KR102100580B1 - 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 Big Bell hippocampus alkalase hydrolyzate, its fraction, or its effective peptide.

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 antihypertension or antioxidant using a Big Valley hippocampus alkalase hydrolyzate, a fraction thereof, or an effective peptide thereof.

Recently, as interest in aging and adult diseases has increased, studies to prevent diseases by eliminating excessively generated reactive oxygen in the living body have been actively conducted. Reactive oxygen is an electron acceptor of oxygen, which is absolutely necessary for the maintenance of life in aerobic organisms, and occurs in the process of continuing biochemical reactions 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 hydrogen peroxide (H ₂ O ₂ ), a non-free species.

Such ROS not only oxidizes lipids constituting cell membranes, but also damages DNA and affects mutagenesis, causing chronic degenerative diseases such as arteriosclerosis, cancer, high blood pressure, 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 diseases including hypertension indicates a close relationship between free radical oxidation and hypertension (American J. of hypertension. 2004, 17(9):809~816; Free radical biology & medicine) 2006, 40:2028~2039).

Hypertension refers to a case in which the average blood pressure measured several times is 140mmHg or more in systolic blood pressure and 90mmHg or more in diastolic blood pressure.It is an important problem for modern people's health as the artificial blood pressure rises during human physiological activities. An abnormal increase in blood pressure due to various causes rather than caused by one reason is called primary hypertension or essential hypertension. In addition, high blood pressure may occur due to the surrounding environment, but blood pressure may increase due to genetic causes, which is called secondary hypertension.

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

The angiotensin converting enzyme inhibitors currently in use are synthetic chemicals or synthetic peptides. Synthetic chemicals and synthetic peptides are useful in that they can be produced by mass synthesis, but itching, headache, and tachycardia , Chest pain, weakness, and other side effects have been reported. Accordingly, there is an urgent need for new angiotensin converting enzyme inhibitors without side effects.

The hippocampus is used worldwide as a drug to treat asthma, heart disease, fractures, etc. In particular, Hippocampus abdominalis is the largest among seahorses and has a beautiful body color and body shape, so it is popular in the international seawater coronary life market due to its high coronal value. There are many. The head is at a right angle to the body, the tail can be wrapped around the substrate and attached, and the male has the habit of protecting the eggs. It is used as a medicinal herb in Asia and elsewhere, and is a fish species with great industrial value due to its unique shape (Technical report series, 2003, 4:15).

However, due to its medicinal value, some countries are overfishing, and furthermore, the number of natural objects is gradually decreasing as the environment that can inhabit due to global warming and environmental pollution on the coast is reduced. In Australia and neighboring countries, China and Korea, artificial insemination is being used, but the number is remarkably insufficient compared to demand (Aquaculture. 2000, 190:377-388). North Seahorse ( Hippocampus kuda Bleeker), Hippocampus guttu - latus ), Short-snouted seahorse ( Hippocampus hippocampus ), etc., but immunophysiological studies of the Big Valley hippocampus are still insufficient.

Therefore, the inventors of the present invention, the Big Belly Hippocampus ( Hippocampus abdominalis ) Alkalase hydrolyzate, its fractions, and the new peptides isolated therefrom show the Alkly radical scavenging activity and ACE (angiotensin converting enzyme) inhibitory activity, thereby becoming a new resource with antihypertensive and antioxidant activity. I want to use it.

An object of the present invention is to provide a composition for antihypertension or antioxidant using a Big Valley hippocampus alkalase hydrolyzate, a fraction thereof, or an effective peptide thereof.

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

The present inventors prepared a Big Belly hippocampus alkalase hydrolyzate, three fractions according to the molecular weight of the hydrolyzate, and four gel filtration fractions of this fraction, as confirmed in the following Examples and Experimental Examples, and gel filtration. Separation of 9 single peptides from the fractions, and evaluation of ACE (angiotensin I converting enzyme) inhibitory activity and alkyl radical scavenging activity for these fractions and peptides. As a result, both these fractions and 9 single peptides have ACE inhibitory activity and alkyl radical scavenging. It was confirmed to have activity.

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

In the present specification, "Big Belly hippocampus alkalase hydrolyzate" refers to an enzyme-treated product obtained by hydrolyzing the Big Belly hippocampus with alkalase, a kind of protease, and refers to a group of substances including amino acids and/or peptides. Such an enzyme-treated product is used as it is or by taking the supernatant after centrifugation, or the enzyme-treated product or its supernatant is concentrated under reduced pressure and/or lyophilized to be used in liquid or powder form, or the enzyme-treated product as it is or its supernatant is used in water or ethanol. It can be used by extraction with an organic solvent such as.

In addition, in the present specification, "a fraction of a Big Belly hippocampus alkalase hydrolyzate" refers to a fraction obtained by using a Big Belly hippocampus alkalase hydrolyzate or an extract of water or an organic solvent of this hydrolyzate using organic solvents having different polarities. , Refers to a fraction obtained by fractionation according to molecular weight or a gel filtration fraction 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, and a mixed solvent thereof, and the like, and the elution solvent during gel filtration may be water or the above organic solvent.

In addition, in the present specification, alkylase ((Alcalase) is an enzyme isolated from Bacillus licheniformis , mainly consisting of subtilisin A, and is a protein having serine endopeptidase activity. It refers to a degrading enzyme.

In addition, in the present specification, the term "active ingredient" refers to an ingredient capable of exhibiting a desired activity alone or exhibiting activity together with a carrier that is not itself active.

In addition, in the present specification, the term "pharmaceutically acceptable salt" refers to a salt that does not inhibit the activity of the peptide compound of the present invention, and is an organic salt that is generally used for the formation of metal salts or acid addition salts (eg, acetate, citrate, Oleate, oxalate, glutonate, etc.) or inorganic salts (eg, chloride, antioxidant, borate, carbonate, etc.) may be used.

In addition, in the present specification, "improvement" is meant to include alleviation of symptoms of a disease, treatment of a disease, and prevention of a disease.

The composition of the present invention may contain the active ingredient in an arbitrary amount (effective amount) as long as it can exhibit the intended hypertension improvement effect, antioxidant effect, etc., depending on the purpose of use, formulation, and combination, and the usual 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. Here, "effective amount" refers to the intended medical and pharmacological effects such as hypertension improvement effect, antioxidant effect, etc., when the composition of the present invention is administered to a mammal, preferably a human, to which the composition of the present invention is administered during the administration period according to the advice of a medical professional, etc. It refers to the amount of the active ingredient contained in the composition of the present invention that can be represented. Such effective amounts can be determined empirically 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 for enhancing or reinforcing hypertension improvement effect, antioxidant effect, etc., or in order to enhance the convenience of intake or intake through the addition of similar activities such as fatigue improvement activity. It may further include any compound or natural extract that has been validated and known to have the corresponding activity.

These compounds or extracts include compounds or extracts, pharmaceuticals, etc. listed in the pharmacopoeia of each country (“Korean Pharmacopoeia” in Korea), health functional food code in each country (“health functional food standards and standards” notified by the Ministry of Food and Drug Safety in Korea), etc. In accordance with the laws of each country governing the manufacture and sale of products (“Pharmaceutical Affairs Act” in Korea), laws governing the manufacture and sale of compounds, extracts, and health functional foods that have been licensed as items (in Korea, “The Act on Health Functional Foods”) ”), the functional compounds or extracts are included. For example, L-glutamic acid-derived GABA-containing powder, which is recognized as a'blood pressure control' in accordance with the Korean Health Functional Food Act, Gatsuobushi oligopeptide, natto bacteria culture powder, Seomoktae peptide complex, salmon peptide, Olive leaf extract, green tea extract, bamboo leaf extract, melon extract, bokbunja extract (powder) recognized as functional as'antioxidant', fermentation-produced amino acid complex that is recognized as a'fatigue improvement', dulcis fruit extract, Hong Gyeongcheon Extracts and the like will correspond to these compounds or extracts.

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

In a specific aspect, the composition of this invention can be grasped as a food composition. When the composition of the present invention is regarded as a food composition, its use may be understood as inhibiting skin hypersensitivity reactions or inhibiting hypersensitivity reactions.

The food composition of the present invention may be prepared in any form, such as beverages such as tea, juice, carbonated drinks, ion drinks, processed oils such as milk, yogurt, gums, rice cakes, Korean sweets, bread, snacks, noodles, etc. Foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrup, gels, jellies, can be prepared as health functional food preparations such as bars.

In addition, the food composition of the present invention can be classified as a product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it is a health functional food according to the Korean 「Health Functional Food Act」, or confectionery, bean, tea, and beverages according to each food type according to the food code of the Korean 「Food Sanitation Act」 (``Food Standards and Standards'' notified by the Ministry of Food and Drug Safety). , Special use food, etc.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to food and mixed or infiltrated in manufacturing, processing, or preserving food. Since they are consumed daily and for a long time with food, their safety must be ensured. Food additives with guaranteed safety are limited in terms of ingredients or functions in the Food Additives Code according to the laws of each country governing the manufacture and distribution of food (the “Food Sanitation Act” in Korea). In the Korean Food Additives Code (“Food Additive Standards and Standards” notified by the Ministry of Food and Drug Safety), food additives are classified into chemical synthetic products, natural additives, and mixed preparations in terms of ingredients.These food additives are sweeteners and flavors in terms of function. It is categorized into agents, preservatives, emulsifiers, acidulants, and thickeners.

Sweeteners are used to impart an appropriate sweet taste to foods, and both natural and synthetic can be used in the composition of the present invention. Preferably, a natural sweetener is used, and 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 taste or aroma, and both natural and synthetic can be used. Preferably, it is the case of using a natural one. In the case of using natural ones, the purpose of nutrient enhancement can be combined in addition to flavor. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, or the like, or from green tea leaves, roundtails, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, you can use those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo. The natural flavoring agent may be a liquid concentrate or a solid extract. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

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

As the thickening agent, a suspending agent, a settling agent, a gel-forming agent, a swelling agent, and the like may be used.

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

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

In the food composition of the present invention, the food additive described above may be included in an appropriate amount to 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 the food code of each country or the food additive code.

The composition of the present invention may be understood as a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention may be prepared in an oral dosage form or a parenteral dosage form according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any suitable route including a local route, an oral route, an intravenous route, an intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case in which two or more formulations of drugs are combined according to the route of administration, for example, when one drug is first administered by an intravenous route and the second drug is administered by a local route.

Pharmaceutically acceptable carriers are well known in the art according to the route of administration or formulation, and specifically, reference may be made to the pharmacopoeia of each country, including the "Korean 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 a method known in the art together with a suitable carrier It can be prepared in a formulation such as. Examples of suitable carriers at this time include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease Serol, etc. are mentioned. In the case of formulation, appropriate binders, lubricants, disintegrants, colorants, and diluents may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferryst, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, etc., and lubricants include oleic acid. Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polydecylene glycol, etc., and as disintegrating agents starch, methyl cellulose , Agar, bentonite, xanthan gum, starch, alginic acid, or a sodium salt thereof. Moreover, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. are mentioned as a diluent.

When the pharmaceutical composition of the present invention is prepared in a parenteral dosage form, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation and a suppository according to a method known in the art together with a suitable carrier. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, triethanol amine-containing PBS (phosphate buffered saline), sterile water for injection, an isotonic solution such as 5% dextrose, etc. may be used. . When formulated as a transdermal administration, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, liniment, air roll, and the like. In the case of nasal inhalants, suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. can be used to form an aerosol spray.When formulated as a suppository, the carrier is Withepsol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like.

The specific formulation of pharmaceutical compositions is known in the art, and for example, Remington's Pharmaceutical Sciences (19th ed., 1995) may be referred to. This document is considered as part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. May be in the /kg range. Administration can be made once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.

In another specific aspect, the antioxidant composition of the present invention can be understood as a cosmetic composition.

Even if the composition of the present invention is identified as a cosmetic composition, the cosmetic composition can be classified as a product for its use and legally, and specifically, functional cosmetics with uses such as improving skin troubles and improving atopic dermatitis, non-functional It may be a general cosmetic or the like. The product form can also take any product form, specifically solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, waxes. It can take the form of a product such as a foundation or spray. In a specific product form, it may be a flexible lotion, a nutritional lotion, a nutritional 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.

In addition to the active ingredient, the cosmetic composition of the present invention may include components commonly used in the cosmetic composition, such as stabilizers, solubilizers, surfactants, vitamins, conventional adjuvants such as pigments and perfumes, and carriers.

When the formulation of the present invention is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. I can.

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

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol 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, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, and crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar, and the like can be used.

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

The cosmetic composition of the present invention can be prepared according to a method for preparing a cosmetic composition conventionally performed 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 antihypertension or antioxidant using a Big Valley hippocampus alkalase hydrolyzate, a fraction, or a peptide isolated therefrom. The composition of the present invention can be commercialized as food, medicine, and the like.

FIG. 1 shows four gel filtration fractions of HAA-III (MW ≤ 5 kDa) of the alkylase hydrolyzate (HAA) of the Big Belly hippocampus and the fractions (HAA-I to III) according to the three molecular weights of the hydrolyzate ( It is the dissolution profile of HAA-Ⅲ-a~d).
2 is a result of separating and confirming 9 peptides through RP-HPLC for the HAA-III-b fraction among the four gel filtration fractions (HAA-III-a to d).
3 to 11 are results of sequence analysis of 9 peptides by a Q-TOF ESI mass spectrometer.
12 to 20 are molecular weight analysis results of 9 peptides by a Q-TOF ESI mass spectrometer.
21 is a result showing the ACE inhibitory activity and the alkyl radical scavenging activity of the fractions (HAA-I to III) according to the three molecular weights of the alkylase hydrolyzate (HAA) and the alkalase hydrolyzate of the Big Belly hippocampus.
22 is a result showing the 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.
23 is a result showing the ACE inhibitory activity and the alkyl radical scavenging activity of nine peptides (HAA-III-b1 to b9) isolated from the HAA-III-b fraction of 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.

<Example> Isolation and Identification of Peptide Derived from Hippocampal Enzyme Hydrolyzate

Big Belly Seahorse ( Hippocampus abdominalis ) was provided with aquaculture species from Haecheonma Co., Ltd. , Bagsvaerd, Denmark) under the optimal activity conditions (pH 8.0, 50° C.) at a substrate-to-enzyme ratio of 1% (w/w). After the hydrolysis reaction, the supernatant was separated by boiling at 100°C for 10 minutes for enzyme inactivation, centrifugation (3,500 rpm, 10 minutes) and filtration under reduced pressure, and then freeze-dried and used in the experiment (the obtained hydrolyzate was used in the experiment. For convenience, referred to as HAA).

The lyophilized enzyme hydrolyzate was 4°C with an ultrafiltration membrane having 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 in 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 subjected to gel filtration. Gel filtration was carried out by dissolving and injecting the third fraction HAA-III at 100 mg/mL using DW in Sephadex G-10 column (φ2.5 X 100 cm), and then distilled water was added at 2 mL/min. It was eluted by flowing at a flow rate, and absorbance was measured at 220 nm. 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 the alkyl radical scavenging activity and the ACE inhibitory activity was excellent. For, using RP-HPLC equipped with ODS C18 column (Waters, Atlantis T3 column, 3.0 Х 150 mm, 3 μm) and linear phase concentration gradient method with acetonitrile and distilled water solution ((0 min-30 min: 0: 100v/v-40:60v/v, 30-40min: 50:50v/v, 40-50min: 100:0v/v and 50-60min: 100:0v/v Then, it was eluted at a flow rate of 0.3 mL/min, and the absorbance was measured at 220 nm and 280 nm. As a result of monitoring, one strong peak and 8 weak peaks were identified (Figure 2), and a total of 9 single peptides (P1 to P9) were fractionated. I did.

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

<Experimental Example> Evaluation of antihypertensive activity and antioxidant activity

1. Experimental method

1.1 Evaluation of alkyl radical scavenging activity

Alkyl radical scavenging activity was performed according to the method suggested in Park et al. (Park PJ, et al. Carbohydrate Polymers, 2004, 55, 17-22). 40 mmol/l of AAPH (2,2'-azobis(2-amidinopropane) hydrochloride), 40 mmol/l of 4-POBN (4-pyridyl-1-oxide)-N-tert-butylnitrone) dissolved in distilled water Samples were treated by concentration in the reaction solution, reacted at 37°C for 30 minutes, transferred to a capillary tube, and the amount of alkyl radicals generated was measured with 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 Evaluation of ACE inhibitory activity

The evaluation of ACE inhibitory activity was performed in 3-hydroxybutyryl-Gly-Gly-Gly, a substrate, according to the method presented in Kang et al. (Kang N, et al., ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY, 2015, 39, 764-772). 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 ACE coenzyme solution was added to 50 μL of the sample solution according to the manufacturer's protocol, followed by pre-reaction at 37°C for 5 minutes, and 50 μL of the substrate was added. It was reacted again at 37°C for 1 hour. Then, 150 μL of 1 N HCl was added to stop the reaction, 750 μL of ethyl acetate was added, stirred for 1 minute, centrifuged (4oC, 5,000 rpm, 10 min), and 500 μL of supernatant was obtained. The supernatant was completely dried at 120oC 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 calculation formula, ACE inhibition (%) = [1-{(CAbs-SAbs)/(CAbs-BAbs)}]×100: "CAbs: control absorbance, SAbs: Sample absorbance, BAbs: Sample-free absorbance calculated by "absorbance".

1.3. Statistical analysis

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

2. Experiment result

Regarding the evaluation results of the alkyl radical scavenging activity and the evaluation results of the ACE inhibitory activity, the fractions (HAA-I to III) according to the three molecular weights of the Big Valley hippocampus hydrolyzate (HAA) and alkalase hydrolyzate are shown in Fig. 21, for the four gel filtration fractions (HAA-III-a to d) of the fraction HAA-III (MW ≤ 5 kDa), Fig. 22, 9 peptides isolated from the gel filtration fraction (HAA-III-b) For (HAA-III-b1 to b9), it is shown in FIG. 23 as 50% inhibitory or scavenging activity (IC ₅₀ ).

Referring to FIG. 21, among the fractions according to the three molecular weights of the alkalase hydrolyzate, the fraction HAA-III (MW ≤ 5 kDa) had high alkyl radical scavenging activity and ACE inhibitory activity. Referring to FIG. 22, the fraction 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 most excellent.

<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)

A composition for improving hypertension, comprising a big belly hippocampus alkalase hydrolyzate or a fraction of the hydrolyzate as an active ingredient.
According to claim 1,
The fraction is a composition having a molecular weight of 5 kDa or less when the supernatant of the Big Valley hippocampus alkalase hydrolyzate is fractionated according to molecular weight.
delete The method according to claim 1 or 2,
The composition is characterized in that the food composition.
The method according to claim 1 or 2,
The composition is characterized in that the pharmaceutical composition.
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