KR20230073772A - Composition for Improving Skin Whiteninig and Skin Wrinkes Using a Mixture of an Extract of Artemisia capillaries and an Extract of Citrus junos - Google Patents

Abstract

The present invention discloses a composition for skin-whitening and skin wrinkle improvement using a mixture of Artemisia capillaries extract and Citrus junos extract having melanin production suppression activity, having intracellular tyrosinase and mushroom tyrosinase inhibition activity, and having elastase and collagenase inhibition activity.

Description

Composition for Improving Skin Whitening and Skin Wrinkes Using a Mixture of an Extract of Artemisia capillaries and an Extract of Citrus junos}

The present invention relates to a composition for skin whitening and a composition for improving skin wrinkles using a mixture of an Artemisia capillaries extract and a Citrus junos extract.

Human hair or skin color is generally determined by the amount of melanin pigment present in hair or skin.

Melanin is a phenolic polymeric natural pigment widely present in living organisms. In the human body, it is synthesized in melanosomes in melanocytes in the basal layer of the epidermis, and is transferred to surrounding keratinocytes to show human skin color.

The synthesis of melanin is achieved by making melanosomes in melanocytes present in the basal layer of the skin, and a series of processes in which melanin is synthesized are collectively referred to as melanogenesis. Melanin production uses tyrosine, one of the amino acids, as a substrate, and DOPA (3,4- After conversion to DOPA quinone through dihydroxyphenylalanine), melanin is synthesized by polymerization with amino acids or proteins after undergoing a non-enzymatic reaction and spontaneous oxidation process (Korean J. FOOD SCI. TECHNOL., 2000. 32(3):736;Journal of Life Science, 2013. 23(12):1445;Pigment Cell Res.1999.12(1):4-12.). The major intracellular signaling pathway is the cAMP/PKA (cyclic monophosphate/protein kinase A) pathway. cAMP promotes the expression of MITF via PKA and CREB1 (cAMP responsive element binding protein 1), and MITF plays an important role in melanin synthesis. As a transcriptional regulator, it promotes the transcription of tyrosinase, TRP-1 and TRP-2 (Pharmazie. 2015 Oct;70(10):646-9.; Int J Mol Sci. 2015 Oct 13;16(10):24219- 42; Curr Pharm Biotechnol. 2015;16(12):1111-9.).

Melanin absorbs light energy from ultraviolet rays in the sunlight and plays a role in protecting cells deep in the skin from damage caused by ultraviolet rays. , Conversely, low production results in vitiligo, depigmented nevus, pseudoleucoderma atopicum, tinea versicolor, ecchymosis, allergy, post-inflammatory depigmentation, idiopathic red hypopigmentation, depigmented nevus, and partial leukoplakia. Melanin hypopigmentation disease (Leukoderma) of the back occurs (J Am Acad Dermatol (1988) 19:217-255; Pinto and Bolognia, Pediatr Clin North Am (1991) 38:991-1017).

Recently, many studies have been conducted on whitening-related functional substances, and substances used as whitening agents include kojic acid, hydroquinone, arbutin, and ascorbic acid, which down-regulate the activity of tyrosinase to form melanin. inhibit the production. However, these substances have low safety, poor skin penetration, and cause inflammation and cytotoxicity when administered for a long time, and whitening agents may cause serious side effects such as albinism and vitiligo. As a result, the need for developing effective functional materials using medicinal plants and natural products with high human safety and little side effects has increased, and research on material development from natural materials has been actively conducted (Kor. J. Pharmacogn., 44(3): 220, 2013).

On the other hand, with aging, skin cells are damaged due to various pollutants, strong ultraviolet rays, stress and nutritional deficiency, and cell proliferation is not smooth, resulting in wrinkles, loss of elasticity, pigmentation and keratinization of the skin. (Gilchrest B.A., J. Am. Acad. Dermatol, 21, 610. 1989).

Collagen is the most important protein for maintaining skin moisture and maintaining skin flexibility and elasticity. It accounts for 90% of the dermal layer (J Am Acad Dermatol, 17(4):610-613. 2001) and provides strength and tension to the skin. It serves to protect the skin from external stimuli. In addition, elastin in the dermis layer accounts for about 2%, which is less than collagen, but plays a role in supporting collagen fibers, and many recent studies have reported that the deficiency, aggregation, and loss of elastin fibers play an important role in the mechanism of skin wrinkle formation. (Weihermann AC et al., Int J Cosmet Sci. 2017 Jun; 39(3): 241-247; Mora Huertas AC et al., Biochimie. 2016 Sep-Oct; 128-129: 163-73).

Collagen and elastin are synthesized in fibroblasts and degraded by collagenase and elastase. As people age or are exposed to ultraviolet light, the activity and cell number of fibroblasts decrease, the amount of synthesis of collagen or elastin decreases, and the activity of collagenase or elastase that decomposes them increases. Once the elasticity of the skin by collagen and elastin is reduced, it is very difficult to recover. Accordingly, studies are being actively conducted to improve skin wrinkles by inhibiting the activities of collagenase and elastase, which increase with age.

The present invention discloses melanogenesis inhibitory activity, tyrosinase inhibitory activity, collagenase inhibitory activity, elastase inhibitory activity, etc. of a mixture of Injin mugwort extract and Citron extract.

An object of the present invention is to provide a composition for skin whitening and skin wrinkle improvement using a mixture of ginseng mugwort extract and citron extract.

Other objects or specific objects of the present invention will be presented below.

As confirmed in the following examples and experimental examples, the present invention has a melanin production inhibitory activity in B16F10 melanoma cells, a melanoma cell line, and intracellular tyrosinase and mushroom tyrosinase. It was completed by confirming that it had inhibitory activity and also had elastase and collagenase inhibitory activity.

Considering the foregoing, in one aspect of the present invention, a mixture of Injin mugwort extract and Citron extract can be identified as a composition for skin whitening containing an active ingredient, and in another aspect, a mixture of Injin mugwort extract and Citron extract is used as an active ingredient. It can be identified as a composition for improving skin wrinkles comprising a.

In the present specification, the term "extract" refers to water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N, Extract obtained by leaching using N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixture of these solvents, using supercritical extraction solvents such as carbon dioxide and pentane Refers to the obtained extract or a fraction obtained by fractionating the extract, and any method such as cooling, reflux, heating, ultrasonic radiation, supercritical extraction, etc. can be applied in consideration of the polarity, degree of extraction, and preservation of the active substance. there is. In the case of the fractionated extract, after suspending the extract in a specific solvent, the fraction obtained by mixing and standing with a solvent of different polarity, and the crude extract are adsorbed on a column filled with silica gel, etc., and then a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof It is meant to include fractions obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract from which the extraction solvent is removed by lyophilization, vacuum drying, hot air drying, spray drying, or the like. Preferably, it refers to an extract obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent.

In addition, in the present specification, "active ingredient" means a component that exhibits the desired activity alone or can exhibit activity in combination with a carrier having no activity itself.

The composition of the present invention may contain the active ingredient in any amount (effective amount) as long as it can exhibit skin whitening effect, skin wrinkle improvement effect, etc. according to the formulation, purpose of blending, etc. It will be determined within the range of 0.001% by weight to 15% by weight. Here, "effective amount" means when the composition of the present invention is administered to a mammal, preferably a human, to which it is applied, during the administration period according to the advice of medical experts, etc. It refers to the amount of the active ingredient included in the composition of the present invention that can exhibit effects and the like. Such an effective amount can be determined empirically within the ordinary skill of the skilled artisan.

In addition to the active ingredients, the composition of the present invention has a skin whitening effect, skin wrinkle improvement effect, or skin hypersensitivity reaction inhibitory activity, skin useful activity (skin moisturizing activity, skin damage protection activity by ultraviolet rays, etc.), antioxidant In order to enhance the convenience of intake, intake, and use through the addition of similar activities such as activity, any compound or natural extract known to have a corresponding activity and whose safety has already been verified in the art may be further included.

These compounds or extracts include each country's Pharmacopoeia (Korean Pharmacopoeia), each country's Health Functional Food Codex (in Korea, it is the Ministry of Food and Drug Safety's notification, "Health Functional Food Standards and Specifications"), each country's Functional Cosmetics Codex (in Korea, the Ministry of Food and Drug Safety notification Compounds or extracts listed in compendial documents such as 「Functional Cosmetics Standards and Test Methods」), compounds or extracts that have been approved as items under the laws of each country governing the manufacture and sale of pharmaceuticals (in Korea, it is the 「Pharmaceutical Affairs Act」), health In accordance with the laws of each country regulating the manufacture and sale of functional foods (in Korea, it is the 「Health Functional Food Act」), the laws of each country regulating the manufacture and sale of compounds or extracts with recognized functionality, and the manufacture and sale of functional cosmetics (in Korea, the 「Cosmetics Act」 ”), compounds or extracts whose functionality has been recognized are included.

These ingredients include, for example, retinol, retinyl palmitate, adenosine, and polyethoxylease recognized as skin wrinkle improvement ingredients in the Cosmetics Codex (Korean Ministry of Food and Drug Safety notification, 「Standards and Test Methods for Functional Cosmetics」) under the Korean 「Cosmetics Act」. tidamide, etc., and drometrizol, drometrizoletrisiloxane, digaloyltrioleate, dimethicodyethylbenzalmalonate, diethylhexylbutamidotriazone, etc. recognized as UV protection ingredients in the Korean Cosmetics Codex. , Arbutin, Niacinamide, Ascorbyl Glucoside, Alpha-Bisabolol, Oil Soluble Licorice (Glycyrrhiza) Extract, etc. recognized as skin whitening ingredients in the same Cosmetics Codex, and Korea 「Health Functional Food Act」 Complexes such as pine bark extract, phosphatidylserine, finger root extract powder, complex extracts such as red ginseng and cornus officinalis, etc. recognized as UV protection ingredients according to the Korea Health Functional Food Act, green tea extract, bamboo leaf extract, melon extract, raspberry extract, beeswax alcohol, ubiquinol, coenzyme Q10, tomato extract, grape seed extract, French maritime pine bark extract, red ginseng concentrate, etc., and also improve sensitive skin conditions according to the Korean Health Functional Food Act. L. sakei Probio 65 recognized as a functional ingredient, gamma-linolenic acid-containing oil, L.plantarum CJLP133, a lactic acid bacterium derived from fruits and vegetables, and probiotics ATP. One or more of these compounds or natural extracts may be included in the composition of the present invention together with the active ingredient.

In a specific aspect, the composition of the present invention can be regarded as a food composition.

The food composition of the present invention can be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, and ionic beverages, processed oils such as milk and yogurt, chewing gum, rice cakes, traditional Korean snacks, bread, snacks, noodles, etc. It can be manufactured into health functional food preparations such as foods, tablets, capsules, pills, granules, liquid, powder, flakes, pastes, syrups, gels, jellies, and bars.

In addition, the food composition of the present invention may take any product classification as long as it conforms to the enforcement regulations at the time of manufacture and distribution in legal and functional classification. For example, health functional food according to the Korean 「Health Functional Food Act」, or snacks, legumes, teas, beverages according to each food type according to the Food Code (MFDS notification 「Food Standards and Specifications」) of the Korea 「Food Sanitation Act」 , special purpose foods, etc.

The food composition of the present invention may include food additives in addition to the active ingredient. Food additives may generally be understood as substances that are added to, mixed with, or infiltrated into food in manufacturing, processing, or preserving food. Since food is consumed daily and for a long period of time, its safety must be guaranteed. According to the Food Additives Code under the laws of each country governing the manufacture and distribution of food (in Korea, it is the 「Food Sanitation Act」), safety-guaranteed food additives are limitedly regulated in terms of ingredients or functions. In the Korean Food Additives Codex (MFDS notification 「Standards and Specifications for Food Additives」), food additives are classified into chemical synthetic products, natural additives and mixed preparations in terms of ingredients and are regulated. It is divided into additives, preservatives, emulsifiers, acidulants, and thickeners.

Sweeteners are used to impart appropriate sweetness to foods, and both natural and synthetic sweeteners can be used in the composition of the present invention. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavors may be used to improve taste or aroma, and both natural and synthetic flavors may be used. Preferably, it is the case of using a natural one. In case of using natural ones, in addition to flavor, the purpose of enhancing nutrition can also be combined. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or obtained from green tea leaves, roundworms, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc. can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavors may be used, and synthetic flavors may include esters, alcohols, aldehydes, terpenes, and the like.

As preservatives, sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used, and as emulsifiers, acacia gum, carboxymethylcellulose, xanthan gum, pectin and the like, and examples of the acidulant include acid salt, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Acidulants may be added to the food composition to have an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to improving taste.

As the thickening agent, a suspending agent, a sedimentation agent, a gel forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may include physiologically active substances or minerals known in the art and whose stability is guaranteed as food additives for the purpose of supplementing or reinforcing functionality and nutrition.

Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, and the like. Examples of minerals include calcium preparations such as calcium citrate and magnesium stearate. magnesium preparations such as the like, iron preparations such as iron citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, and the like.

The food composition of the present invention may include the above-mentioned food additives in an appropriate amount to achieve the purpose of addition according to the type of product.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to national food codes or food additive codes.

The composition of the present invention can be understood as a pharmaceutical composition in another specific aspect. When the composition for skin whitening of the present invention is understood as a pharmaceutical composition, its use may be understood as a composition for improving skin hyperpigmentation.

The pharmaceutical composition of the present invention may be formulated into an oral formulation or a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The route of administration herein may be any appropriate route including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case in which two or more formulations of drugs are combined according to the route of administration, for example, one drug is firstly administered intravenously and the other drug is secondly administered through a topical route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and specifically, reference may be made to the pharmacopoeia of each country including the "Korean Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, suspension, wafer according to a method known in the art together with a suitable carrier. It can be prepared in formulations such as Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, Serol etc. are mentioned. In the case of formulation, appropriate binders, lubricants, disintegrants, coloring agents, diluents, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, and the like, and sodium oleate as a lubricant , sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc. as disintegrants, starch, methyl cellulose, agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, and the like. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it may be formulated in the form of an injection, transdermal administration, nasal inhalation, and suppository along with a suitable carrier according to a method known in the art. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, an isotonic solution such as phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, or 5% dextrose may be used. . When formulated as a transdermal formulation, it may be formulated in the form of ointments, creams, lotions, gels, external solutions, pastas, liniments, air rolls, and the like. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, the carrier is Witepsol ( witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like can be used.

Regarding the specific formulation of the pharmaceutical composition, it is known in the art, and for example, reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995) and the like. These documents are considered as part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, severity of the patient, and route of administration. /kg range. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the present invention in any respect.

In another specific aspect, the composition of the present invention can be identified as a cosmetic composition.

Even when the composition of the present invention is identified as a cosmetic composition, the cosmetic composition may be classified as an arbitrary product in terms of its use and law, and specifically, functional cosmetics and non-functional cosmetics with uses such as improving skin troubles and atopic dermatitis. It may be a general cosmetic or the like. In product form, it may take any product form, specifically, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax It may take the form of a product such as foundation or spray. In a specific product form, it may be a softening lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder formulation.

The cosmetic composition of the present invention may include, in addition to the active ingredient, ingredients commonly used in cosmetic compositions, for example, stabilizers, solubilizers, surfactants, vitamins, conventional adjuvants such as pigments and fragrances, and carriers.

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

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

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, fatty acid esters of sorbitan, and the like may be used.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, microcrystals Star cellulose, aluminum metahydroxide, bentonite, agar, and the like can be used.

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

The cosmetic composition of the present invention may be prepared according to a method for preparing a cosmetic composition conventionally performed in the art, except for including the active ingredient exhibiting skin wrinkle activity.

As described above, according to the present invention, it is possible to provide a composition for skin whitening and a composition for improving skin wrinkles using a mixture of ginseng mugwort extract and citron extract.

These compositions of the present invention can be commercialized as foods, cosmetics, pharmaceuticals, and the like.

1 is a cell viability measurement result of B16F10 melanoma cells.
2 is a melanin content measurement result.
3 is a result of measuring intracellular tyrosinase activity.
4 is a result of measuring mushroom tyrosinase activity.
5 is an elastase activity measurement result.
6 is a specific result of collagenase activity.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

<Example> Preparation of Injin mugwort extract and Citron extract

After adding 10 times the weight of 70% ethanol to each of dried Injin mugwort and citron powder (30 to 50 mesh), extraction was performed at 100° C. for 3 hours using a reflux extractor. After filtering the obtained extract, concentrated under reduced pressure at 60 - 70 ℃ to prepare an extract.

Injin mugwort extract and citron at a weight ratio of 9:1, which are the most active, through preliminary tests on whitening activity (Mushroom tyrosinase inhibitory activity test) and wrinkle improvement activity preliminary test (elastase or collagenase inhibitory activity test) A mixture of extracts (Injin mugwort extract in a weight ratio of 9) was used as a sample in the experiment below.

<Experimental Example> Whitening activity, wrinkle improvement activity and antioxidant activity test

1. Reagents and instruments

For cell culture, Dulbccos's Modified Eagle medium (DMEM, Lonza, Basel Swiss, USA), Phosphate buffered saline (PBS, Lonza, Walkersvile, USA), fetal bovine serum (FBS, GIBCO, California, USA), penicillin/streptomycin (P/S, GIBCO, California, USA) was purchased and used. 1,1-diphenyl-2-picryhydrazyl (DPPH) and 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) used in the antioxidant activity test and 3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), α-melanocyte stimulating hormone (α-MSH), and arbutin were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

ELISA reader (Biochrom, England), UV/vis spectrophotometer (Optizen 2120 UV, Mecasys, Korea), and centrifuge (Combi 514R, Hanil, Korea) were used for the experiments.

2. Whitening activity experiment

2.1 Cell lines

B16F10 melanoma cells, a melanoma cell line used in this experiment, were purchased from ATCC (American Type Culture Collection, USA) and used. The purchased cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO2 conditions.

2.2 Experimental method

2.2.1 Cell viability measurement

The cell viability of the extract was measured using the MTT measurement method. B16F10 melanoma cells were seeded in a 96-well plate and cultured for 24 h in a 37°C, 5% CO2 incubator. Then, the extract was treated with 10 μL of each concentration and cultured for 48 h. Thereafter, the culture medium was removed, treated with an MTT solution prepared at a concentration of 0.5 mg/mL, and cultured for 3 h. The treated MTT solution was removed, DMSO was added to each well, reacted at room temperature for 15 minutes, and absorbance was measured at 570 nm using an ELISA reader.

2.2.2 Measurement of melanin content

B16F10 melanoma cells were divided into 2 × 10 ⁵ cells/well in a 60 mm dish, cultured for 24 h, and then treated with 100 nM of α-MSH to induce melanin production. As a positive control group, arbutin was used. were treated at concentrations of 50, 100, 250, and 500 μg/mL and incubated for 72 h in a 37°C, 5% CO2 incubator. Thereafter, the culture medium was removed, washed with PBS, and cells were lysed using trypsin-EDTA, followed by centrifugation at 12,000 rpm for 15 minutes at 23 °C. Thereafter, 1 N NaOH solution containing 10% DMSO was added to the pellet from which the supernatant was removed, reacted at 80 ° C. for 1 h, and absorbance was measured at 475 nm with an ELISA reader.

2.2.3 Measurement of intracellular tyrosinase activity

B16F10 melanoma cells were divided into 2 × 10 ⁵ cells/well in a 6-well plate and cultured for 24 h, then the culture medium was removed, washed with PBS, and the samples were treated by concentration and cultured for 72 h in a 37 °C, 5% CO₂ incubator. did Thereafter, the cultured cells were washed with cold-PBS on ice, and 200 μl of PRO-PREP lysis solution was added to each well, dissolved on a shaker for 15 min, and then centrifuged for 5 min at 4 ° C. Centrifuge at 13,000 rpm. Then, the same amount of protein and 0.1M sodium phosphate buffer (pH 6.8) were dispensed so that the total amount of 150 μl was added to a 96-well plate, and 0.1% (w / v) = 1 mg / ml L-dopa dissolved in 50 μl was dispensed. After reacting at 37 ° C. for 1 h, the absorbance was measured at 475 nm with an ELISA reader.

2.2.4 Measurement of mushroom tyrosinase activity

Mushroom tyrosinase activity inhibition was measured by measuring absorbance using a 96 well plate. After dispensing 80μ of 67mM sodium phosphate buffer (pH 6.8) into a 96-well plate, 40μ of 10mM L-dopa was dispensed into all wells. After that, 40μ of the sample corresponding to each concentration was added, and mushroom tyrosinase (200U/ml) was dispensed by 40μ, and then reacted in a 37°C incubator for 10min, and the absorbance was measured at 492 nm with an ELISA reader.

3. Wrinkle improvement activity experiment

3.1 Measurement of elastase inhibitory activity

Elastase and substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide were dissolved in Tris-HCl buffer (pH 8.6) and used. Each test solution was prepared to have a certain concentration, and 140 μl of buffer, 2 μl of elastase enzyme solution, 8 μl of sample, and 50 μl of substrate solution were reacted for 30 minutes in an incubator at 37 ° C. The absorbance was measured at 410 nm with an ELISA reader. Elastase inhibitory activity was calculated using the following formula.

Calculation of elastase inhibition rate (%)

Inhibition (%) = (1 - Sample OD / Control OD) x 100

3.2 Measurement of collagenase inhibitory activity

A buffer was prepared by dissolving 4 mM CaCl ₂ in Tris-HCl buffer (100 mM, pH 7.5) and used. In the prepared buffer, collagenase and substrate 4-Phenylazobenzyloxycarbonyl-Pro o-Leu-Gly-Pro-D-Arg were prepared at concentrations of 0.2 mg/ml and 0.3 mg/ml, respectively, and reacted. Each sample was prepared to have a certain concentration, and 100 μl of sample, 150 μl of collagenase, and 250 μl of substrate solution were added and reacted for 20 min in an incubator at 37 ° C. Then, fractionated with ethyl acetate, only the supernatant was taken, and the absorbance was measured at 320 nm. Collagenase inhibitory activity was calculated using the following formula.

Collagenase inhibition rate (%) calculation

Inhibition (%) = [1-(OD ₃₂₀ of sample/OD ₃₂₀ of control)] x 100

4. Experimental results

4.1 Whitening activity

4.1.1 Cell viability

The cell viability measurement results of the melanoma cells of the Example samples are shown in FIG. 1 . No specific cytotoxicity was observed up to 5% (w/v). Based on these cell viability results, the treatment concentration of the sample was set to 5%.

4.1.2 Melanin content

The melanin content measurement results for each concentration of the Example sample are shown in FIG. 2 . Example samples reduced melanin production in a concentration-dependent manner.

4.1.3 Intracellular tyrosinase inhibitory activity

The results of measuring the intracellular tyrosinase inhibitory activity for each concentration of the Example sample are shown in FIG. 3 . Example samples showed intracellular tyrosinase inhibitory activity in a concentration-dependent manner.

4.1.4 Mushroom tyrosinase inhibitory activity

The results of measuring the mushroom tyrosinase inhibitory activity by concentration of the sample in the example are shown in FIG. 4. Example samples showed mushroom tyrosinase inhibitory activity in a concentration-dependent manner.

4.2 Anti-wrinkle activity

4.2.1 Elastase inhibitory activity

The results of measuring the elastase inhibitory activity for each concentration of the Example sample are shown in FIG. 5 . Example samples showed elastase inhibitory activity in a concentration-dependent manner.

4.2.2 Collagenase inhibitory activity

The results of measuring the collagenase inhibitory activity for each concentration of the Example sample are shown in FIG. 6 . Example samples showed collagenase inhibitory activity in a concentration-dependent manner.

Claims (5)

A food composition for skin whitening comprising a mixture of ginseng mugwort extract and citron extract as an active ingredient.
A cosmetic composition for skin whitening comprising a mixture of ginseng mugwort extract and citron extract as an active ingredient.
A food composition for improving skin wrinkles comprising a mixture of ginseng mugwort extract and citron extract as an active ingredient.
A cosmetic composition for improving skin wrinkles comprising a mixture of ginseng mugwort extract and citron extract as an active ingredient.
A pharmaceutical composition for treating or preventing skin hyperpigmentation comprising a mixture of an injin mugwort extract and a citron extract as an active ingredient.

Images