KR20200078803A - An anti-inflammatory or immunological enhancing composition comprising a Halocynthia aurantium tunic lipids as an active ingredient - Google Patents

Abstract

The present invention relates to an anti-inflammatory or immunity function enhancing composition containing Halocynthia aurantium tunic lipid as an active component. Lipid extracted from Halocynthia aurantium tunic of the present invention has effects of inhibiting the production of TNF-α, IL-6, and IL-1β, inhibiting the expression of COX-2, and iNOS, inhibiting the signaling pathways of MAPK, and NF-κB, and producing nitric oxide (NO), thereby being useful as the anti-inflammatory or immunity function enhancing composition.

Description

An anti-inflammatory or immunological enhancing composition comprising a Halocynthia aurantium tunic lipids as an active ingredient}

The present invention relates to a composition for enhancing anti-inflammatory or immune function, which includes a reddish sea urchin tunic lipid as an active ingredient.

The red sea urchin ( Halocynthia aurantium ) belongs to the primary animal family, the off-white river, the lateral black-eyed tree, and the squirrel family, and is called a sea peach because it has a peach-like form. Red sea urchins are mainly distributed in the North Sea of Japan, the Bering Sea of the United States, the Alaskan Peninsula, the northern coast of Canada and the east coast of Korea, and the spawning season is known between September and December. The habitat depth is about 20~100m. In the province, it is called dog sea urchin and silk sea urchin. The red yoke is about 5 to 20 cm in length and 5 to 15 cm in diameter, about the size of a human fist. At the top, there are nipple-like protrusions, and under the body, they are shaped like seaweed roots and inhabit the rocks.

The red yoke is surrounded by a leather-like tunic made of cellulose-like tunisine. There are two holes in the upper part. The shape of the cross (+) is the inlet hole and the shape of the straight (-) is the exit hole. In the case of an adult, it is 18cm long, 10cm in diameter, and weighs up to 700g.

Inflammatory disease is one of the most important health problems in the world, and inflammation is a localized protective response of body tissues to host invasion, usually by foreign substances or harmful stimuli. In most cases, these inflammatory responses lead to the elimination of pathogenic factors using components of innate immunity and induction of specific adaptive immunity, resulting in a continuous immune response (Hawiger J., Innate Immunity and Inflammation: A Transcriptional Paradigm.Immunologic Research .23, pp.99-109, 2001). Causes of inflammation include infectious causes such as bacteria, viruses and parasites, physical causes such as burns and irradiation, chemical causes such as toxins, drugs and industrial agents, immune causes such as allergies and autoimmune reactions, and oxidative stress It has oxidative causes, and is characterized by pain, swelling, fever, and ultimate loss of function in the infected area by a series of complex interactions between immune system cells.

Specifically, proteins such as cytokines, enzymes (protease, peroxidase), major basic proteins, adhesion molecules (ICAM, VCAM), lipid mediators (eicosanoids, prostaglandins, leukotriene, platelet activation factor (PAF)), Several groups of interaction mediators of inflammatory mediators such as reactive oxygen species (hydroperoxide, superoxide anion O2-, nitrogen oxide (NO)) are created, and the lack of an inflammatory response causes damage and infection without host control. It causes autoimmune diseases and inflammatory diseases. On the other hand, infection or autoimmune disease may cause chronic inflammation as a result of continuous immune activation.

The diet and ingredients consumed through it supply the fuel needed for immune cells to maintain energy homeostasis and cellular processes in normal homeostasis, and the health of the human body related to the immune system, adipokines secretion and metabolic pathways Plays an important role in Lipids containing fat-soluble vitamins and essential fatty acids are important components for human health and development, and play an important role in preventing diseases according to changes in composition (fatty acid composition). In particular, free fatty acids (FFA) are related to immune function and regulation, and include saturated fatty acids (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid; PUFA) is known to have a significant association with chronic diseases such as cardiovascular disease (CVD), cancer and diabetes.

On the other hand, red sea urchin contains a fat component called'Plasmarogen', which is known to help prevent senile dementia, and contains a mineral called'Vadium', which helps prevent and treat diabetes. It is known that it contains'cinthiaol', which helps to relieve hangover. It contains'glycogen', which is known to have an excellent effect on preventing asthma as well as preventing colds. There is no known information about the effectiveness of treating inflammatory diseases.

Korean Registered Patent No. 10-0954865 Korean Registered Patent No. 10-0954866

Accordingly, the present inventors confirmed that it is possible to reduce the production of NO in macrophages and to reduce the expression of TNF-α, IL-1β, IL-6 and COX-2 induced by LPS in lipids extracted from the reddish sea urchin tunic. , It was confirmed that it is suitable for use as an anti-inflammatory composition, an immune function enhancing composition, and completed the present invention.

Accordingly, an object of the present invention is to provide an anti-inflammatory composition comprising a lipid extracted from a red sea urchin ( Halocynthia aurantium ) tunic as an active ingredient.

In addition, an object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, including as an active ingredient a lipid extracted from red sea urchin ( Halocynthia aurantium ) tunic.

In addition, an object of the present invention is to provide a health food for preventing or improving inflammatory diseases, including lipids extracted from red sea urchin ( Halocynthia aurantium ) tunic.

In addition, an object of the present invention is to provide a cosmetic composition for preventing or improving inflammation comprising a lipid extracted from a red sea urchin ( Halocynthia aurantium ) tunic as an active ingredient.

Furthermore, an object of the present invention is to provide a composition for enhancing immune function, which includes lipid extracted from a red sea urchin ( Halocynthia aurantium ) tunic as an active ingredient.

In order to achieve the object of the present invention as described above, the present invention provides an anti-inflammatory composition comprising a lipid extracted from a red sea urchin ( Halocynthia aurantium ) tunic (tunic) as an active ingredient.

In one embodiment of the invention, the composition is an inflammatory cytokine tumor necrosis factor-alpha (TNF-α; Tumor Necrosis Factor-α), interleukin 6 (IL-6; interleukin 6) or interleukin 1 beta (IL- 1β; interleukin 1 beta) can be inhibited, and the expression of the inflammatory expression protein cyclooxygenase-2 (COX-2) or inducible nitric oxide synthase (iNOS) can be suppressed.

In one embodiment of the invention, the composition inhibits the signaling pathway of mitogen-activated protein kinase (MAPK) or the signaling pathway of nuclear factor kappa B (NF-κB; Nuclear Factor kappa B) Can be suppressed.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising lipid extracted from red sea urchin ( Halocynthia aurantium ) tunic as an active ingredient.

In one embodiment of the invention, the inflammatory disease is dermatitis, dermatomyositis (dermatomyositis), multiple myositis (polymyositis), allergies, systemic lupus erythematosus, pemphigus, aphthous stomatitis, retinitis, gastritis, hepatitis, bronchitis, esophagitis, enteritis , Pancreatitis, colitis, nephritis, bedsore, lupus, chronic thyroiditis, multiple sclerosis, sepsis, radiation damage, rejection of organ transplantation, systemic edema, and local edema.

In addition, the present invention provides a health food for preventing or improving inflammatory diseases, including lipid extracted from red sea urchin ( Halocynthia aurantium ) tunic as an active ingredient.

In addition, the present invention provides a cosmetic composition for preventing or improving inflammation including lipid extracted from a red sea urchin ( Halocynthia aurantium ) tunic as an active ingredient.

Furthermore, the present invention provides a composition for enhancing immune function, which includes lipid extracted from a red sea urchin ( Halocynthia aurantium ) tunic as an active ingredient.

Lipids extracted from the red sea urchin tunic of the present invention inhibit the production of TNF-α, IL-6, IL-1β, inhibit the expression of COX-2, iNOS, inhibit the signaling pathways of MAPK, NF-κB and NO (nitric oxide, NO) has the effect of producing an anti-inflammatory or immune function enhancing composition that can be usefully used.

1 is a result of confirming the cytotoxicity by varying the concentration to measure the lipid concentration of the red yoke tunic, which is not toxic to the RAW264.7 macrophage.
Figure 2 is a result confirming the effect of lipids of the red snail tunic tunic on the production of nitric oxide in LPS-stimulated RAW264.7 cells.
Figure 3 is a result confirming the effect of red mud tunic lipid on inflammatory cytokine mRNA expression in LPS-stimulated RAW264.7 cells.
Figure 4 is a result confirming the effect of the lipids of the red yoke tunic on LPS-stimulated RAW264.7 cells on the MAPK and NF-κB signal transduction pathway.

Hereinafter, the present invention will be described in detail.

Pharmaceutical composition for preventing or treating inflammatory diseases

The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising lipid extracted from red sea urchin tunic as an active ingredient.

Lipopolysaccharide (LPS), an endogenous substance, is an inducer of inflammation in dendritic cells and promotes the production of pro-inflammatory cytokines that cause an inflammatory reaction. That is, when an external stimulus capable of causing an inflammatory reaction is applied, expression of inflammatory cytokines such as TNF-α is induced, and the resulting inflammatory cytokines stimulate expression of genes encoding iNOS and COX-2, It produces NO and PGE ₂ substances involved in the inflammatory reaction, causing an inflammatory reaction.

Therefore, if the inflammation-causing substances of the inflammatory cytokines of TNF-α, IL-6 or IL-1β are excessively secreted or the cells themselves are activated, they cause serious side effects of tissue damage.

In one embodiment of the present invention, the lipid extracted from the red scab tunic is tumor necrosis factor-alpha (TNF-α; Tumor Necrosis Factor-α) in macrophages (RAW 264.7) stimulated by lipopolysaccharide (LPS). , Interleukin 6 (IL-6; interleukin 6) or interleukin 1 beta (IL-1β; interleukin 1 beta) inhibits the production of inflammatory proteins, cyclooxygenase-2 (COX-2) or inducible nitric oxide Inhibits the expression of protease (iNOS) and inhibits the signaling pathway of mitogen-activated protein kinase (MAPK) or the signaling pathway of nuclear factor kappa B (NF-κB; Nuclear Factor kappa B) Suppress.

In the present invention, the inflammatory disease refers to various diseases arising from inflammation, but is not limited thereto, specifically, dermatitis, dermatomyositis, polymyositis, allergy, systemic lupus erythematosus, pemphigus, Aphthous stomatitis, retinitis, gastritis, hepatitis, bronchitis, esophagitis, enteritis, pancreatitis, colitis, nephritis, bedsore, lupus, chronic thyroiditis, multiple sclerosis, sepsis, radiation damage, rejection of organ transplant, generalized edema and local edema It may be any one selected from the group consisting of.

In the present invention, the lipids (lipids) refers to lipids contained in the red sea urchin tunic, specifically, lipids composed of free fatty acids, pure hydrocarbons, and the like.

In addition, the lipid may mean any biological molecule dissolved in an organic solvent. Most lipids contain fatty acids, and in general, triglycerides and phospholipids (PLs), glycolipids, including two categories, acylglycerols and free fatty acids (FFA), depending on the molecular head group polarity (GL: Glycolipids) can be classified as polar fats.

The reddish sea urchin tunic lipid of the present invention can be administered in various dosage forms, oral and parenteral, during clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. It is prepared using.

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, etc. These solid preparations contain at least one excipient such as starch, carbonic acid in one or more of the present invention. It is prepared by mixing calcium, sucrose, lactose, or gelatin. In addition, lubricants such as magnesium stearate talc are used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions or syrups, etc. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, etc. Can.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspension solutions, emulsions, lyophilized preparations, suppositories, and the like. Non-aqueous solvents, suspension solvents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used.

In addition, the effective dosage of the red bruising tunic lipid of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health status and disease level, and is generally about 0.001 to 100 mg/kg/day. And, preferably 0.01 to 35 mg/kg/day. Based on an adult patient weighing 70 kg, it is generally 0.07 to 7000 mg/day, preferably 0.7 to 2500 mg/day, and once a day at regular time intervals according to the judgment of a doctor or pharmacist It can also be administered in multiple doses.

Cosmetic composition

The present invention provides a cosmetic composition for preventing or improving inflammation including lipid extracted from red sea urchin ( Halocynthia aurantium ) tunic as an active ingredient.

The formulation of the cosmetic composition of the present invention is not particularly limited, but is preferably a formulation for external application for skin, and one preferred form of the formulation for external application for skin is softening lotion, longevity, essence, massage cream, nourishing cream, pack, cleanser , A cosmetic composition having a gel or skin adhesive type cosmetic formulation, and other preferred forms include a transdermal dosage form such as lotion, ointment, gel, cream, patch or spray. In addition, in the composition for external preparation of each formulation, components other than the above-described essential components can be formulated by selecting a suitable one without difficulty by those skilled in the art depending on the formulation or purpose of use of the external formulation.

The cosmetic composition of the present invention preferably contains a cosmetically acceptable medium or base. These are all formulations suitable for topical application, for example solutions, gels, solid or dough anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspension microemulsions, microcapsules, microgranules or ionic (liposomes) and/or bi It can be provided in the form of a warm vesicle dispersant, or in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick. It can also be prepared in the form of a foam or aerosol composition further containing a compressed propellant. In addition, the cosmetic composition of the present invention can be prepared in the form of an adjuvant that can be applied topically or systemically, which is commonly used in dermatology by containing a dermatologically acceptable medium or base, and these compositions are conventional in the art. It can be prepared according to the method.

When the formulation of the present invention is a paste, cream, or gel, animal fibers, plant fibers, wax, paraffin, starch, trakant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. may be used as carrier components. Can.

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

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or 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-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, suspensions such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar or trakant, etc. can be used.

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

Formulations of the present invention may further contain excipients, including fluorescent substances, fungicides, myelogens, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, etc. .

When formulating a cosmetic composition as a pharmaceutical or cosmetic as described above, it may include a known excipient applicable to the skin acting as a carrier for the active ingredient. When formulating into pharmaceuticals, the contents disclosed in [Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA] can be referred to. When formulated into cosmetics, [International cosmetic ingredient dictionary, 6th ed., The cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995. The above documents are included as part of the present specification.

For preventing or improving inflammation Health functional food and health food

The present invention provides a health functional food or health food for preventing or improving inflammatory diseases, including lipid extracted from red sea urchin ( Halocynthia aurantium ) tunic as an active ingredient.

In the present invention, the inflammatory disease refers to various diseases arising from inflammation, but is not limited thereto, specifically, dermatitis, dermatomyositis, polymyositis, allergy, systemic lupus erythematosus, pemphigus, Aphthous stomatitis, retinitis, gastritis, hepatitis, bronchitis, esophagitis, enteritis, pancreatitis, colitis, nephritis, bedsore, lupus, chronic thyroiditis, multiple sclerosis, sepsis, radiation damage, rejection of organ transplant, generalized edema and local edema It may be any one selected from the group consisting of.

When the red sea urchin tunic lipid of the present invention is used as a health functional food and a health food, there are no particular restrictions on the type of food. Examples of foods to which the red sea urchin tunic lipid of the present invention can be added are drink, meat, sausage, bread, biscuit, rice cake, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams Products, various soups, beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, etc., and include both functional health foods and health foods in the ordinary sense.

Health functional foods and health foods containing red sea urchin tunic lipids according to the present invention may be added as such to foods or used with other foods or food ingredients, and may be suitably used according to conventional methods. The mixing amount of the reddish blue seaweed tunic lipid may be appropriately determined according to its purpose of use (for prevention or improvement). In general, the amount of the red yoke tunic lipid in the dietary supplement and health food can be added in 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for the purpose of maintaining health or for the purpose of controlling health, the amount may be below the above range, and since there is no problem in terms of safety, the reddish blue tunic lipid may be used in an amount above the above range. .

The health functional foods and health foods of the present invention are essential ingredients in the indicated proportions, and there are no particular limitations on other ingredients other than those containing the red sea urchin tunic lipid of the present invention, and additional ingredients such as various flavors or natural carbohydrates, such as ordinary drinks It can contain as. Examples of the natural carbohydrates described above include monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, etc.; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (for example, rebaudioside A, glycyrrhizine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 health functional foods and health foods of the present invention.

In addition to the above, the health functional food and health food containing the red sea urchin tunic lipid of the present invention are various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate Etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonic acid used in carbonated beverages, and the like. In addition, the health functional foods and health foods of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice beverages and vegetable beverages.

These ingredients can be used independently or in combination. The proportion of these additives is not so critical, but is generally selected from the range of 0.1 to about 20 parts by weight per 100 parts by weight of dietary supplements and dietary supplements containing the reddish-tuned tunic lipid of the invention.

Immune function enhancing composition

The present invention provides a composition for enhancing immune function, which includes lipid extracted from the red sea urchin ( Halocynthia aurantium ) tunic as an active ingredient.

In one embodiment of the present invention, the red sea urchin tunic lipid of the present invention increases the production of nitric oxide; Increase the expression of immune response-related factors; It is characterized by activating an immune response by increasing phosphorylation of nuclear factor kappa B (NF-κB; Nuclear Factor kappa B) transcriptional activity or mitogen-activated protein kinase (MAPK).

When a living body enters this substance from the outside, macrophages are activated for self-defense, producing phagocytosis and various immunomodulatory substances. NO, one of macrophage activation indicators, is a reactive nitrogen intermediate produced in macrophages under the influence of cytokines or microorganisms. It is combined with NOS (nitric oxide synthase) and oxygen to oxidize L-arginine to produce NO. . The generated NO is reported to control microorganisms and cancer cells that cause intracellular infection, and when NO is generated more than necessary, it is known to cause vascular expansion, tissue damage caused by inflammatory reactions, mutations, etc., and exhibit harmful effects in vivo. . Therefore, NO exhibits a dual property, and it can be judged to increase immune function by promoting NO production at a concentration that does not induce cytotoxicity during drug treatment.

Therefore, before the external stimulation occurs, it is thought that the red bruising tunic lipid of the present invention can stimulate the immune cells to have a beneficial effect on the biological defense in the early stages of the immune response through the primary defense against infection such as foreign substance invasion. .

Hereinafter, the present invention will be described in more detail by examples. The following examples are only intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example 1>

Experimental materials and method

<1-1> Red sea urchin( H. aurantium )Sample

A sample of red sea urchin ( H. aurantium ) was taken from the East Sea near Gangwon-do, Korea. Red sea urchin ( H. aurantium ) tunic was collected for lipid extraction.

<1-2> Lipid extraction

Lipid samples were extracted from the red sea urchin ( H. aurantium ) tunic according to Bligh and Dyer's modified method.

Briefly described, a dry sample of 100 g was homogenized for 2 minutes in a mixture of chloroform and methanol at a ratio of 1: 2, 100 ml and 200 ml in Waring Blendor. Then, an equal volume of chloroform was added to the mixture. After the mixture was blended for 30 seconds, 100 ml of distilled water was added and the mixture was blended for 30 seconds. After centrifugation, homogeneous water was filtered through a Chm filter paper, and the filtrate was collected in a separatory funnel. After separating the filtrate into 2 layers, the volume of the chloroform layer was collected. The chloroform layer was dried and dissolved in DMSO to treat the cells.

<1-3> Macrophage proliferation assay

RAW264.7 cells in RPMI-1640 medium (supplemented with 10% FBS and 1% penicillin/streptomycin) at 1×10 ⁵ cells/well concentration were dispensed into 96-well plates. Plates were incubated for 24 hours at 37° C. in a humidified 5% CO ² atmosphere. Lipids (0.25%, 0.50%, 0.75% or 1.00% total lipid and 0.5%, 1.0%, 2.0% or 4.0% other lipids) were treated and incubated for another 24 hours. The experiment was performed in 3 replicates. The supernatant was discarded and the proliferation of macrophages was confirmed using the EZ-Cytox Cell Viability Assay Kit (Daeil Research Institute, Korea).

Macrophage proliferation rate (%) was calculated by the following formula.

<1-4> Nitrogen monoxide (NO) production analysis

The nitrite accumulation in the medium was used as an indicator of NO production. RAW264.7 cells were seeded in 96-well plates at a density of 1x10 ⁵ cells/well. After incubation for 24 hours, cells were treated with different concentrations of lipids and then stimulated for 24 hours with or without 1 μg/ml lipopolysaccharide (LPS). The nitrate concentration was evaluated using Griess reagent (Sigma-Aldrich, USA).

<1-5> RNA isolation and reverse transcription

RAW264.7 cells were treated according to the NO production analysis. Upon stimulation after 24 h LPS, total RNA was isolated from RAW264.7 cells using Tri reagent ^® (molecular research center, Inc., USA). RNA was precipitated using 100% isopropanol and washed with 75% ethanol. The RNA pellet was dissolved in nuclease-free water and the extracted RNA concentration was measured using a nanophotometer (Implen, Germany). cDNA was synthesized according to the manufacturer's instructions using a high-dose cDNA reverse transcription kit (Applied biosystems, USA).

<1-6> Immune gene expression analysis by quantitative real-time PCR

Immune response gene expression is achieved in a 96-well format with a total reaction volume of 20 μl/well using SYBR ^® Premix Ex Taq ™ II (Takara Bio Inc., Japan) in QuantStudio™ 7 FlexReal-Time PCR System (ThermoFisher scientific, USA) Confirmed. PCR amplification was performed under conditions of repeating 40 times of initial denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, and annealing at 55°C for 5 seconds.

The results were determined by comparing the normalized values to the cycle threshold (Ct) and β-actin values in the melting/dissociation curve step. In addition, the primer sequences used in the experiment of the present invention are shown in Table 1.

<1-7> Western blotting analysis

Raw264.7 cells were treated with various concentrations of red yolk lipids and then stimulated with or without 1 μg/ml LPS. After incubation for 24 hours, cells were harvested with RIPA buffer (Tech &Innovation, China). Protein concentration was measured using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein (30 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. Western blot analysis was conducted by Narayanan et al. Method. The membranes were p-NF-κB p65 (Cell Signaling Technolog), p-p38 (Cell Signaling Technolog), p-ERK1/2 (Cell Signaling Technolog), p-JNK (Cell Signaling Technolog) and α-Tubulin (Abcam), respectively. And incubated with specific antibodies. Protein signals were detected using Pierce® ECL Plus Western Blotting Substrate (Thermo Scientific, USA).

Blots were imaged and quantified using the ChemiDoc XRS+ imaging system (Bio-Rad) and ImageLab software (version 4.1, Bio-Rad).

<1-8> Statistical analysis

Statistical analysis was performed using the'Statistix 8.1' statistical software. Statistical differences were tested using one-way ANOVA, and Duncan's multiple range test was tested at P<0.05.

<Example 2>

Confirmation of the effect of red scab tunic lipid on RAW264.7 cell proliferation

Cytotoxicity was investigated using the EZ-Cytox Cell Viability Assay Kit to measure the lipid concentration of the red monk tunic, which is not toxic to the RAW264.7 macrophage.

As a result, as shown in FIG. 1, the total lipid concentrations of 0.25 and 0.50% did not show toxicity to RAW264.7 cells. Toxicity was lightly detected at 0.75% and more and more at total lipid concentration 1.00%.

<Example 3>

Determination of the effect of red yoke tunic lipids on nitric oxide production in LPS-stimulated RAW264.7 cells

In order to measure the anti-inflammatory activity of red scab tunic lipids, we measured lipid-mediated inhibition of NO production in RAW264.7 cells stimulated with LPS using Griess reagent analysis.

As a result of investigating the inhibition of NO production at various concentrations, as shown in FIG. 2, red lumped tunic lipid gradually inhibited NO production in LPS-stimulated RAW264.7 cells according to lipid concentration. The highest lipid concentration, 1.00%, showed an inhibitory effect on NO production similar to the non-stimulated group (RPMI).

<Example 4>

Determination of the effect of red bruising tunic lipids on inflammatory cytokine mRNA expression in LPS-stimulated RAW264.7 cells

Since NO production was significantly inhibited by the reddish blackened tunic lipids, we measured mRNA expression of inflammatory cytokines in LPS-stimulated RAW264.7 cells.

When 1 μg/ml LPS was added to RAW264.7 cells, expression of inflammatory cytokines was detected by qRT-PCR. In FIG. 3, the expression level of inflammatory cytokines was dose-dependently suppressed according to the concentration of red scab tunic lipid. First of all, the expression level of IL-1β was greatly reduced by the red scab tunic lipid, and the expression levels of other cytokine genes such as IL-6 and TNF-α also decreased dose-dependently. In addition, it was found that the expression level of COX-2, another well-known inflammatory biomarker, was dose-dependently down-regulated according to the concentration of red scab tunic lipid (see FIG. 3).

<Example 5>

Confirmation of the effect of red yoke tunic lipids on MAPK and NF-κB signaling pathways in LPS-stimulated RAW264.7 cells

In order to deepen the understanding of the molecular mechanisms by which red scab tunic lipid exerts anti-inflammatory effects, immune signaling activation such as NF-κB and MAPK was investigated. The phosphorylation level of NF-κB p-65 was measured by analyzing the effect of red lumped tunic lipid on the activation of NF-κB. We also analyzed phosphorylation of JNK, p38 and ERK1/2 to investigate their role in MAPK activation.

As a result, as shown in Figure 4, the red yoke tunic Lipids were found to be dose-dependently inhibited in the phosphorylation of NF-κB p-65 to the NF-κB signaling pathway, particularly in cells treated with phospholipids. Similar to the results of NF-κB signaling, red bruising tunic Lipids inhibited the phosphorylation levels of ERK1/2, JNK and p38 in a dose dependent manner.

The above results indicate that the lipids of the red sea urchin tunic inhibit inflammation through MAPK and NF-κB signaling pathways in RAW264.7 cells stimulated with LPS.

Accordingly, it was confirmed that the lipid extracted from the red sea urchin tunic of the present invention can reduce the production of NO in macrophages and reduce the expression of TNF-α, IL-1β, IL-6 and COX-2 induced by LPS. Thus, there is a technical feature in that it was confirmed that it is suitable for use as an anti-inflammatory composition.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Claims (8)

An anti-inflammatory composition comprising lipids extracted from red sea urchin ( Halocynthia aurantium ) tunic. According to claim 1,
The composition produces the production of tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) or interleukin 1 beta (IL-1β; interleukin 1 beta), which is an inflammatory cytokine. An anti-inflammatory composition characterized by inhibiting and inhibiting the expression of an inflammatory expression protein cyclooxygenase-2 (COX-2) or inducible nitric oxide synthase (iNOS). According to claim 1,
The composition is characterized in that it inhibits the signaling pathway of mitogen-activated protein kinase (MAPK; Mitogen-Activated Protein Kinase) or the signaling pathway of nuclear factor kappa B (NF-κB; Nuclear Factor kappa B). Red sea urchin ( Halocynthia aurantium ) A pharmaceutical composition for preventing or treating inflammatory diseases comprising lipid extracted from a tunic as an active ingredient. According to claim 4,
The inflammatory diseases include dermatitis, dermatomyositis, polymyositis, allergies, systemic lupus erythematosus, pemphigus, aphthous stomatitis, retinitis, gastritis, hepatitis, bronchitis, esophagitis, enteritis, pancreatitis, colitis, nephritis, bedsore, Lupus, chronic thyroiditis, multiple sclerosis, sepsis, radiation damage, rejection of organ transplantation, systemic edema, or edema. Red Lobster ( Halocynthia aurantium ) A health food for preventing or improving inflammatory diseases, which contains lipids extracted from tunic as an active ingredient. Red sea urchin ( Halocynthia aurantium ) A cosmetic composition for preventing or improving inflammation comprising lipid extracted from a tunic as an active ingredient. A composition for enhancing immune function, which contains lipids extracted from red sea urchin ( Halocynthia aurantium ) tunic as an active ingredient.

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