KR101853687B1

KR101853687B1 - Antiinflammatory compositions containing Steron Fraction of Pyropia yezoensis extracts and preparing method of the same

Info

Publication number: KR101853687B1
Application number: KR1020150141318A
Authority: KR
Inventors: 전유진; 이지혁; 고주영
Original assignee: 제주대학교 산학협력단
Priority date: 2015-10-08
Filing date: 2015-10-08
Publication date: 2018-05-04
Also published as: KR20170042390A

Abstract

The antiinflammatory composition of the present invention may be used in combination with Pyropia yezoensis extract containing the active ingredient and the level of NO (nitric oxide) released by iNOS (inducible NO synthase) and the level of IL-1β (Interlukin-1β), an inflammatory cytokine, Lt; / RTI > activity. The method of the present invention for producing sterol fractions of radish sprout extracts can easily and efficiently obtain a sterol fraction containing a large amount of sterol from a radish sprout extract by a centrifugal partition chromatography system.

Description

FIELD OF THE INVENTION [0002] The present invention relates to an antiinflammatory composition comprising a sterol fraction of a radish sprout extract and a method for producing a sterol fraction of a radish sprout extract,

The present invention relates to an anti-inflammatory activity of Pyropia yezoensis < / RTI > extract. The present invention also relates to a simple and efficient method for producing sterile fractions of radish sprout extract.

Inflammation is one of the defense mechanisms of the organism caused by physical stimulation, chemical stimulation, bacterial infection, etc. It is one of various mechanisms to remove external substances and regenerate damaged areas.

When an inflammation-related stimulus is applied, vasoactive substances such as histamine, serotonine, bradykinin, prostaglandin, hydroxyeicosatetraenoic acid (HETE), leukotriene Resulting in increased vascular permeability and inflammation.

Monocytes are transformed into macrophages that are activated by bacterial infection and act as phagocytic cells. Macrophages induce inflammation by secretion of NO, prostaglandin E2, and proinflammatory cytokines, and regulatory cells important in both inflammation and immune response In order for macrophages to function in this way, they must undergo an activation process.

LPS (lipopolysaccharide), one of the cell wall components of Gram-negative bacteria and well known as endotoxin, is the most well-known external factor involved in macrophage activation. LPS is a proinflammatory cytokine such as TNF-α, IL-6 (interleukin-6) and IL-1β (interleukin-1 beta) in monocytes or macrophages such as RAW 264.7 cells pro-inflammatory cytokines. Specifically, when LPS stimulates macrophages, nitric oxide (NO) is converted to L-citrulline in the process of converting L-arginine into L-citrulline by an enzyme called iNOS (inducible nitric oxide synthase) And macrophages produce NO.

In mammals, NO has three types of NO synthase (NOS): nNOS (neuronal NOS), eNOS (endothelial NOS) and iNOS (inducible NOS). Among them, NO produced by iNOS is excessively excessive, and thus shows a harmful effect on the living body such as pathological vasodilation, cytotoxicity and tissue damage. In addition, prostaglandin E2 (PGE2), an inflammatory factor, stimulates phospholipid, which is a component of cell membrane, stimulated by LPS, and arachidonic acid liberated by an enzyme called phospholipase A2 causes the catalytic action of an enzyme called COXs (cyclooxygenase) And an inflammatory reaction is induced.

COX is classified as COX-1 and COX-2. COX-1 acts on normal biological functions such as platelet formation, gastric wall protection and maintenance of renal function in the body, and COX-2 synthesizes PGE2 as an inflammatory mediator .

PGE2 is known to be deeply involved in the development of cancer, such as promoting inflammatory reactions (pain, fever, etc.), immune responses, and angiogenesis. Inhibitors of iNOS are often associated with inhibitors of COXs, and they are often studied as anti-inflammatory agents by identifying iNOS inhibitors that are relatively easy to detect.

Centrifugal partition chromatography (CPC) is a method in which a substance to be separated is separated or extracted by using an affinity for two unmixed solvents having various solvent compositions, and a substance having a different partition coefficient Liquid-Liquid Chromatography (Liquid-Liquid Chromatography), which is characterized by the application of a non-immiscible solvent and the absence of a solid stationary phase.

This method is economical because it does not use a solid phase and does not cause loss of sample and does not require a large amount of expensive solvent. It is possible to mass-produce active fractions in a relatively simple manner, It is a promising method in various industrial fields including

Korean Patent Laid-Open Publication No. 10-2013-0024563, published on Mar. 08, 2013 Korean Patent Laid-Open Publication No. 10-2010-0007521, January 22, 2010

It is an object of the present invention to provide an anti- yezoensis < / RTI > extract. It is a further object of the present invention to provide a process for the production of sterol fractions of radish sprout extract.

In order to achieve the above object, the anti-inflammatory composition according to an embodiment of the present invention contains a sterol fraction of Pyropia yezoensis extract as an active ingredient.

The radiation patterned Kim extract may include ethanol or alcohol extract of lyophilized laver.

The radiopaque Kim extract may be ethanol of the lyophilized lyophilized product or a hexane fraction of the alcohol extract.

The sterol fraction may comprise fractions of the high-speed centrifugal partition chromatography of the radiant spore extract.

The sterol fraction may be comprised of fractions of high-speed centrifugal partition chromatography of the radiolabelled ethanol extract.

The sterol fraction may be a sterol fraction obtained by fractionating the nucleic acid fraction of the radiolabelled ethanol extract with high-speed centrifugal separation chromatography.

The anti-inflammatory composition may comprise a sterol fraction comprising at least 40% by weight of the sterol content.

The sterol fraction, which is an active ingredient of the anti-inflammatory composition, decreases the level of nitric oxide (NO) released by iNOS (inducible NO synthase) and the level of inflammatory cytokine IL-1? (Interlukin-1?) In a concentration- It can be done.

A method for producing a sterol fraction of a radish sprout extract according to another embodiment of the present invention comprises preparing a radish sprout extract using freeze-dried radish sprout and an extraction solvent containing ethanol, alcohol or a mixture thereof Extraction step; And a fractionation step of obtaining a sterol fraction, which is a fraction containing a large amount of sterol, through the centrifugal partition chromatography system from the radiopaque Kim extract.

The sterol fraction has activity to reduce the level of NO (nitric oxide) released by iNOS (inducible NO synthase) and the level of IL-1? (Interlukin-1?), An inflammatory cytokine, and may be included in anti- inflammatory compositions .

The high-speed centrifugal partition chromatography system may be applied with a CPC solvent comprising at least two solvents selected from hexane, chloroform, ethyl acetate, butanol, ethanol, methanol and water.

The centrifugation rate applied to the high-speed centrifuge dispensing chromatography system may be 100 to 2000 rpm. Also, the flow rate of the pump for solvent inflow may be 1 to 10 ml / min.

The hexane is used in a volume ratio of 8 to 10 based on the water 1, the ethylacetate is used in a volume ratio of 0.2 to 1.8 based on the water 1, and the methanol is used in an amount of 8 to 10, May be included in the optimal solvent.

The high-speed centrifugal separation chromatography system may be an apparatus in which an optimal solvent containing hexane, ethyl acetate, methanol, and water is mixed and then the supernatant obtained is applied in a stationary phase and the sub-liquid is used as a mobile phase.

May be injected at a flow rate of 1.5 to 2.5 ml / min in the column applied with the supernatant and applied in the high-speed centrifugal partition chromatography system.

The rotation speed of the column applied in the high-speed centrifugal separation chromatography system may be 800 to 1200 rpm.

As used herein, the term "active ingredient" alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which itself is not active.

As used herein, "anti-inflammatory" means prevention, amelioration, treatment, prevention or delay of the onset of inflammatory diseases. As used herein, "treatment ", unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder , And the term " treatment " means treating the condition when "treating" is defined as described above. (1) inhibiting the development of inflammatory diseases, (2) preventing the spread of inflammatory diseases and diseases associated therewith, (3) preventing inflammatory diseases and disorders, and (4) preventing recurrence of inflammatory diseases and related diseases, and (5) palliating inflammatory diseases and related symptoms.

The inflammatory disease refers to a disease caused by an inflammatory substance (inflammatory cytokine) secreted from immune cells such as macrophages by overexpressing the human immune system due to harmful stimuli such as an inflammatory inducer or radiation. May be defined as diseases accompanied by an inflammatory reaction specified by a local or systemic bio-defense reaction against external physiological stimuli or infections of external infectious agents such as bacteria, fungi, viruses and various allergens. The diseases accompanied by such an inflammatory reaction include various inflammatory mediators and activations of enzymes (e.g., iNOS, COX-2 etc.) associated with immune cells, secretion of inflammatory mediators such as NO, TNF-a, IL-6, , Excretion of PGE2), body fluid infiltration, cell migration, and tissue destruction, and is manifested externally by symptoms such as erythema, pain, swelling, fever, deterioration or loss of specific functions of the body . The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so long as it is included in the definition of inflammatory diseases as above, it may be acute, chronic, ulcerative, allergic, Whether it is necrotic or not.

The inflammatory diseases are specifically exemplified by arthritis, rheumatoid arthritis, rheumatoid polyposis muscle pain, arteriosclerosis, inflammatory visceral disease, ulcerative colitis, osteoporosis, Crohn's disease, encephalomyelitis, meningitis, pancreatitis, peritonitis, osteomyelitis, encephalitis, Acute bronchitis, chronic bronchitis, osteoarthritis, gout, spondyloarthritis, ankylosing spondylitis, psoriatic arthritis, vasculitis, lymphocyte cholinergic meningitis, glomerulonephritis, uveitis, ileitis, liver inflammation, kidney inflammation, asthma, pain, septic shock , Ischemic dermatitis, psoriasis, atopic dermatitis, psoriasis, atopic dermatitis, psoriasis, atopic dermatitis, atopic dermatitis, psoriasis, ischemic heart disease, Alzheimer ' s disease, Parkinson ' s disease, Parkinson ' s disease, cardiovascular disease, Autoimmune diseases, autoimmune diseases, autoimmune diseases, autoimmune diseases and autoimmune diseases.

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

Porphyra yezoensis ) is a seaweed of red algae, Kim peat, and has a length of 5 to 20 cm and a butterfly of 2 to 8 cm. The body is a leaf of a layer of cells, . In Korea, young leaves appear in October and grow into mature individuals until early December. It is a species that flourishes from December to May of the following year.

The inventors of the present invention have found that an extract (crude extract) obtained by using ethanol or a solvent as an extraction solvent is obtained by using radish sprout which is grown or cultured domestically in Korea, and the extract of this extract contains a large amount of components such as a colorant, a sterol and a fatty acid The fractions obtained were fractionated using hexane (n-hexane). The fractions obtained were fractionated using a Centrifugal partition chromatography (CPC) system, a type of liquid-liquid chromatograph The obtained sterol fraction was obtained by a simple and effective method, and the anti-inflammatory activity of this fraction was confirmed, thereby completing the present invention.

The composition according to one embodiment of the present invention comprises Pyropia yezoensis ) extract as an active ingredient and has anti-inflammatory activity.

It is preferable that the radiating pattern is applied in winter (November to February) in domestic Jeolla Province and Chungjeongdo Island in domestic, Japan or China, But is not limited thereto.

The above-mentioned radiation-patterned Kim extract can be obtained by applying a method of extracting the pulverized or lyophilized product obtained by washing, drying and pulverizing the radiant patterned kimchi by immersing it in an extraction solvent, and hot water extraction method, and preferably, And may be obtained by extracting by a decanting method of drying and crushing radiant streaks and immersing them in an extraction solvent for extraction.

The extraction solvent may be water, an organic solvent, or a mixed solvent thereof.

The organic solvent may be selected from the group consisting of alcohol, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, Ethyl acetate, methylene chloride, hexane, and cyclohexane. Specific examples of the solvent include methanol, methanol, ethanol, and the like. ethanol, propanol, isopropanol, and butanol. More specifically, the solvent may be ethanol, alcohol, or a mixture thereof.

The concentration of the organic solvent may be 50% (v / v) to 100% (v / v), 80% (v / v) to 100% (v / v) Extraction solvent can be used to improve the extraction efficiency.

The extraction solvent may be used in an amount of 1 to 20 times as much as the volume of the radial pattern, and the extract obtained by the extraction process may be further subjected to selective decompression, concentration, drying, It can be recovered as a leaf extract.

It is extracted by heating at a temperature of 50 ° C to 100 ° C for 1 hour to 20 hours or immersed at a temperature of 5 ° C to 37 ° C for 1 day to 15 days to prevent the solvent from evaporating, Can be extracted.

The radiant-fimbus Kim extract thus obtained can be further subjected to a step of obtaining fractions of radish-patterned Kim extract using any one fraction solvent selected from the group consisting of hexane, chloroform, ethyl acetate, water and a combination thereof. At this time, the fraction may be prepared by adding the aqueous solution obtained by dissolving the radiopaque extract of Kimchi in water and the fraction solvent at a ratio of 1: 0.1 to 2.0 volume ratio, vigorously stirring, and allowing to stand to recover the fractionated solvent. Chloroform, and ethyl acetate may be sequentially fractionated to obtain fractions.

The sterol fraction may be a fraction of the radiant spore extract with high speed centrifugal partition chromatography. The radiant-fimblane extract may be a radish-patterned Kim extract (crude extract) using the ethanol or alcohol, or a fraction of the crude extract, preferably a hexane fraction of the crude extract.

High-speed centrifugal separation chromatography is a technique that separates or extracts substances using different affinity for a solvent by adding two unmixed solvents composed of various solvents to a sample. It is economical because it does not require the use of a large amount of expensive solvent because there is no loss of the sample because it does not use a solid fixed bed and it is an efficient method capable of mass production of active fractions by a relatively simple method. The advantage of the chromatographic technique is to utilize the extracts of the radiolabelled extract for the fraction of anti-inflammatory active ingredients.

A high-speed centrifugal separation chromatography system for efficiently obtaining a sterol fraction, which is a fraction containing steroi from the radiopaque Kimchi extract, comprises a CPC solvent containing hexane, chloroform, ethyl acetate, butanol, ethanol, methanol and water do.

At this time, the content of each solvent contained in the CPC solvent can be used in a volume ratio of 0 to 20, and the centrifugal speed of high-speed centrifugal partition chromatography can be used in the range of 100 to 2000 rpm. Also, the flow rate of the pump can be 1 to 10 ml / min.

In the preparation of the high-speed centrifugal separation chromatography solvent, the upper layer of the supernatant which has been left to mix with the solvents may be a mobile phase, the lower layer of the supernatant may be a fixed bed, and the upper layer may be a fixed bed and the lower bed may be applied as a moving bed.

In addition, the setting of the high-speed centrifugal separation chromatography may be ascending or descending depending on the states of the mobile phase and the fixed phase.

The UV range of the UV-detector applied to high-speed centrifugal separation chromatography systems may be up to 210 to 600 nm.

Within these conditions it is advantageous to set the conditions of the high-speed centrifuge dispensing chromatography system in order to efficiently obtain the sterol fraction.

Specifically, the high-speed centrifugal partition chromatography system employs a solvent comprising a first solvent and a second solvent that are selectively applied to a stationary phase and a mobile phase, wherein the first solvent and the second solvent are selected from the group consisting of hexane, ethyl acetate, 1 to 5 alcohols and water are mixed, and the mixture is allowed to stand to separate the upper and lower layers of the separated solvent and apply them as the first and second solvents, respectively.

The hexane may be applied in a volume ratio of 8 to 10 based on the volume of ethyl acetate, and the alcohol having 1 to 5 carbon atoms may be applied in a volume ratio of 8 to 10 based on the volume of the water. The water and the hexane may be applied in a volume ratio of 1: 0.5 to 1.5, respectively. When the solvent is used as a CPC solvent at such a ratio, the efficiency of obtaining sterol fractions as an optimal solvent can be maximized.

The optimal solvent of this configuration can be applied as a stationary phase in which the supernatant is placed in the column after mixing and then as the mobile phase injected into the column in the chromatographic process.

At this time, the injection rate in the mobile phase column is also one of the important factors for determining the extraction efficiency. The mobile phase can be injected into the column at a flow rate of 1.5 to 2.5 ml / min, preferably 1.7 to 2.3 ml / min Flow rate. If the flow rate is less than 1.5 ml / min, the sterol may not be sufficiently recovered into the sterol fraction. If the flow rate is more than 2.5 ml / min, a large amount of impurities may be contained in the fraction.

In the high-speed centrifugal separation chromatography system, the injection of the mobile phase can proceed with rotation of the column, at which time the sterol fraction recovery can proceed effectively at 800 to 1200 rpm, and more effective at 900 to 1100 rpm The fraction can be recovered.

Sterol fractions of radiolabeled Kim extracts, which are fractionated in this way and contain a large amount of sterols, contain fractions containing about 40% or more of sterols and containing about 10 times or more sterols compared with those containing less than 5% It can be obtained in a short time in a relatively simple manner.

The fraction of high sterol content thus obtained contains sterol in an amount of at least 40% by weight, and the level of NO (nitric oxide) released by iNOS (inducible NO synthase) and the level of IL-1? (Interlukin-1?) As an inflammatory cytokine In a concentration-dependent manner. Therefore, the above-mentioned Pyropia yezoensis extract of the present invention may be incorporated into an anti-inflammatory pharmaceutical composition, a functional cosmetic composition having anti-inflammatory activity, or a food composition. In addition, the sterol fraction of the radish sprout extract is a natural-derived extract used as a foodstuff, and can be used to support the development of high value-added industries in fishing villages because domestic native seaweeds with little or no toxicity to humans and cultivation are used.

The pharmaceutical composition may comprise a pharmaceutically acceptable carrier or excipient. As used herein, the expression "pharmaceutically acceptable" means that the application (subject) does not have the above-mentioned toxicity that is adaptable without inhibiting the activity of the active ingredient. The "carrier" is defined as a compound that facilitates the addition of a compound into a cell or tissue.

The fractions herein may be administered alone or in admixture with any convenient carrier, etc., and such dosage forms may be single-dose or multiple-dose formulations. The pharmaceutical composition comprising the fraction may be a solid or liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, and the like. Solid form preparations may include, but are not limited to, carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, and the like. Examples of the liquid preparation include water, a solution such as a solution of propylene glycol, a suspension, an emulsion, and the like, but not limited thereto, and it can be prepared by adding a suitable coloring agent, a flavoring agent, a stabilizer, a tackifying agent and the like. For example, the acid can be prepared by simple mixing of a pharmaceutically acceptable carrier such as lactose, starch and microcrystalline cellulose with a fraction which is an active ingredient of the present specification. The granule may be any of the fractions described herein; A suitable pharmaceutically acceptable carrier; And a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropylcellulose, followed by wet granulation using a solvent such as water, ethanol, or isopropanol, or dry granulation using a compressive force . Tablets may also be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate and then tableting using a tableting machine.

The fractions herein may be formulated for oral, injectable (e.g., intramuscular, intraperitoneal, intravenous, infusion, subcutaneous, implants), inhalants, nasal injections, Rectal, rectal, transdermal, topical, and the like, but the present invention is not limited thereto. May be formulated into suitable dosage unit formulations, including those conventionally used and non-toxic, pharmaceutically acceptable carriers, additives, vehicles according to the route of administration.

The pharmaceutical compositions herein may be administered at a daily dose of about 0.0001 mg / kg to about 10 g / kg, with a daily dose of about 0.001 mg / kg to about 1 g / kg being preferred. However, the dosage may vary depending on the degree of purification of the mixture, the condition of the patient (age, sex, weight, etc.), severity of the condition being treated, and the like. For convenience, the total daily dose may be divided as needed and divided into several doses throughout the day.

When the composition of the present invention is used as a pharmaceutical composition, the content of the fraction in the composition can be appropriately adjusted to an effective amount capable of exhibiting anti-inflammatory activity according to the symptoms of the disease, the progress of symptoms, the condition of the patient, The amount may be 0.0001 wt% or more, specifically 0.001 wt% or more, and 80 wt% or less, specifically 50 wt% or less, based on the total weight of the composition, but is not limited thereto.

The composition may be a cosmetic composition, and the cosmetic composition may be prepared in various forms such as emulsion, lotion, cream (underwater type, water type, multiphase), solution, suspension (anhydrous and aqueous) Anhydrous products (oil and glycol), gels, masks, packs or powders, and the like. The cosmetic composition of the present invention may contain, in addition to its active ingredient, an acceptable carrier in cosmetic preparations. As used herein, the term " acceptable carrier for a cosmetic preparation "means a compound or composition that is already known and used in the cosmetic preparation, or a compound or composition to be developed in the future, and which is toxic to the human body. The carrier may be included in the composition herein from about 1% to about 99.99% by weight, specifically from about 50% to about 99% by weight of the composition, based on the total weight of the composition.

However, since the ratio depends on the formulation of the cosmetic composition as described above and its specific application site (face or hands) or its desirable application amount, the ratio is to be understood as limiting the scope of the specification in any aspect It should not be.

Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosifiers, emulsifiers, stabilizers, sunscreens, coloring agents and perfumes. The compounds / compositions which can be used as the carrier and which can be used as alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosifiers, emulsions, stabilizers, sunscreens, A person skilled in the art can select and use appropriate substances / compositions.

The composition may be a food composition and the food composition may comprise an acceptable supplemental food additive and may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of food.

In this specification, food refers to a natural product or a processed product containing one or more nutrients. Specifically, it means that the product can be directly eaten through a certain degree of processing. In general terms, Functional foods, beverages, food additives, and beverage additives. Examples of the food include various foods, beverages, gums, tea, vitamin complex, and functional foods. In addition, the food of the present specification may include special nutritional foods (eg, crude oil, spirit, infant food, etc.), meat products, fish products, tofu, jelly, noodles (eg, (Such as soy sauce, soybean paste, hot pepper paste, mixed sauce), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickled foods But are not limited to, natural flavors (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages and other health supplement foods. The functional food, beverage, food additive or beverage additive may be produced by a conventional production method.

The above-mentioned functional food refers to a food group imparted with added value to function or express the function of the food by using physical, biochemical, biotechnological techniques, etc., or to control the biological defense rhythm of the food composition, Means a food which is processed by being designed so as to sufficiently express the body's controlling function against the living body. Specifically, it may be a health functional food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

In addition, in the food composition, the amount of the fraction may be 0.00001 wt% or more, specifically 0.1 wt% or more, and 80 wt% or less, specifically 50 wt% or less, more specifically 40 wt% or less And when the food is a beverage, it may be contained in a proportion of not less than 0.001 g, specifically not less than 0.01 g, not more than 50 g, specifically not more than 10 g, more specifically not more than 2 g based on 100 ml of the whole volume of the food But is not limited thereto.

The food composition of the present invention may contain sweetening agents, flavoring agents, physiologically active ingredients, minerals and the like in addition to the active ingredients thereof. Sweetening agents may be used in an amount that sweetens the food in a suitable manner, and may be natural or synthetic. Specifically, natural sweeteners are used. Examples of the natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose. Flavors may be used to enhance taste or flavor, both natural and synthetic. Specifically, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. Examples of natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used. Examples of the physiologically active substance include catechins such as catechin, epicatechin, gallocatechin and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine and riboflavin. As the mineral, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium and zinc can be used.

In addition, the food composition of the present invention may contain preservatives, emulsifiers, acidifiers, thickeners and the like as needed in addition to the above sweeteners. Such preservatives, emulsifiers and the like are preferably added in a very small amount as long as they can attain an application to which they are added. The term " trace amount " means, when expressed numerically, in the range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition. Examples of the preservative which can be used include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate and EDTA (ethylenediaminetetraacetic acid). Examples of the emulsifier which can be used include acacia gum, carboxymethyl cellulose, xanthan gum, pectin and the like. Examples of the acidulant that can be used include acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. Such an acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste. Agents that may be used include suspending agents, sedimentation agents, gel formers, bulking agents and the like.

According to another embodiment of the present invention, there is provided a method for producing a radish-patterned Kim extract, comprising the steps of: preparing a radish-patterned Kim extract by using a lyophilized radish-patterned kimchi and an extraction solvent containing ethanol, alcohol or a mixture thereof; The sterol fraction, which is a fraction containing a large amount of sterol, is fractionated from the radiant sprout extract through a Centrifugal partition chromatography system. The sterol fractions are fractionated to obtain NO (nitric acid) released by iNOS (inducible NO synthase) oxide and the level of IL-1? (Interlukin-1?), an inflammatory cytokine, to produce a sterol fraction of radish sprouting extract contained in the anti-inflammatory composition.

The extracting step may further include a step of obtaining a fraction obtained by fractionating the radish sprout extract obtained by using the extraction solvent with a fraction solvent, and the fraction may be applied as a radish sprout extract in the following process. Preferably, It may be a hexane fraction of a patterned roasted ethanol extract.

Also, the setting of the high-speed centrifugal partition chromatography may be ascending or Descending depending on the state of the mobile phase and the fixed phase.

The UV range of the UV-detector applied to high-speed centrifugal separation chromatography systems may be up to 210 to 600 nm. The setting within these conditions is advantageous in that the sterol fraction can be efficiently obtained.

Specifically, the high-speed centrifugal partition chromatography system employs a solvent comprising a first solvent and a second solvent that are selectively applied to a stationary phase and a mobile phase, wherein the first solvent and the second solvent are selected from the group consisting of hexane, ethyl acetate, 1 to 5 of alcohol and water are mixed, and the separated solvent is separated from the upper and lower layers to be applied as the first and second solvents, respectively.

The antiinflammatory composition of the present invention may be used in combination with Pyropia yezoensis extract containing the active ingredient and the level of NO (nitric oxide) released by iNOS (inducible NO synthase) and the level of IL-1β (Interlukin-1β), an inflammatory cytokine, And can be utilized as a pharmaceutical composition, a cosmetic composition, or a food composition having anti-inflammation and antioxidative activity. The method of the present invention for producing sterol fractions of radish sprout extracts can easily and efficiently obtain a sterol fraction containing a large amount of sterol from a radish sprout extract by a centrifugal partition chromatography system. When the method of the present invention is applied, a sterol-containing fraction having a high content of about 40% by weight or more can be obtained in a relatively simple and easy manner in a large amount, using radiant stain with a sterol content of about 1% by weight as a raw material .

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view for explaining a process for obtaining a sterol-containing fraction from a radish-patterned Kim extract in an example of the present invention (Production Example 1); FIG.
FIG. 2 shows the results of evaluating NO production using RAW 264.7 cells (* P <0.05, ** P <0.01, + P <0.05) in the anti-inflammatory effect of PYSRF obtained in the example of the present invention P < 0.05).
FIG. 3 shows the anti-inflammatory effect of PYSRF obtained in the example of the present invention (Production Example 1) as evaluated by PGE2 production using RAW 264.7 cells in which LPS was introduced (* P <0.05, ** P <0.01, P < 0.05).
4 shows the anti-inflammatory effect of PYSRF obtained in the example of the present invention (preparation example 1) by Western blotting of RAW 264.7 cells into which LPS was introduced (* P <0.05, ** P <0.01, + P < 0.05).
FIG. 5 is a graph (* P <0.05, ** P <0.01, + P <0.05) obtained by quantifying the results of FIG.
FIG. 6 shows the results of confirming the inhibitory effect of PYSRF on IL-1β expression in the examples of the present invention using an ELISA kit (* P <0.05, ** P <0.01, + P <0.05).
FIG. 7 shows the results (* P <0.05, ** P <0.01, + P <0.05) of the inhibitory effect of PYSRF on the expression of TNF-α as determined in the examples of the present invention using an ELISA kit.
8 shows the results of the in vivo test using the zebrafish embryo (7 dpf) (* P <0.05, ** P <0.01, + P &Lt; 0.05).
FIG. 9 shows the results (* P <0.05, ** P <0.01, P <0.05) of the toxicity (cell death level) of PYSRF obtained in the example of the present invention (Production Example 1) by an in vivo test using a zebrafish embryo + P < 0.05).
FIG. 10 is a graph (* P <0.05, ** P <0.01, + P <0.05) obtained by quantifying the results of FIG.
Fig. 11 shows the result of in vivo test using zebrafish embryo (3 dpf) (* P < 0.05, **) for anti-inflammatory activity (NO production inhibitory effect) of PYSRF obtained in the example of the present invention P < 0.01, + P < 0.05).
12 is a graph (* P <0.05, ** P <0.01, + P <0.05) obtained by quantifying the results of FIG.
13 is an experimental result for setting a solvent of an optimal CPC system for obtaining a sterol-containing fraction in Production Example 1 of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

<Preparation of experimental materials>

Pyropia yezoensis was collected from Wando Island in Korea from November 2014 to March 2015. Solvents from extraction and CPC (Centrifugal partition chromatography) were purchased from Sigma (St. Louis, Mo., USA). DMEM (Dulbecco's modified Eagle's medium), fetal bovine serum (FBS), penicillin-streptomycin and trypsin-EDTA were purchased from Gibco / BRL (Grand Island, NY, USA).

The ELISA kit used for the analysis of TNF-α, IL-1β and PGE2 production was purchased from R & D Systems Inc. (Minneapolis, MN, USA). The BAC kit was purchased from Bio-Rad (Richmond, CA, USA) and LPS, DMSO, DAF-DM-DA and other solvents or reagents were purchased from Sigma (St. Louis, Mo., USA) .

<Test method: Fraction Production method and method for evaluating sterol content>

One. Radiant patterned kimchi Method of producing extract

In the examples of the present invention, fractions as shown in FIG. 1, which is a drawing for explaining a process for obtaining a sterol-containing fraction from a radish sprout extract, were prepared.

Radiophore was washed with water to remove salt and various foreign matter, dried for 3 hours or more, frozen and freeze-dried at -80 ° C. The lyophilized radiant streaks were pulverized and prepared as powders, and a stock solution of radial pattern extract was prepared using 100% by weight of ethanol, and the extract solution was evaporated using a vacuum concentrator to obtain a radiolabelled ethanol extract .

The fractions were prepared by using the radiolabelled ethanolic crude extracts in hexane (n-hexane), chloroform (chloroform), ethyl acetate, or water, The fractions were collected by using a separatory funnel (stored for 6-12 hours until separated at 25 ° C), and the fractions were collected by adding hexane, chloroform, Fractions containing the following sterols were prepared using hexane fractions having the highest activity among them.

2. Manufacturing example 1: Sterol content Fraction Manufacturing method

To separate the sterol-containing fractions, a Centrifugal partition chromatography (CPC) system was applied. The CPC optimum solvent conditions were set in the two-phase solvent system using n-hexane: ethyl acetate (EtOAc): methanol (MeOH): water (9: 1: 9: acending mode.

After mixing the above four kinds of solvents, they were allowed to stand at room temperature and separated into two layers as a solvent layer. At this time, the lower layer was used as a mobile phase and the upper layer was used as a fixed phase. The CPC column was filled with upper layer solvent and the lower layer solvent was injected while the column was rotating at 1000 rpm. The CPC was operated in a descending mode at a flow rate of 2 ml / min. Columns and flow rates were monitored using a UV detector (254 nm) and the fractions were collected from fractions F1 to F3 using a fraction collector (Gilson FC 203 B) and the sterol rich fraction of Pyropia yezoensis extract, PYSRF).

* Setting the optimum solvent condition

The optimum CPC solvent conditions were set as follows. In order to select the solvent of CPC, four different solvents were used, two solvents were mixed, and the mixture was mixed with the radiolabelled ethanol extract. After separating the CPC solvent into two layers, (N-hexane: ethyl acetate = 7: 3) were loaded in a thin layer chromatography (TLC).

The target component, sterol, becomes purple when it comes into contact with H ₂ SO ₄ (sulfuric acid). This principle was used to set the optimal solvent conditions for CPC. Sikyeotgo reacting the TLC and the H ₂ SO _4, and heated until the purple sterol is sensibly reacts with H ₂ SO ₄ eseo 105 ℃. When the TLC bands of the sterol dissolved in the upper layer (indicated by B in Fig. 13) and the lower layer (indicated by T in Fig. 13) of the TLC confirmed by the purple color appear to be similar to each other, (See Fig. 13).

Containing 3 sterols Fraction Sterol content analysis method

The Liebermann Burchard test was used for the quantitative analysis of the sterol content in fractions F1 to F3 obtained above.

Cholesterol was dissolved in glacial acetic acid as a reference standard, and 2 mg of each sample was dissolved in 1 ml of glacial acetic acid to prepare a sample for analysis. Absorbance was measured at 625 nm using a microplate reader (BioTek, SYNERGY, HT, USA) mf. The prepared Liebermann Burchard regent and analytical samples were mixed and allowed to proceed in the dark for 10 min. Standard curves were derived from the measurement results and the sterol content in the fractions was quantitated.

<Test method: Evaluation of anti-inflammatory activity using cell line>

The anti-inflammatory efficacy was assessed by measuring the levels of nitroglycerin (NO) and PGE 2 produced by lipopolysaccharide-stimulated mouse-derived macrophages RAW 264.7 using the Griess regent and competitive enzyme immunoassay (ELISA) Respectively.

1. Cell culture

RAW 264.7 cells are 10% heat-inactivated FBS, streptomycin (100 g / mL) and penicillin ^- in an incubator to maintain the 5% CO ₂ wet-containing atmosphere and the conditions of 37 ℃ in DMEM containing (100 unit.mL ¹⁾ Lt; / RTI >

2. LPS Induction NO Production capacity evaluation

Cells were treated with various concentrations of PYSRF, incubated for 1 hour, treated with LPS (1 μg / ml), incubated for 24 hours and then reacted with Griess reagent. The amount of nitrite accumulated in the culture medium was quantitated by measuring absorbance using a microplate reader of 540 nm wavelength and using the following equation (1).

In the above equation, G _NO is the NO generation percentage of Raw 264.7 cells, A _{S is} the absorbance value of Raw 264.7 cells treated with PYSRF sample and LPS, A _C is the absorbance value of Raw 264.7 cells not treated with PYSRF sample or LPS And A _L is the absorbance value of Raw 264.7 cells in which only LPS is not treated.

3. pro-inflammatory cytokines ( TNF -α, IL- 1β )and PGE2 measurement of production

PMSRF solution was prepared with minimum amount of DMSO and diluted to various concentrations using PBS (phosphate buffer saline). How does PYSRF affect pro-inflammatory cytokines TNF-α, IL-1β, and PGE2 production? LPS-induced RAW 264.7 cells were evaluated using an ELISA kit.

4. Western blotting (Western blot analysis)

RAW 264.7 cells were plated in 6-well plates and treated with different concentrations of PYSRF for 1 hour before introducing LPS.

Cells were lysed by lysis buffer, and the cell lysates were centrifuged and electrophoresed using 10% (or 15%) sodium dodecyl sulfate-polyacrylamide gels. The cells were incubated with nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were treated with COX-2, iNOS, and β-actin for 1 hour to allow the primary antibody to be introduced, exposed to X-ray films using ECL Western blotting detection kit.

<Test method: Jeffrey Fish Evaluation of anti-

One. Zebra fish Ready

Ten zebrafish were kept in three lacrylic tanks maintained at 28.5 ℃ and 14/10 h light / dark cycle. The feeding was provided 3 times a day for 6 days a day and provided with tetramin flake food supplemented with live brine shrimps ( Artemiasalina ). The embryos got natural spawning.

2. Zebra fish Embryonic Cytotoxicity PYSRF Impact Assessment Method

Cells were assessed using live zebrafish larvae (7 dpf) using acridine orange staining. The evaluation was carried out by treating the embryos treated with various concentrations of PYSRF with 10 μg / mL of LPS.

The treated larvae were transferred to 96-well plates and treated with 7 μg / mL acridine orange solution, and then 28.5 ± 1 Lt; 0 > C for 30 minutes. The treated and cultured larvae were washed with embryo media and observed after anesthesia with 2-phenoxyethanol (1/500 dilution sigma). Observations were taken using SNAP-Procolor digital camera (Olympus, Japan) and fluorescence intensity was quantified using image J program. The results are shown in Fig. The cytotoxicity evaluation results are shown in FIG. 9 and FIG.

3. Zebra fish In embryo in vivo NO production evaluation method

The evaluation was carried out by treating the embryos treated with various concentrations of PYSRF with 10 μg / mL of LPS. The treated larvae were transferred to a 96-well plate and stained with 5 μM of DAF-DM-DA solution, followed by 28.5 ± 1 Lt; 0 > C for 30 minutes. The treated and cultured larvae were washed with embryo media and anesthetized with 2-phenoxyethanol (1/500 dilution sigma) and observed with fluorescence microscopy. Observations were taken using SNAP-Procolor digital camera (Olympus, Japan) and fluorescence intensity was quantified using image J program. The results are shown in FIG. 11 and FIG.

<Statistical Analysis>

Experimental results were expressed as mean ± standard error (SE), and mean values were compared using one-way ANOVA test (using SPSS 11.5 statistical software). Significant differences were assessed using student's t- test ( p <0.05, p <0.01).

<Experimental Results: Radiant patterned kimchi Sterol content of extract Unadulterated Acquisition>

Sterol fractions were successfully isolated from radiolabelled (500 mg) using CPC. Using CPC, the alcohol extract of radish sprout was separated into three fractions, labeled F1 to F3. The results of the TLC showed that F2 contained the largest amount of sterols. The fraction (F2) containing the separated sterols was 40 mg, and the sterol content was 45%, showing a yield of 8%. The evaluation was then carried out using F2 as PYSRF.

<Test results: Sterol-containing Fraction Anti-inflammatory effects on cells>

1. NO production and PGE ₂ production evaluation result

The results of evaluation of anti-inflammatory activity using RAW 264.7 cells in which LPS was introduced are shown in FIG. 2 and FIG. 3, respectively. Referring to the results of FIG. 2 and FIG. 3, it can be seen that NO production and PGE ₂ production decrease in a dose-dependent manner.

PYSRF did not show cytotoxicity up to 25 μg / ml, and in the subsequent experiments, PYSRF was applied at a concentration of 25 μg / ml or less.

2. Proinflammatory Cytokines and PGE2 production evaluation result

The degree of expression of the inflammatory cytokines TNF-α and IL-1β measured by ELISA kit is shown in FIG. 6 and FIG. 6 and 7, IL-1β was significantly decreased in a concentration-dependent manner among the inflammatory cytokines Interlukin-1β (IL-1β) and TNF-α, while TNF-α decreased at 25 μg / ml .

3. Experimental Results: Western blotting result

Referring to FIGS. 4 and 5, NO released by iNOS, an inflammatory mediator induced by LPS, was greatly decreased in a concentration-dependent manner by PYSRF, while PGE2 released by COX-2 was relatively decreased. In addition, the expression of iNOS was significantly decreased in a concentration-dependent manner, and the degree of COX-2 expression was relatively small.

4. Survival , Cell death And NO generation in vivo Evaluation results

The zebrafish model was used to confirm cytotoxic and antiinflammatory effects of PYSRF in vivo. Referring to the results of FIGS. 8 to 12, PYSRF appears to exhibit toxicity to zebrafish at 12 μg / ml, and the cytotoxic and NO inhibitory effects of 1 to 6 μg / ml, Respectively.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims

An antiinflammatory composition comprising, as an active ingredient, a fraction (sterol rich fraction) containing 40% or more by weight of a sterol obtained from a Pyropia yezoensis extract by high speed centrifugal partition chromatography.

The method according to claim 1,
Wherein the radiant sprout extract is ethanol or a hexane fraction of a lyophilized laver.

delete

The method according to claim 1,
Wherein said fraction is a concentration-dependent decrease in the level of NO (nitric oxide) released by iNOS (inducible NO synthase) and the level of IL-1? (Interlukin-1?), An inflammatory cytokine.

An extracting step (1) of preparing a radish-patterned Kim extract by using a lyophilized radish stalk and an extraction solvent containing ethanol, a spirulina or a mixture thereof;
A CPC solvent having a ratio (v / v) of hexane, ethyl acetate, methanol, and water of 9: 1: 9: 1 was mixed through the Centrifugal Partition Chromatography (CPC) Step (2); And
And fractionation step (3) of obtaining a sterol rich fraction of the sterol obtained by the high-speed centrifugal separation chromatography from the radiopaque kimchi extract with a sterol content of not less than 40% by weight. By weight of a sterol rich fraction obtained by the method according to the present invention.

6. The method of claim 5,
The flow rate of the pump for introducing the CPC solvent applied to the high-speed centrifugal separation chromatography system is 1 to 10 ml / min, and the mobile phase is injected at a flow rate of 1.5 to 2.5 ml / min into the column in which the supernatant is injected By weight of a sterol rich fraction.

6. The method of claim 5,
Wherein the sterol rich fraction of the sterol is present in an amount of not less than 40% by weight, the sterol rich fraction having an anti-inflammatory activity.

delete

7. An antiinflammatory composition comprising a sterol rich fraction comprising at least 40% by weight of a sterol produced according to the method of any one of claims 5-7.