured.

The third step was detected at 254 nm absorbance using a cation exchange HPLC column (TSK-gel SP-5PW, 7.5 × 7.5 mm, Tosoh, Japan). 10 mM phosphate buffer (pH 6.0) was used as the solvent A, and 10 mM phosphate buffer (pH 6.0) containing 1M NaCl was used as the solvent. The concentration gradient was linear gradient under the condition of 0 to 1.0M B solvent / 100min, and the flow rate was partially purified under the condition of 1 mL / min. Angiotensin inhibitory capacity of 1/50 of each fraction was measured.

In a fourth step, an aliquot was collected using a reverse phase HPLC column (Watchers 120 ODS-AP, 4.6 × 250 mm, 5 μm, Daiso, Japan). As solvent A, H ₂ O containing 0.1% TFA was used, and as solvent B, CH ₃ CN containing 0.1% TFA was used, and the absorbance was detected at 220 nm. The concentration gradient was linear gradient under the condition of 0 to 20% B solvent / 60min, and the flow rate was partially purified under the condition of 1 mL / min. Angiotensin inhibitory capacity of 1/25 of each fraction was measured.

In the fifth step, detection was performed at 220 nm absorbance using the same reversed phase HPLC column used in the fourth step. 12% CH ₃ CN containing 0.1% TFA was used as a solvent and partially purified at a flow rate of 1 mL / min under isocratic conditions. Angiotensin inhibitory capacity of 1/25 of each fraction was measured.

In the sixth step, the fraction obtained in the fifth step was partially purified using the same reversed phase HPLC column used in the fourth step under the same conditions. Angiotensin inhibitory capacity of 1/20 of each fraction was measured.

The seventh step was purified using a size exclusion FPLC column (Superdex Peptide HR 10/30, 10 × 300 mm, Pharmacia, Sweden), solvent was used with 12% CH ₃ CN, detected at 226 nm absorbance, flow rate was It was performed at 0.5 ml / min conditions. Angiotensin inhibitory capacity of 1/10 of each fraction was measured.

Example 2: Determination of Angiotensin Converting Enzyme Inhibitory Activity of Active Fraction

Angiotensin converting enzyme inhibitory ability of each active fraction prepared in Example 1-4 was measured in the same manner as in Example 1-2.

First, when comparing the active fraction of 1 kDa or more and 1 kDa or less obtained in Example 1-4, the angiotensin converting enzyme inhibitory ability was confirmed that the IC ₅₀ value of 4.5157 mg / ml at 1 kDa or more, and less than 1 kDa In the case of IC ₅₀ value of 0.6142 mg / ml was confirmed that the active fraction of less than 1 kDa has a remarkably excellent inhibition.

According to the above confirmation results, fractions of 1 kDa or less active fractions were fractionated using the first size exclusion chromatography of Examples 1-4, and were identified as a total of four main fractions. The results are shown in Table 3 below.

[Table 3]

Fraction IC ₅₀ (mg / ml) Elution volume / void volume Molecular Weight (Da) SX1 0.2642 0.95-1.95 > 1,000 SX2 0.0790 2.35 200-500 SX3 0.1591 2.70 100-200 SX4 - 3.10 <100

As shown in Table 3, three fractions whose inhibitory activity was confirmed were named SX1, SX2, and SX3. Each IC ₅₀ value was found to be 0.2642 mg / ml, 0.0790 mg / ml and 0.1591 mg / ml.

The SX2, which was found to have the best IC ₅₀ value, was concentrated with a rotary vacuum evaporator (N-1, EYELA, Japan), and the step of detecting it by the second fractionation method described in Examples 1-4 was performed. , Angiotensin converting enzyme fractions with excellent inhibitory activity, specifically fractions having more than 50% activity was isolated. The separated fractions were named RP9 (9.5 min, 4-5% CH ₃ CN) and RP17 (15.7 min, 10-11% CH ₃ CN), respectively, with activity of 55.67% and 50.54%, respectively.

The RP9 and RP17, which were found to have excellent IC ₅₀ values, were concentrated with a rotary vacuum evaporator, and the third fractionation method described in Examples 1-4 was carried out. The RP9 was divided into eight fractions and the RP17 was five fractions. Aliquots were fractionated to determine the angiotensin converting enzyme inhibitory ability. Among them, those having excellent inhibition were named RP9CX1 (2.68 min, 10 mM NaCl), RP17CX1 (2.40 min, 8 mM NaCl), RP17CX3 (4.23 min, 26 mM NaCl), and RP17CX4 (5.09 min, 35 mM NaCl), respectively. , Activity was 54.58%, 76.11%, 72.68% and 70.20%, respectively. Each of the active fractions confirmed to have excellent inhibitory ability was removed by volatilizing the solvent with a vacuum freeze drying apparatus (FDU-540, EYELA, Japan).

The fractions from which the solvent was removed were subjected to the fourth fractionation method described in Examples 1-4, and in the case of RP9CX1, all fractions except the two fractions showed absorbances of 254 nm or 280 nm higher than those of 220 nm, and the two fractions. Did not show angiotensin converting enzyme inhibitory ability and was excluded from later experiments.

Meanwhile, RP17CX1 was aliquoted into 6 fractions, RP17CX3 was aliquoted into 8 fractions, and RP17CX1 was aliquoted into 4 fractions to measure angiotensin converting enzyme inhibition. Among them, it was confirmed that the excellent inhibitory activity, RP17CX1RP1 (43.22min, 11-12% CH ₃ CN), RP17CX1RP4 (46.72min, 12-13% CH ₃ CN) and RP17CX1RP5 (47.68min, 13% CH ₃ CN), respectively At 55.20%, 69.79%, and 76.02%, respectively, RP17CX3RP4 (44.13min, 12% CH ₃ CN), RP17CX3RP5 (45.02min, 12% CH ₃ CN) and RP17CX3RP6 (45.61min, 12% CH ₃ CN) ) Showed 52.57%, 54.99%, and 71.25% of inhibitory activity, respectively, and RP17CX4RP1 (43.26min, 12% CH ₃ CN) and RP17CX4RP2 (44.53min, 12% CH ₃ CN) inhibited 43.13% and 32.62%, respectively. Indicated.

The fifth fractionation method described in Examples 1-4 was performed on the fractions, and the results are as follows. Specifically, RP17CX1RP1 was aliquoted into three fractions, RP17CX1RP4 was aliquoted into six fractions, RP17CX1RP5 was aliquoted into seven fractions, RP17CX3RP5 was aliquoted into six fractions, and RP17CX3RP6 was divided into three fractions. In the case of RP17CX4RP1, a single peak was detected, and in the case of RP17CX4RP2, four fractions were collected to measure angiotensin converting enzyme inhibitory ability. In the case of RP17CX3RP4, both fractions were found to have no angiotensin converting enzyme inhibition.

Of these, when it was confirmed that the excellent inhibitory ability, RP17CX1RP1RP2 (8.80min) showed an inhibitory capacity of 58.65%, but it was confirmed that it is difficult to further distinguish the clear fraction.

In addition, RP17CX1RP4RP6 (11.10min) showed 73.62% inhibition, RP17CX1RP5RP5 (13.27min) showed 54.17% inhibition, RP17CX3RP5RP2 (9.60min) showed 64.11% inhibition, and RP17CX3RP5RP6 (12.03). 53.53% inhibition, RP17CX3RP6RP4 (12.12min) showed 58.97% inhibition, single peak of RP17CX1RP1 (12.80min) showed 53.46% inhibition, and RP17CX4RP2RP4 (11.80min) was fractionated from RP17CX4RP2 fraction Among them, 59.60% of the inhibitory effect was shown.

The fractionation was carried out for the sixth fractionation described in Examples 1-4 and the results are as follows. Specifically, in the case of RP17CX1RP4RP6 it was confirmed that there is no angiotensin converting enzyme inhibitory ability in all fractions. From the results, it was determined that the fraction was divided into several fractions having low activity. On the other hand, in the case of RP17CX1RP5RP5, it was divided into a total of three fractions, of which RP17CX1RP5RP5RP3 (15.60min) showed an inhibition of 15.60%. In addition, for RP17CX3RP5RP2 (11.17 min), RP17CX3RP5RP6 (14.24 min), RP17CX3RP6RP3 (12.60 min), RP17CX4RP1RP1 (12.57 min) and RP17CX4RP2RP4 (13.74 min), all separated into a single fraction.

Finally, the seventh fractionation method described in Examples 1-4 was performed on the fractions, and the results are shown in FIGS. 2 to 5 and Table 4 below.

[Table 4]

Fraction number % Inhibition (A) RP17CX1RP5RP5RP3SX1 66.11 RP17CX1RP5RP5RP3SX2 22.90 RP17CX1RP5RP5RP3SX3 0.37 (B) RP17CX3RP5RP2RP1SX1 93.67 (C) RP17CX3RP5RP6RP1SX1 57.66 (D) RP17CX4RP1RP1RP1SX1 92.76

As shown in Table 4, in the case of RP17CX1RP5RP5RP3, it was divided into a total of three fractions, of which 66.11% of angiotensin inhibition was confirmed in the RP17CX1RP5RP5RP3SX1 fraction. On the other hand, RP17CX3RP5RP2RP1, RP17CX3RP5RP6RP1 and RP17CX4RP1RP1RP1 were shown as single fractions, and in the case of (12.60 min), RP17CX4RP1RP1 (12.57 min) and RP17CX4RP2RP4 (13.74 min) were all separated into a single fraction.

Example 3: Amino Acid Sequencing

The amino acid sequence included in the fraction finally selected in Example 2 was analyzed. The amino acid sequence analysis was performed using the electrospray ion ring (ESI) method using a QTOF2 (Micormass, UK) mass spectrometer to obtain an amino acid sequence and accurate molecular weight results, Edmnan method was performed to confirm the sequence Amino acid sequence analysis was performed with peptides that matched the results. The amino acid sequences analyzed above are shown in FIG. 6 and Table 5 below.

TABLE 5

designation Amino acid sequence SEQ ID NO: Cpep1 Pro-Asn (P-N) One Cpep2 Arg-His-Asp (R-H-D) 2

As shown in FIG. 6 and Table 5, the amino acids Cpep1 and Cpep2 included in the fraction identified as the best in Example 2 were confirmed to have a mass of 215 Da and 397 Da, respectively, and two and three amino acid residues, respectively. It is a peptide consisting of, the sequence was confirmed as shown in Table 5.

In order to confirm the inhibitory ability of the angiotensin converting enzyme of the peptides described in Table 5, according to the angiotensin converting enzyme inhibitory measuring method, IC ₅₀ was measured at a concentration capable of inhibiting angiotensin converting enzyme by 50%, and the results are shown in the following table. 6 is shown.

TABLE 6

designation Amino acid sequence IC ₅₀ ([mu] M) Cpep1 Pro-Asn (P-N) 0.19 Cpep2 Arg-His-Asp (R-H-D) 2.96

As shown in Table 6, it was confirmed that the two peptides have an excellent inhibitory ability of angiotensin converting enzyme, in particular, Cpep1 (Pro-Asn) of SEQ ID NO: 1 inhibited 50% of angiotensin converting enzyme even at a content of 0.19 μM. Since it can be confirmed, it is expected that it can be used for various purposes, such as a therapeutic agent for cardiovascular diseases related to angiotensin converting enzyme.

1 is a diagram illustrating a step of separating and purifying an active ingredient by performing chromatography on an oyster protein enzyme treatment according to an embodiment of the present invention and the result thereof, wherein TGPN is a transglutaminase and a protein. Treated with hydrolase (Protamex and Neutrase) in order, SX is size exclusion chromatography, RP is reversed phase chromatography and CX is the result of performing cation-exchange chromatography, respectively.

Figure 2 shows the active fraction of the result of the final fractionation method according to an embodiment of the present invention, a graph showing the results of RP17CX1RP5RP5RP3SX1 to RP17CX1RP5RP5RP3SX3.

Figure 3 shows the active fraction of the result of the final fractionation method according to an embodiment of the present invention, a graph showing the results of RP17CX3RP5RP2RP1SX1.

Figure 4 shows the active fraction of the result of the final fractionation method according to an embodiment of the present invention, a graph showing the results of RP17CX3RP5RP6RP1SX1.

Figure 5 shows the active fraction of the result of the final fractionation method according to an embodiment of the present invention, a graph showing the results of RP17CX4RP1RP1RP1SX1.

Figure 6 is a graph showing the mass spectrum results of analyzing the molecular weight of the peptide amino acid extracted from the active fraction of the result of the final fractionation method according to an embodiment of the present invention by electrospray ionization method using a mass spectrometer.

<110> KONG, JAI-YUL <120> A ANTIHYPERTENSIVE PEPTIDE <130> KP200080200 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 2 <212> PRT <213> Cpep1 <400> 1 Pro asn   One <210> 2 <211> 3 <212> PRT <213> Cpep2 <400> 2 Arg His Asp   One  

Claims (8)

Peptides having the amino acid sequence of SEQ ID NO: 2 or acid addition salts thereof. The method of claim 1, The peptide is a peptide or acid addition salts thereof having an inhibitory ability of angiotensin converting enzyme. A nucleotide encoding a peptide having the amino acid sequence of SEQ ID NO: 2. A vector comprising the nucleotide of claim 3. A composition for treating or improving cardiovascular disease, comprising the peptide according to claim 1 or 2 as an active ingredient. The method of claim 5, wherein the cardiovascular disease is any one selected from the group consisting of hypertension, heart disease, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, atherosclerosis and arteriosclerosis. delete delete

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