KR102008620B1 - Composition for Anti-hypertension or Anti-oxidation Using Alcalase Hydrolysates of Hippocampus abdominalis, Fractions Thereof, or Effective Peptides Thereof - Google Patents

Abstract

The present invention discloses a composition for antihypertension or antioxidant using alcalase hydrolysate of Hippocampus abdominalis, fractions thereof or active peptides thereof. According to the present invention, it is possible to provide the composition for antihypertension or antioxidant using alcalase hydrolysate of Hippocampus abdominalis, fractions thereof or peptides isolated therefrom. The composition of the present invention can be commercialized into food, drugs and the like.

Description

Composition for Anti-hypertension or Anti-oxidation Using Alcalase Hydrolysates of Hippocampus abdominalis, Fractions Thereof, or Effective Peptides Thereof}

The present invention relates to a composition for antihypertensive blood pressure or antioxidant using the Big Valley hippocampal alkalase hydrolyzate, fractions thereof or an effective peptide thereof.

Recently, as interest in aging and adult diseases increases, studies are being actively conducted to prevent disease by eliminating excessively generated reactive oxygen in vivo. Free radicals occur in aerobic organisms where oxygen, which is absolutely necessary to maintain life, is an electron acceptor and a continuous biochemical reaction is carried out to supply energy. 2-3% of the oxygen introduced into the body is is converted into active oxygen (Reactive Oxigen Species) by the electron transport processes and energy metabolism, and a variety of external stimuli in ^{_{cells, superoxide radical (O 2 -)}} , hydroxyl radical ( and OH) and the non-free species hydrogen peroxide (H ₂ O ₂ ).

These ROS not only oxidize the lipids that make up the cell membrane, but also damage DNA and affect the production of mutations resulting in chronic degenerative diseases such as atherosclerosis, cancer, hypertension and diabetes (IJBCB. 2007, 39 (1): 44 ~). 84; J. Hepatology. 2007, 46 (2): 193-197). Reports that ROS is an important factor involved in cardiovascular disease, including hypertension, show a close association between free radical oxidation and hypertension (American J. of hypertension. 2004, 17 (9): 809-816; Free radical biology & medicine 2006, 40: 2028-2039).

Hypertension (hypertension) refers to a case where the average blood pressure measured several times is more than 140mmHg systolic blood pressure, more than 90mmHg diastolic blood pressure, and artificially elevated blood pressure during human physiological activity has emerged as an important problem for the health of modern people. Abnormally elevated blood pressure due to various causes rather than caused by one reason is called primary hypertension or essential hypertension. In addition, hypertension may be caused by the environment, but blood pressure may increase due to genetic causes, which is called secondary hypertension.

Meanwhile, angiotensin converting enzyme (ACE), an important enzyme in the renin-angiotensin system (RAS), an important mechanism of blood pressure elevation, is an enzyme that converts angiotensin I (AngI) to angiotension II (AngII), AngII type 1 It is known to increase blood pressure by acting on (AT1) receptors to constrict blood vessels and increase the release of aldosterone. In addition, AngII increases the production of reactive oxygen species and nitrogen species by activating the NADH / NADPH oxidase system to induce oxidative stress of endothelial cells and induce inflammatory reactions of blood vessels through oxidation of low density lipoproteins (Am J.Cardiol 2003, 91 (12A).

Angiotensin converting enzyme inhibitors currently in use are synthetic chemicals or synthetic peptides. Although synthetic chemicals and synthetic peptides can be produced in large quantities, it is useful, but itching, headache, and tachycardia can be achieved. Side effects such as chest pain, chest pain and weakness have been reported. There is an urgent need for new angiotensin converting enzyme inhibitors without side effects.

The hippocampus is used worldwide for the treatment of asthma, heart disease, and fractures. Especially, the Hippocampus abdominalis is the largest and most beautiful body and body in the hippocampus. There are many. The head is at right angles to the body, the tail can be attached to the substrate, and the males tend to protect their eggs. It is used as a Chinese herbal medicine in Asia and is popular because of its unique shape. It is a fish species with high industrial value (Technical report series, 2003, 4:15).

However, because of its medicinal value, it is overfished in some countries, and the number of natural objects is gradually decreasing as the environment that can be inhabited by global warming and coastal pollution is reduced. Australia and its neighboring countries, China and Korea, are becoming aquaculture, but their numbers are significantly short of demand (Aquaculture. 2000, 190: 377-388). Although immune responses and physiological studies are being conducted in the hippocampus kuda Bleeker, the hippocampus guttu-latus , and the short-snouted seahorse ( Hippocampus hippocampus ), the immunophysiological studies of the Big Valley hippocampus are still insufficient.

Accordingly, the present inventors, by confirming that the Hippocampus abdominalis alkalase hydrolyzate, its fractions, and the alkyl radical agonizing activity of the new peptide isolated therefrom and the inhibitory activity of angiotensin converting enzyme (ACE), It will be used as a new resource with antihypertensive and antioxidant activity.

An object of the present invention is to provide an antihypertensive or antioxidant composition using a Big Belly Hippocampus alcalase hydrolyzate, a fraction thereof or an effective peptide thereof.

Other and specific objects of the present invention will be presented below.

The inventors have prepared three fractions according to the molecular weight of the Big Valley hippocampus alcalase and the hydrolyzate and four gel filtration fractions of this fraction, as confirmed in the following examples and experimental examples, and further gel filtration. Nine single peptides were isolated from the fractions, and ACE (aniotensin I converting enzyme) inhibitory activity and alkyl radical scavenging activity were evaluated for these fractions and peptides. It was confirmed to have activity.

The present invention is presented based on the results of these experiments, and in one aspect, the present invention provides a Big Valley hippocampal alkalase hydrolyzate, a fraction of the hydrolyzate, any one of the nine peptides of Table 1 below, or a peptide thereof. The present invention relates to a composition for improving hypertension, including a pharmaceutically acceptable salt as an active ingredient, and in another aspect, any one of the nine peptides of Table 1 below is effective. It relates to an antioxidant composition comprising as a component.

In the present specification, "Big Belly Hippocampus Alkalase Hydrolyzate" refers to a group of substances including amino acids and / or peptides as an enzyme treatment obtained by hydrolyzing Big Belly Hippocampus with alkalase, a kind of protease. Such enzyme treatment may be used as it is or by taking the supernatant after centrifugation, or the enzyme treatment as it is or under reduced pressure and / or lyophilized to be used in liquid or powder form, or the enzyme treatment as it is or as a supernatant of water or ethanol. It can be extracted and used with an organic solvent.

In the present specification, the "fractions of the Big Belly Hippocampus alkalase hydrolyzate" is a fraction obtained by using an organic solvent having different polarities from the Big Belly Hippocampus alkalase hydrolyzate or an extract of water or an organic solvent of this hydrolyzate. , Fractions obtained by fractionation according to molecular weight or gel filtration fractions of these fractions. The organic solvent is a lower alcohol having 1 to 4 carbon atoms including ethanol, methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N, N- dimethylformamide (DMF), dimethyl sulfoxide (DMSO ), 1,3-butylene glycol, propylene glycol, a mixed solvent thereof, and the like, and as the elution solvent in the gel filtration, water or the organic solvent may be used.

In the present specification, an alkylase ((Alcalase) is an enzyme mainly composed of subtilisin A, isolated from Bacillus licheniformis , a protein having serine endopeptidase activity. Refers to a degrading enzyme.

In addition, in the present specification, the "active ingredient" means a component that can exhibit the desired activity alone or in combination with a carrier which is itself inactive.

In addition, in the present specification, "pharmaceutically acceptable salt" means a salt that does not inhibit the activity of the peptide compound of the present invention, and organic salts commonly used in the formation of metal salts or acid addition salts (for example, acetate, citrate, Oleate, oxalate, glutonate, etc.) or inorganic salts (such as chloride, antiacid, borate, carbonate, etc.) may be used.

In addition, in this specification, "improvement" is meant to include symptom relief of disease, treatment of disease and prevention of disease.

The composition of the present invention may include the active ingredient in any amount (effective amount) as long as it can exhibit the intended hypertension-improving effect, antioxidant effect, etc., depending on the use, formulation, formulation purpose, etc., a typical effective amount is the total weight of the composition It will be determined within the range of 0.001% to 15% by weight based on the above. Here, the "effective amount" refers to the intended medical and pharmacological effects, such as an antihypertensive effect, an antioxidant effect, when the composition of the present invention is administered to a mammal, preferably a human, during the administration period according to the suggestion of a medical professional or the like. Refers to the amount of the active ingredient included in the composition of the present invention, which can be represented. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.

In addition to the active ingredient, the composition of the present invention is already safe in the art in order to increase or enhance hypertension improvement effect, antioxidant effect, etc., or to enhance the convenience of taking or ingesting by adding a similar activity such as fatigue improving activity. This may further include any compound or natural extract that has been validated and known to have the corresponding activity.

Such compounds or extracts include compounds, extracts, and medicines contained in procedures such as the Pharmacopoeia of each country ("Korea Pharmacopoeia" in Korea) and the Health Functional Foods Code of Korea ("Health Functional Food Standards and Standards" in Korea). Laws governing the manufacture and sale of compounds, extracts or health functional foods licensed under the laws of each country governing the manufacture and sale of pharmaceutical products ("Pharmaceutical Acts in Korea"). And compounds or extracts whose functionality has been recognized. For example, L-glutamic acid-derived GABA-containing powders, Katsuobushi oligopeptides, NATO cultured powders, Seomoktae peptides, salmon peptides, Olive leaf extract, green tea extract, bamboo leaf extract, melon extract, bokbunja extract (powder), etc., which have been recognized as an antioxidant; Extracts and the like will correspond to such compounds or extracts.

One or more such compounds or natural extracts may be included with the active ingredient in the composition of the present invention.

The composition of this invention can be grasped | ascertained as a food composition in a specific aspect. When the composition of the present invention is identified as a food composition, its use may be understood as inhibiting skin hypersensitivity or inhibiting hypersensitivity.

The food composition of the present invention may be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, ionic beverages, processed oils such as milk, yogurt, gums, rice cakes, sweets, bread, sweets, noodles, and the like. Foodstuffs, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, and the like can be prepared as functional food preparations.

In addition, the food composition of the present invention can distinguish any product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in the legal and functional divisions. For example, confectionery, soybeans, teas, beverages according to each food type, or health functional foods in accordance with the Act on Health Functional Foods, or in the Food Code of the Korean Food Sanitation Act , Special purpose foods, and the like.

The food composition of the present invention may include food additives in addition to the active ingredient. Food additives can generally be understood as substances which are added to the food, mixed or infiltrated in the manufacture, processing or preservation of the food, which must be ensured because of its daily and long-term intake with the food. The Food Additives Code of Korea ("Food Sanitation Act" in Korea) governs the manufacture and distribution of foods, and food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korean Food Additives Code (KFDA Notification and Standards of Food Additives), food additives are classified into chemical synthetics, natural additives, and mixed preparations in terms of ingredients. These food additives are sweeteners, flavors in terms of function. Agent, preservative, emulsifier, acidulant, thickener and the like.

Sweeteners are used to impart a suitable sweetness to foods, either natural or synthetic, which can be used in the compositions of the present invention. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavoring agents can be used to enhance the taste or aroma, both natural and synthetic. It is the case of using a natural thing preferably. In addition to flavors, the use of natural ones can be combined with nutritional purposes. The natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like. In addition, ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo and the like can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

Sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used as a preservative, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin etc. can be mentioned, As acidic acid, acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. can be used. The acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste.

As the thickener, suspending implementers, sedimenting agents, gel formers, swelling agents and the like can be used.

In addition to the food additives described above, the food composition of the present invention may include a bioactive substance or minerals known in the art for the purpose of supplementing and reinforcing the functionality and nutritional properties and ensuring the stability as a food additive.

Examples of such physiologically active substances include catechins, vitamin B1, vitamin C, vitamin E, vitamin B12 and the like, tocopherol, dibenzoylthiamine, and the like contained in green tea. Examples of the minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron, iron preparations such as iron citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc and the like.

In the food composition of the present invention, the food additive as described above may be included in an amount that can achieve the purpose of addition according to the product type.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to each country's food or food additives.

The composition of the present invention may be regarded as a pharmaceutical composition in another specific embodiment.

The pharmaceutical compositions of the present invention may be prepared in oral or parenteral formulations according to the route of administration by conventional methods known in the art, including pharmaceutically acceptable carriers in addition to the active ingredient. The route of administration here can be any suitable route, including the topical route, the oral route, the intravenous route, the intramuscular route, and direct absorption through mucosal tissue, and may be used in combination of two or more routes. An example of a combination of two or more routes is where a drug of two or more formulations according to the route of administration is combined, for example when one drug is administered first by the intravenous route and the other by the local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specific reference may be made to the pharmacopoeia of each country, including "Korea Pharmacopoeia."

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to methods known in the art with suitable carriers It may be prepared in a formulation such as. Examples of suitable carriers include lactose, glucose, sucrose, dextrose, sorbitol, sugars such as mannitol, xylitol, starch such as corn starch, potato starch, wheat starch, cellulose, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, and Cerrol etc. are mentioned. If formulated, appropriate binders, lubricants, disintegrants, colorants, diluents and the like may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweeteners, sodium alginates, polyethylene glycols, waxes, and the like. Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts thereof, calcium salts, polydetylene glycol and the like, and the disintegrants include starch, methyl cellulose And agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt and the like. Moreover, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine etc. are mentioned as a diluent.

When the pharmaceutical compositions of the present invention are prepared in parenteral formulations, they may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories with suitable carriers according to methods known in the art. When formulated as an injection, a suitable carrier may be an aqueous isotonic solution or suspension. Specifically, an isotonic solution such as phosphate buffered saline (PBS) containing triethanol amine, sterile water for injection, or 5% dextrose may be used. . When formulated as a transdermal administration, it may be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, linen, aerosol, and the like. Nasal inhalants can be formulated in the form of aerosol sprays using suitable propellants, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters and the like.

Specific formulations of pharmaceutical compositions are known in the art and can be found, for example, in Remington's Pharmaceutical Sciences (19th ed., 1995). The document is considered part of this specification.

Preferred dosages of the pharmaceutical compositions of the present invention range from 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g, depending on the condition, body weight, sex, age, severity of the patient and route of administration. It can range from / kg. Administration can be done once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.

The antioxidant composition of this invention can be grasped | ascertained as a cosmetic composition in another specific aspect.

Even when the composition of the present invention is identified as a cosmetic composition, the cosmetic composition can be classified into any product according to its purpose and law, and specifically, a functional cosmetic having a use such as skin trouble improvement, atopic dermatitis improvement, and non-functionality. General cosmetics, and the like. The product form can also be in any product form, specifically solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, waxes You can find product types such as foundation and spray. In a specific product form, it may be a softening lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder formulation.

The cosmetic composition of the present invention may include, in addition to the active ingredient, components conventionally used in the cosmetic composition, for example, conventional adjuvants such as stabilizers, solubilizers, surfactants, vitamins, pigments and pharmaceuticals, and carriers.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as the carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan and the like can be used.

When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, and microcrystals are used as carrier components. Castle cellulose, aluminum metahydroxy, bentonite, agar and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention can be prepared according to the method for producing a cosmetic composition commonly carried out in the art, except for including the active ingredient.

As described above, according to the present invention, it is possible to provide a composition for antihypertensive or anti-hypertension using a Big Valley hippocampal alkalase hydrolyzate, fraction or peptide isolated therefrom. The composition of the present invention can be commercialized into food, drugs and the like.

Figure 1 shows four gel filtration fractions of the fraction HAA-III (MW ≤ 5 kDa) of the alkylase hydrolyzate (HAA) of Big Belly Hippocampus and the fractions (HAA-I-III) according to the molecular weight of the hydrolyzate. Elution profile of HAA-III-a ~ d).
Figure 2 shows the results of separating and confirming nine peptides through RP-HPLC for the HAA-III-b fractions of the four gel filtration fractions (HAA-III-a ~ d).
3 to 11 show sequencing results by Q-TOF ESI mass spectrometer of nine peptides.
12 to 20 are molecular weight analysis results by Q-TOF ESI mass spectrometer of nine peptides.
Figure 21 shows the results of ACE inhibitory activity and alkyl radical scavenging activity of the fractions (HAA-I ~ III) according to the molecular weight of the alkylase hydrolyzate (HAA) and alkalase hydrolyzate of the Big Valley hippocampus.
FIG. 22 shows ACE inhibitory activity and alkyl radical scavenging activity of four gel filtration fractions (HAA-III-a ~ d) of HAA-III (MW ≤ 5 kDa) fractions according to molecular weight.
FIG. 23 shows the ACE inhibitory activity and alkyl radical scavenging activity of the nine peptides (HAA-III-b1 to b9) isolated from the HAA-III-b fraction in the gel filtration fraction.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

EXAMPLES Isolation and Identification of Peptides from Hippocampus Enzyme Hydrolysates

Hippocampus abdominalis was provided with aquaculture species from Haecheon horse (Jeju), and the whole hippocampus including internal organs was lyophilized, ground 1 g finely, mixed with 100 mL of distilled water, and alcalase 2.4. Enzymatic hydrolysis was performed at 1% (w / w) substrate-to-enzyme ratio at optimum activity conditions (pH 8.0, 50 ° C.) of L FG, Novozyme Nordisk, Bagsvaerd, Denmark). After hydrolysis, boil at 100 ° C. for 10 minutes for enzyme inactivation, centrifugal separation (3,500 rpm, 10 minutes) and vacuum filtration to separate the supernatant, which was then lyophilized and used in the experiment (the obtained hydrolyzate was used). For convenience, it is called HAA).

The lyophilized enzyme hydrolyzate was 4 ° C. using an ultrafiltration membrane with a molecular weight cut-off (MWCO) of 5 kDa and 10 kDa, respectively, using a Millipore's Lab scale TFF system (Millipore Corporation, Bedford, Massachusetts, USA). Fractionation was performed at to obtain three fractions HAA-I (MW 10 kDa), HAA-II (MW 5-10 kDa) and HAA-III (MW 5 kDa).

Among them, the third fraction HAA-III having the most excellent alkyl radical scavenging activity and ACE inhibitory activity was carried out by gel filtration. Gel filtration was performed by dissolving the third fraction HAA-III at 100 mg / mL using DW in Sephadex G-10 column (φ2.5 × 100 cm) and distilled water at 2 mL / min. The solution was eluted by flowing at a flow rate, and the absorbance was measured at 220 nm. The four fractions (HAA-III-a ~ d) eluted according to the measured absorbance were recovered (FIG. 1), and the second fraction (HAA-III-b) having both excellent alkyl radical scavenging activity and ACE inhibitory activity was obtained. For ethanol, linear concentration gradient method (0 min-30 min: 0 :) using RP-HPLC equipped with an ODS C18 column (Waters, Atlantis T3 column, 3.0 150 mm, 3 μm) and acetonitrile and distilled water solution. 100 v / v-40: 60 v / v, 30-40 min: 50:50 v / v, 40-50 min: 100: 0 v / v and 50-60 min: 100: 0 v / v) Eluting at a flow rate of 0.3 mL / min and monitoring the absorbance at 220 nm and 280 nm to identify one strong peak and eight weak peaks (FIG. 2) to fractionate a total of nine single peptides (P1 to P9). It was.

Nine single peptides were analyzed by amino acid sequence with a Q-TOF ESI mass spectrometer (Bruker Daltonics, 255748 Germany) and are shown in Table 1 below, and the results of each analysis are shown in FIGS. 3 to 11. In addition, the molecular weight (Fig. 12 to Fig. 20) measured by the Q-TOF ESI mass spectrometer (Bruker Daltonics, 255748 Germany) and the molecular weight of the theoretical value, it was confirmed that all nine peptides match.

Experimental Example Evaluation of Antihypertensive Activity and Antioxidant Activity

1. Experiment Method

1.1 Evaluation of Alkyl Radical Scavenging Activity

Alkyl radical scavenging activity was performed according to the method presented in Park et al. (Park PJ, et al. Carbohydrate Polymers, 2004, 55, 17-22). 40 mmol / l AAPH (2,2'-azobis (2-amidinopropane) hydrochloride), 40 mmol / l 4-POBN (4-pyridyl-1-oxide) -N-tert-butylnitrone) were dissolved in distilled water. Samples were treated in the reaction solution by concentration and reacted at 37 ° C. for 30 minutes, and then transferred to a capillary tube to measure alkyl radical generation using an ESR spectrometer. At this time, the measurement conditions were: central field: 3475 G, modulation frequency: 100 kHz, modulation amplitude: 2 G, microwave power: 10 mW, gain: 6.3 × 10 ⁵ , temperature: 298 K.

1.2 Assessment of ACE Inhibitory Activity

The evaluation of ACE inhibitory activity was carried out in Kang et al. (Kang N, et al., ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY, 2015, 39, 764-772), according to the method of 3-hydroxybutyryl-Gly-Gly-Gly. The enzyme activity was measured by determining the amount of hyroxybutylic acid.

 Specifically, ACE kit-WST (Dojindo Inc., Japan) was used, and 50 μL of the ACE coenzyme solution was added to 50 μL of the sample solution according to the manufacturer's protocol, followed by preliminary reaction at 37 ° C. for 5 minutes, and then 50 μL of the substrate was added thereto. It was made to react at 37 degreeC again for 1 hour. Then, 150 μL of 1 N HCl was added to stop the reaction, 750 μL of ethyl acetate was added thereto, stirred for 1 minute, centrifuged (4 ° C., 5,000 rpm, 10 min), and 500 μL of supernatant was obtained. The supernatant was completely dried at 120 ° C. for 30 minutes, 2 mL of methanol was added, and the absorbance was measured at 228 nm. As a control, 50 μL of the extraction solvent was added instead of the extract, and the inhibitory activity was calculated by the formula, ACE inhibition (%) = [1-{(CAbs-SAbs) / (CAbs-BAbs)}] × 100: "CAbs: control absorbance, SAbs: absorbance of sample, BAbs: absorbance of sample-free sphere ".

1.3. Statistical analysis

Statistical processing of the experimental results is expressed as the mean ± standard deviation for each sample. One-way ANOVA-test was conducted using the SPSS program (SPSS Inc. Ver. 12.0) to verify the significance of the survey items using the Turkeys multiple range test. P-value is *; p <0.05, **; p <0.005.

2. Experimental Results

Regarding the evaluation result of the alkyl radical scavenging activity and the evaluation result of the ACE inhibitory activity, the fractions (HAA-I to III) according to the molecular weights of the hydrolyzate (HAA) and the alkalase hydrolyzate of the Big Valley hippocampus are shown in FIG. In Fig. 21, nine peptides separated from the gel filtration fraction (HAA-III-b) for the four gel filtration fractions (HAA-III-a to d) of the fraction HAA-III (MW ≤ 5 kDa) are shown in FIG. (HAA-III-b1 to b9) are shown in FIG. 23 as 50% inhibitory or scavenging activity (IC ₅₀ ).

Referring to FIG. 21, among the fractions according to the molecular weight of the alkalase hydrolyzate, fraction HAA-III (MW ≤ 5 kDa) had high alkyl radical scavenging activity and ACE inhibitory activity. Among the four gel filtration fractions (HAA-III-a ~ d) of HAA-III (MW ≤ 5 kDa), the ACE inhibitory activity was high in HAA-III-a and the alkyl radical scavenging activity was high in HAA-III-d. , HAA-III-b was excellent in both ACE inhibitory activity and alkyl radical scavenging activity. Referring to FIG. 23, among the nine peptides (HAA-III-b1 to b9), HAA-III-b5 and b6 were the best.

<110> YOU JIN, JUN <120> Composition for Improving Hypertension or Anti-oxidant          Composition Using a Fraction or its Petptide of Alcalase          Hydrolysates of Hippocampus abdominalis <130> PP18-223 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HA-III-b-P1 <400> 1 Gln Ile Gly Ile Gly Gly Val Gly Gln   1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HA-III-b-P2 <400> 2 Ala Gly Ser Met Trp Asn Leu Lys   1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HA-III-b-P3 <400> 3 Gln Ile Gly Phe Ile Gly Gly Asp Gln   1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HA-III-b-P4 <400> 4 Gly Ile Ile Gly Pro Ser Gly Ser Pro   1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HA-III-b-P5 <400> 5 Ile Gly Thr Gly Ile Pro Gly Ile Trp   1 5 <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> HA-III-b-P6 <400> 6 Gln Ile Gly Phe Ile Trp   1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HA-III-b-P7 <400> 7 Ile Gly Ile Gly Pro Ser Gly Ala Ser   1 5 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HA-III-b-P8 <400> 8 Thr Ser Ile Gly Ile Gly Thr Gly Ile Pro   1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HA-III-b-P9 <400> 9 Gln Gly Ile Gly Asn Ile Gly Phe Ala Asn   1 5 10

Claims (12)

Table 1 below any one of the P4 to P6 peptide, or a composition for improving hypertension comprising a pharmaceutically acceptable salt of the peptide as an active ingredient.
TABLE 1

delete delete The method of claim 1,
The composition is a composition, characterized in that the food composition.
The method of claim 1,
Wherein said composition is a pharmaceutical composition.
Food composition for antioxidant comprising any one of P5 and P6 peptides of Table 1 as an active ingredient.
TABLE 1

delete delete delete delete Table 1 below, an antioxidant cosmetic composition comprising any one of P5 and P6 peptide as an active ingredient.
TABLE 1

Peptide of Table 1 or a pharmaceutically acceptable salt thereof.
TABLE 1

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