KR102566545B1 - Composition for anti-inflammation comprising extract of Racomitrium canescens as effective component - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition comprising an extract of Racomitrium canescens as an active ingredient. Since the effect of reducing the protein expression of IL-6 and IL-1β and the mRNA expression of iNOS and IL-6 increased by , or can be usefully used as a material for pharmaceuticals for treatment, health functional foods for anti-inflammatory, and cosmetics for anti-inflammatory.

Description

Composition for anti-inflammation comprising extract of Racomitrium canescens as effective component}

The present invention frost moss ( Racomitrium canescens ) Relates to an anti-inflammatory composition comprising an extract as an active ingredient.

Inflammation is a normal biological response that occurs when a harmful stimulus, that is, a physical wound or infection with microorganisms, is a protective response involving immune cells, blood vessels, and inflammatory mediators as one of the defense mechanisms in vivo. The purpose of such an inflammatory response is to inhibit cell damage at an early stage, neutralize or remove pathogens, and at the same time regenerate damaged tissue to achieve normal structure and function.

However, most diseases accompanied by inflammation can reduce quality of life by causing symptoms such as heat, swelling, pain and itching. In addition, in addition to acute inflammation caused by infection, physicochemical damage, etc., chronic inflammation may persist due to stress, including arthritis, asthma, autoimmune disease, heart disease, and the like, and lifestyle habits such as smoking and drinking.

In the case of acute inflammation, the immune system is activated as immune cells such as macrophages, dendritic cells, histiocytes, and Kupffer cells recognize stimuli. There are pathogen-associated molecular types (PAMPs) and damage-associated molecular types (DAMPs) on the surface of stimulated cells. When tissues are damaged, the immune cells recognize these molecules and transmit signals through transcription factors such as NF-κB　path begins. As a result, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 (IL-1), interleukin-8 (interleukin-8) , IL-8) and other pro-inflammatory cytokines are secreted, followed by the secretion of inflammatory transmitters that induce inflammatory symptoms. In particular, inflammatory factors such as nitric oxide (NO) and prostaglandin E2 (PGE2) are produced by macrophages. is formed, followed by increased NO. When NO is produced in excess, it can cause problems such as DNA structure damage, nerve damage, edema, and epithelial cancer.

In addition, when there is an inflammatory response, free radicals are generated along with such inflammation-related factors. In general, they are involved in maintaining homeostasis such as differentiation, growth, and survival of cells, but among free radicals, reactive oxygen species ( Reactive oxygen species (ROS) are continuously generated through oxidation and reduction processes of oxygen by respiration and immune reactions, and their reactivity can cause harmful effects to the body. However, when the balance between production and removal is disrupted, oxidative stress occurs and problems such as inflammation, aging, tissue damage, and cancer may occur.

For the above reasons, despite the positive action of inflammation, in some cases, anti-inflammatory drugs are required. Since most anti-inflammatory drugs are synthetic, efforts to treat inflammation using natural substances that are safer for the human body are required. It is becoming.

As prior art related to natural products with anti-inflammatory action, Korean Patent No. 1694662 discloses a method for preparing an ultrasonic extract of Chickweed moss having an anti-inflammatory effect, and Korean Patent No. 1093015 discloses a method for producing an extract or fraction containing a pine moss extract. Although an anti-inflammatory or anti-cancer composition is disclosed, there is no disclosure of an anti-inflammatory composition comprising the frost moss extract of the present invention as an active ingredient.

The present invention has been derived from the above needs, and provides an anti-inflammatory composition comprising a frost moss extract as an active ingredient, wherein the frost moss extract suppresses increased NO production by LPS treatment, and IL-6 , The present invention was completed by confirming that the protein expression of IL-1β and mRNA expression of iNOS and IL-6 were reduced.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a frost moss extract as an active ingredient.

In addition, the present invention provides a cosmetic composition for preventing or improving inflammatory diseases comprising a frost moss extract as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving inflammatory diseases comprising the frost moss extract as an active ingredient.

The present invention relates to an anti-inflammatory composition comprising an extract of Racomitrium canescens as an active ingredient. Since the effect of reducing the protein expression of IL-6 and IL-1β and the mRNA expression of iNOS and IL-6 increased by Or it can be used as a pharmaceutical composition for treatment, anti-inflammatory health functional food or anti-inflammatory cosmetic composition.

Figure 1 is the result of confirming the cytotoxicity of the frost moss extract in the HaCaT cell line. CON is a group treated with 1% (v/v) methanol instead of frost moss extract.
Figure 2 is the result of confirming the NO (nitric oxide) production inhibitory effect of the frost moss extract in the HaCaT cell line. CON and 0 are groups treated with 1% (v/v) methanol instead of frost moss extract. ### means that the concentration of NO production in the LPS-treated group compared to CON increased statistically significantly, p <0.001. *** means that the concentration of NO production in the frost moss extract treatment group was statistically significantly decreased compared to the LPS alone treatment group (0), p <0.001.
Figure 3 is the result of confirming the effect of inhibiting the mRNA production of inflammation-related markers (iNOS and IL-6) by treatment with the frost moss extract in the HaCaT cell line. CON and 0 are groups treated with 1% (v/v) methanol instead of frost moss extract. ### means that the expression of iNOS and IL-6 mRNA in the LPS-treated group compared to CON increased statistically significantly, p <0.001, and * and *** indicate frost moss compared to the LPS-only treated group (0). It means that the mRNA expression of iNOS and IL-6 in the extract treatment group decreased statistically significantly, * is p <0.05, and *** is p <0.001.
Figure 4 is a result confirming the effect of inhibiting protein production of inflammation-related markers (IL-6 and IL-1β) by treatment with the frost moss extract in the HaCaT cell line. CON and 0 are groups treated with 1% (v/v) methanol instead of frost moss extract. ### means that the IL-6 and IL-1β protein expressions of the LPS-treated group compared to CON increased statistically significantly, p <0.001, and *** indicates frost moss compared to the LPS-only treated group (0). It means that the protein expression of IL-6 and IL-1β in the extract treatment group decreased statistically significantly, p <0.001.

The present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a frost moss extract as an active ingredient.

In one embodiment of the present invention, the extraction solvent of the frost moss extract is preferably a C ₁ ~ C ₄ lower alcohol, water or a mixture thereof, more preferably using methanol as a solvent, but is not limited thereto .

As used herein, the term inflammatory disease refers to arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis, and degenerative nerve inflammation. It means above, but is not limited thereto.

The frost moss extract is characterized in that it inhibits NO production and reduces the protein expression of IL-6 and IL-1β and mRNA expression of iNOS and IL-6.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient, and such carriers are commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, Starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.

A suitable dosage of the pharmaceutical composition according to the present invention is variously prescribed depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity. It can be.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.

The concentration of the active ingredient included in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the required period, etc., and is not limited to a specific range of concentration.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier or excipient according to a method that can be easily performed by those skilled in the art, or It can be prepared by incorporating into a dose container. At this time, the formulation may be prepared as any one formulation selected from injections, creams, patches, sprays, ointments, warnings, lotions, liniments, pastas, and cataplasmas, and may additionally include a dispersing agent or a stabilizer. there is.

In addition, the present invention relates to a cosmetic composition for preventing or improving inflammatory diseases comprising a frost moss extract as an active ingredient.

In the cosmetic composition according to one embodiment of the present invention, the cosmetic composition is a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion, It may be prepared in any one formulation selected from foundation, wax foundation and spray, but is not limited thereto.

The cosmetic composition composed of each of these formulations may contain various bases and additives necessary and appropriate for formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.

When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fiber, vegetable fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide as a carrier component etc. can be used.

When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydro propellants such as carbon, propane-butane or dimethyl ether.

When the formulation of the cosmetic composition 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 fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, 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, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.

When the formulation of the cosmetic composition of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, and sarcosinate , fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters.

The cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrotropes inducing substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, and the like. .

In addition, the present invention relates to a health functional food composition for preventing or improving inflammatory diseases comprising a frost moss extract as an active ingredient.

The health functional food composition may be prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The amount of the active ingredient can be appropriately used depending on the purpose of its use (prevention or improvement). In general, when preparing food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term intake for health purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

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

In addition, the health functional food composition of the present invention can be made into food, particularly functional food. The functional food of the present invention includes components commonly added during food preparation, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to active ingredients. The natural carbohydrates are monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrins, cyclodextrins, etc.) or sugar alcohols (eg, maltose, sucrose, etc.) , xylitol, sorbitol, erythritol, etc.) are preferred. As the flavoring agent, natural flavoring agents (eg, thaumatin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.

In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may further contain a carbonation agent used in beverages and the like. The ratio of the components to be added is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

Hereinafter, the present invention will be described in more detail using preparation examples and examples. These Preparation Examples and Examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

Preparation Example 1. Preparation of frost moss extract

The collected frost moss was refrigerated at a temperature of 4 ℃. Thereafter, the soil of the frost moss, which was refrigerated for the preparation of the extract, was washed and then dried at room temperature. After drying the frost moss, the dried frost moss was pulverized using a grinder (HANIL HMF-3260S, Hanil Electric Co., Seoul, Korea) to a size of 0.6 mm, and 4 L of methanol was added to 1 kg of the crushed frost moss for 2 days. After the first extraction, it was filtered. Then, the filtrate was concentrated using a rotary vacuum concentrator (EYELA NE-1101, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) at a temperature of 40 ° C., and 50 mL of distilled water was added to the concentrate to obtain a secondary extracted. Frost moss secondary extract was dried using a freeze dryer (FD5505, ILSHINBIOBASE, Dongduchon, Korea), and dissolved in methanol at 10 mg/mL, and used as a frost moss extract sample.

Example 1. Cell viability analysis

A cytotoxicity test was conducted on the frost moss extract of the present invention. The cells used in the cytotoxicity test were human epidermal fibroblasts (HaCaT cells), 100 unit/mL penicillin-streptomycin (P/S) and 10% (v/v) fetal bovine serum (fetal bovine serum). Bovine serum, FBS) was mixed with DMEM medium (Dulbecco's modified Eagle's medium, DMEM) at a temperature of 37° C. and cultured under 5% CO ₂ conditions.

Cytotoxicity was confirmed through quantitative analysis of MTT (3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Cells were treated with 12.5 to 100 μg/mL of frost moss extract and incubated for 24 hours, then treated with MTT solution and reacted at 37°C for 4 hours, confirming that purple formazan was formed inside the cells. did Then, the solution in each well was completely removed, and the purple formazan crystals were mixed with DMSO (dimethyl sulfoxide) and isopropyl alcohol at a ratio of 1:1 (v/v) (100 μl/v). well), the absorbance of the lysate was measured at 540 nm, and the relative cell viability of the frost moss extract treatment group was calculated based on the 100% survival rate of the group treated with 1% (v / v) methanol (CON). .

As a result, as shown in Figure 1, the frost moss extract of Preparation Example 1 did not significantly affect cell viability, and it was found that the frost moss extract of the present invention had little cytotoxicity.

Example 2. NO (nitric oxide) production inhibitory effect of frost moss extract

To verify the anti-inflammatory effect, HaCaT cells were treated with 30 or 100 μg/mL of frost moss extract, treated with 1 μg/mL lipopolysaccharide (LPS) 1 hour later, and then cultured for 24 hours. Then, the degree of nitrite accumulation in the culture medium was measured using a grease reagent. After mixing the supernatant (100 μl) of the culture medium with grease reagent (100 μl) for 10 minutes, the absorbance was measured at 540 nm, and the amount of nitrite in the medium was determined by referring to the sodium nitrite (NaNO ₂ ) standard graph. .

As a result, as shown in FIG. 2, when the frost moss extract was treated, it was confirmed that the NO production increased by the LPS treatment was reduced, and in particular, when the frost moss extract was treated with 100 μg / mL, NO production was reduced. The effect was excellent.

Example 3. Inhibitory effect of inflammatory marker production of frost moss extract

To verify the anti-inflammatory effect, enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction were performed.

HaCaT cells were treated with 30 or 100 μg/mL of frost moss extract, and 1 hour later, 1 μg/mL of LPS (lipopolysaccharide) and cultured for 24 hours. Then, the amounts of IL-6 and IL-1β in the culture medium were measured by ELISA, and the mRNA expression of iNOS and IL-6 was confirmed by RT-PCR. ELISA was carried out according to the manufacturer's protocol. For RT-PCR, total RNA (1μg) was extracted from HaCaT cells (using RNAiso PLUS), then cDNA was synthesized by reverse transcription (All-in-One first-Strand cDNA Synthesis SuperMix), and the synthesized cDNA was used as a template for qRT-PCR (using QuantStudio 3).

As a result of dissociation curve analysis for iNOS, IL-6, and β-actin, a single peak was identified, and the expression level for each target gene was quantified by overlapping measurement, and then 2 Calibration was performed using ^{the ΔΔCT} method.

As a result, as shown in FIGS. 3 and 4, the mRNA expression of iNOS and IL-6 and the protein expression of IL-6 and IL-1β were decreased in the group treated with the frost moss extract, and 100 μg / mL was treated. It was confirmed that the reduction rate was more significant in the group.

Claims (10)

Among arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis and degenerative nerve inflammation containing frost moss extract as an active ingredient A pharmaceutical composition for preventing or treating one or more selected inflammatory diseases. The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 1, wherein the extraction solvent of the extract is C ₁ ~ C ₄ lower alcohol, water or a mixture thereof. delete The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the extract. According to claim 1, wherein the frost moss extract inhibits NO production, IL-6, IL-1β protein expression and iNOS, IL-6 for preventing or treating inflammatory diseases, characterized in that reducing the mRNA expression pharmaceutical composition. A cosmetic composition for preventing or improving burn inflammation or dermatitis, comprising a frost moss extract as an active ingredient. The cosmetic composition according to claim 6, wherein the cosmetic composition is selected from solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsions, foundations, wax foundations and sprays. A cosmetic composition for preventing or improving burn inflammation or dermatitis, characterized in that it is prepared in any one selected formulation. Among arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis and degenerative nerve inflammation containing frost moss extract as an active ingredient A health functional food composition for preventing or improving one or more selected inflammatory diseases. The functional food composition for preventing or improving inflammatory diseases according to claim 8, wherein the composition is prepared in any one formulation selected from powders, granules, pills, tablets, capsules, candies, syrups and beverages. delete