Composition for improving skin itch or calming skin

Abstract

The present invention relates to a composition for improving skin, which comprises beta-sitosterol and betulin and, in the case of the composition, reduces dermatoneuropathic stimulation-inducing factors, increases the expression of moisturizing-related factors, increases skin absorbance, improves skin penetration, inhibits mast cell activity, and improves skin quality. Thus, it has been confirmed that the composition has excellent skin-calming and skin-irritation-relieving effects, and thus can be usefully used in skin-improving cosmetics, pharmaceuticals, and the like.

Description

Technical Field

[0001] This invention relates to a skin-improving composition comprising β-sitosterol and betulin. The composition is characterized by its ability to improve skin itching, its skin-soothing effect, and its significant improvement in skin permeability. Background Technology

[0002] Human skin, as the body's first line of defense, protects internal organs from external environmental stimuli such as changes in temperature and humidity, ultraviolet radiation, and pollutants. It also acts as a sensor, sensitively responding to external stimuli. While skin sensitivity is influenced by genetic factors, it is also significantly affected by external factors. For example, environmental pollution, ultraviolet radiation, dry air, chemicals, and stress can all trigger skin sensitivity, and using inappropriate skincare products can worsen it. Therefore, it is recommended that people with sensitive skin use preparations with appropriate skin-soothing or irritation-reducing effects to protect their skin.

[0003] Excessive hypersensitivity, along with skin irritation and itching, is induced by a variety of physiological mechanisms. First, damage to the skin barrier mediates various inflammatory responses, leading to a range of pathological phenomena such as irritation and itching (James Del Rosso, J Clin Aesthet Dermatol. 2016). In atopic dermatitis and psoriasis, the most prominent problem is damage to the skin barrier; it has been confirmed that repairing the skin barrier reduces inflammatory responses and irritation. Therefore, skin humidity and dryness are also related to skin sensitivity. In dry environments, damage to the skin barrier and increased penetration of external substances (M Denda, J Invest Dermatol. 1998) induce more inflammatory responses (Y. Ashida, Br J Dermatol. 2003). Thus, skin hydration or moisture retention capacity is also a crucial factor in managing skin sensitivity.

[0004] Recent studies have confirmed that the activity of TRPV1 (Transient Receptor Potential Vanilloid 1) receptors in skin cells and nerve cells has a significant impact on skin inflammation and sensitivity (Michelle D. Bagood, Int. J. Mol. Sci. 2021). TRPV1 receptors act as ion channel receptors that regulate neurotransmission in response to temperature and chemical stimuli. They respond to temperature, pH, and chemical stimuli, allowing calcium cations to flow into the cell, thereby inducing neurotransmission signals. In the skin, TRPV1 performs physiological functions such as regulating body temperature, sensing pain, and sensing heat. Studies have shown that TRPV1 is overexpressed in individuals with sensitive skin (Adriana Ehnis-Pιrez, J Cosmet Dermatol. 2016), and it is known that treating TRPV1 with antagonists (agonists) can alleviate this skin irritation (Thomas Kueper. Exp Dermatol, 2010).

[0005] Mast cells in skin tissue produce various inflammatory mediators, such as histamine, in response to external stimuli, inducing allergic reactions and itching. It is understood that inhibiting this reaction can suppress skin inflammation and itching (Tiphaine Voisin, Immunity. 2019).

[0006] Although a variety of cosmetic or topical skin ingredients have been developed, there are limited combinations that exhibit all of the above effects or demonstrate a satisfactory level of effectiveness.

[0007] In response, the inventors, while researching substances that can relieve pruritus while also improving skin condition, discovered that the combination of betulin, sitosterol, and troxerutin effectively relieves and soothes skin itching.

[0008] The outer layer of the skin contains 10–15 corneocytes, with a thickness of 10–45 μm. It is composed of structures called mortar and bricks. The brick structures are made up of keratin-rich corneocytes, filled with ceramides, fatty acids, waxes, etc., acting as mortar. This prevents the loss of internal moisture and protects against external invasion. Therefore, although external substances have difficulty penetrating the skin layer, low-molecular-weight components below 500 Da can be transported into the skin through diffusion (Exp. Dermatol., 2000, 9, 165-9.). Alternatively, they can be transported through sweat glands, pores, sebaceous glands, etc., without penetrating the corneocyte layer.

[0009] For this reason, research is being actively conducted to develop a method that can penetrate the skin regardless of the size or properties of molecules and can be uniformly delivered throughout the skin.

[0010] Against this backdrop, the inventors discovered that β-sitosterol, betulin, and troxerutin have excellent effects on improving the skin, specifically relieving skin itching, calming the skin, and enhancing skin penetration, and thus completed this invention. Summary of the Invention

[0011] The problem to be solved

[0012] The purpose of this invention is to provide a skin-improving composition comprising β-sitosterol and betulin as active ingredients.

[0013] The composition described above is characterized by relieving skin itching, calming the skin, and significantly improving skin permeability.

[0014] Another object of the present invention is to provide a cosmetic composition for improving skin, comprising β-sitosterol and betulin as active ingredients.

[0015] Another object of the present invention is to provide a topical pharmaceutical composition for improving skin, comprising β-sitosterol and betulin as active ingredients.

[0016] Methods for solving problems

[0017] The specific details are explained below. Furthermore, the various descriptions and embodiments disclosed in this invention can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Additionally, the scope of this invention should not be considered limited by the specific descriptions below.

[0018] Furthermore, those skilled in the art can recognize or identify multiple equivalents to specific aspects of the invention described herein using only conventional experiments. Moreover, such equivalents are intended to be included in this invention.

[0019] One aspect of the present invention for achieving the above objectives provides a skin-improving composition comprising β-sitosterol and betulin as active ingredients.

[0020] The term "β-sitosterol" in this invention has a chemical structure similar to cholesterol and is a type of phytosterol. β-sitosterol has been used as a medicine for treating burns or wounds on the skin. The term β-sitosterol in this invention can be used interchangeably with the term sitosterol.

[0021] The β-sitosterol of the present invention may be a substance isolated or purified from an extract or fraction thereof obtained from a source plant containing it as an active ingredient, but is not limited to the method of obtaining it.

[0022] In this invention, the composition may contain 0.00001 to 3% (w / w) of β-sitosterol relative to the total weight of the composition. Specifically, it may contain 0.00001 to 3%, 0.001 to 2%, 0.01 to 1%, 0.05 to 1%, or 0.1 to 1% of β-sitosterol, but is not limited thereto.

[0023] The term "betulin" in this invention refers to a triterpene compound obtained from the bark or sap of *Betula spp.*. It is known to possess anticancer, sedative, and anti-aging effects.

[0024] The betulinol of the present invention can be a substance isolated or purified from an extract or fraction thereof obtained from a source plant containing it as an active ingredient, but is not limited to the method of obtaining it.

[0025] In this invention, the composition may contain 0.00001 to 3% (w / w) betulin relative to the total weight of the composition. Specifically, it may contain 0.00001 to 3%, 0.001 to 2%, 0.01 to 1%, 0.05 to 1%, or 0.1 to 1% betulin, but is not limited thereto.

[0026] In addition, in this invention, β-sitosterol and betulinol can be mixed in a weight ratio of 10:1 to 1:10, specifically 7:1 to 1:7, more specifically 5:1 to 1:5 or 3:1 to 1:3, but are not limited thereto.

[0027] The term "skin penetration" in this invention refers to the ability to penetrate the skin through a biological membrane. This biological membrane can refer to cell permeation, skin absorption, and / or mucosal permeability, but is not limited thereto. In this invention, "skin permeability" refers to the measurement of the skin permeability of drugs (ointments, plasters, sprays, etc.) and cosmetics applied to the skin.

[0028] The term "improvement" in this invention refers to all behaviors that at least reduce parameters related to the relief or treatment of a condition, such as the degree of symptoms, and may encompass all of the following concepts: selected from any one or more of moisturizing skin, calming skin, regenerating skin, improving skin inflammation, protecting skin from external stimuli, calming sensitive skin, improving sensitive skin, relieving erythema, relieving pruritus, strengthening skin barrier function, relieving skin irritation, improving skin permeability, and relieving skin pain.

[0029] In one embodiment of the present invention, it was confirmed that the combination of β-sitosterol and betulinol has a significant effect on improving the skin compared with betulinol alone, specifically improving neuroinflammatory, calming the skin, improving pruritus, enhancing moisturizing, anti-allergy and skin penetration effects, and also improving the stability in solvents.

[0030] The above composition can be used to improve skin penetration, but is not limited thereto.

[0031] In this invention, the composition comprising β-sitosterol and betulinol is characterized by having anti-inflammatory, skin-soothing, pruritus-relieving, moisturizing, anti-allergic, and skin-irritation-relieving effects.

[0032] In this invention, the term "inflammation" refers to an in vivo response to damage or infection of a specific tissue, primarily mediated by immune cells. The aim of such inflammation is to maximally suppress tissue damage, remove infectious agents, and regenerate tissue. In this invention, inflammation may refer to neuroinflammatory inflammation or inflammatory skin diseases, but is not limited to these.

[0033] The aforementioned "neurogenic inflammation," "neurogenic inflammatory diseases," or "neurogenic inflammatory skin diseases" refer to inflammation limited to the peripheral nervous system (PNS) and central nervous system (CNS), or skin diseases that occur or are associated with these systems. Neurogenic inflammation is induced by nerve activation, leading to pain, itching, rashes, edema, and swelling of the skin. It is known to induce diffuse lesions in a short period and mediate long-term inflammatory responses. The aforementioned neurogenic inflammatory diseases or neurogenic inflammatory skin diseases can include atopic dermatitis, eczema, psoriasis, rashes, prurigo nodularis (PN), etc., but are not limited to these. Furthermore, the aforementioned neurogenic inflammation is considered a major cause of sensitive skin.

[0034] In this invention, "TRPV1 (transient receptor potential vanilloid 1) receptor or cutaneous neurostimulation induced receptor TRPV1" is a receptor that is abundantly distributed in skin cells and nerve cells within the skin. Its function is to sense external stimuli or pain and induce an inflammatory response in surrounding tissues. When TRPV1 is overactivated, the threshold for itch decreases, which means that itching sensations such as pruritus increase, potentially leading to sensitive skin.

[0035] Therefore, inhibiting TRPV1 can improve sensitive skin. In one embodiment of the present invention, the improvement effect on sensitive skin was confirmed by inhibiting TRPV1 with the composition of this application.

[0036] Specifically, in one embodiment of the present invention, it was confirmed that the combination of β-sitosterol and betulinol exhibited significantly superior TRPV1 inhibition compared to β-sitosterol and betulinol alone.

[0037] The term "soothing skin" in this invention not only means relieving and calming erythema, itchy skin due to irritation, or sensitive skin, but also means the prevention of such symptoms, and can encompass the concept of relieving skin irritation. For example, relieving pruritus, relieving skin pain, and reducing redness can be listed, but are not limited to these.

[0038] The term "pruritus" in this invention, also known as itchy sensation, refers to an uncomfortable feeling that causes the urge to scratch or rub the skin. Generally, it may occur independently of external stimuli, but it can also be induced by slight mechanical contact with external substances, changes in ambient temperature, chemical substances, or electrical stimulation. In this invention, "improvement of pruritus" can be used interchangeably with the term anti-pruritus.

[0039] In this invention, the term "moisturizing" refers to all actions that maintain skin's suppleness and smooth surface by supplying moisture to the skin or preventing moisture evaporation, while "dryness" refers to this state of insufficient skin moisturization. It is understood that dry skin contributes to skin barrier collapse and inflammation, and increased skin hydration, in addition to its moisturizing effect, can also be expected to have a soothing effect on the skin.

[0040] In this invention, hyaluronic acid can contain 300 to 1000 times its own weight in water, thus exhibiting a moisturizing effect on the skin by binding with water.

[0041] In one embodiment of the present invention, it was confirmed that the composition of the present invention increases the expression of the hyaluronic acid synthesis gene (HAS3), thereby confirming an increase in skin moisture and a moisturizing effect.

[0042] More specifically, it was confirmed that the HAS3 gene expression level of the combination of β-sitosterol and betulinol of the present invention was significantly increased compared with β-sitosterol and betulinol alone.

[0043] Moreover, the excellent moisturizing effect described above indicates that the composition of the present invention can prevent, improve or alleviate skin diseases such as psoriasis, xerosis or eczema.

[0044] The term "anti-allergy" in this invention encompasses the improvement (reduction of symptoms), treatment, and prevention (inhibition or delay of onset) of allergic diseases. Allergic diseases refer to pathological symptoms caused by allergic reactions mediated by mast cell activation, such as mast cell degranulation. Such allergic diseases may include, but are not limited to, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, allergic contact dermatitis, and allergic keratitis.

[0045] The aforementioned anti-allergy measures can inhibit the secretion / expression level of β-hexosaminidase in inflammatory cells by inhibiting the degranulation of inflammatory cells, or inhibit the secretion / expression level of histamine and other substances.

[0046] In one embodiment of the present invention, it was confirmed that the combination of β-sitosterol and betulin, and the combination of β-sitosterol, betulin, and troxerutin have superior inhibitory effects on the secretion of β-hexosaminease compared to β-sitosterol, betulin, and troxerutin alone.

[0047] The composition of the present invention comprising β-sitosterol and betulin may further comprise troxerutin.

[0048] The term "Troxerutin" in this invention refers to a hydroxyethyl ether derivative of rutin extracted from the Sophora japonica tree, also known as vitamin P. It has the effects of protecting blood vessels, improving capillary permeability and circulation, and preventing leakage of vascular fluids; therefore, it is used to treat hemorrhoids and varicose veins.

[0049] The troxerutin of the present invention can be a substance isolated or purified from an extract or fraction thereof obtained from a source plant containing it as an active ingredient, but is not limited to the method of obtaining it.

[0050] In this invention, the composition may contain 0.00001 to 3% (w / w) of troxerutin relative to the total weight of the composition. Specifically, it may contain 0.00001 to 3%, 0.001 to 2%, 0.01 to 1%, 0.05 to 1%, or 0.1 to 1% of troxerutin, but is not limited thereto.

[0051] Another aspect of the present invention provides a cosmetic composition for improving skin, comprising β-sitosterol and betulin as active ingredients.

[0052] The terms β-sitosterol, betulinol, skin improvement, etc. in this invention are as described above.

[0053] The above cosmetic composition may further contain troxerutin.

[0054] In addition, the cosmetic composition described above may contain 0.00001 to 3% (w / w) of β-sitosterol relative to the total weight of the composition, may contain 0.00001 to 3% (w / w) of betulin relative to the total weight of the composition, and may contain 0.00001 to 3% (w / w) of troxerutin relative to the total weight of the composition, but is not limited thereto.

[0055] In addition, the above-mentioned cosmetic composition can be used to improve skin penetration, but is not limited thereto.

[0056] In this invention, the above-mentioned cosmetic composition can be prepared into a dosage form selected from the group consisting of solutions, topical ointments, creams, foams, nourishing lotions, softening lotions, masks, softening waters, lotions, makeup bases, serums, liquid cleansers, bath additives, sunscreens, sunscreen oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, cleansers containing surfactants, oils, powder foundations, emulsion foundations, wax foundations, patches, and sprays, but is not limited thereto.

[0057] In this invention, the cosmetic composition may further include one or more cosmetically acceptable carriers incorporated into general skin cosmetics. As conventional ingredients, such as oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., may be appropriately incorporated, but are not limited thereto.

[0058] The cosmetically acceptable carriers contained in the cosmetic compositions of the present invention vary depending on the dosage form of the cosmetic composition.

[0059] When the dosage form of this invention is an ointment, paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin wax, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc., can be used as the carrier component, but are not limited thereto. These can be used alone or in combination of two or more.

[0060] When the dosage form of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc., can be used as the carrier component. Especially in the case of a spray, propellants such as chlorofluorocarbons, propane / butane, or dimethyl ether can be additionally included, but are not limited thereto. These can be used alone or in combination of two or more.

[0061] When the dosage form of the present invention is a solution or emulsion, the carrier component can be a solvent, solubilizer, or emulsifier, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylethylene glycol oil, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol aliphatic esters, polyethylene glycol, or fatty acid esters of sorbitol, but is not limited thereto. These can be used alone or in combination of two or more.

[0062] When the dosage form of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester, and polyoxyethylene dehydrated sorbitan ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, or tragacanth gum can be used as the carrier component, but are not limited thereto. These can be used alone or in combination of two or more.

[0063] When the dosage form of this invention is soap, the carrier component can be alkali metal salts of fatty acids, fatty acid half-esters, fatty acid protein hydrolysates, hydroxyethyl sulfonates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerin, sugars, etc., but is not limited to these. These can be used alone or in combination of two or more.

[0064] Another object of the present invention is to provide a topical pharmaceutical composition for improving skin, comprising β-sitosterol and betulin as active ingredients.

[0065] In this invention, the above composition may further contain troxerutin.

[0066] The terms “β-sitosterol,” “betulin,” and “skin improvement” used in this invention are as described above.

[0067] In addition, the above-mentioned pharmaceutical composition may contain 0.00001 to 3% (w / w) of β-sitosterol relative to the total weight of the composition, may contain 0.00001 to 3% (w / w) of betulin relative to the total weight of the composition, and may contain 0.00001 to 3% (w / w) of troxerutin relative to the total weight of the composition, but is not limited thereto.

[0068] In addition, the above-mentioned topical pharmaceutical compositions can be used to improve skin penetration, but are not limited thereto.

[0069] In addition to the above-mentioned components, the pharmaceutical composition of the present invention may further include, as needed, a pharmaceutically acceptable carrier, excipient, or diluent. The aforementioned pharmaceutically acceptable carrier, excipient, or diluent is not limited, as long as it does not impair the effects of the present invention, and may include, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, flavorings, preservatives, etc.

[0070] Examples of the pharmaceutical external product compositions of the present invention include disinfectant cleaners, bath foams, ointments, wet wipes, and balms, but they are not limited thereto. The formulation method, dosage, application method, and constituent components of the pharmaceutical external product can be appropriately selected from conventional techniques known in the art.

[0071] Another aspect of the invention provides a method for improving skin, comprising administering a composition containing β-sitosterol and betulin as active ingredients to an individual.

[0072] In this invention, the above composition may further contain troxerutin.

[0073] The terms “β-sitosterol,” “betulin,” and “skin improvement” used in this invention are as described above.

[0074] As used in this invention, the term "administration" refers to the introduction of the composition of this invention into a patient by any suitable method. The route of administration of the composition can be any general route, as long as it reaches the target tissue. Based on the properties of the composition of this invention to soothe or relieve skin irritation, the route of administration of the composition can be by applying it to the skin.

[0075] The compositions of the present invention can be administered daily or intermittently, and the number of administrations per day can be divided into one or two to three doses. Furthermore, to soothe the skin or relieve skin irritation, the compositions of the present invention can be used alone or in combination with other drug treatments. Considering all the above factors, it is important to administer the composition in an amount that yields the maximum effect with the minimum dosage and without side effects, which can be readily determined by those skilled in the art.

[0076] In this invention, the term "individual" refers to all animals, including humans, such as rats, mice, and livestock, that have developed or may develop skin irritation. Specifically, it can include mammals, including humans.

[0077] Another aspect of the present invention provides a method for improving neuroinflammatory symptoms, a method for sedating skin, a method for improving pruritus, a method for enhancing hydration, a method for treating allergies, or a method for relieving skin irritation, including the step of administering a composition containing β-sitosterol and betulin as active ingredients to an individual.

[0078] The above-described compositions of the present invention may further contain troxerutin.

[0079] The terms “β-sitosterol”, “betulinol”, “skin improvement”, “neuroinflammatory improvement”, “skin calming”, “pruritus improvement”, “enhanced moisturizing”, “anti-allergy” and “skin irritation relief” in the present invention are as described above.

[0080] One aspect of the present invention for achieving the above objectives provides a composition comprising β-sitosterol and betulin as active ingredients for use in improving skin; improving neuroinflammatory function; calming skin; improving pruritus; enhancing moisturization; anti-allergy; or relieving skin irritation.

[0081] The above-mentioned composition containing β-sitosterol and betulin may further contain troxerutin.

[0082] The terms “β-sitosterol”, “betulinol”, “skin improvement”, “neuroinflammatory improvement”, “skin calming”, “pruritus improvement”, “enhanced moisturizing”, “anti-allergy” and “skin irritation relief” in the present invention are as described above.

[0083] In order to achieve the above-mentioned objectives, another aspect of the present invention provides the use of β-sitosterol and betulin as active ingredients in the preparation of compositions for improving skin; the use of β-sitosterol and betulin as active ingredients in the preparation of compositions for improving neuroinflammatory disorders; the use of β-sitosterol and betulin as active ingredients in the preparation of compositions for calming the skin; the use of β-sitosterol and betulin as active ingredients in the preparation of compositions for improving pruritus; the use of β-sitosterol and betulin as active ingredients in the preparation of compositions for enhancing moisturization; the use of β-sitosterol and betulin as active ingredients in the preparation of compositions for treating allergies; or the use of β-sitosterol and betulin as active ingredients in the preparation of compositions for relieving skin irritation.

[0084] The above-mentioned composition containing β-sitosterol and betulin may further contain troxerutin.

[0085] The terms “β-sitosterol”, “betulinol”, “skin improvement”, “neuroinflammatory improvement”, “skin calming”, “pruritus improvement”, “enhanced moisturizing”, “anti-allergy” and “skin irritation relief” in the present invention are as described above.

[0086] Invention Effects

[0087] In the case of the skin-improving composition according to the present invention containing β-sitosterol and betulin as active ingredients, it is found to have excellent effects in calming the skin and relieving skin irritation by reducing skin neurostimulation inducing factors, increasing the expression of moisturizing-related factors, increasing skin absorption, and inhibiting mast cell activity. Therefore, it can be usefully used in skin-improving cosmetics, quasi-medicinal products, etc. Detailed Implementation

[0088] The present invention will now be described in more detail through embodiments and experimental examples. However, these embodiments and experimental examples are only for illustrative purposes, and the scope of the present invention is not limited to these embodiments and experimental examples.

[0089] Example 1. Transient receptor potential (TRPV1) induced by skin neurostimulation Evaluation of vanilloid activity inhibition effect

[0090] HEK293 cell lines overexpressing TRPV1 were suspended in DMEM medium and aliquoted into 96-well plates, then cultured at 37°C in a 5% CO2 incubator for 24 hours. Afterward, the cells were washed with PBS buffer and treated with calcium-binding dye using the Invitrogen Fluo-4 Direct™ Calcium Assay Kit. The experimental materials were then treated as shown in Table 1 below, and the cells were cultured at 37°C in a 5% CO2 incubator for 40 minutes. To measure TRPV1 activation, the fluorescence intensity was measured using a fluorescence microscope 2 minutes after treatment with 0.5 μM capsaicin. The activation level (fluorescence) of TRPV1 was then calculated using the background and peak values ​​according to the following mathematical formula (Table 1).

[0091]

[0092] Table 1 below shows the results of the reduction in TRPV1 activation relative to the untreated group treated only with capsaicin on the prepared cell lines, expressed as inhibition capacity (%) when the experimental subject was treated separately.

[0093] [Table 1]

[0094]

[0095] As shown in Table 1 above, the TRPV1 inhibitory capacity of β-sitosterol alone did not differ statistically significantly from the standard value. Conversely, when β-sitosterol was combined with betulin, a significant TRPV1 inhibitory capacity was confirmed.

[0096] Therefore, it can be seen that the β-sitosterol and betulin of the present invention have excellent effects in improving skin neuroinflammation, reducing nerve stimulation / pain, calming the skin, improving pruritus, and improving the skin.

[0097] Example 2. HAS3 (hyaluronic acid synthase 3), a marker of skin moisturizing function Synthase 3) Activity Enhancement Effect Evaluation

[0098] Keratinocytes were suspended in Ca 2+After adding 0.01 mM DMEM culture medium, the cells were aliquoted into 12-well plates and cultured at 37°C in a 5% CO2 incubator for 24 hours. The cultured cells were washed with PBS and treated as shown in Table 2 below. RNA was then extracted from the cells using an RNA extraction kit (AccuPrep® Universal RNA Extraction Kit), and RT-qPCR was performed using a TOPscript™ RT DryMIX (dN6) and TOPreal™ SYBR Green RT-qPCR Kit to measure HAS3 gene expression levels (Table 2).

[0099] Table 2 below shows the relative values ​​(percentage %) of HAS3 expression levels compared to the untreated group when the experimental subjects were treated.

[0100] [Table 2]

[0101]

[0102] As shown in Table 2 above, it was confirmed that the HAS3 gene expression level was significantly increased when β-sitosterol and betulinol were combined compared with β-sitosterol and betulinol alone.

[0103] Therefore, it can be seen that the β-sitosterol and betulin of the present invention increase the water content in the skin, thereby exhibiting excellent effects in moisturizing the skin.

[0104] Example 3. Skin permeability evaluation

[0105] To confirm the skin penetration efficiency of the active ingredients, skin penetration was evaluated using cream compositions containing β-sitosterol or betulin. The cream compositions contained the following ingredients in both the oil and aqueous phases.

[0106] <Cream Composition>

[0107] Oil phase: cetyl stearyl alcohol, glyceryl stearate, PEG-40 stearate, ceteareth-20, beeswax, C14-22 alcohols, C12-20 alkyl glucoside, lecithin, tocopherol, caprylic / capric triglyceride, squalene, cyclopentasiloxane, cyclohexasiloxane, dimethicone / vinyl dimethicone crosspolymer, isocetyl myristate.

[0108] Aqueous phase: dipropylene glycol, glycerin, betaine, 1,2-hexanediol, EDTA-3Na, xanthan gum, carbomer, tromethamine, water.

[0109] Add all the oil phase components to a beaker and slowly heat to 60°C while stirring. Add all the aqueous phase components to another beaker and slowly heat to 60°C while stirring. Then, while stirring the oil phase, gradually pour the aqueous phase into the oil phase to emulsify. After emulsification is complete, allow the mixture to stand and cool to room temperature.

[0110] In the above process, betulin was added to the oil phase at 0.1% of the final composition's mass. Similarly, sitosterol was added to the oil phase of each cream composition at the concentrations shown in Table 3, based on 100% of the final composition's mass.

[0111] 100 μL of the prepared cream was applied to the upper end (corneal side) of pigskin (Apures, 2.5 cm × 2.5 cm). The pigskin was then adhered to a Franz diffusion cell and incubated at 37°C for 24 hours. The reservoir solution was prepared using ethanol / PBS (1:1). The pigskin was then homogenized, and 1 mL of ethanol / PBS (1:1) was added to fully dissolve the betulinol. The mixture was then centrifuged at 12,000 rpm for 10 minutes. After centrifugation, the supernatant was collected, and the total mass of betulinol contained in the supernatant and reservoir was quantified. The sum of these values ​​was taken as the total betulinol permeation. Quantification of betulinol was performed using RP-HPLC under the following conditions.

[0112] - Mobile phase: Acetonitrile-water (86:14, v / v), isocratic elution.

[0113] -Injection volume 20ul, flow rate 1ml / min

[0114] - Measure absorbance at 210 nm

[0115] [Table 3]

[0116]

[0117] As shown in Table 3 above, the combination of β-sitosterol and betulinol was found to have increased skin penetration compared to betulinol alone.

[0118] Therefore, it can be seen that the β-sitosterol and betulin of the present invention increase skin permeability, thereby showing excellent effects in improving the skin.

[0119] Example 4. Stability Evaluation

[0120] Betulin and sitosterol have low solubility not only in water but also in many other solvents, making them prone to precipitation or stability issues. This experiment investigated the effect of simultaneously containing two poorly soluble components on their respective solubility.

[0121] First, to simulate a composition containing large amounts of organic solvent and water, a solvent was prepared by mixing DPG and distilled water in a 3:4 ratio. For experimental convenience, poloxamer 188, acting as a solubilizer, was added to this solvent at 1% by weight. The experimental material and the prepared solvent were then added to 20 mL glass vials, ensuring the experimental material was contained in the specified weight ratio as shown in Table 4. At this point, it was ensured that the final composition in each vial was identical, totaling 10 g. The vials were then stirred for 30 minutes, allowed to stand for 1 minute, and the presence of any precipitate was observed. The results are shown in Table 4.

[0122] [Table 4]

[0123]

[0124] The results confirm that precipitates are formed when β-sitosterol and betulin are included individually, but no precipitates are formed when they are included in combination.

[0125] This confirms that in compositions containing a large amount of organic solvent or water, the solubility is improved when the components are used in combination compared to using each component alone.

[0126] Example 5. Evaluation of the inhibitory effect on mast cell activity

[0127] Mast cells (RBL-2H3) were suspended in DMEM medium and aliquoted into 24-well plates, then cultured at 37°C in a 5% CO2 incubator for 15 hours. The experimental materials were then treated with the cells for 2 hours according to Tables 5-7. Afterward, the cells were washed with Siraganian buffer and reacted in C48 / 80 (50 μg / ml in Siraganian buffer) for 30 minutes. The reaction was then terminated by placing the culture plates on ice. Here, 50 μl of reaction supernatant was obtained and transferred to a 96-well plate, followed by the addition of 50 μl of substrate buffer (2 mM 4-nitrophenyl N-acetyl-β-D-glucosaminidide, 0.05 M sodium citrate, pH 4.5), and the reaction was carried out for 90 minutes. Then, 200 μl of stop buffer (0.1 M sodium carbonate / sodium bicarbonate, pH 10.5) was added to terminate the reaction. Next, the absorbance was measured at 405 nm to quantify the degranulation index, β-hexosaminidase. The amount of β-hexosamine released was calculated as a relative value to the amount released in the untreated group (negative control group), as shown in the following formula. The β-hexosamine release inhibition rate was calculated as the percentage (%) of the amount of release reduced when the experimental material was treated (experimental group) to the amount of release in the positive control group that was treated with C48 / 80 (50 μg / ml, in siraganian buffer) but not the experimental material, and is shown in Tables 5 to 7.

[0128] β-hexosaminease release inhibition rate = (positive control group - experimental group) ÷ positive control group × 100

[0129] [Table 5]

[0130]

[0131] [Table 6]

[0132]

[0133] [Table 7]

[0134]

[0135] As shown in Tables 5 to 7 above, it was confirmed that the combination of β-sitosterol and betulinol exhibited superior inhibitory effects on the secretion of β-hexosamine enzyme compared to β-sitosterol and betulinol alone. Furthermore, compared to β-sitosterol, betulinol, and troxerutin alone, the combination of troxerutin with β-sitosterol and betulinol significantly enhanced the inhibitory effects on the secretion of β-hexosamine enzyme.

[0136] The results show that the combination of β-sitosterol and betulin, and the combination of β-sitosterol, betulin, and troxerutin effectively inhibit mast cell degranulation, thus exhibiting excellent anti-allergic, pruritus-relieving, skin-soothing, and skin-irritation-relieving effects.

[0137] Based on the foregoing description, those skilled in the art should understand that the present invention can be implemented in other specific forms without altering its technical concept or essential features. Relatedly, it must be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be interpreted to include the meaning and scope of the following claims and all modifications or variations derived from their equivalents, and should not be construed as including only the detailed description above.

Claims

1. A skin-improving composition comprising β-sitosterol and betulin as active ingredients.

2. The composition according to claim 1, wherein, The composition contains 0.00001 to 3% (w / w) of the β-sitosterol relative to the total weight of the composition.

3. The composition according to claim 1, wherein, The composition comprises 0.00001 to 3% (w / w) of the betulin relative to the total weight of the composition.

4. The composition according to claim 1, characterized in that, The composition has the effects of improving neuroinflammatory reactions, soothing the skin, relieving pruritus, enhancing moisturization, anti-allergy, and relieving skin irritation.

5. The composition according to claim 1, wherein, The composition also contains troxerutin.

6. The composition according to claim 5, wherein, The composition contains 0.00001 to 3% (w / w) of the troxerutin relative to the total weight of the composition.

7. A cosmetic composition comprising the composition according to any one of claims 1 to 6.

8. A quasi-pharmaceutical composition comprising the composition according to any one of claims 1 to 6.

9. A skin-improving use of a composition comprising β-sitosterol and betulin as active ingredients.

10. The use according to claim 9, wherein, The skin improvement is selected from any one of the following: calming the skin, enhancing hydration, and relieving skin irritation.

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