KR102493443B1 - Cosmetic composition comprising extracts extracted with an eco-friendly natural eutectic solvent - Google Patents

Abstract

The present invention relates to a cosmetic composition which is obtained by extracting natural products using a eutectic solvent of betaine and citric acid, thereby having low cytotoxicity compared to organic solvent extracts, and having improved safety and stability by preventing the discoloration of the extract. Therefore, the cosmetic composition of the present invention can be usefully used as a cosmetic product for improving skin conditions and an external skin preparation.

Description

Cosmetic composition comprising extracts extracted with an eco-friendly natural eutectic solvent

It relates to a cosmetic composition.

Generally, organic solvents are used for liquid phase extraction. Organic solvents have the advantages of being inexpensive and having good extraction efficiency, but have a problem in that they are harmful to the user's health or cause environmental pollution. Accordingly, as interest in sustainable development increases, studies related to extraction solvents that can replace organic solvents are being actively conducted.

Among them, eutectic solvent is proposed as an alternative. A eutectic solvent is a eutectic mixture in which two types of components are mixed and exhibit a lower melting point than each component. In the early stage of development of eutectic solvents, ionic liquids were used as an alternative to organic solvents, but they were considered unsuitable as environmentally friendly solvents due to low biodegradability, toxicity, and the amount of pollutants emitted during the manufacturing process. There is a report. Since then, research on deep eutectic solvents, natural eutectic solvents, and the like, which compensate for the disadvantages of conventional ionic liquids, has been conducted. However, deep eutectic solvents and natural eutectic solvents not only contain many ingredients that cannot be used in cosmetics, such as choline chloride, but also have problems such as high viscosity and discoloration of the solvent during the manufacturing process of the eutectic solvent. Therefore, there is a limit to the application of cosmetics and external skin products.

Therefore, in order to solve the above problems, it is necessary to develop an eco-friendly natural eutectic solvent.

One aspect is to provide a composition for improving skin conditions containing, as an active ingredient, a natural extract extracted with an eco-friendly natural eutectic solvent.

Another aspect is to provide a method for preparing an environmentally friendly natural eutectic solvent.

One aspect provides a composition for improving skin condition comprising, as an active ingredient, a natural extract extracted with an eco-friendly natural eutectic solvent.

The natural eutectic solvent may be at least one selected from the group consisting of amino acids or their derivatives, and organic acids.

Such amino acids or derivatives thereof include, for example, betaine, alanine, cysteine, aspartic acid, glutamine, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine , tryptophan, tyrosine, and the like.

In addition, the organic acids are, for example, citric acid, lactic acid, malic acid, maleic acid, pyruvic acid, fumaric acid, succinic acid, acetic acid, aconitic acid, tartric acid, ascorbic acid, malonic acid, oxalic acid, glucuronic acid, neuraminic acid, sialic acid, shikimic acid, phytic acid, galacturonic acid, iduronic acid, hyaluronic acid, hydroxycitric acid, lactone derivatives and the like.

In one embodiment, the natural eutectic solvent may be a mixture of betaine and citric acid. The betaine and citric acid may be mixed in a weight ratio of 1:0.001 to 10. The betaine and citric acid are, for example, 1:0.001 to 10, 1:001 to 8, 1:0.05 to 7, 1:0.05 to 5, 1:0.01 to 10, 1:0.01 to 6, 1:0.1 to 4, 1:0.1 to 3, 1:1 to 10, 1:1 to 4, or 1:1 to 2 in weight ratio. At this time, when the mixed weight ratio of betaine and citric acid is less than or exceeds the above range, there are problems in that betaine and citric acid are precipitated, or the eutectic solvent is suspended or discolored.

In another embodiment, the natural eutectic solvent may be pH 2.5 to pH 8. Generally, when betaine and citric acid are mixed, an acidic solution with a pH less than 2.5 is prepared. Since the acidity of the extract extracted with an acidic solvent is low, it is highly likely to cause irritation or skin damage when applied to the skin. Therefore, the pH of the natural eutectic solvent can be adjusted to 2.5 to 8 by adding a pH adjusting agent to the mixture of betaine and citric acid. The natural eutectic solvent is, for example, pH 2.5 to pH 8, pH 2.5 to pH 7.5, pH 3 to pH 7, pH 3 to pH 6.5, pH 3.5 to pH 6, pH 4 to pH 6 or pH 4.5 to pH 5.5 can be At this time, when the pH of the natural eutectic solvent is less than the above range, there is a problem of causing skin irritation due to high acidity. There is a problem with not being able to do it.

The pH adjusting agent may be a base or a buffering agent, for example, trisodium citrate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, arginine, tro Tromethamine, triethanol amine, aminomethyl propanol, dipotassium phosphate, potassium phosphate, sodium anisate, sodium lactate , sodium carbonate, and urea.

The natural products are figs, kwonbaek, black rice, blood, black beans, jacaranda, seolryeon, rowan, pollen, water frog rice, juniper, bongmicho, dicentra, mistletoe, sedum, sedum, Sanghwang mushroom, tomato, houttuynia cordata, dermis, peony, chestnut flower It may be selected from the group consisting of kudzu, kudzu, and walnut.

The natural product extract may be extracted by cutting the dried natural product and adding a natural eutectic solvent in an amount of 2 to 20 times the weight of the natural product. The extraction temperature may be performed at 30 °C to 100 °C. Extraction time may be performed for 1 hour to 10 hours. As the extraction method, cooling, ultrasonic extraction, or reflux cooling extraction method may be used. The number of extractions may be repeated 1 to 5 times.

In one embodiment, the natural product extract extracted with a natural eutectic solvent prepared by mixing betaine and citric acid not only has improved safety and stability compared to the natural product extract extracted with an organic solvent, but also has excellent active oxygen and free radical scavenging ability and UV It was confirmed that the cell damage defense effect by was excellent. In addition, the natural product extract extracted with the natural eutectic solvent significantly suppressed the expression of hyaluronidase, inflammatory cytokines and/or inflammatory mediators, and inhibited tyrosinase activity and melanin synthesis, compared to the natural product extract extracted with an organic solvent. In addition, it was confirmed that the expression of skin barrier or moisturizing, elasticity-related factors was increased, and the expression of acne or sebum, or hair loss-related factors was suppressed.

The skin condition improvement or skin beauty improvement may be suppression or improvement of skin aging, wrinkles, elasticity, strengthening of skin barrier, skin moisturizing, suppression or improvement of skin acne or sebum. In addition, it may prevent, improve, or treat hair loss, or promote hair growth or hair growth.

As used herein, the term "skin aging" refers to both tangible and intangible changes that appear in the skin with age, such as thinning of the epidermis, number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, It refers to reduction in wound healing, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage defense, chemical substance elimination ability, immune response, sensory function, and thermoregulation.

The composition may be used to improve skin aging caused by exogenous factors or endogenous factors. The extrinsic factor refers to various external factors, such as ultraviolet rays (light), and the endogenous factor is also referred to as a chronological factor and refers to a factor mainly caused by the passage of time. That is, the skin aging is not only premature aging symptoms induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemicals, etc., but also a natural aging phenomenon that occurs as the proliferation of skin cells decreases with age. It is a concept that includes wrinkles, loss of elasticity, skin sagging and dryness. In addition, wrinkles include stimulation caused by changes in internal and external factors that cause wrinkles by changing components constituting skin tissue.

The aging may be aging by active oxygen or photoaging.

The "reactive oxygen species" are produced by normal metabolic processes, ultraviolet rays, harmful toxins such as pesticides, cigarette smoke, air pollution, and the like. Active oxygen leads to skin cell and tissue damage and destroys the skin's antioxidant defense network, resulting in lipid peroxidation, protein oxidation, activation of proteolytic enzymes that destroy cellular interstitial components, chain severing and abnormal cross-linking of collagen and elastin, which are elastic fibers, and hyaluronic acid. It causes damage to biological components such as severing of the ronic acid chain, promotion of melanin production, and DNA oxidation.

The "photoaging" is a phenomenon caused by external environmental factors, and the most representative factor is ultraviolet rays. Ultraviolet rays cause damage to biocomponents such as activation of proteolytic enzymes, chain scission of matrix proteins, and abnormal cross-linking, and repetition of these mechanisms results in apparent skin aging.

In this specification, the term "wrinkle" refers to a state in which elasticity of the skin is lost and loosened, and for example, the skin may be folded. The "prevention or improvement of skin wrinkles" may mean any action of preventing or improving wrinkles by suppressing the expression of wrinkle-related factors or increasing the total amount of collagen.

The "strengthen the skin barrier" may refer to any action that enhances the function of the skin barrier, which is located at the outermost layer of the skin and prevents loss of moisture and nutrients.

The "moisturizing skin" may refer to any action that maintains skin moisture or prevents moisture loss.

The "skin disease" may be a disease caused by impaired skin barrier function, skin aging, skin wound, skin scar, or skin inflammation. The term "prevention" includes inhibiting the development of a disease. The term "treatment" includes the inhibition, alleviation, or elimination of the development of a disease.

The skin barrier function impairment may refer to any change that occurs in the skin due to a decrease or damage of the skin barrier function. Examples include increased skin wrinkling, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, and the like.

The skin inflammatory disease may be any one selected from the group consisting of skin wound, dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, drug rash, and acne.

As used herein, the term "alopecia" refers to a phenomenon in which hair falls out from the scalp or a condition in which hair becomes sparse or thin, and "prevention or improvement of hair loss" not only promotes the generation of new hair, but also reduces existing hair. It means growing up healthy.

The hair loss is, for example, androgenetic alopecia, alopecia areata, hereditary androgenetic alopecia, telogen effluvium, traumatic alopecia, trichotillomania, pressure alopecia ( Pressure Alopecia), anagen alopecia, pityroid alopecia, syphilitic alopecia (Alopecia Syphlltiac), seborrheic alopecia (Alopecia Seborrhecia), symptomatic alopecia, scarring alopecia, congenital alopecia, and the like.

As used herein, the term "hair growth" means that hair grows on the scalp, and "hair growth" means that the length of the hair is lengthened (ie, hair growth), and is another term used in the art, "wool" and used with the same meaning.

The composition is effective in improving hair loss caused by exogenous or endogenous factors. The extrinsic factor includes various external factors, such as ultraviolet rays, and the endogenous factor is a factor caused by the passage of time and includes oxidative stress due to cell death of dermal papilla cells due to telomeres and deterioration in cell function. can do.

As used herein, the term "promotion of hair growth or hair growth" means to promote the generation and growth of hair to ultimately increase the specific gravity of anagen hair in total hair. In addition, the hair growth or hair growth promotion means suppressing hair loss caused by a decrease in the specific gravity of hair in the anagen phase.

The composition is 0.001% to 80% by weight, for example, 0.01% to 60% by weight, 0.01% to 40% by weight, 0.01% to 30% by weight, 0.01% to 20% by weight, based on the total weight of the composition. %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20%, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight % to 10%, or 0.1% to 5% by weight of natural eutectic solvent extract. At this time, when the content of the extract is less than the above range, there is a problem that the skin condition improvement effect, for example, skin aging inhibition, wound improvement, skin barrier strengthening, skin wrinkle inhibition, or skin moisturizing effect is not sufficiently exhibited.

As used herein, the term "included as an active ingredient" means that the natural extract of the present specification is added to the extent that the above-mentioned effects can be exhibited, and various components are added as subcomponents for drug delivery and stabilization in various forms. It means that it is formulated as (formulation).

In another embodiment, the composition may be a cosmetic composition.

The cosmetic composition may have, for example, softening lotion, nutrient lotion, massage cream, nutrient cream, essence, pack, gel, ampoule, or skin-adhesive cosmetic formulation.

Ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions other than the composition as active ingredients, for example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and flavors. can include

In another embodiment, the composition may be a composition for external application for skin.

In the present specification, 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. In addition, the external skin preparations are scalp tonic, scalp lotion, scalp cream, scalp serum, scalp essence, scalp ampoule, scalp treatment, scalp conditioner, scalp shampoo, scalp pack, hair tonic, hair lotion, hair cream, hair spray, hair Mousse, hair gel, hair conditioner, hair shampoo, hair conditioner, hair pack, hair treatment, eyebrow hair growth agent, eyelash growth agent, eyelash nourishment, pet shampoo, pet conditioner, hand sanitizer, detergent soap, soap, disinfectant, It can be wet wipes, masks, ointments, patches or filter fillers, and the like. 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.

In another embodiment, the composition can be a pharmaceutical composition.

The pharmaceutical composition may additionally include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and magnesium stearate, talc, or a combination thereof as a lubricant. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated for oral or parenteral administration. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or combinations thereof. Parenteral dosage forms may be injections.

In another embodiment, the composition may be a health functional food composition.

The health functional food composition may be used alone or in combination with the strain or its culture medium or other food or food component, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the type of health functional food. Among the types of health functional foods, beverage compositions may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The health food composition may also contain 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, and carbonated beverages. carbonation agent used, or a combination thereof. The health functional food composition may also contain natural fruit juice, fruit juice beverages, fruit flesh for preparing vegetable beverages, or a combination thereof.

Another aspect also provides a method of preventing, ameliorating, or treating a condition in a subject comprising treating or administering to a subject in need thereof an effective amount of a composition described above.

The condition of the subject may be a skin-related condition or an inflammation-related condition.

As used herein, the terms "administering," "introducing," and "implanting" are used interchangeably and according to one embodiment into a subject by a method or route that results in at least partial localization of a composition to a desired site. It may mean the arrangement of the composition according to one embodiment of.

Administration may be administered by a method known in the art. Administration can be administered directly to a subject by any means, for example, by routes such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. can The administration may be administered systemically or locally.

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 improving skin beauty, for example, moisturizing the skin, strengthening the skin barrier, inhibiting skin inflammation, and improving skin wrinkles.

The administration is 0.1 mg to 1,000 mg per day of the composition according to one embodiment, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg , 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art can Dosage can be appropriately adjusted in consideration of these factors. The number of administrations can be once a day or two or more times within the range of clinically acceptable side effects, and administration can be performed at one or two or more sites, daily or at intervals of 2 to 5 days. The number of administration days may be administered from 1 day to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For non-human animals, the same dosage per kg as for humans is used, or the above dosage is converted by the volume ratio (eg, average value) of the organ (heart, etc.) between the target animal and the human. A single dose can be administered.

In another aspect, preparing a mixture by mixing betaine and citric acid; In the mixture, sodium citrate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, arginine, tromethamine, triethanolamine , aminomethyl propanol, dipotassium phosphate, potassium phosphate, sodium anisate, sodium lactate, sodium carbonate, and urea It provides a method for producing a natural eutectic solvent, comprising the step of adjusting the pH by adding any one selected from the group consisting of.

In addition, another aspect provides a method for preparing a natural product extract extracted with a natural eutectic solvent, including preparing an extract by extracting the natural product with an amino acid, an organic acid, or a mixture thereof. Details of the natural product, natural eutectic solvent, etc. are as described above.

The natural extract prepared according to the above method has excellent active oxygen and free radical scavenging rate, inhibits melanin synthesis, and increases the expression of SPT, AQP3, and RNase7, thereby strengthening the skin barrier, improving skin moisturization, elasticity, and skin aging It can be usefully used to improve skin conditions such as In addition, by reducing the expression of inflammatory cytokines, it can be usefully used for preventing, improving or treating inflammatory skin diseases.

In the cosmetic composition according to one aspect, by extracting a natural product using a eutectic solvent of betaine and citric acid, cytotoxicity is lower than that of an organic solvent extract, and safety and stability are improved by preventing discoloration of the extract, skin condition It can be usefully used for improving cosmetics and skin external preparations.

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.

[Production Example]

Preparation Example 1. Preparation of Natural Eutectic Solvent Containing Amino Acids and Organic Acids

A natural eutectic solvent containing betaine as an amino acid and citric acid as an organic acid was prepared. Specifically, betaine and citric acid were mixed in equal amounts and stirred at 60,100 °C and 150 rpm for 2 hours to prepare a natural eutectic solvent having a pH of 2. .

Production Examples 2 to 4. Preparation of pH-adjusted natural eutectic solvents

A pH-adjusted natural eutectic solvent was prepared. Specifically, 1, 5, and 10 kg of 1 M sodium citrate aqueous solution were added to the natural eutectic solvent of Preparation Example 1, respectively, to pH 3 (Preparation Example 2), pH 5 (Preparation Example 3) and pH 7 ( A natural eutectic solvent of Preparation Example 4) was prepared.

[Example]

Example 1. Preparation of fig fruit eutectic solvent extract

A natural eutectic solvent extract of Ficus carica fruit was prepared. Specifically, 1 kg of dried and pulverized fig fruits were extracted with the natural eutectic solvent of Preparation Example 1 for 6 hours. Thereafter, a fig fruit eutectic solvent extract was prepared by filtering with a Whatman #5 filter paper.

Example 2. Preparation of eutectic solvent extract

A eutectic solvent extract of Selaginella tamariscine was prepared in the same manner as in Example 1, except that Selaginella tamariscine was used.

Example 3. Preparation of germinated black rice eutectic solvent extract

A eutectic solvent extract of germinated black rice was prepared in the same manner as in Example 1, except that germinated black rice (Oryza sativa) was used.

Example 4. Preparation of blood eutectic solvent extract

A Daemonorops draco extract was prepared in the same manner as in Example 1, except that Daemonorops draco was used.

Example 5. Preparation of Eutectic Solvent Extracts of Fig Fruits, Peonbaek, Germinated Black Rice, and Blood Brown Mixtures

A eutectic solvent extract of a mixture of fig fruit, puffed rice, germinated black rice, and blood stem was prepared in the same manner as in Example 1, except that the same amounts of fig fruit, puffed rice, germinated black rice, and blood were used.

Examples 6-8. Preparation of pH-controlled fig fruit natural eutectic solvent extract

A pH-controlled fig fruit natural eutectic solvent extract was prepared in the same manner as in Example 1, except that the natural eutectic solvent of Preparation Examples 2 to 4 was used.

Examples 9-11. Preparation of pH-controlled eutectic solvent extract

A pH-controlled eutectic natural eutectic solvent extract was prepared in the same manner as in Example 1, except that the natural eutectic solvent of Preparation Examples 2 to 4 was used.

Examples 12-14. Preparation of pH-controlled germinated black rice eutectic solvent extract

A pH-controlled germinated black rice eutectic solvent extract was used in the same manner as in Example 1, except that the natural eutectic solvent of Preparation Examples 2 to 4 was used.

Production Examples 15 to 17. Preparation of natural eutectic solvent extract of pH controlled blood gall

A pH-adjustable natural eutectic solvent extract was prepared in the same manner as in Example 1, except that the natural eutectic solvent of Preparation Examples 2 to 4 was used.

Production Examples 18 to 20. Preparation of eutectic solvent extracts of pH-adjustable fig fruit, kerosene, germinated black rice and blood brown mixture

Eutectic solvent extracts of fig fruit, corn white, germinated black rice, and blood stem mixture were prepared in the same manner as in Example 1, except that the natural eutectic solvent of Preparation Examples 2 to 4 was used.

[Comparative example]

Comparative Example 1. Preparation of fig fruit ethanol extract

A fig fruit ethanol extract was prepared in the same manner as in Example 1, except that 70% (v/v) ethanol was used.

Comparative Example 2. Preparation of Kwonbaek Ethanol Extract

An ethanol extract was prepared in the same manner as in Example 1, except that 70% (v / v) ethanol was used.

Comparative Example 3. Preparation of Germinated Black Rice Ethanol Extract

An ethanol extract of germinated black rice was prepared in the same manner as in Example 1, except that 70% (v/v) ethanol was used.

Comparative Example 4. Preparation of Blood Gall Ethanol Extract

An ethanol extract was prepared in the same manner as in Example 1, except that 70% (v / v) ethanol was used.

[Experimental example]

Experimental Example 1. Confirmation of Active Ingredient Content

The contents of phenols and flavonoids included in the natural eutectic solvent extract according to one embodiment were confirmed. Specifically, 1 ml of each of Examples 1 to 20 and Comparative Examples 1 to 4 were added to 10 ml of distilled water, and then 2 ml of Folin-Ciocalteu phenol reagent (Sigma) was added and mixed. Then, after reacting at room temperature for 5 minutes, 2 ml of 20% sodium carbonate was added and further reacted at room temperature for 1 hour. Total phenol content was determined by measuring absorbance at 680 nm. Gallic acid was used as an indicator material.

Next, 1.5 ml each of Examples 1 to 20 and Comparative Examples 1 to 4 and the same amount of 2% AlCl ₃ H ₂ O dissolved in methanol were mixed and reacted at room temperature for 10 minutes. Thereafter, absorbance was measured at 367 nm to determine the total flavonoid content. Catechin was used as an indicator material.

Total polyphenol content
(mg/kg of powder) Total flavonoid content
(mg/kg of powder) Example 1 148.9 59.1 Example 2 212.5 73.8 Example 3 163.5 51.5 Example 4 184.1 53.7 Example 5 189.9 56.5 Example 6 204.4 49.7 Example 7 221.4 71.6 Example 8 220.1 57.2 Example 9 241.8 64.7 Example 10 254.6 88.4 Example 11 245.4 88.2 Example 12 254.7 63.4 Example 13 268.9 73.5 Example 14 264.1 68.9 Example 15 210.4 74.1 Example 16 238.6 94.9 Example 17 229.5 87.1 Example 18 213.5 63.2 Example 19 253.6 72.5 Example 20 240.7 69.4 Comparative Example 1 255.8 73.4 Comparative Example 2 262.3 102.5 Comparative Example 3 279.4 103.8 Comparative Example 4 234.5 99.4

As a result, as shown in Table 1, in the case of Examples 6 to 20 in which the pH was adjusted, it was confirmed that the total polyphenol and total flavonoid contents were significantly higher than those in Examples 1 to 5 in which the pH was not adjusted. . Specifically, in Examples 7, 10, 16 and 19 adjusted to pH 5, it was confirmed that the total polyphenol and total flavonoid content was higher than that of the natural eutectic solvent adjusted to pH 3 or pH 7.

That is, the natural eutectic solvent according to one embodiment can maximize the active ingredient of the extraction target at a specific pH.

Experimental Example 2. Confirmation of cytotoxicity

Cytotoxicity to skin cells of the natural eutectic solvent extract according to one embodiment was confirmed. Specifically, fibroblasts diluted in cell culture medium supplemented with 10% FBS in DMEM were placed in a 96-well plate at 1x10 ⁵ cells/mL and adhered for 24 hours. Thereafter, Examples 1 to 20 and Comparative Examples 1 to 4 were diluted to an appropriate concentration, treated in each well, and cultured for 24 hours. After 24 hours, the medium was removed, and cell culture medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution (2.5 mg/ml) per well. After putting 200 μl, it was incubated for 2 hours at 37° C. in a CO ₂ incubator. The medium was removed and 100 μl of DMSO (Dimethyl sulfoxide) was added. After lysing the cells by shaking for 5 minutes, absorbance at 565 nm was measured in a microplate reader. The cell viability (%) was determined by Equation 1 below, and the concentration of the sample that did not affect cell survival was determined, and the results are shown in Table 2 below.

Bo: Absorbance at 565 nm of wells in which only the cell culture medium developed color reaction

Bt: Absorbance at 565 nm of a color reaction well without sample treatment

S: Absorbance at 565 nm of the well where the sample was treated and the color reaction occurred

100% cell viability concentration Example 1 0.25% Example 2 0.25% Example 3 0.25% Example 4 0.25% Example 5 0.25% Example 6 One% Example 7 2% Example 8 One% Example 9 One% Example 10 2% Example 11 2% Example 12 One% Example 13 2% Example 14 2% Example 15 One% Example 16 2% Example 17 2% Example 18 One% Example 19 2% Example 20 2% Comparative Example 1 0.05% Comparative Example 2 0.05% Comparative Example 3 0.05% Comparative Example 4 0.05%

As a result, as shown in Table 2, it was confirmed that the 100% cell survival concentration was 5 to 40 times higher in Examples 1 to 20 compared to Comparative Examples 1 to 4. In particular, in the case of pH 3 to pH 7 of the natural eutectic solvent, it was confirmed that the cell survival concentration was significantly higher than that of pH 2.5. Specifically, in the case of Examples 6 to 20 where the pH was adjusted, the cytotoxicity tended to decrease as the pH increased, and in particular, it was confirmed that the 100% cell survival concentration was the highest in the natural eutectic solvent extract adjusted to pH 5. could

Therefore, the natural eutectic solvent according to one embodiment has a low possibility of causing skin irritation and has a wide useful concentration range, so it can be used as a safe and efficient solvent.

Experimental Example 3. Room temperature stability confirmation

Room temperature stability of the natural eutectic solvent extract according to one embodiment was confirmed. Specifically, samples were prepared by mixing 60% by weight of dipropylene glycol with Examples 1 to 20 and Comparative Examples 1 to 4. Thereafter, the sample was placed in a thatched container, and stored for 4 weeks in a thermostat maintained at a constant temperature of 45° C. to measure the degree of discoloration. After measuring the color change (ㅿE) using a chromameter (Minolta CR300), the color change of the sample was evaluated for stability, and the results are shown in Table 3 below.

ㅿE Example 1 19.84 Example 2 28.16 Example 3 22.98 Example 4 25.47 Example 5 25.81 Example 6 15.34 Example 7 8.32 Example 8 9.11 Example 9 23.47 Example 10 8.05 Example 11 11.36 Example 12 15.54 Example 13 9.51 Example 14 9.17 Example 15 19.57 Example 16 8.97 Example 17 9.24 Example 18 18.47 Example 19 8.16 Example 20 8.03 Comparative Example 1 18.35 Comparative Example 2 13.84 Comparative Example 3 9.44 Comparative Example 4 15.21

As a result, as shown in Table 3, in the case of Examples 6 to 20 compared to the organic solvent extracts of Comparative Examples 1 to 4, it was confirmed that the color change at room temperature was relatively small. Specifically, it was confirmed that the natural product extracts (Examples 7, 10, 14, 16, and 19), which were extracted with a natural eutectic solvent adjusted to pH 5, exhibited the highest stability as the color change was small.

According to Experimental Examples 1 to 3, Comparative Examples 1 to 4 had excellent extraction efficiency compared to Examples 1 to 20, but had problems with poor cytotoxicity and stability at room temperature. However, the natural eutectic solvent according to one embodiment can be usefully used as a solvent for the preparation of cosmetics and external skin preparations in that it improves the problems of cell toxicity and room temperature stability that may occur due to organic solvent extraction.

Experimental Example 4. Confirmation of antioxidant activity (1)

According to the results of Experimental Example 3, in the following Experimental Example, an activity test of a natural product extract extracted with a natural eutectic solvent of pH 5 was performed.

The antioxidant activity of the natural eutectic solvent extract according to one embodiment was confirmed using the NBT (Nitro Blue Tetrazolium) method. Specifically, 2.4 ml of 0.05 M Na2CO3, 0.1 ml of 3 mM xanthine solution, 0.1 ml of 3 mM EDTA solution, 0.1 ml of BSA solution, and 0.1 ml of 0.72 mM NBT solution were added to a Bayer bottle, and the same as in Examples 7, 10, and 13 , 16 and 19 and Comparative Examples 1 to 4 were added at 0.1% by weight, respectively, and then left at 25 ° C. for 10 minutes. Thereafter, 0.1 ml of a xanthine oxidase solution was added, stirred rapidly, incubated at 25 ° C. for 20 minutes, and 0.1 ml of a 6 mM CuCl ₂ solution was added to stop the reaction, and the absorbance (St) at 560 nm was measured. measured. As a negative control group, distilled water (Bt) was used instead of the extract of the above example. For the blank of the sample solution, distilled water (Bo) was used instead of the reaction enzyme xanthine oxidase solution, and the measurement result was calculated by Equation 2 below. BHT (Butylated hydroxytoluene) was used as a positive control.

St: absorbance at 560 nm after enzymatic reaction of the sample

Bt: absorbance at 560 nm after enzymatic reaction of negative control

So: Absorbance at 560 nm before enzyme reaction of sample without enzyme addition

Bo: Absorbance at 560 nm before the enzyme reaction of the negative control group without enzyme addition

Active oxygen scavenging rate (antioxidant effect, %) positive control 86.15 Example 7 74.57 Example 10 91.15 Example 13 83.32 Example 16 90.47 Example 19 90.07 Comparative Example 1 70.16 Comparative Example 2 88.64 Comparative Example 3 79.57 Comparative Example 4 82.45

As a result, as shown in Table 4, it was confirmed that Examples 7, 10, 13, 16 and 19 had a significantly higher active oxygen scavenging rate compared to Comparative Examples 1 to 4 in which the same extraction target was extracted with an organic solvent. .

Therefore, since the natural eutectic solvent extract according to one embodiment has excellent antioxidant activity, it can be usefully used for preventing or improving aging.

Experimental Example 5. Confirmation of antioxidant activity (2)

The antioxidant activity of the natural eutectic solvent extract according to one embodiment was confirmed using the DPPH method [2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl free radical]. Specifically, Examples 7, 10, 13, 16, and 19 and Comparative Examples 1 to 4 were diluted to a concentration of 0.1%, and then placed in a 96-well plate. Thereafter, DPPH prepared as a 100 μl methanol solution was added so that the total volume of the solution was 200 μl, and then left at 37° C. for 30 minutes, and then absorbance was measured at 560 nm. The free radical scavenging activity (%) was calculated by Equation 3 below, and the results are shown in Table 5 below. BHT (Butylated hydroxytoluene) was used as a positive control.

A: Absorbance of untreated control wells

B: Absorbance of experimental wells

Free radical scavenging rate (antioxidant effect, %) positive control 81.33 Example 7 70.51 Example 10 89.67 Example 13 81.35 Example 16 74.51 Example 19 88.87 Comparative Example 1 61.34 Comparative Example 2 78.24 Comparative Example 3 69.47 Comparative Example 4 66.50

As a result, as shown in Table 5, it can be confirmed that Examples 7, 10, 13, 16 and 19 have significantly higher free radical scavenging rates compared to Comparative Examples 1 to 4 in which the same extraction target was extracted with an organic solvent. there was.

Therefore, since the natural eutectic solvent extract according to one embodiment has excellent antioxidant activity, it can be usefully used for preventing or improving aging.

Experimental Example 6. Confirmation of skin cell defense effect by ultraviolet rays

The cytotoxicity mitigation effect of the natural eutectic solvent extract according to one embodiment was confirmed by ultraviolet light. Specifically, fibroblasts were placed in 1x10 ⁵ cells/ml each in a 96-well plate and adhered for 24 hours. After 24 hours, Examples 7, 10, 13, 16, and 19 and Comparative Examples 1 to 4 were treated and further cultured for 24 hours under the same culture conditions. Thereafter, the medium was removed, each well was washed once with PBS, and 100 μl of PBS was added to each well. Thereafter, 10 mJ/cm2 of ultraviolet light was irradiated using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), and then PBS was removed and 200 μl of DMEM with 10% FBS was added. . Here, each sample was treated with a concentration that does not exhibit cytotoxicity, and then cultured for 24 hours. After 24 hours, the medium was removed, and 200 μl of MTT solution (2.5 mg/ml) containing cell culture medium was added to each well, followed by incubation at 37° C. for 2 hours in a CO ₂ incubator. After removing the medium, 100 μl of DMSO (Dimethyl sulfoxide) was added thereto, shaken for 2 minutes to lyse the cells, and absorbance at 565 nm was measured in a microplate reader. Cell viability (%) and cytotoxicity reduction rate by ultraviolet light were calculated by Equations 4 and 5, respectively, and the results are shown in Table 6 below.

Bo: Absorbance at 565 nm of wells in which only the cell culture medium developed color reaction

Bt: Absorbance at 565 nm of a color reaction well without sample treatment

St: Absorbance at 565 nm of the well where the sample was treated and the color reaction occurred

Bo: cell viability of wells without UV irradiation and sample treatment

Bt: Cell viability of wells irradiated with UV light and not treated with samples

St: Cell viability of wells irradiated with UV light and treated with samples

Cytotoxicity relief rate (%) Example 7 25.9 Example 10 28.5 Example 13 32.7 Example 16 30.1 Example 19 35.4 Comparative Example 1 12.8 Comparative Example 2 11.5 Comparative Example 3 14.6 Comparative Example 4 11.1

As a result, as shown in Table 6, Examples 7, 10, 13, 16 and 19 were confirmed to have significantly higher cytotoxicity reduction rates compared to Comparative Examples 1 to 4 in which the same extraction target was extracted with an organic solvent. .

Therefore, since the natural eutectic solvent extract according to one embodiment has excellent cell damage protection activity by ultraviolet rays, it can be usefully used for preventing or improving aging.

Experimental Example 7. Inhibition of inflammation-related enzyme activity confirmation

The effect of preventing, improving or treating inflammation of the natural eutectic solvent extract according to one embodiment was confirmed. Specifically, 50 μl of a hyaluronidase solution (type Sigma, 400 U/ml) was added to 100 μl of Examples 7, 10, 13, 16, and 19 and Comparative Examples 1 to 4 in each well, and 37° C. After reacting for 20 minutes, 100 μl of an enzyme activation solution (compound 48/80 CaCl ₂ 2H ₂ O, Sigma, 0.1 mg/ml) was added and further reacted at 37° C. for 20 minutes. Thereafter, 250 μl of a hyaluronic acid solution (0.4 mg/ml) was added and reacted at 37° C. for 40 minutes, and then 100 μl of 0.4N NaOH was added to terminate the reaction. 100 μl of a potassium borate solution was added, reacted at 95° C. for 3 minutes, cooled, and then 3 mg of a ρ-dimethylaminobenzaldehyde solution was added and further reacted at 37° C. for 20 minutes, followed by color development. Thereafter, the absorbance was measured at 585 nm to measure the inhibition rate of hyaluronidase activity. An oil-soluble licorice extract was used as a positive control. The activity inhibition rate (%) of hyaluronidase was calculated by Equation 6 below, and the results are shown in Table 7 below.

A: Enzyme activity of wells to which no sample was added

B: Enzyme activity of the well to which the sample was added

Hyaluronidase activity inhibition rate (%) positive control 78.6 Example 7 65.2 Example 10 74.1 Example 13 59.2 Example 16 62.7 Example 19 66.8 Comparative Example 1 53.7 Comparative Example 2 43.4 Comparative Example 3 46.7 Comparative Example 4 41.3

As a result, as shown in Table 7, it was confirmed that Examples 7, 10, 13, 16 and 19 had significantly higher inhibition of hyaluronidase activity compared to Comparative Examples 1 to 4 in which the same extraction target was extracted with an organic solvent. could

Therefore, the natural eutectic solvent extract according to one embodiment can be usefully used for preventing, improving or treating inflammatory diseases.

Experimental Example 8. Confirmation of inflammatory cytokine inhibitory effect by ultraviolet light

It was confirmed that the natural eutectic solvent extract according to one embodiment had an effect of preventing, improving or treating inflammation caused by ultraviolet rays. Specifically, 3 ml of a keratinocyte line diluted to 3X10 ⁵ cells/ml was dispensed into a 35φ dish in a medium supplemented with 10% FBS and cultured for 24 hours. Thereafter, after irradiation with UVB of 15 mJ/cm ² and diluting Examples 7, 10, 13, 16, and 19 and Comparative Examples 1 to 4 in a medium containing 2% FBS at a concentration that does not exhibit cytotoxicity, 24 hours during further incubation. After incubation, total RNA was extracted using an RNeasy mini kit (Qiagen, Hilden), and 1 μg of total RNA was mixed with oligo (dT)15 primer, dNTP (0.5uM), 1 unit RNase inhibitor and 4 unit Omniscript reverse transcriptase (Qiagen , Hilden, Germany) to synthesize cDNA by heating at 37°C for 60 minutes and at 93°C for 5 minutes, and then stopping the reaction. PCR was performed to amplify IL-1α, TNF-α, IL-1β, IL-6, COX-2, and GAPDH from the cDNA. The products generated by PCR were electrophoresed on a 2% agarose gel to confirm gene expression with an image analyzer, and the density of each band was measured using the Image J program (NIH Image software, Maryland, USA). Madecassoside was used as a positive control. The expression inhibition rate of inflammatory cytokines and inflammatory mediators was calculated by Equation 7 below, and the results are shown in Table 8 below.

Bo: Production amount of inflammatory cytokines or inflammatory mediators in wells that were not irradiated with ultraviolet rays and treated with samples

Bt: production amount of inflammatory cytokines or inflammatory mediators in wells irradiated with UV light and not sample treated

St: production amount of inflammatory cytokines or inflammatory mediators in wells irradiated with ultraviolet light and treated with samples

Inhibition of inflammatory cytokine expression (%) Inhibition rate of inflammatory mediator expression (%) IL-1α IL-1β IL-6 TNF-α COX-2 positive control 32 31 21 44 54 Example 7 44 37 45 56 64 Example 10 40 42 50 58 57 Example 13 34 42 28 63 55 Example 16 37 41 34 53 56 Example 19 48 52 41 54 63 Comparative Example 1 25 21 25 36 37 Comparative Example 2 29 32 32 45 43 Comparative Example 3 27 29 24 43 48 Comparative Example 4 23 27 29 41 40

As a result, as shown in Table 8, Examples 7, 10, 13, 16 and 19 showed a significant rate of inhibition of expression of inflammatory cytokines and inflammatory mediators compared to Comparative Examples 1 to 4 in which the same extraction target was extracted with an organic solvent. I was able to confirm that it was quite high.

Therefore, the natural eutectic solvent extract according to one embodiment inhibits the expression of inflammatory cytokines and/or inflammatory mediators, and thus can be usefully used to prevent, improve, or treat inflammatory diseases caused by ultraviolet rays.

Experimental Example 9. Confirmation of tyrosinase activity inhibitory effect

The whitening improvement activity of the natural eutectic solvent extract according to one embodiment was confirmed. Specifically, after dissolving L-tyrosine (Sigma) in 0.05M sodium phosphate buffer solution (pH 6.8) to prepare a 0.1 mg/ml solution, 0.5 ml of the prepared solution was placed in a test tube. Here, 0.5 ml of each of Examples 7, 10, 13, 16, and 19 and Comparative Examples 1 to 4 dissolved by adjusting the appropriate concentration in 0.05M sodium phosphate buffer solution (pH 6.8) was added and incubated in an incubator at 37 ° C for 10 left for a minute. Thereafter, 0.5 ml of 200 unit/ml tyrosinase was added and reacted at 37° C. for 10 minutes, followed by rapid cooling on ice to stop the reaction. The absorbance at a wavelength of 475 nm was measured with a spectrophotometer. measured. As a negative control group, 0.5 ml of the buffer solution was used, and as a positive control group, an arbutin solution dissolved at an appropriate concentration in 0.05M sodium phosphate buffer solution (pH 6.8) was used. The inhibition rate (%) of tyrosinase was calculated by Equation 8 below, and the results are shown in Table 9 below.

A: Absorbance before reaction of the well to which the sample was added

B: Absorbance after reaction of the well to which the sample was added

C: Absorbance before reaction of wells to which no sample was added

D: Absorbance after reaction of wells to which no sample was added

Tyrosinase activity inhibition rate (%) positive control 92.4 Example 7 64.1 Example 10 56.8 Example 13 73.1 Example 16 68.9 Example 19 63.3 Comparative Example 1 49.7 Comparative Example 2 51.2 Comparative Example 3 62.7 Comparative Example 4 52.4

As a result, as shown in Table 9, it was confirmed that Examples 7, 10, 13, 16 and 19 had a significantly higher tyrosinase activity inhibition rate compared to Comparative Examples 1 to 4 in which the same extraction target was extracted with an organic solvent.

Therefore, the natural eutectic solvent extract according to one embodiment can be usefully used to improve skin whitening.

Experimental Example 10. Confirmation of melanin synthesis inhibitory effect

The whitening improvement activity of the natural eutectic solvent extract according to one embodiment was confirmed. Specifically, melanoma cells, B16F10 (accession number: ATCC 6323), were dispensed in a 6-well plate at a concentration of 2 x 10 ⁶ cells/ml per well, attached to the cells, and at a concentration that did not induce toxicity, as described in Example 7, 10, 13, 16, and 19 and Comparative Examples 1 to 4 were treated and cultured for 72 hours. Thereafter, the cells were detached with trypsin-EDTA, the number of cells was measured, and the cells were recovered by centrifugation. Quantification of intracellular melanin was performed by slightly modifying the method of Rotan [Lotan: Cancer Res., 40, 3345-3350 (1980)]. After washing the cell pellet once with PBS (phosphate buffered saline), 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF (phenyl methane sulphonyl fluoride)) was added and vortexed for 5 minutes. Cells were disrupted. 1N NaOH 10% DMSO (Dimethyl sulfoxide) was added to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes) to dissolve the extracted melanin. Then, after measuring the absorbance of melanin at 405 nm with a microplate reader, melanin was quantified to measure the melanin production inhibition rate (%) of the sample. The melanin production inhibition rate was calculated by Equation 9 below, and the results are shown in Table 10 below. Arbutin was used as a positive control.

A: amount of melanin in wells to which no sample was added

B: the amount of melanin in the well to which the sample was added

Melanin production inhibition rate (%) positive control 59.4 Example 7 65.3 Example 10 54.5 Example 13 38.5 Example 16 48.3 Example 19 61.1 Comparative Example 1 45.3 Comparative Example 2 35.4 Comparative Example 3 27.6 Comparative Example 4 31.1

As a result, as shown in Table 10, it was confirmed that Examples 7, 10, 13, 16 and 19 had a significantly higher melanin production inhibition rate compared to Comparative Examples 1 to 4 in which the same extraction target was extracted with an organic solvent.

Therefore, the natural eutectic solvent extract according to one embodiment can be usefully used to improve skin whitening.

Experimental Example 11. Confirmation of skin barrier strengthening effect

The skin barrier strengthening effect of the natural eutectic solvent extract according to one embodiment was confirmed. Specifically, HaCaT cells, a human keratinocyte, were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 11965-092) containing 10% fetal bovine serum, and all cultures were performed at 37°C 5 % CO ₂ was performed in an incubator. The cultured cell lines were treated with Examples 7, 10, 13, 16 and 19 and Comparative Examples 1 to 4 and further cultured for 24 hours. Thereafter, total RNA was isolated from the cells of each sample using an RNeasy mini kit (Qiagen, Germany), RNA was quantified at 260 nm by nanodrop, and cDNA was synthesized using an amplifier using 2 μg of RNA ( C1000 Thermal Cycler, Bio-Rad, USA). Using the synthesized cDNA as a template, a real-time PCR machine (Mic qPCR, Mic qPCR, Real-time polymerase chain reaction was performed in a biomolecular system (Australia). The expression level of the gene was finally analyzed through correction for the GAPDH gene, and the results are shown in Table 11 below.

SPT mRNA expression level AQP3 mRNA expression level RNase 7 mRNA expression level 5% FBS 1.440 4.200 2.600 0% FBS 1.000 1.000 1.000 Example 7 1.343 3.547 1.943 Example 10 1.452 2.979 1.644 Example 13 1.389 2.714 2.145 Example 16 1.411 3.009 1.840 Example 19 1.402 3.248 1.742 Comparative Example 1 1.151 2.684 1.640 Comparative Example 2 1.217 1.746 1.216 Comparative Example 3 1.270 2.357 1.574 Comparative Example 4 1.187 2.246 1.157

As a result, as shown in Table 11, Examples 7, 10, 13, 16 and 19 compared to Comparative Examples 1 to 4 in which the same extraction target was extracted with an organic solvent, SPT, a ceramide synthase, skin moisturizing, barrier strengthening and It was confirmed that the expression of related AQP and skin antibacterial peptide RNase7 was significantly high.

Therefore, the natural eutectic solvent extract according to one embodiment can be usefully used to strengthen the skin barrier.

Experimental Example 12. Confirmation of skin wrinkles and moisturizing effect

The wrinkle and moisturizing activity of the natural eutectic solvent extract according to one embodiment was confirmed. Specifically, human dermal fibroblasts (Modern Tissue Technology, Korea) were cultured at a concentration of 1Х10 ⁴ cell/cm ² by adding 10% fetal bovine serum (FBS) to DMEM/F-12 (3:1) medium. When they grew to about 70-80%, they were divided and subcultured at a ratio of 1:3, and cells subcultured for the 3rd and 4th passages were used in the experiment. After culturing the fibroblasts in a 48-well plate to 90% or more, the Examples 7, 10, 13, 16, and 19 and Comparative Examples 1 to 4 were added at a concentration that did not exhibit cytotoxicity, and after 24 hours The amount of procollagen released in the medium was measured using a procollagen type-1 C-peptide EIA kit (MK101, Takara, Japan). Ascorbic acid was used as a positive control.

Type 1 procollagen production rate (%) positive control 65.4 Example 7 43.5 Example 10 35.3 Example 13 63.1 Example 16 41.8 Example 19 56.8 Comparative Example 1 25.4 Comparative Example 2 21.5 Comparative Example 3 43.6 Comparative Example 4 24.7

As a result, as shown in Table 12, it was confirmed that Examples 7, 10, 13, 16 and 19 had a significantly higher procollagen production rate compared to Comparative Examples 1 to 4 in which the same extraction target was extracted with an organic solvent.

Therefore, the natural eutectic solvent extract according to one embodiment can be usefully used for preventing or improving skin wrinkles and moisturizing.

Experimental Example 13. Acne and sebum improvement or hair loss prevention or improvement effect confirmation

The effect of preventing or improving acne, sebum, or hair loss of the natural eutectic solvent extract according to one embodiment was confirmed. Specifically, after adding 0.1 μCi of testosterone radiolabeled with 3H (tritium) and Examples 7, 10, 13, 16, and 19 and Comparative Examples 1 to 4 per microplate hole, Fibroblasts were inoculated and cultured so that 10,000 cells were entered. After culturing for 24 hours, the supernatant was obtained and steroids were obtained with 1 ml of ethyl acetate-cyclohexane (1:1) extraction solvent. The steroid was placed on a thin film chromatography plate and developed with a mixture of chloroform/methanol (98/2 (v/v)). The radioactive activity of the points corresponding to testosterone and dihydrotestosterone (DHT) was measured using a densitometer to calculate the conversion rate, and the result was compared with the negative control group to obtain 5-alpha-reductase according to Equation 10 Inhibition rates were evaluated.

A: Conversion rate of testosterone to dihydrotestosterone when sample is not added

B: Conversion rate of testosterone to dihydrotestosterone upon addition of sample

5alpha-reductase inhibition rate (%) Example 7 57.1 Example 10 61.3 Example 13 30.2 Example 16 51.4 Example 19 55.3 Comparative Example 1 38.5 Comparative Example 2 46.7 Comparative Example 3 28.6 Comparative Example 4 34.8

As a result, as shown in Table 13, Examples 7, 10, 13, 16 and 19 confirmed that the 5 alpha-reductase inhibition rate was significantly higher compared to Comparative Examples 1 to 4 in which the same extraction target was extracted with an organic solvent. could

Therefore, the natural eutectic solvent extract according to one embodiment inhibits the conversion of dihydrotestosterone by 5-alpha-reductase, thereby preventing or improving acne and sebum, as well as preventing, improving or treating hair loss, or hair growth or hair growth. It can be useful for promotion.

Experimental Example 14. Confirmation of skin elasticity improvement effect

The skin elasticity improvement effect of the cosmetic composition containing the natural eutectic solvent extract according to one embodiment was confirmed. First, a creamy cosmetic composition containing the components and contents of Table 14 below was prepared. Specifically, phase B of Table 14 below was heated and stored at 70° C., followed by pre-emulsification by adding phase A. Thereafter, the mixture was uniformly emulsified with a homomixer, and then slowly cooled to prepare a cosmetic composition. In this specification, unless otherwise indicated, content is % by weight.

Twenty women aged 20 to 35 years old were asked to use Formulation Examples 1 to 6 and Comparative Formulation Examples 1 to 4 twice a day for 2 months, respectively. Thereafter, skin elasticity was measured before and after using Formulation Examples 1 and 5 and Comparative Formulation Example 1 using a skin elasticity meter (cutometer SEM 575, C+K Electronic Co., Germany). The measured value was described as the R7 value of Cutometer SEM 575, and the R7 value represents the viscoelasticity of the skin.

Formulation example 1 Formulation Example 2 Formulation Example 3 Formulation Example 4 Formulation Example 5 Comparative Formulation Example 1 Comparative Formulation Example 2 Comparative Formulation Example 3 Comparative Formulation Example 4 A Stearyl Alcohol 8 8 8 8 8 8 8 8 8 stearic acid 2 2 2 2 2 2 2 2 2 cholesterol stearate 2 2 2 2 2 2 2 2 2 squalane 4 4 4 4 4 4 4 4 4 2-Octyldodecyl alcohol 6 6 6 6 6 6 6 6 6 Polyoxyethylene (25 mol added) alcohol ester 3 3 3 3 3 3 3 3 3 Glyceryl Monostearate Aster 2 2 2 2 2 2 2 2 2 B Example 7 One - - - - - - - - Example 10 - One - - - - - - - Example 13 - - One - - - - - - Example 16 - - - One - - - - - Example 19 - - - - One - - - - Comparative Example 1 - - - - - One - - - Comparative Example 2 - - - - - - One - - Comparative Example 3 - - - - - - - One - Comparative Example 4 - - - - - - - One propylene glycol 5 5 5 5 5 5 5 5 5 Purified water balance

skin viscoelasticity Formulation Example 1 0.33 Formulation Example 5 0.31 Comparative Formulation Example 1 0.11

As a result, as shown in Table 15, it was confirmed that Formulation Examples 1 and 5 significantly increased skin viscoelasticity compared to Comparative Formulation Example 1.

Therefore, the natural eutectic solvent natural product extract according to one embodiment can be usefully used to improve skin elasticity.

Experimental Example 15. Confirmation of skin wrinkle improvement effect

The skin wrinkle improvement effect of the cosmetic composition containing the natural eutectic solvent extract according to one embodiment was confirmed. Specifically, Formulation Examples 1 and 5 of Experimental Example 14 and Comparative Formulation Example 1 were used in the same way for the same subjects. Then, after manufacturing a replica made of silicon, using a visiometer (SV60, C+K Electronic Co., registered patent 10-1905517-13-Germany), which is an image analyzer, the formulation example 1, 5 and Comparative Formulation Example 1 were used to measure skin wrinkles. The measured value was expressed as an average after subtracting the parameter value before use from each parameter value after 2 months of use, and the more negative the measured value, the higher the anti-wrinkle effect.

R1 R2 R3 R4 R5 Formulation example 1 -0.23 -0.19 -0.13 -0.17 -0.07 Formulation Example 5 -0.24 -0.15 -0.13 -0.14 -0.08 Comparative Formulation Example 1 -0.17 -0.16 -0.06 -0.14 -0.02

R1: difference between the highest and lowest values of the wrinkle contour

R2: The average of the R1 values after randomly dividing the wrinkle contour lines into 5 cells

R3: highest value among R1 values divided by 5

R4: the average value of subtracting the value of each peak and valley from the baseline of the wrinkle contour

R5: average value obtained by subtracting each wrinkle contour from the baseline of the wrinkle contour

As a result, as shown in Table 16, it was confirmed that Formulation Examples 1 and 5 had significantly lower parameter values representing skin wrinkles compared to Comparative Formulation Example 1.

Therefore, the natural eutectic solvent natural product extract according to one embodiment can be usefully used to improve skin wrinkles.

Experimental Example 16. Confirmation of skin whitening improvement effect

The skin whitening improvement effect of the cosmetic composition containing the natural eutectic solvent extract according to one embodiment was confirmed. Specifically, Formulation Examples 1 and 5 of Experimental Example 14 and Comparative Formulation Example 1 were used in the same way for the same subjects. Thereafter, the change in brightness (L) of the skin color was measured using an image analyzer and a chromameter (Minolta CR300) on the skin of the application area. In addition, objective visual observation by a plurality of experts and subjective visual observation by subjects were conducted to evaluate the degree of whitening efficacy according to the following grades.

-3: very worse, -2: worse, -1: slightly worse, 0: no change, 1: slightly improved, 2: improved, 3: very improved

Brightness change of skin color (L) Objective evaluation by experts Subjective assessment of the subject Formulation Example 1 3.14 2.0 2.3 Formulation Example 5 3.79 3.0 3.0 Comparative Formulation Example 1 2.15 2.0 2.0

As a result, as shown in Table 17, it was confirmed that Formulation Examples 1 and 5 significantly increased the change in skin color brightness compared to Comparative Formulation Example 1. Specifically, in Formulation Example 5 in which the acidity of the natural eutectic solvent was adjusted to pH 5, it was confirmed that the change in skin color brightness was significantly increased.

Therefore, the natural eutectic solvent natural product extract according to one embodiment can be usefully used to improve skin whitening.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (13)

A cosmetic composition for improving skin condition comprising a natural extract extracted with a natural eutectic solvent as an active ingredient, wherein the natural eutectic solvent includes betaine and citric acid, and the natural eutectic solvent has a pH of 3 to pH 7. The natural product according to claim 1, wherein the natural product is fig, black rice, black bean, black bean, jacaranda, seolryeon, rowan tree, pollen, water frog rice, juniper tree, bongmicho, dicentra, mistletoe, sedum, sedum, Sanghwang mushroom, tomato, houttuynia cordata, A composition selected from the group consisting of dermis, peony, chestnut flower, kudzu, and walnut. The composition of claim 1, wherein the natural eutectic solvent has a pH of 3. The composition of claim 1 , wherein the natural eutectic solvent further comprises sodium citrate. The composition according to claim 1, wherein the natural product extract is obtained by extracting figs, kwonbaek, black rice, blood brown, or mixtures thereof with betaine and citric acid. The composition according to claim 1, wherein the betaine and citric acid are mixed in a weight ratio of 1:0.001 to 10. The method according to claim 1, wherein the skin condition is skin aging, moisturizing, wrinkles, elasticity, skin barrier strengthening, acne or sebum secretion of the composition. The method according to claim 7, wherein the skin aging is due to ultraviolet rays or active oxygen, the composition. A composition for external application for skin for improving skin condition comprising a natural extract extracted with a natural eutectic solvent as an active ingredient, wherein the natural eutectic solvent includes betaine and citric acid, and the natural eutectic solvent has a pH of 3 to pH 7. A pharmaceutical composition for preventing or treating inflammatory skin diseases comprising a natural extract extracted with a natural eutectic solvent as an active ingredient, wherein the natural eutectic solvent includes betaine and citric acid, and the natural eutectic solvent has a pH of 3 to 7 composition. The method according to claim 10, wherein the inflammatory disease is selected from the group consisting of skin wounds, dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, drug rash, and acne, composition. A pharmaceutical composition for preventing or treating hair loss, or promoting hair growth or hair growth, comprising a natural extract extracted with a natural eutectic solvent as an active ingredient, wherein the natural eutectic solvent includes betaine and citric acid, and the natural eutectic solvent has a pH of 3 to 3 A composition having a pH of 7. Preparing a mixture by mixing betaine and citric acid;
In the mixture, sodium citrate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, arginine, tromethamine, triethanolamine , aminomethyl propanol, dipotassium phosphate, potassium phosphate, sodium anisate, sodium lactate, sodium carbonate, and urea A method for preparing a natural eutectic solvent, comprising adding a pH adjusting agent selected from the group consisting of, wherein the natural eutectic solvent has a pH of 3 to 7.