KR20210038157A - Composition for preventing or treating inflammatory disease comprising Hippocampus abdominalis extract or fractions thereof - Google Patents

Abstract

A Hippocampus abdominalis extract according to the present invention inhibits NO production induced by LPS in RAW 264.7 cells, and effectively inhibits the expression of iNOS and COX-2 proteins, and pro-inflammatory cytokines TNF-α, IL-6, and IL-12. Therefore, the Hippocampus abdominalis extract can be used for preventing, alleviating or treating inflammatory diseases and/or alleviating skin inflammation.

Description

Composition for preventing or treating inflammatory diseases comprising Hippocampus abdominalis extract or fractions thereof}

The present invention relates to a pharmaceutical composition, quasi-drug composition, food composition, feed additive composition for preventing, improving or treating inflammatory diseases comprising a Big Valley hippocampus extract or a fraction thereof.

In addition, the present invention relates to a composition for relieving skin inflammation comprising a Big Valley hippocampus extract or a fraction thereof.

Inflammation is a physiological reaction that protects a living body from the invasion of foreign substances such as bacteria and mechanical damage to the harmful surrounding environment. Although various biochemical phenomena are involved in the inflammatory response, it is known that macrophages in particular play an important role in the inflammatory response by producing nitric oxide (NO) and various inflammatory cytokines by chemical stimulation.

In the inflammatory response, nuclear factor-kB (NF-kB), a core transcription factor, is essentially activated by various stresses, which promotes the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). do. The activation of iNOS and COX-2 triggers mass production of coral nitrogen (NO) and prostagladin E2 (PGE2), which are known as key factors inducing inflammation. In the case of nitric oxide synthase (NOS) in mammalian cells, there are three similar forms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Among them, iNOS is particularly involved in the inflammatory response. In the case of iNOS, it is expressed when stimulated by interferon-gamma, lipopolysaccharide (LPS), and various inflammatory cytokines. PGE ₂ is produced from arachidonic acid by cyclooxygenase (COX). In other words, the expression of COX-2 and iNOS is a representative inflammatory factor of immune cells that promotes the production _{of PGE 2 and NO.}

In general, steroidal or nonsteroidal anti-inflammatory drugs are used to treat inflammatory diseases. As a typical example, nonsteroidal anti-inflammatory drugs (NSAIDs) are used to relieve inflammation, and if inflammation is severe or NSAIDs are ineffective, steroids are used, and if intractable, immunosuppressants or surgical therapy are performed. In this case, the NSAIDs used in this case mainly inhibit COX-2, thereby inhibiting the production of prostaglandins, which are involved in the inflammatory response, thereby exhibiting anti-inflammatory action, but it is difficult to use for a long time because it causes side effects such as gastrointestinal disorders, liver disorders, and kidney dysfunction There is. Compared to NSAIDs, steroids are known to be the fastest method available to patients, and are known to have rapid and dramatic anti-inflammatory effects. However, as is widely known, steroid preparations weaken resistance to bacterial infections, cause worsening of diabetes, adrenal insufficiency, and mental dysfunction. It is known to require and should be avoided if possible. Synthetic anti-inflammatory drugs like this are often accompanied by various side effects. Therefore, the development of anti-inflammatory drugs with excellent effects and relatively few side effects is constantly required.

The Big Belly Seahorse ( Hippocampus abdominalis ) belongs to the Syngnathidae family, inhabits temperate and tropical seas, has the largest size among seahorses, has a beautiful body color and body shape, and is popular in the international seawater ornamental life market due to its high ornamental value. Seahorse cultivation has already been attempted in New Zealand, Australia, and China, and in Jeju recently, the complete cultivation of the Big Belly Seahorse, which has been designated for food, has been successful. However, compared to the North Seahorse ( Hippocampus kuda Bleeker ) and Hippocampus guttulatus species, which have been previously studied for physiological activity, the Big Belly Hippocampus is lacking in physiological activity studies.

Korean Patent Registration No. 10-1757827

An object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory disease containing the Big Valley hippocampus (Hippocampus abdominalis) extracts or fractions thereof.

Another object of the present invention is to provide a quasi-drug composition for preventing or improving inflammatory diseases comprising a Big Valley hippocampus (Hippocampus abdominalis) extract or a fraction thereof.

Another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases, including the Big Belly Hippocampus (Hippocampus abdominalis) extract or a fraction thereof.

Another object of the present invention is to provide a feed additive composition for preventing or improving inflammatory diseases, including the Big Belly Hippocampus (Hippocampus abdominalis) extract or a fraction thereof.

Another object of the present invention is to provide a cosmetic composition for relieving skin inflammation comprising a Big Valley hippocampus (Hippocampus abdominalis) extract or a fraction thereof.

In order to achieve the above object, the present inventors have confirmed that the Big Valley hippocampus extract reduces the production of NO in RAW 264.7 macrophages in which the inflammatory reaction is induced with LPS, PGE ₂ , iNOS, COX-2, TNF-α, By inhibiting the expression of IL-6 and IL-12 and inhibiting the activity of NF-κB, it was confirmed that it has an anti-inflammatory effect, and the present invention was completed.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, including , in one aspect, an extract of Hippocampus abdominalis or a fraction thereof.

In the present invention, " Hippocampus abdominalis " belongs to the family Syngnathidae , lives in temperate and tropical seas, has the largest and most beautiful body color and body shape among seahorses, and has a high ornamental value, so the international seawater ornamental market It is also very popular. Seahorse cultivation has already been attempted in New Zealand, Australia, and China, and in Jeju recently, the complete cultivation of the Big Belly Seahorse, which has been designated for food, has been successful.

In the present invention, "extract" refers to an extract obtained by an extraction treatment, a diluted solution or a concentrate of the extract, a dried product obtained by drying the extract, a preparation or purified product of the extract, or a mixture thereof, the extract itself and the extract. It includes extracts of all formulations that can be formed.

In the present invention, drying of the extract may be performed by a known method within a range in which useful components are not destroyed from the collected sample, for example, it may be performed by a method of freeze-drying in a vacuum state. Further, the crushing or pulverization may be pulverized or pulverized to a degree that sufficient components of the sample can be sufficiently extracted during the subsequent extraction process. The drying and crushing or pulverizing process may be performed in reverse order or repeatedly performed as necessary.

In the extraction of the present invention, the method of extracting is not particularly limited, and may be extracted according to a method commonly used in the art.

In the present invention, the Big Valley hippocampus (Hippocampus abdominalis ) extract can be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof. Specifically, it may be obtained by using an alcohol _{having 1 (C 1} ) to 4 (C ₄ ) carbon atoms such as water, methanol, ethanol, and butanol, or a mixed solvent thereof. In addition, the alcohol may be ethanol.

In the example of the present invention, 60% (v/v) ethanol was added to the lyophilized Big Belly hippocampus, extracted at 60° C. for 2 hours, and filtered to obtain an ethanol extract of the Big Belly hippocampus.

Therefore, the Big Valley hippocampus extract may be specifically extracted with 50 to 70% ethanol solvent, and specifically may be extracted at 50 to 70°C with 60% ethanol solvent.

In the present invention, the term "fraction" means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents.

The fractionation method for obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a method of obtaining a fraction from the extract by treating the extract of Hippocampus abdominalis with a predetermined solvent.

After suspending the extract of the Hippocampus abdominalis of the present invention in distilled water (water), using _{an alcohol having 1 (C 1} ) to 4 (C ₄ ) carbon atoms, ethyl acetate, n-hexane, or a mixed solvent thereof It can be obtained by fractionation.

In an embodiment of the present invention, it was confirmed that the ethanol extract of the Big Valley hippocampus inhibits the production of NO induced by LPS and inhibits _{the expression of prostaglandin E 2} (prostaglandin E ₂ , PGE _{2 ).} In addition, it was confirmed that the expression of the inflammation regulator iNOS or COX-2 was suppressed in a concentration-dependent manner, and the expression of the inflammatory cytokines IL-12, IL-6, and TNF-α was also decreased in a concentration-dependent manner.

In addition, it was confirmed that the ethanol extract of the Big Valley hippocampus inhibits the activity of NF-kB induced by LPS.

That is, it was confirmed that the Big Belly hippocampus extract has excellent anti-inflammatory effect, and the Big Belly hippocampus extract or its fractions can be used in pharmaceuticals, foods, quasi-drugs for improving, preventing, and treating inflammatory diseases, and for relieving skin inflammation. It can be used as a cosmetic composition.

In the present invention, after analyzing the ethanol extract of the Big Belly hippocampus as a general component, it was confirmed that the crude protein content of the ethanol extract of the Big Belly hippocampus was 81.8%, and it was confirmed that most of the ethanol extract of the Big Belly hippocampus was made of protein, and amino acid analysis As a result, the content of glycine was the highest at 21.5%. Subsequently, it was confirmed that 12.5% of glutamic acid and 11.4% of alanine were included.

Therefore, the ethanol extract of the Big Belly hippocampus of the present invention may have a protein content of 70 to 90%, specifically, a glycine content of 15 to 25%, a glutamic acid of 10 to 15%, and alanine. It may be included in 10 to 13%.

In the present invention, "inflammatory diseases" are TNF-α (tumor necrosis factor-alpha), IL-12 (interleukin-alpha) secreted by immune cells such as macrophages by excessively promoting the human immune system due to harmful stimulation such as inflammation inducing factors. 12), IL-6 (interleukin-6), prostaglandin (prostagladin), or nitric oxide (nitric oxide) refers to a disease caused by an inflammatory substance such as.

In the present invention, the inflammatory diseases are allergy, dermatitis, atopic, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, Any one selected from the group consisting of psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder joint inflammation, tendinitis, tendonitis, tendonitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases. Can be

In the present invention, "prevention" means any action of inhibiting or delaying the onset of the inflammatory disease by administration of the composition according to the present invention.

In the present invention, "treatment" refers to any action in which the symptoms of the inflammatory disease are improved or advantageously changed by administration of the composition according to the present invention.

The pharmaceutical composition of the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions other than the active ingredient. At this time, the Big Valley hippocampus ( Hippocampus abdominalis ) extract or a fraction thereof included in the composition is not particularly limited thereto, but is 0.001% to 99% by weight, preferably 0.01% to 50% by weight, based on the total weight of the composition. Can include.

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

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

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

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

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

In another aspect of the present invention, it provides a quasi-drug composition for preventing or improving inflammatory diseases, including a Big Belly Hippocampus (Hippocampus abdominalis) extract or a fraction thereof.

In the present invention, Big Valley hippocampus ( Hippocampus abdominalis ), extract, fraction, inflammatory disease, description of the prevention is as described above.

In the present invention, “improvement” refers to any action in which symptoms of an individual with suspicion of an inflammatory disease and symptoms of an inflammatory disease are improved or benefited by using a composition containing the Big Valley hippocampus extract or a fraction thereof as an active ingredient.

In the present invention, “quasi-drugs” are fibers, rubber products or similar materials used for the purpose of treating, alleviating, treating or preventing diseases of humans or animals. A product corresponding to one of the preparations used for sterilization, insecticide, and similar purposes to prevent infection, and for the purpose of diagnosing, treating, alleviating, treating or preventing diseases of humans or animals. Means non-instruments, machines, or devices, and those used for the purpose of pharmacologically affecting the structure and function of humans or animals, excluding those that are not devices, machines, or devices. Skin external preparations and personal hygiene products are also used. Includes.

When the Big Belly hippocampus extract of the present invention or a fraction thereof is added to a quasi-drug composition for the purpose of preventing or improving inflammatory diseases, the extract or fraction may be added as it is or used with other quasi-drug components, and it is appropriate according to a conventional method. Can be used. The mixing amount of the active ingredient can be appropriately determined according to the intended use.

The external preparation for skin is not particularly limited thereto, but may be preferably prepared and used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel.

The personal hygiene product is not particularly limited thereto, but preferably includes soap, wet tissue, tissue paper, shampoo, toothpaste, air freshener gel, cleaning gel, disinfectant cleaner, detergent, shower foam, mouthwash, hand wash, or mask. .

In another aspect of the present invention, it provides a food composition for preventing or improving inflammatory diseases, including the Big Belly Hippocampus (Hippocampus abdominalis) extract or a fraction thereof.

In the present invention, the Big Belly Hippocampus ( Hippocampus abdominalis ), extract, fraction, inflammatory disease, prevention and improvement are as described above.

The food composition of the present invention may include the form of pills, powders, granules, needles, tablets, capsules, liquids, etc., and types of foods to which the Big Belly Seahorse ( Hippocampus abdominalis ) extract or fractions thereof of the present invention can be added There is no particular limitation on this, and for example, there are various beverages, gums, teas, vitamin complexes, and dietary supplements.

In addition to the Big Belly Hippocampus ( Hippocampus abdominalis ) extract or a fraction thereof, other ingredients may be added to the food composition, and the kind is not particularly limited. For example, as an additional component, various herbal extracts, food additives, or natural carbohydrates that are acceptable for food may be included as an additional component, but are not limited thereto.

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

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

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

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

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

In another aspect of the present invention, there is provided a feed additive composition for preventing or improving inflammatory diseases, including a Big Belly Hippocampus (Hippocampus abdominalis) extract or a fraction thereof.

In the present invention, the Big Belly Hippocampus ( Hippocampus abdominalis ), extracts, fractions, inflammatory diseases, prevention and improvement are as described above.

In the present invention, the term "feed additive" is a generic term for a substance added in trace amounts to feed for nutritional or specific purposes, and in the present invention, it means a substance added for the purpose of preventing or improving inflammatory diseases. Here, an animal is a concept including livestock and pets.

The feed additive of the present invention may further include a binder, emulsifier, preservative, etc. added to prevent quality degradation, and amino acid, vitamin, enzyme, probiotic, flavoring, non-protein nitrogen added to increase utility A compound, a silicate, a buffer, a colorant, an extractant, an oligosaccharide, etc. may be additionally included, and in addition, a feed mixture may be additionally included, but the present invention is not limited thereto.

In another aspect of the present invention, there is provided a cosmetic composition for relieving skin inflammation comprising a Big Valley hippocampus (Hippocampus abdominalis) extract or a fraction thereof.

In the present invention, the description of the Big Valley hippocampus (Hippocampus abdominalis ), extracts, and fractions is as described above.

Relieving skin inflammation in the present invention may be relieving inflammatory skin degeneration caused by irritation causing inflammation.

The inflammatory skin degeneration refers to the degeneration of the skin caused by external inflammatory stimulation. More specifically, it may include hypercytosis, intercellular edema, and spongiform epithelial in which the thickness of the epidermal layer increases, but is not limited thereto.

In the present invention, the skin inflammation refers to all skin inflammatory diseases accompanied by an inflammatory reaction occurring in the skin. More specifically, it may be any one selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhea, and acne. Sebaceous gland nevus, seborrheic keratosis, melanocytosis, actinic keratosis, cutaneous fibroids, psoriasis, chronic eczema, tuberculosis or deep mycosis, infectious granulomatous granulocyte tumors, acute contact dermatitis, monetary eczema mainly due to lichen planus and spongiform spongiformis , Herpes zoster, hydrophobic pseudosensitization, hydrophobic dermatophytes, but are not limited thereto.

The cosmetic composition of the present invention may include, without limitation, ingredients that are generally permitted in addition to the active ingredients, and may include conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and a carrier. have.

The cosmetic composition according to the present invention is a solution, external ointment, cream, foam, nutrient lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleaning agent, bathing agent, sunscreen cream, sun oil, suspension, Emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, can be prepared in one or more formulations selected from the group consisting of patches and sprays, but It is not limited.

In addition, the cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers blended in general skin cosmetics, and as common ingredients, for example, oil, water, surfactants, moisturizers, lower alcohols, A thickener, a chelating agent, a colorant, a preservative, a fragrance, and the like may be appropriately blended, but the present invention is not limited thereto. The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation.

When the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures of these can be used.

When the formulation of the present invention is a powder, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component.

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, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty ester, polyethylene glycol or fatty acid ester of sorbitan may be used. have.

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 and polyoxyethylene sorbitan ester, micro Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

When the formulation of the present invention is a soap, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolyzate, isethionate, a lanolin derivative, an aliphatic alcohol, vegetable oil, glycerol, sugar, etc. may be used as a carrier component. I can.

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, sarcositate, 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 Big Valley hippocampus extract according to the present invention inhibits NO production induced by LPS in RAW 264.7 cells, and expresses iNOS and COX-2 proteins and pro-inflammatory cytokines TNF-α and IL-6, IL-12. Effectively inhibited. Therefore, the Big Valley hippocampus extract can be used for preventing, ameliorating or treating inflammatory diseases or/and alleviating skin inflammation.

1 is a result of analysis of cytotoxicity according to the treatment concentration of the ethanol extract of Big Valley hippocampus in Raw 264.7 cells.
2 is a result of analyzing the inhibition of NO production according to the treatment concentration of the ethanol extract of the Big Valley hippocampus.
3 is a result of ELISA analysis of the protein expression change of _{PGE 2} according to the treatment concentration of the ethanol extract of Big Valley hippocampus.
4 is a result of RT-PCR analysis of the mRNA expression change of inflammation regulators (iNOS, COX-2) according to the treatment concentration of the ethanol extract of the Big Valley hippocampus.
5 is a result of Western blot analysis of protein expression changes of inflammation regulators (iNOS, COX-2) according to the treatment concentration of the ethanol extract of the Big Valley hippocampus.
6 is a result of RT-PCR analysis of the change in mRNA expression of inflammatory cytokines (IL-6, IL-12, TNF-α) according to the treatment concentration of the ethanol extract of Big Valley hippocampus and IL-6, IL-12 and TNF This is the result of ELISA analysis of the expression change of -α protein.
7 is a result of analyzing the effect of inhibiting the activity of NF-κB in the cell nucleus according to the treatment concentration of the ethanol extract of the Big Valley hippocampus.
8 is a result of analyzing the amino acid composition of the ethanol extract of Big Valley hippocampus.

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

<Materials and methods>

1. Preparation of ethanol extract of Big Valley hippocampus

The pulverized Big Belly hippocampus ( Hippocampus abdominalis ) 23g was added 230ml of 60% ethanol, extracted for 2 hours at 60 ℃, this was repeated three times. The ethanol extract of H. abdominalis (EEHA) from Big Valley hippocampus was filtered, concentrated in a vacuum condition, and lyophilized (yield 26.9%).

2. Analysis of Components of Ethanol Extract of Big Valley Hippocampus

2.1 Analysis of crude protein, crude lipid, ash and carbohydrate content

Crude protein content was analyzed using automatic Kjeltec Analyzer Unit 2300 (FOSS, Sweden).

The crude lipid (fatty acid) content was determined by Folch et al. Total lipids were extracted using a mixed solvent of chloroform/methanol (2:1, v/v) according to the method of (1957), and fatty acids were methylated with a 14% BF3-methanol (Sigma, USA) solution. Then, a gas chromatography (PerkinElmer, Clarus 600, USA) equipped with a capillary column (SPTM-2560, 100 m × 0.25 mm id, film thickness 0.20 um, USA) was performed and analyzed.

The ash content was analyzed by burning at 550° C. for 4 hours in a muffle furnace. The content of carbohydrates was analyzed using the phenol-sulfuric acid method.

2.2 Amino acid composition analysis

The amino acid content of the Big Valley hippocampus extract was analyzed using Dionex UltiMate 3000 HPLC and UHPLC system (Thermo Fisher Scientific, Sunnyvale, CA) at the Institute of Agricultural Life Sciences (Seoul National University).

For the analysis of the amanic acid content, 30 ml of 6N HCl was added to a sample having a protein amount of 2 to 3 mg, followed by hydrolysis at 130°C for 24 hours. After the hydrolysis was completed, it was diluted with ultrapure water, filtered through a 0.45 μm aqueous syringe filter, neutralized, diluted with tertiary distilled water, and analyzed by HPLC.

3. Cytotoxicity

Flow cytometric analysis was performed using the Muse® Count & Viability Assay Kit (Millipore, Billerica, MA) to measure the total number of cells, cell viability, and apoptotic cells.

First, Raw 264.7 cells ^{were inoculated at a density of 1×10 5} cells/ml, and cultured at 37° C. for 12 hours. Next, the ethanol extract of Big Valley hippocampus (EEHA) was treated at a concentration of 0 to 1000 μg/ml and incubated for 24 hours.

The cells were recovered, washed with cold PBS, reacted for 5 minutes with Muse® Count & Viability Assay Kit, and analyzed using Muse® cellcycler.

Apoptosis induction control was _{treated with H 2} O ₂ (100 μM).

4. NO generation analysis

RAW 264.7 cells were inoculated into a 24-well plate at ^{1×10 5 cells/ml and cultured.} Big Valley hippocampus ethanol extract (EEHA) 0 to 500 µg/ml or/and LPS 500 ng/ml were treated for 24 hours. Thereafter, the culture supernatant was recovered and the amount of NO production was analyzed using Griess reagent.

Griess reagent was prepared by mixing 5% phosphoric acid with 1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride.

Each sample was mixed with the same volume of Griess reagent, and reacted at room temperature for 15 minutes. Thereafter, absorbance was measured at 540 nm using a microplate reader. The concentration of nitrite was calculated using the standard concentration of sodium nitrite.

5. RNA isolation and quantification (Reverse transcriptase polymerase chain reactions, RT-PCR)

RT-PCR was performed to analyze the effect of the ethanol extract of Big Valley hippocampus on the mRNA expression of iNOS, COX-2, TNF-α, IL-6 and IL-12.

RAW 264.7 cells were inoculated into a 24-well plate at ^{1×10 5 cells/ml and cultured.} The cells were treated with ethanol extract of Big Valley hippocampus (EEHA) from 0 to 500 µg/ml or/and LPS 500 ng/ml for 9 hours.

Total RNA was isolated from Raw 264.7 cells using Easy Blue (iNtRON Biotechnology, Seongnam, Korea). The isolated RNA was reverse transcribed using the M-MLV reverse transcriptase kit (Promega, Madison, WI).

For cDNA, PCR was performed under the following conditions using a primer set specific to iNOS, COX-2, IL-6, TNF-α, IL-12p35 or GAPDH (Table 1).

1) iNOS, COX-2, IL-6

PCR was performed by repeating 25 cycles of denaturation at 95°C for 45 seconds, annealing at 55°C for 45 seconds, and extending for 1 minute at 72°C.

2) TNF-α

PCR was performed by repeating 25 cycles of denaturation at 95°C for 45 seconds, annealing at 53°C for 45 seconds, and extending for 1 minute at 72°C.

3) IL-12p35

PCR was performed by repeating 25 cycles of denaturation at 95°C for 45 seconds, annealing at 61°C for 45 seconds, and extending for 1 minute at 72°C.

At this time, GAPDH was used as an internal control to evaluate the relative expression of iNOS, COX-2, TNF-α, IL-6 and IL-12.

Gene Forward primers (5'→3') Reverse primers (5'→3') iNOS CCT CCT CCA CCC TAC CAA GT
(SEQ ID NO: 1) CAC CCA AAG TGC TTC AGT CA
(SEQ ID NO: 2) COX-2 TGC TGT ACC AGC AGT GGC AA
(SEQ ID NO:3) GCA GCC ATT TCC TTC TCT CC
(SEQ ID NO: 4) TNF-α ATG AGC ACA GAA AGC ATG AT
(SEQ ID NO: 5) TAC AGG CTT GTC ACT GA AT
(SEQ ID NO: 6) IL-6 AAG TGC ATC ATC GTT GTT TTC A
(SEQ ID NO: 7) GAG GAT ACC ACT CCC AAC AG
(SEQ ID NO: 8) IL-12p35 AAG ACA TCA CAC GGG ACCC AA
(SEQ ID NO: 9) GAG GAT ACC ACT TCC CAA CAG
(SEQ ID NO: 10) GAPDH AGG TCG GTG TGA ACG GAT TTG
(SEQ ID NO: 11) TGT AGA CCA TGT AGT TGA GGT CA
(SEQ ID NO: 12)

6. Western Blot Analysis

RAW 264.7 cells were inoculated and cultured at ^{1×10 5 cells/ml.} Big Valley hippocampus ethanol extract (EEHA) 0 to 500 ㎍ / ㎖ or / and LPS 500 ng / ㎖ was treated and incubated for 24 hours.

Cell extracts were prepared using pro-prep protein extraction solution (iNtRON Biotechnology). After recovering the cultured cells and lysing the cells by treating the pro-prep protein extraction solution on ice for 30 minutes, the lysates were centrifuged at 14,000 × g at 4° C. for 10 minutes to obtain supernatants. I did.

The separated extract was measured for protein concentration by Bradford analysis (Bio-Rad, Hercules, CA), and the sample was stored at -80°C or immediately used for Western blot analysis.

Protein was separated by electrophoresis on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and 5% in TBS-T (1 x TBS, 0.1% Tween-20, pH 7.4). The membrane was blocked with skim milk at room temperature for 12 hours and then incubated overnight with primary antibody at 4°C. After washing the unreacted primary antibody, it was reacted with HRP (horseradish peroxidase)-bound secondary antibody for 2 hours at room temperature, and protein expression was confirmed using an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL). . At this time, GAPDH was used as an internal control.

In addition, in order to analyze the inhibitory effect of NF-kB activity, the protein expression level of p50 or p65 in the cell nucleus was analyzed by Western blot. Specifically, cytosolic and nuclear extracts were prepared from cells using NE-PER nuclear and cytosolic extraction reagents (Pierce, Rockford, IL), and Western blot was performed in the same manner as above.

7. Enzyme-linked immunosorbent assay (ELISA)

RAW 264.7 macrophages were treated with ethanol extract of Big Valley hippocampus or/and 500 ng/ml of LPS at a concentration of 0 to 500 μg/ml. The culture supernatant without cells was recovered, and the concentration of PGE2, TNF-α, IL-6 or IL-12 was measured using an ELISA kit (R&D Systems, Minneapolis, MN).

<Result>

1. Cytotoxicity analysis

In order to analyze the cytotoxicity of the ethanol extract of the Big Valley hippocampus, RAW 264.7 macrophages were treated with extracts of 0, 125, 250, 500 and 1000 µg/ml and cultured for 24 hours and then cytotoxicity was measured. At this time, the control group was treated with _{H 2} O _2.

The ethanol extract of the Big Valley hippocampus did not show cytotoxicity for 24 hours even at a concentration of 500 μg/ml or less, but it was confirmed that it exhibited higher cytotoxicity than the _{control H 2} O _{2 at 1000 μg/ml.} Therefore, in the subsequent experiment, a concentration of 500 μg/ml or less, which does not exhibit cytotoxicity, was used (Fig. 1).

2. Checking the effect of inhibiting inflammation

2.1 NO and PGE ₂ Regulation of expression of

Next, in order to confirm the inflammation inhibitory effect of the ethanol extract of Big Valley hippocampus, the changes in the expression _{of NO and PGE 2, which are inflammatory factors, were analyzed.}

As a result of NO production analysis, it was confirmed that only the ethanol extract of Big Valley hippocampus did not affect NO production in the cells treated with 500 µg/ml. On the other hand, it was confirmed that NO production increased in the cells induced inflammation with LPS (500 ng/ml), and it was confirmed that NO production induced by LPS was inhibited as the treatment concentration of the ethanol extract of the Big Valley hippocampus increased (FIG. 2) ).

In addition _{, as a result of ELISA analysis of the PGE 2} protein expression, similarly to this, it was confirmed that the protein expression of _{PGE 2} induced by LPS was inhibited as the concentration of the ethanol extract of the Big Valley hippocampus increased (FIG.

2.2 Inflammation regulator iNOS and COX-2 expression regulation

In addition, as a result of analyzing the mRNA and protein expression of inducible nitric oxide synthase (iNOS) involved in NO production and _{cyclooxygenase-2 (COX2) that regulates the expression of PGE 2} by RT_PCR and Western blot, treatment with ethanol extract of the Big Valley hippocampus As the concentration increased, the expression of iNOS or COX2 mRNA induced by LPS decreased (FIG. 4), and it was confirmed that the expression of these proteins decreased (FIG. 5).

3. Inhibition of the expression of inflammatory cytokines

Next, the effect of inhibiting the expression of inflammatory cytokines IL-6, IL-12, and TNF-α of the ethanol extract of Big Valley hippocampus was confirmed through RT-PCR and ELISA analysis.

As a result of the analysis, it was confirmed that the mRNA or protein expression of the inflammatory cytokines IL-6, IL-12, and TNF-α was increased during LPS treatment, and the IL-6 in cells treated with both LPS and Big Valley hippocampus ethanol extract , IL-12, TNF-α mRNA or protein expression was confirmed to be significantly reduced compared to the cells treated with LPS alone (Fig. 6).

That is, it was confirmed that the ethanol extract of the Big Valley hippocampus has an anti-inflammatory effect by inhibiting the expression of inflammatory cytokines.

4. Inhibition of NF-kB activity in the nucleus

For transcription mediated by NF-kB after exposure to LPS, the NF-kB p50/p65 heterodimer must be transferred to the nucleus. Therefore, when NF-κB is activated, its concentration increases in the nucleus.

In order to confirm the NF-kB inhibitory effect of the ethanol extract of Big Valley hippocampus, the protein expression level of p50 or p65 in the cell nucleus according to the concentration of the ethanol extract of Big Valley hippocampus in RAW264.7 macrophages stimulated with LPS was analyzed by Western blot. . At this time, Nucleolin was used as an internal control.

As a result of the analysis, it was confirmed that the expression of p50 and p65 hardly appeared in the nucleus of the cells not treated with LPS, and the expression of p50 and p65 was increased by the LPS treatment. On the other hand, as the concentration of the ethanol extract of the Big Belly hippocampus increased, the expression of p50 and p65 decreased, and the expression of p50 and p65 was hardly observed in the nuclei of the cells treated with the ethanol extract of 500 ng/ml. Therefore, it was confirmed that the ethanol extract of the Big Valley hippocampus effectively inhibited the activity of NF-kB induced by LPS (FIG. 7).

5. Analysis of composition

The component analysis results of the ethanol extract of the Big Valley hippocampus are shown in Table 2. Crude protein showed the highest content of 81.8% ± 1.2%, crude lipid 3.5% ± 0.2%, ash content 12.2% ± 0.2%, and carbohydrate content 4.6% ± 1.2% (Table 2).

EEHA (%) Crude protein 81.8 ± 1.2 Crude lipid 3.5 ± 0.2 Crude ash 12.2 ± 0.2 Carbohydrate 4.6 ± 1.2

Therefore, it was confirmed that the ethanol extract of the Big Belly hippocampus was rich in protein. The content of each amino acid component in the ethanol extract of the Big Belly hippocampus was analyzed, and the concentration of the total amino acids in the ethanol extract was calculated as %. As a result of the analysis, the content of glycine was the highest at 21.5%. Subsequently, it was confirmed that 12.5% of glutamic acid and 11.4% of alanine were included (Table 3).

amino acid density(%) Aspartic acid 6.5 Glutamic acid 12.5 Serine 5.3 Histidine 1.7 Glycine 21.5 Threonine 3.4 Arginine 8.7 Alanine 11.4 Taurine 0.6 Tyrosine 1.0 Valine 4.1 Methionine 2.3 Phenylanine 3.2 Isoleucine 2.0 Leucine 3.5 Lysine 3.6 Proline 8.7

<110> Jeju National University Industry-Academic Cooperation Foundation <120> Composition for preventing or treating inflammatory disease comprising Hippocampus abdominalis extract or fractions thereof <130> DP20190215 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS forward primer <400> 1 cctcctccac cctaccaagt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS reverse primer <400> 2 cacccaaagt gcttcagtca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 forward primer <400> 3 tgctgtacca gcagtggcaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 reverse primer <400> 4 gcagccattt ccttctctcc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha forward primer <400> 5 atgagcacag aaagcatgat 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha reverse primer <400> 6 tacaggcttg tcactgaat 19 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL-6 forward primer <400> 7 aagtgcatca tcgttgtttt ca 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 reverse primer <400> 8 gaggatacca ctcccaacag 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-12p35 forward primer <400> 9 aagacatcac acgggaccca a 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-12p35 reverse primer <400> 10 gaggatacca cttcccaaca g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 11 aggtcggtgt gaacggattt g 21 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 12 tgtagaccat gtagttgagg tca 23

Claims (7)

Big Valley hippocampus ( Hippocampus abdominalis ) extract or a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a fraction thereof. In claim 1, wherein the extract is a pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that the extract is extracted using _{water, an alcohol having 1 (C 1} ) to 4 (C _{4) carbon atoms or a mixture thereof as a solvent.} The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 2, wherein the alcohol is ethanol. Big Valley hippocampus ( Hippocampus abdominalis ) extract or a quasi-drug composition for the prevention or improvement of inflammatory diseases comprising a fraction thereof. Big Belly hippocampus ( Hippocampus abdominalis ) extract or a food composition for preventing or improving inflammatory diseases comprising a fraction thereof. Big Belly hippocampus ( Hippocampus abdominalis ) feed additive composition for preventing or improving inflammatory diseases comprising an extract or a fraction thereof. A cosmetic composition for relieving skin inflammation comprising a Big Valley hippocampus extract or a fraction thereof.

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