KR102253996B1 - Anti-hypertensive composition comprising peptides derived from hydrolysates of Paralichthys olivaceus - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or food composition for treating or preventing hypertension, comprising a peptide isolated from a hydrolyzate of flounder (Paralichthys olivaceus) meat as an active ingredient.
Since the peptide isolated from the hydrolyzate of the flounder ( Paralichthys olivaceus ) meat has an angiotensin I converting enzyme (ACE) inhibitory activity, using peptides isolated from the hydrolyzate of the flounder (Paralichthys olivaceus) meat , It can be used in a pharmaceutical composition or food composition for the treatment or prevention of hypertension.

Description

Anti-hypertensive composition comprising peptides derived from hydrolysates of Paralichthys olivaceus}

The present invention relates to a pharmaceutical composition or a food composition for treating or preventing hypertension comprising a peptide isolated from a hydrolyzate of flounder (Paralichthys olivaceus) meat.

High blood pressure is one of the most frequent chronic circulatory diseases that 15-20% of adults in the world suffer from, and is relatively asymptomatic.However, due to high blood pressure, organs that have maintained normal function gradually lose their function. Since it can cause fatal complications such as stroke, heart failure, and coronary artery disease, it is a disease that requires more active and consistent patient management and treatment. The World Health Organization (WHO) defines hypertension when the highest blood pressure is 160 mmHg or higher and the lowest blood pressure is 95 mmHg or higher. As for types of hypertensive diseases, essential hypertension and secondary hypertension due to the causative disease are largely unclear. More than 90% of these are known to belong to essential hypertension, and more than 600 million people worldwide suffer from hypertension, and 3 million people die every year from this hypertension, which is emerging as a global task.

Inactivated angiotensin I present in the living body is converted to Angiotensin II, which has a vasoconstrictive action by the C-terminal His-Leu being separated by angiotensin I converting enzyme (ACE). It is converted to constrict blood vessels, and increases the amount of fluid by increasing the secretion of aldosterone from the kidneys, thereby increasing blood pressure. Therefore, by inhibiting the activity of ACE, blood pressure can be suppressed by lowering the production of angiotensin II, which causes an increase in blood pressure.

For the purpose of improving hypertension, ACE inhibitors such as enelapril or Ramipril have been developed and are commercially available. However, side effects from the use of these drugs include taste disorders, white blood cell reduction, angioedema, and liver dysfunction. Was found.

Therefore, the demand for research having antihypertensive effect from natural substances continued, and ACE inhibitors isolated from soy protein hydrolysates, chitosan, and milk casein were recognized as functional foods individually recognized by the Ministry of Food and Drug Safety.

Bioactive peptides can be made through various proteolytic enzymes, and natural peptides can function as potential physiological regulators in metabolic processes while food is digested in the intestine. In addition, depending on the structure, composition, and amino acid sequence, antioxidant, antibacterial, and antihypertensive effects can be expected.

As high blood pressure preparations manufactured through conventional chemical synthesis methods or the like have problems such as side effects, recent efforts to obtain substances having antihypertensive effects from natural substances have been continued in countries around the world. In addition, in each country, in order to search for new substances that can be used for various purposes such as food materials such as enzymes and health supplements, as well as medicines, studies on living organisms in their natural state, especially those that are easy to cultivate and reproduce in large quantities, are actively conducted. Is proceeding.

On the other hand, marine organisms have the ability to search for various new physiologically active substances due to their unique metabolic processes and unique environments that are not found in terrestrial organisms. In addition, many studies on terrestrial organisms have already been conducted, but marine organisms have not yet been sufficiently studied, so it is also a field with high expectations for the development of natural materials with new usefulness.

Domestic registered patent No. 10-1150425

An object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of hypertension, comprising as an active ingredient at least one peptide selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 8.

Another object of the present invention is to provide a food composition for improving or preventing hypertension, comprising as an active ingredient at least one peptide selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 8.

Another object of the present invention is a first hydrolyzate preparation step of performing the first hydrolysis by adding a hydrolytic enzyme to flounder; A second hydrolyzate preparation step of performing secondary hydrolysis by adding a hydrolytic enzyme to the prepared first hydrolyzate; And

Heating the secondary hydrolyzate to perform enzyme inactivation and filtering to prepare a hydrolyzate of flounder comprising one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 8 as an active ingredient. It is to provide a method for preparing a composition for treating, preventing or improving hypertension, including the step.

The present inventors prepared the first hydrolyzate of flounder (Paralichthys olivaceus ) meat using the hydrolytic enzyme Protamex, and then added pepsin to the first hydrolyzate to prepare a second hydrolyzate. And, it was confirmed that the peptide having a specific amino acid sequence isolated from the secondary hydrolyzate had an angiotensin I converting enzyme (ACE) inhibitory activity.

Accordingly, it was confirmed that the peptides isolated from the hydrolyzate of the flounder ( Paralichthys olivaceus ) meat can be used in a pharmaceutical composition or food composition for treating or preventing hypertension, and the present invention was completed.

As an aspect for achieving the above object, the present invention provides a pharmaceutical composition for treating or preventing hypertension, comprising as an active ingredient one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 8 to provide.

The peptide in the present invention may be isolated from the hydrolyzate of flounder (Paralichthys olivaceus), can be specifically isolated from the hydrolyzate of flounder (Paralichthys olivaceus) meat.

The term "flour meat" refers to a portion of the flounder after the head, skin, intestines, blood, and bones have been removed.

The hydrolyzate is pepsin, trysin, neutrase, protamex, alcalase, kojizyme, flavozyme, and combinations thereof. It may be a hydrolyzate of an enzyme selected from the group consisting of. The hydrolyzate may be a secondary hydrolyzate obtained by performing hydrolysis twice or more, more specifically, hydrolyzing twice.

In one embodiment of the present invention, after removing the head, skin, intestines, blood, and bones of flounder, the remaining flounder meat is ground and washed, and then the first hydrolyzate is performed by adding a hydrolytic enzyme to obtain a first hydrolyzate. Was prepared, and a second hydrolyzate was prepared by adding a hydrolyzate to the prepared first hydrolyzate, and the second hydrolyzate was heated to inactivate the enzyme, followed by filtration. A hydrolyzate of flounder was prepared comprising one or more peptides selected from the group consisting of amino acid sequences represented by 1 to 8 as an active ingredient. Specifically, for the production of the first hydrolyzate, protamex was used as the hydrolyzate, and pepsin was used for the production of the second hydrolyzate.

Eight kinds of peptides were isolated from the hydrolyzate of flounder obtained by performing the secondary hydrolysis, and the sequence and molecular weight of the peptide were analyzed using Q-TOF mass spectrometer (Micromass, Altrincham, UK) electrospray ionization (ESI). As a result, Leucine-Aspartic acid-Aspartic acid (Leu-Asp-Asp, LDD) (SEQ ID NO: 1), Aspartic acid-Leucine-Glutamine-Arginine (Asp-Leu-Gln-Arg, DLQR) (SEQ ID NO: 2), Leucine -Glutamic acid (Leu-Glu, LE) (SEQ ID NO: 3), Leucine-Valine-Histidine (Leu-Val-His, LVH) (SEQ ID NO: 4), Leucine-Glutamic acid-Arginine (Leu-Glu-Arg, LER ) (SEQ ID NO: 5), Isoleucine-Glutamic acid (Ile-Glu, IE) (SEQ ID NO: 6), Isoleucine-Glutamic acid-Arginine (Ile-Glu-Arg, IER) (SEQ ID NO: 7) or Isoleucine-Aspartic acid- It was confirmed that it has an amino acid sequence of aspartic acid (Ile-Asp-Asp, IDD) (SEQ ID NO: 8), and each molecular weight is 361.15Da, 531.28Da, 260.14Da, 367.23Da, 416.24Da, 260.14Da, 416.24Da, or It was confirmed to have 361.15 Da.

In addition, as a result of evaluating the inhibitory effect of ACE (Angiotensin I converting enzyme) using the peptides, Aspartic acid-Leucine-Glutamine-Arginine (Asp-Leu-Gln-Arg, DLQR) (SEQ ID NO: 2), Leucine-Valine -Histidine (Leu-Val-His, LVH) (SEQ ID NO: 4), Leucine-Glutamic acid-Arginine (Leu-Glu-Arg, LER) (SEQ ID NO: 5), Isoleucine-Glutamic acid-Arginine (Ile-Glu-Arg , IER) (SEQ ID NO: 7) was confirmed to have excellent antihypertensive activity due to high ACE inhibitory effect.

Therefore, the pharmaceutical composition for the treatment or prevention of hypertension is Aspartic acid-Leucine-Glutamine-Arginine (Asp-Leu-Gln-Arg, DLQR) (SEQ ID NO: 2), Leucine-Valine-Histidine (Leu-Val-His, LVH) (SEQ ID NO: 4), Leucine-Glutamic acid-Arginine (Leu-Glu-Arg, LER) (SEQ ID NO: 5), and Isoleucine-Glutamic acid-Arginine (Ile-Glu-Arg, IER) (SEQ ID NO: 7) Contains one or more peptides selected from the group as an active ingredient, or Leucine-Aspartic acid-Aspartic acid (Leu-Asp-Asp, LDD) (SEQ ID NO: 1), Aspartic acid-Leucine-Glutamine-Arginine (Asp-Leu-Gln -Arg, DLQR) (SEQ ID NO: 2), Leucine-Glutamic acid (Leu-Glu, LE) (SEQ ID NO: 3), Leucine-Valine-Histidine (Leu-Val-His, LVH) (SEQ ID NO: 4), Leucine- Glutamic acid-Arginine (Leu-Glu-Arg, LER) (SEQ ID NO: 5), Isoleucine-Glutamic acid (Ile-Glu, IE) (SEQ ID NO: 6), Isoleucine-Glutamic acid-Arginine (Ile-Glu-Arg, IER ) (SEQ ID NO: 7) and Isoleucine-Aspartic acid-Aspartic acid (Ile-Asp-Asp, IDD) (SEQ ID NO: 8).

The ACE (Angiotensin I converting enzyme) is an enzyme that serves to convert a dipeptide (His-Leu) from angiotensin I, which is a decapeptide, to angiotensin II having vasoconstriction. The increase of angiotensin Ⅱ increases blood pressure by increasing circulating blood volume by promoting the secretion of aldosterone, an anti-diuretic hormone, and suppressing the excretion of water and sodium. In addition, ACE plays a role of inducing blood pressure increase by decomposing and inactivating bradykinin, which acts to relax blood vessels.

In the present invention, the scientific name of the "flour" is Paralichthys olivaceus , is called a flounder, its body length is about 60cm, and its shape is a long oval that is wide up and down. The mouth is large and the teeth are well developed, and the eyes are on the left side of the body. The side with eyes has black and white spots scattered on a dark yellowish brown background, but the side without eyes is white. It lives in the sand or pearl area of the coast of 10-200m water depth, and spawns in February-June. It appears in all coasts of Korea, and is distributed in the Kuril Islands, Japan, and the South China Sea. It is often called'flour' because of its flat body shape, and because the eye is on the left, it is generally easily distinguished from the flatfish with the eye on the right.

Regarding the term "hypertension" of the present invention, the World Health Organization (WHO) defines a case where the maximum blood pressure is 160 mmHg or more and the lowest blood pressure is 95 mmHg or more as hypertension, and the type of hypertensive disease is largely It is divided into essential hypertension of which the cause is unknown and secondary hypertension due to the causative disease, of which more than 90% are known to belong to essential hypertension.

The term "prevention" as used in the present invention may mean any action of inhibiting or delaying the onset of hypertension by administering the composition according to the present invention to an individual. The term "treatment" used in the present invention may refer to any action in which the composition of the present invention is administered to an individual suspected of developing hypertension to improve or benefit from symptoms caused by hypertension.

In an embodiment of the present invention, in the group consisting of the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 8 isolated from the hydrolyzate of flounder (Paralichthys olivaceus ) meat remaining after removing the head, skin, intestines, blood, and bone It was confirmed that one or more of the selected peptides have an angiotensin I converting enzyme (ACE) inhibitory activity, and that the peptides can be used in a pharmaceutical composition for treating or preventing hypertension.

The pharmaceutical composition comprising the peptides of the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions. At this time, the peptide included in the composition is not particularly limited thereto, but may include 0.001% by weight to 99% by weight, preferably 0.01% by weight to 50% by weight, based on the total weight of the composition.

The pharmaceutical composition may have any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, freeze-dried agents, and suppositories. , May be oral or parenteral in various dosage forms. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin. It can be prepared by mixing and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the individual, age, sex, and disease. It can be determined according to the type, activity of the drug, sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors, and can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention is not particularly limited as long as it is an individual for the purpose of treating hypertension, and any pharmaceutical composition can be applied. For example, non-human animals such as monkeys, dogs, cats, rabbits, mormons, rats, mice, cows, sheep, pigs, goats, etc., humans, birds and fish, etc. can be used, and the pharmaceutical composition may be parenterally or subcutaneously , Intraperitoneal, intrapulmonary and intranasal administration, and for local treatment, if necessary, may be administered by a suitable method including intralesional administration. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the individual, the severity of the disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or cerebrovascular injection, but is not limited thereto.

A suitable total daily use amount can be determined by the physician within the range of correct medical judgment, and is generally in an amount of 0.001 to 1000 mg/kg, preferably 0.05 to 200 mg/kg, more preferably 0.1 to 100 mg/ The amount of kg can be divided and administered once to several times a day.

As another aspect, the present invention provides a food composition for improving or preventing hypertension, comprising as an active ingredient at least one peptide selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 8.

The food composition for improving or preventing hypertension of the present invention is Aspartic acid-Leucine-Glutamine-Arginine (Asp-Leu-Gln-Arg, DLQR) (SEQ ID NO: 2), Leucine-Valine-Histidine (Leu-Val-His, LVH) (SEQ ID NO: 4), Leucine-Glutamic acid-Arginine (Leu-Glu-Arg, LER) (SEQ ID NO: 5), and Isoleucine-Glutamic acid-Arginine (Ile-Glu-Arg, IER) (SEQ ID NO: 7) Contains one or more peptides selected from the group as an active ingredient, or Leucine-Aspartic acid-Aspartic acid (Leu-Asp-Asp, LDD) (SEQ ID NO: 1), Aspartic acid-Leucine-Glutamine-Arginine (Asp-Leu-Gln -Arg, DLQR) (SEQ ID NO: 2), Leucine-Glutamic acid (Leu-Glu, LE) (SEQ ID NO: 3), Leucine-Valine-Histidine (Leu-Val-His, LVH) (SEQ ID NO: 4), Leucine- Glutamic acid-Arginine (Leu-Glu-Arg, LER) (SEQ ID NO: 5), Isoleucine-Glutamic acid (Ile-Glu, IE) (SEQ ID NO: 6), Isoleucine-Glutamic acid-Arginine (Ile-Glu-Arg, IER ) (SEQ ID NO: 7) and Isoleucine-Aspartic acid-Aspartic acid (Ile-Asp-Asp, IDD) (SEQ ID NO: 8).

In the present invention, the peptide, hypertension, flatfish, and prevention are as described above.

In the present invention, the term "improvement" refers to hypertension that is prevented or treated using one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 7. It refers to any action that improves or benefits the symptoms of a suspected or diseased individual.

Specifically, one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 8 of the present invention may be added to a food composition for the purpose of preventing or improving hypertension.

The food composition of the present invention may include a form such as a pill, powder, granule, needle, tablet, capsule or liquid, and there is no particular limitation on the type of food to which the peptides of the present invention can be added, for example There are various beverages, gums, teas, vitamin complexes, and health supplements.

In addition to the one or more peptides selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 8, other ingredients may be added to the food composition, and the kind is not particularly limited. For example, it may contain as an additional component, such as various herbal extracts, food additives or natural carbohydrates that are acceptable in food, as in ordinary foods, but is not limited thereto.

In the present invention, the term "food supplementary additive" refers to a component that can be added auxiliary to food, and is added to the manufacture of health functional foods of each formulation, and can be appropriately selected and used by those skilled in the art. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. are included, but the types of the food additives of the present invention are not limited by the above examples.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; And polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, etc.), stevia extract (rebaudioside A, glysir) Hibiscus) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used advantageously.

The food composition of the present invention may contain a health functional food, and in the present invention, the food composition may be a health functional food. The term "health functional food" as used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body. Here, the term "functionality" refers to obtaining useful effects for health purposes such as controlling nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material, and it may be excellent in portability.

The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food, the peptide of the present invention may be added in an amount of 1 to 50% by weight, preferably 5 to 10% by weight of the raw material composition, but is not limited thereto. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be used even below the above range.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include soft meat, soft products, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages. , Tea, drinks, alcoholic beverages and vitamin complexes, and includes all health functional foods in the usual sense.

In another aspect, the present invention provides a step of preparing a first hydrolyzate for performing first hydrolysis by adding a hydrolytic enzyme to flounder; A second hydrolyzate preparation step of performing secondary hydrolysis by adding a hydrolytic enzyme to the prepared first hydrolyzate; And

Heating the secondary hydrolyzate to perform enzyme inactivation and filtering to prepare a hydrolyzate of flounder comprising one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 8 as an active ingredient. It provides a method for preparing a composition for treating, preventing or improving hypertension, comprising the steps of.

The manufacturing method of the present invention may further include grinding and washing the flatfish before adding the hydrolytic enzyme. Specifically, the head, shell, intestines, blood, and bones are removed, and the remaining flatfish meat is ground and washed with water.

In one embodiment of the present invention, after removing the head, skin, intestines, blood, and bones of flounder, the remaining flounder meat is ground and washed, and then the first hydrolyzate is performed by adding a hydrolytic enzyme to obtain a first hydrolyzate. Was prepared, and a second hydrolyzate was prepared by adding a hydrolyzate to the prepared first hydrolyzate, and the second hydrolyzate was heated to inactivate the enzyme, followed by filtration. A hydrolyzate of flatfish containing a peptide consisting of an amino acid sequence represented by 1 to SEQ ID NO: 8 was prepared. Specifically, for the production of the first hydrolyzate, protamex was used as the hydrolyzate, and pepsin was used for the production of the second hydrolyzate.

It was confirmed that the hydrolyzate of flounder prepared above contained a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 8, and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 8 was ACE As it was confirmed that it has inhibitory activity, using a hydrolyzate of flounder containing one or more peptides selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 8, a composition for treating, preventing or improving hypertension Manufacturing is possible.

In the present invention, the hydrolyzate, hydrolytic enzyme, and hypertension of the flounder are as described above, and the composition includes the above-described pharmaceutical composition and food composition.

Since the peptide isolated from the hydrolyzate of the flounder ( Paralichthys olivaceus ) meat of the present invention has an angiotensin I converting enzyme (ACE) inhibitory activity, the peptides isolated from the hydrolyzate of the flounder (Paralichthys olivaceus) meat By using, it can be used in a pharmaceutical composition or food composition for the treatment or prevention of hypertension.

1 shows the hydrolysis process of flatfish meat of the present invention.
Figure 2 shows the measurement results of the Hydrogen peroxide scavenging ability of the hydrolyzate of flounder.
3 shows the results of measuring the ACE inhibitory activity of hydrolyzed flounder.
4 is an LC/MS analysis result of the first hydrolyzate.
5 is an LC/MS analysis result of a secondary hydrolyzate.
6 shows the molecular weight distribution diagram of the hydrolyzate of flatfish.
7 shows the MS chromatogram of the LDD peptide.
8 shows the MS chromatogram of the DLQR peptide.
9 shows the MS chromatogram of the LE peptide.
10 shows the MS chromatogram of the LVH peptide.
11 shows the MS chromatogram of the LER peptide.
12 shows the MS chromatogram of the IE peptide.
13 shows the MS chromatogram of the IER peptide.
14 shows the MS chromatogram of the IDD peptide.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art may easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

<A. Experiment method>

1. Preparation of hydrolyzate of flounder meat

1-1. Preparation of primary hydrolyzate of flatfish meat

The material used in the present invention was flounder ( Paralichthys olivaceus ) purchased from the fish market in Jeju Island, and the head, skin, intestines, blood, and bones of the flounder were removed, ground using a mincer, and then added to purified water. Washing with water was repeated twice to remove contaminants such as blood. After that, it was stored in a -50℃ freezer and used for the experiment.

In order to prepare the primary hydrolyzate of flatfish, the frozen flatfish meat from which the head, skin, intestines, blood, and bones were removed was thawed at low temperature (5°C) for 24 hours, and 10 times of distilled water was added. After that, Protamex enzyme was added 1% compared to flatfish meat, the pH was adjusted to 6.0, and the mixture was stirred at 40°C for 24 hours. After completion of the hydrolysis, the enzyme was inactivated by heating at 95° C. for 10 minutes, and the first hydrolyzate was obtained through centrifugation (4000 rpm, 20 minutes) and filtration. The primary hydrolyzate was concentrated, lyophilized, and stored frozen, and used in the experiment.

1-2. Preparation of secondary hydrolyzate of flatfish meat

In order to prepare a secondary hydrolyzate of flatfish, frozen flatfish meat from which the head, skin, intestines, blood, and bones were removed was thawed at low temperature (5°C) for 24 hours, and 10 times of distilled water was added. After that, Protamex enzyme was added 1% compared to flatfish meat, the pH was adjusted to 6.0, and the mixture was stirred at 40°C for 24 hours. Thereafter, the pH was adjusted to about 2.5, and 1% of pepsin enzyme was added compared to the flatfish meat, followed by stirring at 36° C. for 24 hours. After completion of the hydrolysis, the enzyme was inactivated by heating at 95° C. for 10 minutes, and a secondary hydrolyzate was obtained through centrifugation (4000 rpm, 20 minutes) and filtration. The secondary hydrolyzate was concentrated, lyophilized, and stored frozen, and used in the experiment. The hydrolysis process according to the present invention is summarized in FIG. 1.

2. Measurement of antioxidant activity

To measure the hydrogen peroxide radical scavenging activity, 100 μL of 0.1M phosphate buffer (pH 5.0) and a sample were mixed in a 96 well plate, and then 20 μL of hydrogen peroxide (100 mM) was added and reacted at 37° C. for 5 minutes. After the reaction was completed, 30 μL of 1.25 mM ABTS and peroxidase (1 unit/mL) were added respectively, and finally reacted at 37° C. for 10 minutes, and the absorbance value was measured at 405 nm using an ELISA reader.

3. Antihypertensive activity measurement (ACE inhibitory ability measurement)

In order to measure antihypertensive activity, ACE inhibitory activity was measured. ACE kit-WST (Dojindo inc., Japan) was used, and it was confirmed through a colorimetric method that measures 3-hyroxybutylic acid generated by the action of ACE on 3-hydroxybutyryl-Gly-Gly-Gly, a substrate. After each of the substrate buffer and the sample were dispensed into a 96 well plate, an enzyme solution was added and reacted. At this time, a solvent was added to Blank1 and Blank2 instead of the sample, and after reacting at 37°C for 1 hour, an indicator solution was added to each well. The absorbance was measured at 450 nm for the amount of 3-hyroxybutylic acid generated after the reaction to calculate the ACE-inhibition rate of the sample.

4. Measurement of molecular weight distribution

After dissolving 20 mg of a sample in 1 mL of DW, 100 µl was taken, and 500 µl of methanol was added and stirred. The stirred sample was reacted on ice for 5 minutes, centrifuged at 13,000 rpm for 10 minutes, and the supernatant was transferred to a new microtube, followed by vacuum drying. The dried sample was dissolved in 100 µl of DW, filtered through a 0.2 µm syringe filter, and used as a sample.

The analytical LC used for the analysis was an UltiMate 3000 system (Dionex, US), and a ZORBAX 300SB-C18 (1.0 X 150mm, 3.5 μm, Agilent) column was used. For the mobile phase, distilled water with 0.2% formic acid and acetonitrile with 0.2% formic acid were used. The mobile phase was flowed at a rate of 100 μl/min. For MS, a MicroQ-TOF III mass spectrometer (Bruker Daltonics, 255748 Germany) was used, and the ESI+ mode was used. The scan range was set to m/z 200-2000.

5. Separation and purification of functional peptides derived from flounder hydrolysates

To separate and purify the functional peptide derived from the secondary hydrolyzate of flatfish meat, the hydrolyzate is dissolved in distilled water to a concentration of 200 mg/mL to prepare a high-concentration sample, and then 3 times the amount of ethanol is added to the sample and refrigerated at 5°C. It was stored and precipitated. Thereafter, the precipitate was collected by centrifugation at 10000 rpm for 20 minutes, dried and used as an analysis sample. After that, the analysis conditions were the same as the molecular weight distribution measurement method.

<B. Experiment result>

Experimental Example 1: Antioxidant Activity of Hydrolyzate of Flounder Meat

The antioxidant effect of the hydrolyzate was compared by measuring the scavenging ability for hydrogen peroxide (H ₂ O _{2 ).} Samples at a concentration of 0.5 to 4 mg/mL were measured, and the secondary hydrolyzate showed slightly excellent antioxidant effect up to 0.5 to 1 mg. When compared to the IC ₅₀ value, the secondary hydrolyzate removed 50% of hydrogen peroxide at a lower concentration of 1.34 mg/mL, and the antioxidant effect of the secondary hydrolyzate was superior to that of the primary hydrolyzate.

Experimental Example 2: Measurement of antihypertensive activity of hydrolyzate of flounder meat

In order to confirm the antihypertensive activity of the hydrolyzate of flounder meat, the ACE inhibitory activity was measured, and it was measured at a concentration of 0.33 to 1.33 μg/ml. At all concentrations, the secondary hydrolyzate showed superior ACE inhibitory activity than the primary hydrolyzate, and _{when converted to IC 50} values, the first hydrolyzate was 559.96 μg/ml and the second hydrolyzate was 432.41 μg/ml. It was confirmed that the secondary hydrolyzate had a better ACE inhibitory effect.

Experimental Example 3: Analysis of molecular weight distribution of hydrolyzed flounder meat

The first hydrolyzate and the second hydrolyzate were analyzed by LC/MS analysis, and the first hydrolyzate was analyzed by dividing into a total of 7 parts according to the peak of the analysis sample (Fig. 4), and a total of 10 secondary hydrolysates. It was analyzed in parts (Fig. 5). The molecular weight and information of the peptides detected as a result of the analysis are shown in Tables 1 and 2.

The two hydrolysates were shown in FIG. 6 according to the molecular weight of the peptide, and at a molecular weight of 300 to 599 Da, about 27% was distributed in the first hydrolyzate, and about 47% was distributed in the second hydrolyzate. In the range of 600 to 899 Da, the first hydrolyzate was about 37%, which was higher than that of the second hydrolyzate about 33%. In the range of 900~1199 Da, the primary hydrolyzate was about 22%, which was higher than that of the secondary hydrolyzate about 18%. Peptides were detected at a molecular weight of 1200 Da or higher in the primary hydrolyzate, but no peptide was detected in the secondary hydrolyzate. In addition, peptides were detected only in the secondary hydrolyzate at a molecular weight of less than 300 Da.

Therefore, in the case of the secondary hydrolyzate, it was confirmed that many peptides having a molecular weight of 599 Da or less were distributed, and the peptide was not detected at a molecular weight of 1200 Da or higher, so that the hydrolyzate obtained by the secondary hydrolysis was It was found that the molecular weight was lower than that of the first hydrolyzate obtained by the first hydrolysis.

List of molecular weights of peptides in the primary hydrolyzate of flounder No. m/z Charge Intensity Calc. mass(M+H) No. m/z Charge Intensity Calc. mass(M+H) One 316.21 1+ 63419 316.21 33 412.17 2+ 10870 823.34 2 323.15 1+ 11684 323.15 34 415.23 2+ 10019 829.46 3 342.17 1+ 12312 342.17 35 423.73 2+ 44783 846.46 4 390.18 1+ 23080 390.18 36 848.34 1+ 36711 848.34 5 430.24 1+ 32712 430.24 37 424.70 2+ 41338 848.40 6 461.22 1+ 10522 461.22 38 440.21 2+ 19910 879.42 7 461.22 1+ 15550 461.22 39 441.20 2+ 20804 881.40 8 475.24 1+ 29304 475.24 40 442.24 2+ 15909 883.48 9 475.24 1+ 38331 475.24 41 455.71 2+ 12252 910.42 10 504.22 1+ 11556 504.22 42 460.22 2+ 23523 919.40 11 505.21 1+ 14487 505.21 43 465.73 2+ 10219 930.46 12 507.22 1+ 10634 507.22 44 469.19 2+ 62636 937.38 13 519.22 1+ 23764 519.22 45 971.49 1+ 11895 971.49 14 526.28 1+ 30209 526.28 46 488.25 2+ 45624 975.50 15 555.19 1+ 13430 555.19 47 501.75 2+ 14799 1002.50 16 585.32 1+ 12003 585.32 48 518.75 2+ 15900 1036.50 17 590.26 1+ 16520 590.26 49 521.27 2+ 12720 1041.54 18 600.32 1+ 29673 600.32 50 535.24 2+ 10607 1069.48 19 604.28 1+ 27942 604.28 51 554.72 2+ 17477 1108.44 20 637.30 1+ 15297 637.30 52 575.25 2+ 26144 1149.50 21 639.36 1+ 15824 639.36 53 587.78 2+ 11916 1174.56 22 648.27 1+ 16412 648.27 54 590.75 2+ 59373 1180.50 23 653.24 1+ 44938 653.24 55 611.27 2+ 19512 1221.54 24 655.23 1+ 15410 655.23 56 642.78 2+ 30577 1284.56 25 328.12 2+ 16556 655.24 57 428.86 3+ 14609 1284.58 26 658.30 1+ 20992 658.30 58 670.80 2+ 10639 1340.60 27 355.16 2+ 21981 709.32 59 690.30 2+ 10200 1379.60 28 357.18 2+ 17663 713.36 60 583.63 3+ 10211 1748.89 29 763.29 1+ 19516 763.29 61 437.98 4+ 11618 1748.92 30 766.32 1+ 18983 766.32 62 686.31 3+ 15024 2056.93 31 806.33 1+ 13443 806.33 63 756.02 3+ 12227 2266.06 32 810.31 1+ 16117 810.31

List of molecular weights of peptides in secondary hydrolysates of flatfish No. m/z Charge Intensity Calc. mass(M+H) No. m/z Charge Intensity Calc. mass(M+H) One 261.14 One 149608 261.14 34 637.31 One 29062 637.31 2 261.15 One 16052 261.15 35 328.13 2 12916 655.26 3 316.21 One 61129 316.21 36 658.3 One 34551 658.3 4 323.15 One 11048 323.15 37 680.23 One 12078 680.23 5 362.15 One 56931 362.15 38 697.34 One 12494 697.34 6 362.16 One 91531 362.16 39 357.18 2 20597 713.36 7 368.23 One 256056 368.23 40 719.31 One 11772 719.31 8 374.23 One 10820 374.23 41 730.41 One 15659 730.41 9 390.19 One 16872 390.19 42 368.22 2 10842 735.44 10 417.24 One 113532 417.24 43 763.3 One 11202 763.3 11 417.25 One 125180 417.25 44 415.24 2 15430 829.48 12 430.24 One 51567 430.24 45 423.73 2 25625 846.46 13 461.23 One 18228 461.23 46 424.7 2 25006 848.4 14 461.23 One 32013 461.23 47 429.25 2 13808 857.5 15 475.24 One 53844 475.24 48 871.44 One 15621 871.44 16 475.24 One 82414 475.24 49 442.24 2 11727 883.48 17 489.25 One 19308 489.25 50 451.76 2 19078 902.52 18 490.28 One 19178 490.28 51 463.74 2 20782 926.48 19 505.22 One 10149 505.22 52 468.74 2 20284 936.48 20 507.22 One 10401 507.22 53 468.75 2 33532 936.5 21 519.23 One 17949 519.23 54 940.57 One 27760 940.57 22 523.24 One 11036 523.24 55 489.77 2 12232 978.54 23 523.24 One 10651 523.24 56 521.28 2 12758 1041.56 24 526.28 One 21503 526.28 57 528.31 2 65767 1055.62 25 527.29 One 10901 527.29 58 553.8 2 17420 1106.6 26 529.26 One 10405 529.26 59 586.81 2 38517 1172.62 27 531.28 One 188,181 531.28 60 590.76 2 31422 1180.52 28 550.26 One 11474 550.26 29 574.31 One 14550 574.31 30 577.26 One 15902 577.26 31 600.32 One 17871 600.32 32 604.28 One 38180 604.28 33 622.34 One 13925 622.34

Experimental Example 4: Identification of functional peptide derived from secondary hydrolyzate of flounder meat

As a result of analyzing the peptides in the secondary hydrolyzate of flatfish meat, a total of eight peptides were identified, and the peptides are shown in Table 3.

Sequence number name order Calc. mass (M+H) Molecular weight (Da) One LDD Leucine-Aspartic acid-Aspartic acid 362.15 361.37 2 DLQR Aspartic acid-Leucine-Glutamine-Arginine 531.28 530.89 3 LE Leucine-Glutamic acid 261.14 260.31 4 LVH Leucine-Valine-Histidine 368.23 367.48 5 LER Leucine-Glutamic acid-Arginine 417.24 416.51 6 IE Isoleucine-Glutamic acid 261.15 260.31 7 IER Isoleucine-Glutamic acid-Arginine 417.25 416.51 8 IDD Isoleucine-Aspartic acid-Aspartic acid 362.16 361.37

Further, the molecular weight and amino acid sequence of the peptides were analyzed using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) electrospray ionization (ESI), and the results are shown in FIGS. 7 shows the MS chromatogram of the LDD peptide. 8 shows the MS chromatogram of the DLQR peptide. 9 shows the MS chromatogram of the LE peptide. 10 shows the MS chromatogram of the LVH peptide. 11 shows the MS chromatogram of the LER peptide. 12 shows the MS chromatogram of the IE peptide. 13 shows the MS chromatogram of the IER peptide. 14 shows the MS chromatogram of the IDD peptide.

Experimental Example 5: Measurement of ACE inhibitory activity of functional peptide derived from secondary hydrolyzate of flatfish

Table 4 shows the results of measuring the ACE inhibitory activity of each peptide isolated from the secondary hydrolyzate. As a result, LDD, LE, and IE had IC ₅₀ values higher than 66.67, so they did not show significant ACE inhibitory activity. However, DLQR, LVH, LER and IER peptides _{showed ACE inhibitory IC 50} values of 22.64, 39.78, 63.60 and 20.13, respectively. Therefore, it can be used as an active ingredient in a composition for treating or preventing hypertension by using the DLQR, LVH, LER and IER.

LDD DLQR LE LVH LER IE IER IDD IC ₅₀ value
(μg/mL) > 66.67 22.64
± 0.62 > 66.67 39.78
± 0.16 63.60
± 4.56 > 66.67 20.13
± 1.61 > 66.67

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention defined in the following claims are also present. It belongs to the scope of rights of

<110> Jeju National University Industry-Academic Cooperation Foundation <120> Anti-hypertensive composition comprising peptides derived from hydrolysates of Paralichthys olivaceus <130> DP20190076 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 3 <212> PRT <213> Paralichthys olivaceus <400> 1 Leu Asp Asp One <210> 2 <211> 4 <212> PRT <213> Paralichthys olivaceus <400> 2 Asp Leu Gln Arg One <210> 3 <211> 2 <212> PRT <213> Paralichthys olivaceus <400> 3 Leu Glu One <210> 4 <211> 3 <212> PRT <213> Paralichthys olivaceus <400> 4 Leu Val His One <210> 5 <211> 3 <212> PRT <213> Paralichthys olivaceus <400> 5 Leu Glu Arg One <210> 6 <211> 2 <212> PRT <213> Paralichthys olivaceus <400> 6 Ile Glu One <210> 7 <211> 3 <212> PRT <213> Paralichthys olivaceus <400> 7 Ile Glu Arg One <210> 8 <211> 3 <212> PRT <213> Paralichthys olivaceus <400> 8 Ile Asp Asp One

Claims (10)

A pharmaceutical composition for treating or preventing hypertension, comprising as an active ingredient at least one peptide selected from the group consisting of amino acid sequences represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 7. The pharmaceutical composition of claim 1, wherein the peptide is isolated from the hydrolyzate of flatfish. The method of claim 2, wherein the hydrolyzate is pepsin, trysin, neutrase, protamex, alcalase, kojizyme, flabozyme ( flavourzyme) and a hydrolyzate of an enzyme selected from the group consisting of a combination thereof. The pharmaceutical composition according to claim 2, wherein the hydrolyzate is obtained by performing hydrolysis twice. A food composition for improving or preventing hypertension, comprising as an active ingredient at least one peptide selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 7. The food composition of claim 5, wherein the peptide is isolated from the hydrolyzate of flatfish. The food composition according to claim 6, wherein the hydrolyzate exhibits antioxidant activity. The method of claim 6, wherein the hydrolyzate is pepsin, trysin, neutrase, protamex, alcalase, kojizyme, flabozyme ( flavourzyme) and a hydrolyzate of an enzyme selected from the group consisting of a combination thereof. The food composition according to claim 6, wherein the hydrolyzate is obtained by performing hydrolysis twice. A step of preparing a first hydrolyzate for performing a first hydrolysis by adding a hydrolytic enzyme to the flatfish;
A second hydrolyzate preparation step of performing secondary hydrolysis by adding a hydrolytic enzyme to the prepared first hydrolyzate; And
The secondary hydrolyzate is heated to perform enzyme inactivation and filtered to contain one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 7 as an active ingredient. A method for preparing a composition for treating, preventing or improving hypertension, comprising the step of preparing a hydrolyzate of flounder.

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