WO2023128481A1 - Composition including inotodiol and chaga mushroom extract containing same for improving skin condition - Google Patents

Abstract

The present invention relates to a composition for improving a skin condition. Having the activity of inhibiting skin inflammation, moisturizing the skin, strengthening the skin barrier, delaying skin aging, reducing skin wrinkles, alleviating psoriasis, and ameliorating atopic dermatitis, the inotodiol or the chaga mushroom extract containing inotodiol at a high concentration according to one aspect can be advantageously used for the prevention, improvement, or treatment of skin conditions.

Description

Composition for improving skin condition containing inotodiol and chaga mushroom extract containing the same

It relates to a composition for improving skin conditions.

The function of the epidermis, located at the outermost layer of the skin, is to defend against various stimuli from the outside (physical and chemical stimulants such as chemicals, air pollutants, dry environments, and ultraviolet rays) and to release excessive moisture in the body through the skin. This protective function can be maintained only when the stratum corneum composed of keratinocytes is normally formed. Among the epidermis, the outermost stratum corneum is formed from keratinocytes, and is composed of fully differentiated keratinocytes and a lipid layer surrounding them (J. Invest. Dermatol. 1983; 80:44-49). After a certain period of time, old keratinocytes are eliminated from the skin and new keratinocytes take over their functions, and this repetitive series of changes is called 'differentiation of epidermal cells' or 'keratinization'. During the keratinization process, keratinocytes form the stratum corneum while producing natural moisturizing factor (NMF) and intercellular lipids (ceramide, cholesterol, fatty acids), making the stratum corneum firm and flexible, thereby forming a skin barrier. ) function as

However, this stratum corneum can easily lose its function due to lifestyle factors such as excessive washing or bathing, environmental factors such as dry air and pollutants, and endogenous diseases such as atopic skin or senile skin. In addition, the production-turnover rate of the stratum corneum is slowed, the ability of keratinocytes to synthesize lipids is reduced, or normal cell division, maturation, and differentiation in the epidermis are not smooth, resulting in the loss of moisturizing factors and lipids in the stratum corneum. There is an increasing trend in cases where the amount is reduced and the skin in a state where the function of the stratum corneum is not maintained, that is, the skin barrier function is broken.

Atopic dermatitis, which occurs when the skin barrier function is disrupted, is a chronic, recurrent skin disease that is often accompanied by itching. Although the etiology of atopic dermatitis has not yet been clearly identified, it is known that it is caused by an immunologic abnormality mediated by type 2 T helper lymphocytes, and is also caused by environmental allergens, genetic predispositions, psychological factors or pharmacological factors.

Due to such abnormal division and differentiation of epidermal cells, the skin causes various skin diseases such as xeroderma, atopy, and psoriasis, and it is difficult to improve these diseases with only conventional moisturizers having only a water retention function.

Therefore, it is necessary to develop a material capable of fundamentally improving skin conditions by strengthening the skin barrier.

One aspect relates to inotodiol or an acceptable salt thereof; Or to provide a cosmetic composition for improving skin conditions comprising a chaga mushroom ( Inonotus obliquus ) extract containing inotodiol at a high concentration.

Another aspect is inotodiol or an acceptable salt thereof; Or to provide a composition for external application for skin for the prevention or improvement of inflammatory skin diseases, comprising an extract of chaga mushroom ( Inonotus obliquus ) containing high concentration of inotodiol as an active ingredient.

Another aspect relates to inotodiol or an acceptable salt thereof; Or to provide a pharmaceutical composition for the prevention or improvement of inflammatory skin diseases comprising an extract of chaga mushroom ( Inonotus obliquus ) containing high concentration of inotodiol as an active ingredient.

Another aspect is inotodiol or an acceptable salt thereof for improving skin conditions for preventing, improving, or treating skin diseases (eg, skin inflammatory diseases); Or to provide the use of a chaga mushroom ( Inonotus obliquus ) extract containing inotodiol in high concentration.

Another aspect relates to an effective amount of inotodiol or an acceptable salt thereof; Or, it relates to a method for improving a skin condition comprising administering an extract of chaga mushroom ( Inonotus obliquus ) containing inotodiol at a high concentration to a subject, and a method for preventing, improving, or treating a skin disease.

One aspect relates to inotodiol or an acceptable salt thereof; Or, it relates to a cosmetic composition for improving skin condition comprising an extract of chaga mushroom ( Inonotus obliquus ) containing inotodiol at a high concentration.

The inotodiol may be organically synthesized or isolated from plant extracts. The plant extract may be chaga mushroom. The chaga extract can be extracted by any extraction method such as hot water extraction, solvent extraction, distillation extraction, supercritical extraction, etc., which have been conventionally used to extract natural plants, and preferably water, organic solvents, or mixed solvents thereof. Characterized in that it is extracted as. The organic solvent may be any one or more selected from the group consisting of alcohols having 1 to 4 carbon atoms, such as ethanol, methanol, isopropanol, and butanol, etc., preferably ethanol, and more preferably fermented alcohol can be used The alcohol concentration of the aqueous alcohol solution is 1 to 99.5 (v / v)%, for example, 10 to 99.5 (v / v)%, 1 to 70 (v / v)%, 1 to 40 (v / v)% , 5 to 25 (v/v)%, 7 to 20 (v/v)%, 5 to 25 (v/v)%, or 10 to 20 (v/v)%.

The extraction is 3 to 50 (volume / weight) times, for example, 3 to 40 (volume / weight) times, 3 to 30 (volume / weight) times, 5 to 50 (volume / weight) times the extraction solvent with respect to the chaga mushroom / weight) times, or 5 to 40 (volume / weight) times. For example, it may include adding 3 to 50 kg of the extraction solvent based on 1 kg of the material derived from the chaga mushroom.

In one embodiment, the chaga mushroom may contain inotodiol at a high concentration. Specifically, the chaga mushroom extract contains at least 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% inotodiol in the total extract. (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 11% (w/w), 12% (w/w), 13% (w/w) /w), 14% (w/w), 15% (w/w), 16% (w/w), 17% (w/w), 18% (w/w), 19% (w/w) ), 20% (w/w), 21% (w/w), 22% (w/w), 23% (w/w), 24% (w/w), 25% (w/w), 26% (w/w), 27% (w/w), 28% (w/w), 29% (w/w), 30% (w/w), 31% (w/w), 32% (w/w), 33% (w/w), 34% (w/w), 35% (w/w), 36% (w/w), 37% (w/w), 38% (w/w) /w), 39% (w/w), 40% (w/w), 41% (w/w), 42% (w/w), 43% (w/w), 44% (w/w) ), 45% (w/w), 46% (w/w), 47% (w/w), 48% (w/w), 49% (w/w), 50% (w/w), 55%(w/w), 60%(w/w), 65%(w/w), 70%(w/w), 75%(w/w), 80%(w/w), 85% (w/w), 90% (w/w), 95% (w/w), or 98% (w/w).

The chaga extract containing the inotodiol at a high concentration may be prepared by a method comprising preparing a primary extract by extracting the chaga extract with an organic solvent. In addition, the method comprises the steps of preparing a secondary extract by performing secondary extraction of the precipitate obtained from the primary extract with an organic solvent; and mixing, concentrating, and filtering the primary and secondary extracts.

According to one embodiment, the chaga extract containing the inotodiol at a high concentration comprises extracting the chaga extract with an organic solvent to prepare a primary extract; preparing a secondary extract by performing secondary extraction of the precipitate obtained from the primary extract with an organic solvent; And it may be prepared by a method comprising the steps of mixing, concentrating, and filtering the primary and secondary extracts.

The inotodiol compound may be a fraction of the chaga mushroom extract. The fraction refers to a material containing a specific component separated from the extract. The inotodiol compound may be separated and purified using column chromatography. The chromatography is silica gel column chromatography, HP-20 column chromatography, RP-18 column chromatography, LH-20 column chromatography ( LH-20 column chromatography), high-performance liquid chromatography, or a combination thereof can be selected and used.

The skin improvement may be at least one selected from the group consisting of skin barrier improvement, skin wrinkle improvement, skin aging, skin aging by light, skin moisturizing, skin inflammation inhibition, skin soothing, and skin regeneration.

In the present specification, the term "skin barrier improvement" means to include both skin barrier strengthening and protective functions. It is made up of corneocytes. The multi-lamellar lipid layer formed of intercellular lipids such as ceramide, cholesterol, and fatty acids synthesized by keratinocytes of the skin barrier, maintained through normal epidermal cell division and differentiation, is a protective barrier to prevent evaporation of moisture in the skin. play a role On the other hand, among these intercellular lipids, omega hydroxy ceramide is chemically covalently linked to involucrin, a protein of the outer layer of corneocytes, to form a corneocyte lipid envelope (CLE), thereby forming a multilayer lipid membrane. It plays a role in strengthening the barrier function by physically stabilizing the intercellular lipid in the form. The skin barrier improvement is a skin disease caused by weakening of the skin barrier function, such as psoriasis, contact dermatitis, eczematous dermatitis, actinic dermatitis, seborrheic dermatitis, dermatitis herpetiformis, lichen planus, lichen sclerosus, pyoderma gangrenosum, pemphigus, bullous epidermis It can mean any action that alleviates exfoliation, systemic sclerosis or leprosy or improves the damaged skin barrier function.

The improvement of the skin condition may be any one or more selected from the group consisting of skin inflammatory diseases, skin diseases, for example, skin wounds, dermatitis, atopic dermatitis, pruritus, eczema, erythema, urticaria, psoriasis, and drug rash.

As used herein, the term "acceptable salt" refers to a salt prepared using a specific compound according to one aspect and a relatively non-toxic acid or base. When the compounds contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include salts of sodium, potassium, calcium, ammonium, organic amines, or magnesium or similar salts. When the compounds contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of an acid in neat solution or in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include salts of inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate ion, phosphoric acid, hydrogen phosphate ion, dihydrogen phosphate ion, sulfuric acid, hydrogen sulfate ion, hydroiodic acid or phosphorous acid; and salts of organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, and methanesulfonic acid. and salts of amino acids (eg, arginine) and salts of organic acids such as glucuronic acid.

The acceptable salts can be synthesized by conventional chemical methods from parent compounds containing acidic or basic moieties. Generally, these salts are prepared by reacting the free acid or base form of these compounds with an appropriate stoichiometric amount of the base or acid, either in water or in an organic solvent or in a mixture of the two.

The inotodiol compound or chaga extract containing inotodiol at a high concentration may be included in an amount of 0.001 to 10% by weight based on the total weight of the cosmetic composition. For example, 0.001 to 5% by weight, 0.001 to 3% by weight, 0.001 to 1% by weight, 0.01 to 10% by weight, 0.01 to 5% by weight, 0.01 to 3% by weight, 0.01 to 1% by weight, 0.1 to 10% by weight %, 0.1 to 5% by weight, 0.1 to 3% by weight, or 0.1 to 1% by weight.

The cosmetic composition includes lotion (skin lotion), toner, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, moisture cream, hand cream, hand sanitizer, foundation, essence, Nutritional essence, packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, suspensions, gels, powders, pastes, mask packs, and can be prepared into formulations including sheets. Compositions of such formulations may be prepared according to conventional methods in the art. The blending amount of the additional ingredients such as the moisturizing agent can be easily selected by those skilled in the art within a range that does not impair the objects and effects of the present invention.

The cosmetic composition may further include functional additives and ingredients included in general cosmetic compositions in addition to the active ingredients disclosed herein, and commonly used purified water, thickeners, preservatives, stabilizers, solubilizers, surfactants, carriers, A flavoring agent or a combination thereof may be further included. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular peptides, high-molecular polysaccharides, sphingolipids, and seaweed extracts. Alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, UV scattering agents, UV absorbers, coloring agents, perfumes, and the like may be exemplified as the carrier. Compounds/compositions that can be used as the alcohol, oil, surfactant, fatty acid, silicone oil, wetting agent, humectant, viscosity modifier, emulsion, stabilizer, UV scattering agent, UV absorber, coloring agent, fragrance, etc. are already known in the art. Since there is, those skilled in the art can select and use an appropriate corresponding material / composition. In addition, the cosmetic composition, if necessary, sunscreen, antioxidants (butylhydroxyanisole, gallic acid propyl, ellisorbic acid, tocopheryl acetate, butylated hydroxytoluene, etc.), preservatives (methylparaben, butylparaben , propylparaben, phenoxyethanol, imidazolidinylurea, chlorphenesin, etc.), colorant, pH adjuster (triethanolamine, citric acid, citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid , sodium succinate, sodium hydroxide, sodium hydrogen phosphate, etc.), humectants (glycerin, sorbitol, propylene glycol, butylene glycol, hexylene glycol, diglycerin, betaine, glycereth-26, methylglue Seth-20, etc.) and lubricants may be further added.

In addition, in the cosmetic composition of each formulation, appropriate components may be selected and blended according to the cosmetic formulation or purpose of use. Since the formulation components and methods may follow conventional techniques, detailed descriptions thereof are omitted herein.

Another aspect relates to inotodiol or an acceptable salt thereof; Or it provides a pharmaceutical composition for the prevention or improvement of skin inflammatory diseases comprising an extract of chaga mushroom ( Inonotus obliquus ) containing high concentration of inotodiol as an active ingredient.

Another aspect is inotodiol or an acceptable salt thereof; Alternatively, a composition for external application for skin for preventing or improving inflammatory skin disease is provided, which includes, as an active ingredient, an extract of chaga mushroom (Inonotus obliquus) containing inotodiol at a high concentration.

The term "pharmaceutical composition" can refer to a molecule or compound that imparts some beneficial effect upon administration to a subject. Beneficial effects may include enabling diagnostic determination; amelioration of a disease, symptom, disorder or condition; reducing or preventing the occurrence of a disease, condition, disorder or condition; and responding to a disease, symptom, disorder or condition in general.

The term "prevention" refers to partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its attendant symptoms, preventing the acquisition or re-acquisition of a disease or disorder, or the risk of acquiring a disease or disorder. tells how to reduce For example, the prevention refers to any action that inhibits or delays the occurrence of skin damage or symptoms by administration of the composition according to the present invention.

The term "treatment" includes the inhibition, alleviation or elimination of the development of a disease.

The term "improvement" may refer to any action that at least reduces a parameter associated with alleviation or treatment of a condition, eg, the severity of a symptom.

The pharmaceutical composition can be administered parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation. Parenteral administration may refer to administration through an administration route other than oral, such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal, inhalation, ocular and subcutaneous administration. When the pharmaceutical composition of the present invention is used as a medicine, it may additionally contain one or more active ingredients exhibiting the same or similar functions.

Types of pharmaceutically active ingredients capable of delivering the active ingredient into the subject include anticancer agents, contrast agents (dye), hormones, anti-hormonal agents, vitamins, calcium agents, mineral preparations, saccharide preparations, organic acid preparations, protein amino acid preparations, detoxifying agents, and enzymes. Preparations, metabolic preparations, diabetes concomitant medicines, tissue regeneration medicines, chlorophyll preparations, pigment preparations, tumor medicines, tumor treatment agents, radiopharmaceuticals, tissue cell diagnostic agents, tissue cell therapy agents, antibiotics preparations, antiviral agents, complex antibiotics preparations, chemicals Therapeutic agents, vaccines, toxins, toxoids, antitoxins, leptospira serum, blood products, biological agents, analgesics, immunogenic molecules, antihistamines, allergy medications, non-specific immunogenic agents, anesthetics, stimulants, psychoactive agents, small molecule compounds, nucleic acids, It may include aptamers, antisense nucleic acids, oligonucleotides, peptides, siRNAs, and micro RNAs.

The pharmaceutical composition may be prepared by further including one or more pharmaceutically acceptable carriers. A pharmaceutically acceptable carrier may be a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and, if necessary, antioxidants and buffers. , bacteriostatic agents and other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component by an appropriate method in the art.

When formulating the pharmaceutical composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, liurine fat, glycerogeratin and the like may be used.

The pharmaceutical composition may include carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, and small molecules in order to increase stability or absorption. Proteins or other Stabilizers can be used as pharmaceuticals.

The external skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The external skin preparation is a component usually used in external preparations for skin such as cosmetics or pharmaceuticals, for example, water-based components, oil-based components, powder components, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or combinations thereof and may be suitably blended as needed. The external skin preparations include metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridin, and calin. Hot-water extracts of fruits, various herbal medicines, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts and other drugs, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can also be mix|blended suitably.

The skin includes all skin areas of the body, including the face, hands, arms, legs, feet, chest, stomach, back, buttocks, and scalp.

Another aspect provides a method for preventing, improving or treating inflammatory skin diseases, including administering the composition to a subject, and a method for improving skin conditions.

Another aspect provides the use of the composition for improving a skin condition, for preventing, ameliorating or treating a skin inflammatory disease.

The term “administering” means providing a pharmaceutical composition to a subject by any suitable method.

A pharmaceutically effective amount and an effective dosage of the pharmaceutical composition may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition. In addition, the type and degree of response to be achieved by administration of the pharmaceutical composition, the type of subject to be administered, age, weight, general health condition, symptom or degree of disease, gender, diet, excretion, simultaneous or It may vary according to various factors including drugs and other components of the composition used together in this case, and similar factors well known in the medical field. A person of ordinary skill in the art can readily determine and prescribe an effective dosage for the desired treatment. Administration of the pharmaceutical composition according to the present invention can be administered once a day, divided into several administrations. Therefore, the dosage is not intended to limit the scope of the present invention in any way. The dosage of the pharmaceutical composition may be 1 ug/kg/day to 1,000 mg/kg/day.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be a subject in need of healing of skin damage.

Another aspect relates to inotodiol or an acceptable salt thereof; Or it provides a health functional food for preventing or improving skin inflammatory diseases or improving skin conditions, which contains an extract of chaga mushroom ( Inonotus obliquus ) containing inotodiol at a high concentration as an active ingredient.

When using the inotodiol or chaga extract containing inotodiol as a food additive, the compound may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). For example, 0.0001% to 99.0% by weight, for example, 0.01% to 60% by weight, 0.01% to 40% by weight of the inotodiol or chaga mushroom extract containing inotodiol in high concentration, based on the total weight of the composition. %, 0.01% to 30%, 0.01% to 20%, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, It may be included in 0.05% by weight to 30% by weight, or 7 to 10% by weight. However, in the case of long-term intake for the purpose of health and hygiene or health control, it may be below 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.

The inotodiol or chaga mushroom extract containing inotodiol in high concentration may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

There is no particular limitation on the type of food. Examples of foods to which the above substances may be added include powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, leached teas, and formulations selected from the group consisting of health drinks, etc. It includes all health foods in the usual sense.

The health beverage composition of the present invention may include various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

The health functional food may include food additives acceptable in food science, and may include appropriate carriers commonly used in the manufacture of health functional food.

In addition to the above, the composition of the present invention contains 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, carbonation agents used in carbonated beverages; and the like. In addition, the composition of the present invention may include fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination.

The terms, methods, and effects described for the above invention are equally applied between each invention.

According to the chaga mushroom extract containing inotodiol or inotodiol in high concentration according to one aspect, skin inflammation inhibitory activity, skin moisturizing activity, skin barrier strengthening activity, skin aging inhibitory activity, skin wrinkle inhibitory activity, psoriasis, atopic dermatitis It has an improvement activity and has an effect that can be usefully used for prevention, improvement or treatment of skin conditions.

Figure 1 is a graph analyzing the content of inotodiol in chaga mushroom extract according to one embodiment: Figure 1a is a graph analyzing the content of inotodiol in semi-inotodiol according to one embodiment, Figure 1b is one It is a graph analyzing the content of inotodiol in the chaga mushroom extract containing 5% inotodiol according to an embodiment.

2 is a graph showing the content of inotodiol in pure inotodiol according to one embodiment.

3 is a graph showing the expression of IL-8 after TNF-α-treated HaCaT cells were treated with semiinotodiol according to an embodiment.

4 is a graph showing the expression of IL-6 after TNF-α-treated HaCaT cells were treated with semiinotodiol according to an embodiment.

5 is a graph showing the expression of IL-1β after TNF-α-treated HaCaT cells were treated with semiinotodiol according to an embodiment.

6 is a graph showing the expression of TNF-α after TNF-α-treated HaCaT cells were treated with semiinotodiol according to an embodiment.

7 is a graph showing the expression of IL-8 after UVB-irradiated HaCaT cells were treated with semiinotodiol according to an embodiment.

8 is a graph showing the expression of IL-6 after UVB-irradiated HaCaT cells were treated with semiinotodiol according to an embodiment.

9 is a graph showing the expression of TNF-α after UVB-irradiated HaCaT cells were treated with semiinotodiol according to an embodiment.

10 is a graph showing the expression of HAS-2 according to treatment with semiinotodiol according to one embodiment.

11 is a graph showing the expression of HAS-3 according to treatment with semiinotodiol according to one embodiment.

12 is a graph showing the expression of COL1A1 according to treatment with semiinotodiol according to one embodiment.

13 is a graph showing the expression of hyaluronic acid degrading enzyme according to the treatment of semiinotodiol according to one embodiment.

14 is a graph showing the expression of TSLP according to treatment with semiinotodiol according to one embodiment.

15 is a graph showing the expression of IL-8 after TNF-α-treated HaCaT cells were treated with pure inotodiol according to an embodiment.

16 is a graph showing the expression of IL-6 after TNF-α-treated HaCaT cells were treated with pure inotodiol according to an embodiment.

17 is a graph showing the expression of IL-1β after TNF-α-treated HaCaT cells were treated with pure inotodiol according to one embodiment.

18 is a graph showing the expression of TNF-α after TNF-α-treated HaCaT cells were treated with pure inotodiol according to an embodiment.

19 is a graph showing the expression of IL-6 after UVB-irradiated HaCaT cells were treated with pure inotodiol according to an embodiment.

20 is a graph showing the expression of TNF-α after UVB-irradiated HaCaT cells were treated with pure inotodiol according to an embodiment.

21 is a graph showing the expression of HAS-2 according to treatment with pure inotodiol according to one embodiment.

22 is a graph showing the expression of HAS-3 according to treatment with pure inotodiol according to one embodiment.

23 is a graph showing the expression of COL1A1 according to treatment with pure inotodiol according to one embodiment.

24 is a graph showing the expression of COL1A1 according to treatment with pure inotodiol according to one embodiment in UVB-irradiated HaCaT cells.

25 is a graph showing the expression of hyaluronic acid degrading enzyme according to treatment with pure inotodiol according to one embodiment.

26 is a graph showing the expression of TSLP according to treatment with pure inotodiol according to one embodiment.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

Example 1. Preparation of extract containing high concentration of inotodiol

1.1. Preparation of Chaga Mushroom Extract Containing 20% Inotodiol

A chaga mushroom extract (hereinafter referred to as 'semi-inotodiol') containing 20% inotodiol was prepared.

Specifically, 30 kg of chaga mushroom powder was mixed with 300 L of 100% alcohol and extracted at 50 ° C. for 20 hours to prepare a primary extract. In order to prepare an extract containing inotodiol at a high concentration, a secondary extraction process was performed. Specifically, the prepared primary extract was cooled, and the precipitate was obtained by filtration. The prepared precipitate was again mixed with 300 L of 100% alcohol and extracted at 50 ° C. for 20 hours to prepare a secondary extract. Next, the first and second extracts were mixed, first filtered with a non-woven fabric filter (10um) and secondly filtered with a membrane filter (1um), and then concentrated under reduced pressure. The concentrate was purified by filtering through a membrane filter (1 um), and lyophilized to obtain semiinotodiol as a final product. The solid content and yield in each step are shown in Table 1 below.

Sample	Solid content (g)
1st extract	814.0
2nd extract	197.6
1 tea + 2 extract	1011.6
sample	Total amount (g)	Yield for extract (%)
Semiinotodiol (refined yield)	184.3	18.2

Subsequently, the content of inotodiol in the prepared semiinotodiol was confirmed by LC-ELSD. Specific LC conditions are shown in Table 2 below, and chromatogram analysis results are shown in FIG. 1a.

LC condition
Solvent	A: DW, B: Methanol
Column	YMC-Triart C18 (4.6 mm x 250 mm, 5 μm, YMC)
Flow rate	1mL/min
Injection volume	10 µL
Column temp	50℃
Detector	ELSD

As shown in FIG. 1A, it was found that the content of inotodiol in semiinotodiol was about 20% or more.

1.2 Preparation of Chaga Mushroom Extract Containing 5% Inotodiol

A chaga mushroom extract containing 5% of inotodiol was prepared.

Specifically, 50 kg of chaga powder was mixed with 500 L of 80% alcohol and extracted at 50 ° C. for 22 hours to prepare an extract. Thereafter, the extract was first filtered through a non-woven fabric filter (10um) and secondly filtered through a membrane filter (1um), and then concentrated under reduced pressure. The concentrate was purified by filtering through a membrane filter (1um), and lyophilized to obtain a chaga mushroom extract containing 5% of inotodiol as a final purification. The solid content and yield in each step are shown in Table 3 below.

Sample	Solid content (g)
extract	674.0
sample	Total amount (g)	Yield for extract (%)
Extracts containing inotodiol (containing more than 5% of inotodiol)	448.0	66.5

Subsequently, the content of inotodiol in the chaga mushroom extract prepared above was confirmed by LC-ELSD. Specific LC conditions are shown in Table 4 below, and chromatogram analysis results are shown in FIG. 1B.

LC condition
Solvent	A: DW, B: Methanol
Column	YMC-Triart C18 (4.6 mm x 250 mm, 5 μm, YMC)
Flow rate	1mL/min
Injection volume	10 µL
Column temp	50℃
Detector	ELSD

As shown in Figure 1b, it was found that the content of inotodiol in the chaga mushroom extract was about 5% or more.

Example 2. Preparation of high-purity inotodiol

In order to isolate high-purity inotodiol (hereinafter referred to as 'pure inotodiol'), inotodiol was separated and purified from the semi-inotodiol prepared above.

Specifically, the semiinotodiol prepared above was obtained through the following fractionation process using a mass preparative chromatography device (YMC LC-FORTE/R), and the purified product was obtained, and the LC conditions are shown in Table 5 below.

LC condition
Solvent	A: DW B: Methanol
Column	YMC column (ODS-AQ, 50*500, 20 µm)
Flow rate	30mL/min

HPLC-ELSD analysis was performed to analyze the purity of the purified inotodiol, and the LC conditions are shown in Table 6. The results of HPLC-ELSD analysis are shown in FIG. 2 .

LC condition
Solvent	A: DW B: Methanol
Gradient	Min	A(%)	B(%)
	0	10	90
	10	8	92
	15	0	100
	20	0	100
	20.1	10	90
	25	10	90
Flow rate	1mL/min
Colum Temp.	50 ℃
Injection volume	10 µL
ELSD Drift tube Temp.	60 ℃
ELSD Spray Chamber tube temp.	58 ℃

As shown in FIG. 2, it was found that the content of inotodiol in pure inotodiol was 95 or more.

Experimental Example 1. Activity analysis of semiinotodiol

1.1. Anti-inflammatory or anti-aging activity of semiinotodiol

The anti-inflammatory activity of semiinotodiol in HaCaT cells was confirmed.

Specifically, HaCaT cells were seeded with 0.5X10 ⁶ cells/well 2 ml (10% FBS+DMEM) and cultured for one day. Thereafter, semiinotodiol was treated in each well at a dose of 2.5, 5, 10, and 20 ug/ml, and incubated for 18 hours under CO ₂ conditions. Next, each well was treated with 20 ng/ml of TNF-α and cultured for 18 hours under CO ₂ conditions. In order to analyze the expression of IL-8, IL-6, IL-1β and TNF-α, which are inflammation-related factors in the semiinotodiol-treated cells, cells were recovered and PCR analysis was performed, and the results are shown in Figs. 6, respectively.

As shown in Figures 3 to 6, semiinotodiol according to one embodiment, at a dose of 20 ug / ml, the expression of IL-8, IL-6, IL-1β and TNF-α by 54% and 95.4%, respectively , 83.8%, and 46.6% were found to decrease. The above results mean that semiinotodiol according to one embodiment can be usefully used to improve inflammatory skin conditions such as dermatitis and atopic dermatitis.

Subsequently, in order to analyze the activity of semiinotodiol against skin inflammation or photoaging caused by ultraviolet rays, cells were irradiated with 10 mJ/cm ² of UVB for 10 seconds, and IL-8, IL-6, and TNF were irradiated in the same manner as above. Expression of -α was analyzed. The above results are shown in Figures 7 to 9.

As shown in FIGS. 7 to 9, semiinotodiol according to one embodiment increased the expression of IL-8, IL-6, and TNF-α in HaCaT cells by UV irradiation at a dose of 20 ug/ml, respectively. 35.6%, 94.2%, and 68.1% reduction was found. The above results mean that semiinotodiol according to one embodiment can be usefully used for suppressing skin aging such as photoaging as well as suppressing skin inflammation.

1.2. Skin moisturizing and skin barrier strengthening activity of semi-inotodiol

The skin barrier strengthening activity of semi-inotodiol in HaCaT cells was confirmed.

Specifically, HaCaT cells were seeded with 1.3X10 ⁶ cells/well 2 ml (10% FBS+DMEM) and cultured for one day. Thereafter, each well was treated with semiinotodiol at a dose of 1.25, 2.5, 5, 10, or 20 ug/ml, and incubated for 24 hours under CO ₂ conditions. In order to analyze the expression of HAS-2, HAS-3, and COL1A1, which are factors related to skin moisturizing and skin barrier strengthening in the cells treated with semi-inotodiol, cells were recovered and PCR analysis was performed, and the results are shown in FIGS. 12, respectively.

As shown in Figures 10 to 12, semiinotodiol according to one embodiment increased the expression of HAS-2, HAS-3, and COL1A1 by 188%, 138.2%, and 129%, respectively, at a dose of 20 ug/ml Sikkim was known. The above results mean that semiinotodiol according to one embodiment can be usefully used for improving inflammatory skin conditions such as skin moisturizing and skin barrier strengthening.

1.3. Analysis of anti-wrinkle and anti-aging activity of semi-inotodiol

The anti-wrinkle and anti-aging activities of semiinotodiol in HaCaT cells were confirmed.

Specifically, the experiment was performed in the same manner as in Experimental Example 1.2, and the expression of hyaluronic acid degrading enzyme (HYAL-3) was confirmed, and the results are shown in FIG. 13 .

13 is a graph showing the expression of hyaluronic acid degrading enzyme according to the treatment of semiinotodiol according to one embodiment.

As shown in Figure 13, it was found that semiinotodiol according to one embodiment reduced the expression of HYAL-3 by 74.8% at a dose of 20 ug/ml. The above results indicate that semiinotodiol according to one embodiment inhibits hyaluronic acid degrading enzymes and increases the content of hyaluronic acid in the skin, thereby effectively improving the skin barrier, suppressing wrinkles, and anti-aging.

1.4. Analysis of the therapeutic activity of semi-inotodiol in psoriasis and atopic dermatitis

The therapeutic activity of semiinotodiol on psoriasis and atopic dermatitis in HaCaT cells was confirmed.

Specifically, the experiment was performed in the same manner as Experimental Example 1.1., expression of TSLP was confirmed, and the results are shown in FIG. 14 .

14 is a graph showing the expression of TSLP according to treatment with semiinotodiol according to one embodiment.

As shown in FIG. 14, it was found that semiinotodiol according to one embodiment reduced the expression of TSLP by 86% at a dose of 20 ug/ml. The above results mean that semiinotodiol according to one embodiment can be usefully used for the treatment or improvement of psoriasis, atopy, skin itching, and the like.

Experimental Example 2. Activity analysis of pure inotodiol

2.1. Anti-inflammatory or anti-aging activity of pure inotodiol

The anti-inflammatory activity of pure inotodiol in HaCaT cells was confirmed.

Specifically, the experiment was performed in the same manner as in 1.1. except for using pure inotodiol at doses of 0.55, 1.1, 2.2, and 4.4 ug/ml, and the results are shown in FIGS. 15 to 18 .

As shown in Figures 15 to 28, the pure inotodiol according to one embodiment at a dose of 4.4 ug / ml IL-8, IL-6, IL-1β and TNF-α expressions were reduced by 67.2% and 57.3%, respectively. , 72.8%, and 50% reduction. The above results mean that the pure inotodiol according to one embodiment can be usefully used to improve inflammatory skin conditions such as dermatitis and atopic dermatitis.

Subsequently, in order to analyze the activity of pure inotodiol against skin inflammation or photoaging caused by ultraviolet rays, cells were irradiated with 10 mJ/cm ² of UVB for 10 seconds, and the expression of IL-6 and TNF-α in the same manner as above. was analyzed. The above results are shown in FIGS. 19 and 20.

As shown in FIGS. 19 and 20, Pure Inotodiol according to one embodiment, at a dose of 4.4 ug/ml, increased the expression of IL-6 and TNF-α in HaCaT cells by 47% and 57.7, respectively, by UV irradiation. % reduction was found. The above results mean that the pure inotodiol according to one embodiment can be usefully used for suppressing skin aging such as photoaging as well as suppressing skin inflammation.

2.2. Skin moisturizing and skin barrier strengthening activity of Pure Inotodiol

The skin barrier strengthening activity of Pure Inotodiol in HaCaT cells was confirmed.

Specifically, the experiment was performed in the same manner as in 1.2. except for using pure inotodiol at doses of 0.55, 1.1, 2.2, and 4.4 ug/ml, and the results are shown in FIGS. 21 to 23 .

In addition, the expression of collagen protein (COL1A1) according to UVB irradiation was tested in the same manner as in 1.1 above, and the results are shown in FIG. 24 .

As shown in Figures 21 to 23, pure inotodiol according to one embodiment increased the expression of HAS-2, HAS-3, and COL1A1 by 160.6%, 145%, and 432%, respectively, at a dose of 4.4 ug/ml Sikkim was known. In addition, as shown in FIG. 24, it was found that pure inotodiol according to one embodiment increased the expression of COL1A1 by 150% in UVB-irradiated cells at a dose of 1.1 ug/ml. The above results mean that the pure inotodiol according to one embodiment can be usefully used to improve inflammatory skin conditions such as skin moisturizing and skin barrier strengthening.

2.3. Analysis of anti-wrinkle and anti-aging activity of pure inotodiol

The anti-wrinkle and anti-aging activities of Pure Inotodiol in HaCaT cells were confirmed.

Specifically, the experiment was performed in the same manner as in 1.3. except for using pure inotodiol at doses of 0.55, 1.1, 2.2, and 4.4 ug/ml, and the results are shown in FIG. 25 .

As shown in Figure 25, it was found that pure inotodiol according to one embodiment reduced the expression of HYAL-3 by 65.4% at a dose of 4.4 ug/ml. The above results indicate that the pure inotodiol according to one embodiment inhibits hyaluronic acid degrading enzymes to increase the content of hyaluronic acid in the skin, thereby improving the skin barrier, suppressing wrinkles, and anti-aging.

2.4. Analysis of the therapeutic activity of pure inotodiol in psoriasis and atopic dermatitis

The therapeutic activity of pure inotodiol on psoriasis and atopic dermatitis in HaCaT cells was confirmed.

Specifically, the experiment was performed in the same manner as in 1.4. except for using pure inotodiol at doses of 0.55, 1.1, 2.2, and 4.4 ug/ml, and the results are shown in FIG. 26 .

As shown in FIG. 26, it was found that pure inotodiol according to one embodiment reduced the expression of TSLP by 64.2% at a dose of 4.4 ug/ml. The above results mean that the pure inotodiol according to one embodiment can be usefully used for the treatment or improvement of psoriasis, atopy, skin itching, and the like.

Experimental Example 3. Activity analysis of chaga mushroom extract containing 5% of inotodiol

In order to analyze the activity of the chaga extract containing 5% inotodiol prepared in Example 1.2, anti-inflammatory activity, skin moisturizing activity, and skin barrier strengthening activity of the chaga extract containing 5% inotodiol , anti-wrinkle activity and atopic dermatitis therapeutic activity were confirmed.

Specific experiments were performed in the same manner as in Experimental Example 1.1, Experimental Example 1.2, Experimental Example 1.3, and Experimental Example 1.4, except that the chaga mushroom extract (10 μg/ml) containing 5% inotodiol was used. . And the results are shown in Tables 7 to 10 below. As a control group, a group not treated with chaga extract was used.

	TNF-α	IL-8	IL-6	IL-1β
Chaga mushroom extract containing 5% inotodiol (10 μg/ml)	4.5±0.2	1.0±0.01	5.5±0.5	0.3±0.5
control group	6.2±1.1	1.3±0.2	9±0.8	1.18±0.1

	HAS-2	HAS-3	COL1A1
Chaga mushroom extract containing 5% inotodiol (10 μg/ml)	1.2±0.1	1.2±0.2	1.3±0.1
control group	1.0±0.06	1.0±0.05	1.0±0.1

	HYAL-3
Chaga mushroom extract containing 5% inotodiol (10 μg/ml)	1.1±0.6
control group	1.5±0.3

	TSLP
Chaga mushroom extract containing 5% inotodiol (10 μg/ml)	10.5±0.3
control group	15±1.5

As shown in Tables 7 to 10, the chaga mushroom extract containing 5% of inotodiol showed significantly improved anti-inflammatory activity, skin moisturizing activity, skin barrier strengthening activity, anti-wrinkle activity and therapeutic activity for atopic dermatitis. Confirmed.

More specifically, the chaga mushroom extract containing 5% of inotodiol reduced the expression of IL-8, IL-6, IL-1β and TNF-α by about 30%, 40%, 75%, and 30%, respectively, compared to the control group. % reduction was found.

In addition, it was found that the chaga mushroom extract containing 5% of inotodiol increased the expression of HAS-2, HAS-3, and COL1A1 by about 120%, 120%, and 130%, respectively, compared to the control group.

In addition, it was found that the chaga mushroom extract containing 5% of inotodiol reduced the expression of HYAL-3 by about 27% and the expression of TSLP by about 30% compared to the control group.

Claims (14)

1. inotodiol or an acceptable salt thereof; Or a cosmetic composition for improving skin conditions comprising a chaga mushroom ( Inonotus obliquus ) extract containing inotodiol at a high concentration.
2. The method of claim 1,

   The chaga extract is a cosmetic composition comprising inotodiol in a concentration of at least 3% (w / w) of the total extract.
3. The method of claim 1,

   The chaga extract containing the inotodiol at a high concentration is prepared by extracting the chaga extract with an organic solvent to obtain a primary extract; preparing a secondary extract by performing secondary extraction of the precipitate obtained from the primary extract with an organic solvent; And the cosmetic composition prepared by a method comprising the step of concentrating and filtering the mixture of the primary and secondary extracts.
4. The method of claim 3,

   The organic solvent is a cosmetic composition comprising an alcohol of C1 to C4.
5. The method of claim 1,

   The improvement of the skin condition is at least one selected from the group consisting of skin barrier improvement, skin wrinkle improvement, skin aging, skin aging by light, skin moisturizing, skin inflammation inhibition, skin soothing and skin regeneration, cosmetic composition.
6. The method of claim 1,

   The improvement of the skin condition is to prevent or improve any one or more selected from the group consisting of skin wounds, dermatitis, atopic dermatitis, pruritus, eczema, erythema, urticaria, psoriasis, and drug rash, cosmetic composition.
7. inotodiol or an acceptable salt thereof; Or, a composition for external application for skin for preventing or improving inflammatory skin disease, comprising an extract of chaga mushroom ( Inonotus obliquus ) containing high concentration of inotodiol as an active ingredient.
8. The method of claim 7,

   The chaga extract contains inotodiol in a concentration of at least 3% (w / v) of the total extract, a composition for external application for skin.
9. The method of claim 7,

   The chaga extract containing the inotodiol at a high concentration is prepared by extracting the chaga extract with an organic solvent to obtain a primary extract; preparing a secondary extract by performing secondary extraction of the precipitate obtained from the primary extract with an organic solvent; And A composition for external application for skin prepared by a method comprising mixing, concentrating, and filtering the primary and secondary extracts.
10. The method of claim 7,

    The skin inflammatory disease is any one or more selected from the group consisting of skin wounds, dermatitis, atopic dermatitis, pruritus, eczema, erythema, urticaria, psoriasis, and drug rash, composition for external application for skin.
11. inotodiol or an acceptable salt thereof; Or a pharmaceutical composition for the prevention or improvement of skin inflammatory diseases comprising an extract of chaga mushroom ( Inonotus obliquus ) containing high concentration of inotodiol as an active ingredient.
12. The method of claim 11,

    The chaga extract is a pharmaceutical composition comprising inotodiol in a concentration of at least 3% (w / v) of the total extract.
13. inotodiol or an acceptable salt thereof for preventing, ameliorating, or treating inflammatory skin diseases, or improving skin conditions; Or use of a chaga mushroom ( Inonotus obliquus ) extract containing inotodiol in high concentration.
14. an effective amount of inotodiol or an acceptable salt thereof; Or a method for improving skin conditions, preventing, improving, or treating skin inflammatory diseases, comprising administering to a subject an extract of chaga mushroom containing inotodiol at a high concentration.

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