KR102313646B1 - Cosmetic composition for skin whitening containing complex extracts of Swertia Japonica, Hypericum Perforatum and Trichosanthes Kirilowii prepared by a method based on natural deep eutectic solvents - Google Patents

Abstract

The present invention relates to a cosmetic composition for skin whitening containing a mixed extract of Swertia japonica, Hypericum perforatum and Trichosanthes kirilowii and, more specifically, to a cosmetic composition that contains a mixed extract of Swertia japonica, Hypericum perforatum and Trichosanthes kirilowii and exhibits excellent skin whitening efficacy, wherein the mixed extract of Swertia japonica, Hypericum perforatum and Trichosanthes kirilowii is prepared by extracting Swertia japonica, Hypericum perforatum and Trichosanthes kirilowii using a natural eutectic solvent, and then performing a subcritical water extraction using the same.

Description

Cosmetic composition for skin whitening containing complex extracts of Swertia Japonica, Hypericum Perforatum and Trichosanthes Kirilowii prepared by a method based on natural deep eutectics }

The present invention relates to a cosmetic composition for skin whitening containing a mixed extract of bitter grass, red pepper sprouts and Hanultari extracted with a natural eutectic solvent. Specifically, bitter grass, red pepper herbs and Hanultari extract are extracted using a natural eutectic solvent, and the It relates to a cosmetic composition which contains a mixed extract of bitter grass, western red pepper and Haneultari produced by subcritical extraction again and exhibits excellent skin whitening efficacy.

The skin plays a very important role as a barrier function to protect the individual from the outside. The barrier function is a protective function against various external stimuli such as chemicals, air pollutants, dry environment, ultraviolet rays, etc. Stratum corneum, horney layer) can maintain its function only when it is normally formed. Such skin is histologically composed of three layers of epidermis, dermis, and subcutis. The epidermis is also the subject of the most intensive research.

The components that make up the stratum corneum, the outermost layer of the epidermis, are keratinocytes and skin lipids, which are responsible for the barrier function of the skin. It can be said to be an important function to protect, prevent leakage of body substances, and prevent evaporation of moisture in the skin.

To have fair and fair skin is everyone's unchanging desire. In general, the color of human skin, hair, eyes, etc. is determined by melanin, carotene, hemoglobin, and the like. Human skin color is determined by several factors, such as the active distribution of blood vessels of melanocytes that make melanin, the thickness of the skin, and the presence or absence of pigments outside the body such as carotenoids and bilirubin. The most important is the black pigment called melanin, which is produced by the action of several enzymes. The formation of melanin is influenced by genetic factors, physiological factors related to hormone secretion and stress, and environmental factors such as UV irradiation. Melanin is present in the skin and has an important function to protect the body from ultraviolet rays, but it is known that melanin is produced in excess and not only forms spots or freckles, but also promotes skin aging and plays an important role in inducing skin cancer.

The main function of melanin is to protect the skin from damage by removing these harmful radicals. Therefore, having a large amount of melanin means having an effective countermeasure system that can protect the skin from physical or chemical toxic substances. Factors that promote the production of melanin include estrogen or prostaglandin as well as sunlight ultraviolet rays.

The biosynthesis of melanin is the result of specific enzymes such as tyrosinase, tyrosinase related protein-1 (TRP-1), and dopachrome tautomerase (DCT) in the melanosome of melanocytes present in the basal layer of the epidermis. It occurs by a continuous oxidation reaction, and among them, an experiment to inhibit the activity of tyrosinase, a major enzyme in melanin synthesis, is being adopted at the initial stage of the development of melanin polymer inhibitors. However, in addition to the oxidation reaction by tyrosinase, among the factors affecting the production pathway of melanin in the skin, there is an Advanced glycation end products (AGEs) reaction or a non-enzymatic glycation reaction. The glycation reaction (glycation) is a non-enzymatic reaction between sugar and protein, which reacts with the carbonyl group of a reducing sugar and lysine or arginine, which is a free amino group of a protein, to form a schiff base, an initial glycation product. It is a reaction in which the formed compounds continuously undergo a series of reactions such as rearrangement and cross-linking to produce irreversible advanced glycation end products (AGEs), which are browning compounds.

This is a reaction between lysine or arginine, a free amino group of a protein, and a carbonyl group of a reducing sugar to form a shiff base, which is an initial glycation product. This reaction produces a yellowish-brown compound (melanoidine). When the process of generation and accumulation of AGEs proceeds, the production and accumulation of melanin pigment occurs in the skin. The production of such melanin is synthesized in an organelle called melanosome in melanocytes of the basal layer. When AGEs are generated, the receptor for AGE (RAGE) acts on the receptor site. AGE promotes the expression of the enzyme tyrosinase, which plays the most important role in the production of melanin, by promoting Microphthalmia associated transcription factor (MITF), which is an important transcriptional regulator in the process of melanin synthesis after binding to RAGE. Finally, melanin is synthesized. The synthesized melanin pigment is delivered to adjacent cells, keratinocytes, through the dendrites of melanocytes, and the delivered melanin pigment is distributed to various parts of the skin through the keratinocytes.

Ascorbic acid, kojic acid, arbutin, hydroquinone, glutathione or derivatives thereof have been previously used to treat or alleviate skin pigmentation symptoms such as spots, freckles or pigmentation, and excessive melanin pigmentation caused by UV exposure. Substances with rosinase inhibitory activity have been mixed and used in cosmetics and pharmaceuticals, but their use is limited due to insufficient whitening effect, safety issues for the skin, and formulation stability issues when formulated in cosmetics.

On the other hand, organic solvents have been generally used for liquid extraction for a long time. Organic solvents have advantages of low cost and good extraction efficiency, but have disadvantages that are harmful to the health of the experimenter and may cause environmental pollution problems. Recently, studies on various green solvents have been conducted to overcome the disadvantages of these organic solvents. Accordingly, the use of ionic liquids or deep eutectic solvents has been proposed as an alternative to organic solvents, and research is being actively conducted.

Ionic liquids are salts composed of a combination of a large heterocyclic cation and various inorganic anions, and have a low melting point and exist as a liquid at room temperature. Ionic liquids have been applied in extraction and separation fields because of their low boiling point, low vapor pressure, non-volatile properties, low melting point, and high ionic conductivity. However, on the other hand, these ionic liquids are toxic to the human body, their biodegradability is low, and the physical properties of ionic liquids can be affected even by trace impurities. Since many salts and solvents are used, it is not suitable as an environmentally friendly solvent and has a disadvantage in that the price is high.

To overcome these shortcomings, a deep eutectic solvent (DES) has been developed. These DESs are inexpensive, non-toxic, harmless to the human body, and many physicochemical properties such as density, viscosity, and ionic conductivity are similar to those of ionic liquids, so many studies are being conducted as new cleaning solvents.

The present inventors have studied natural product extraction using Natural Deep Eutectic Solvents (NADES), which are eutectic solvents made of natural compounds among the eutectic solvents, and through this, they tried to develop a new natural cosmetic that has secured skin safety. . As a result, when a mixed extract of bitter grass, western red pepper and Hanultari was prepared by using a specific natural eutectic solvent as an extraction solvent, the activity was greatly increased and it was confirmed that it exhibits excellent skin whitening efficacy, thereby completing the present invention.

(0001) Republic of Korea Patent Registration No. 10-2162377 (2020.09.25) (0002) Republic of Korea Patent Publication No. 10-2020-0073851 (2020.06.24) (0003) Republic of Korea Patent Publication No. 10-2016-0036122 (2016.04.04)

An object of the present invention is to provide a cosmetic composition that contains bitter grass extracted using a natural eutectic solvent, a mixed extract of western red pepper and Hanultari as an active ingredient, thereby exhibiting an excellent skin whitening effect.

In addition, another object of the present invention is to provide a method for preparing a mixed extract of bitter grass, western red pepper and Hanultari with excellent skin whitening activity by using the natural eutectic solvent.

In order to achieve the above object, according to the present invention, bitter grass, Western red pepper produced by extracting a mixture of bitter grass, red pepper sprouts and Hanultari with a natural eutectic solvent consisting of betaine and saccharose, and then subcritical extraction again. There is provided a cosmetic composition for skin whitening containing a mixed extract of herbs and Hanultari as an active ingredient in an amount of 0.1 to 10% by weight based on the total weight of the composition.

The mixture is made by mixing bitter grass, red pepper greens, and Hanultari, respectively, in a weight ratio of 1: 1-3: 1-3.

The natural eutectic solvent is preferably made by mixing betaine and saccharose in a weight ratio of 1:1.

The subcritical extraction is characterized in that it consists of extracting for 10 to 30 minutes at a subcritical condition of 120 ~ 200 ℃, a pressure of 0.1 ~ 15 MPa by adding an extraction solvent.

According to the present invention in order to achieve the above other object, (A) preparing a mixture by mixing bitter grass, red pepper greens and Hanultari in a ratio of 1:1 to 3: 1-3, respectively; (B) extracting the mixture using a natural eutectic solvent consisting of betaine and saccharose and water as a solvent; (C) a subcritical extraction step of adding an extraction solvent to the extract prepared in step (B) and then extracting for 10 to 30 minutes at a subcritical condition of 120 to 200° C. and a pressure of 0.1 to 15 MPa; And (D) there is provided a method for producing a mixed extract of bitter grass, western red pepper and Hanultari mixed extract comprising the step of concentrating after filtration.

In the step (B), it is more preferable that the solvent be mixed in a ratio of 5% by weight of betaine, 5% by weight of saccharose, and 90% by weight of water.

The mixed extract of bitter grass, red pepper herbs and Hanultari produced according to the present invention exhibits excellent skin whitening activity due to its synergistic effect, and thus can be usefully used as a skin whitening cosmetic.

Hereinafter, the present invention will be described in more detail.

According to the present invention, after extracting a mixture of bitter grass, red pepper herbs and Haneultari with a natural eutectic solvent consisting of betaine and saccharose, then subcritical extraction is performed to obtain a mixed extract of bitter grass, western red pepper herbs, and Hanultari. A cosmetic composition containing as an active ingredient is provided.

Bitter grass (Swertia japonica) is an annual or biennial plant of the dicotyledonous plywood family, Gentianaceae. The height is 15-30 cm. The leaves are opposite in the shape of a thread. Purple flowers bloom at the end of the stalk from the upper leaf axil in September-October. The fruit is a capsule (果). Both leaves and stems, including flowers, are used medicinally. It contains Swertiamarin and has a strong bitter taste.

Hypericum perforatum (Hypericum perforatum) is a perennial plant in the family Molese. It is one of the herbs native to Europe and Western Asia. It grows to about 1 m in height. The rhizome is thick and short, and many stems come out of it. Stems are hard at the bottom and branched at the top. From June to August, bright yellowish orange flowers hang in umbels. According to folk remedies, it relieves depression, reduces skin inflammation, and promotes wound healing.

Haneultari (Trichosanthes Kirilowii) is a creeping perennial plant of the dicotyledonous gourd family. It grows below the foot of the mountain. The root becomes as thick as a sweet potato, and the stem rises while winding other objects with tendrils. The leaves are alternate phyllotaxis, split into 5-7 like maple leaves, and have sawtooths on the forked pieces. Flowers bloom in July-August, are bivalent, and yellow. In oriental medicine, the root is called wanggwageun (王瓜根), the fruit is called fruit fruit (土瓜實), and the seed is called togwain (土瓜仁) and is used medicinally.

The active ingredients of the present invention, bitter grass, western red pepper herbs, and Hanultari mixed extract are prepared by natural eutectic solvent extraction.

According to a preferred embodiment of the present invention, first, a mixture of bitter grass, western red pepper sprouts and Hanultari is extracted using a natural eutectic solvent. The mixture is made by mixing bitter grass, red pepper herbs, and Hanultari in a weight ratio of 1:1 to 3: 1-3, respectively, and exhibits superior whitening activity when extracted by mixing in a ratio of 1:3:2, respectively. Therefore, it is more preferable

Eutectic solvents (DES, Deep Eutectic Solvents) are in a state in which salt and HBD are eutectic with hydrogen bonds, and are prepared by mixing salt and HBD (hydrogen bond donors, hydrogen bond donors) in a certain ratio. In the preparation of a specific bioactivity fortified extract, the selection of the DES type is very important. This is because diffusion, solubility, viscosity, surface tension, polarity, and physicochemical interactions between the solid and liquid phases greatly affect the extraction yield.

In the present invention, betaine was used as a salt. Trimethylglycine is a natural compound and is a quarternary ammonium compound with three methyl groups attached to the N atom of glycine. In addition, as HBD (hydrogen bond donor), saccharose was used. Saccharose (C ₁₂ H ₂₂ O ₁₁ ) is also called sucrose or sucrose, and can be obtained from sugar cane or sugar beet.

In the present invention, when the vitamin and saccharose are mixed in a weight ratio of 1:1, the extraction efficiency is high and the activity of the extract is high, so it is more preferable. The eutectic solvent can be used directly as an extraction solvent, but since it has high viscosity, it is mixed with other solvents to increase extraction efficiency. In the present invention, it is preferably mixed with water and used as a solvent. 5 to 15 times the weight of water is added to the eutectic solvent composed of betaine and saccharose and used as an extraction solvent. In the present invention, it is most preferable to use a mixture of 5% by weight of betaine, 5% by weight of saccharose and 90% by weight of water.

According to a preferred embodiment of the present invention, the bitter grass, red pepper sprouts and Hanultari mixture are put into a mixed solvent consisting of 90% by weight of water, 5% by weight of betaine, and 5% by weight of saccharose, and extracted at 40 to 45° C. for 3 to 4 hours. do.

Then, in order to more efficiently extract the active ingredient, the obtained eutectic solvent extract is subjected to subcritical extraction again. According to a preferred embodiment of the present invention, an extraction solvent is added to the eutectic eutectic extract of bitter grass, western red pepper sprouts and Haneultari, extracted under subcritical extraction conditions of 120-200° C. and pressure of 0.1-15 MPa for 10 to 30 minutes, followed by filtration. Concentrate with a reduced pressure concentrator to prepare a mixed extract of bitter grass, western red pepper greens, and Haneultari.

In this case, the extraction solvent is water, anhydrous alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, glycerin, ethylene glycol, propylene glycol, butylene glycol, caprylic/capric triglyceride, dimethicone, mineral oil, cyclo At least one selected from the group consisting of methicone, octyldodecanol, cetylethylhexanoate, trirethylhexanoin, isopropylmyristate and vegetable oil is used. The vegetable oil means an oil extracted by a method such as pressing the seeds or flesh of a plant, and olive oil, sunflower seed oil, palm oil, avocado oil, etc. may be used.

As described above, after extraction with natural eutectic solvent, the mixed extract of bitter grass, red pepper herb and Hanultari mixed extract of the present invention prepared by subcritical extraction has better skin whitening activity than each extract, natural eutectic solvent mixed extract, or subcritical mixed extract was shown (Test Example).

The bitter grass, western red pepper herbs and Hanultari mixed extract as active ingredients is contained in an amount of 0.1 to 10% by weight based on the total weight of the composition.

The cosmetic composition of the present invention may be prepared in any conventionally prepared formulation, for example, lotion, cream, essence, cleansing foam, cleansing water, pack, body lotion, body oil, body gel, shampoo, conditioner, hair. conditioners, hair gels, foundations, lipsticks, mascaras, makeup bases, and the like.

[Example]

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited by the following examples.

Preparation Examples 1 to 3: Preparation of bitter grass, western red pepper and Haneultari extract

To 100 g each of dried bitter grass, red pepper herbs, and Haneultari root, add 5 times the weight of purified water as an extraction solvent and heat to 100°C in an extractor equipped with a cooling condenser (Cosmos-660, Gyeongseo Machinery) to extract 3 times each for 2 hours. did.

After extraction by the above method, the mixture was left at room temperature for 3 days, and the precipitate was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. Then, using a vacuum concentrator (Coolace CCA-1100, EYELA), it was concentrated at a temperature of 40°C to 50°C, and then, using a spray dryer (B-290, BUCHI), the inlet temperature was 180°C, and the inhaler (Aspirator) Drying under the conditions of 100% efficiency (35㎤/hour), 25% pump efficiency (7.5ml/min), Nozzle Cleaner 4 and Rotameter: 30mm (357 liters/hour) to obtain the title extract powder.

Preparation Examples 4 to 6: Preparation of eutectic solvent extracts of bitter grass, western red pepper and Haneultari

For extraction by the NADES method as an extraction solvent 10 times the weight of 100 g of dried bitter grass, red pepper herbs outpost, and Haneultari root each, 90% by weight of water, 5% by weight of betaine and 5% by weight of saccharose were used in a hot water bath at 45℃ for 3 hours. extracted.

After extraction by the above method, the mixture was left at room temperature for 3 days, and the precipitate was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. Then, using a vacuum concentrator (Coolace CCA-1100, EYELA), it was concentrated at a temperature of 40°C to 50°C, and then, using a spray dryer (B-290, BUCHI), the inlet temperature was 180°C, and the inhaler (Aspirator) Drying under the conditions of 100% efficiency (35㎤/hour), 25% pump efficiency (7.5ml/min), Nozzle Cleaner 4 and Rotameter: 30mm (357 liters/hour) to obtain the title extract powder.

Preparation Examples 7 to 9: Preparation of bitter grass, red pepper sprouts and Haneultaria subcritical extract

Purified water was added 10 times by weight as an extraction solvent to 100 g of dried bitter grass, red pepper herb outpost, and Haneultari root, and extracted for 15 minutes at 121° C., a subcritical water condition, and a pressure of 1 MPa.

After extraction by the above method, the mixture was left at room temperature for 3 days, and the precipitate was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. Then, using a vacuum concentrator (Coolace CCA-1100, EYELA), it was concentrated at a temperature of 40°C to 50°C, and then, using a spray dryer (B-290, BUCHI), the inlet temperature was 180°C, and the inhaler (Aspirator) Drying under the conditions of 100% efficiency (35㎤/hour), 25% pump efficiency (7.5ml/min), Nozzle Cleaner 4 and Rotameter: 30mm (357 liters/hour) to obtain the title extract powder.

Preparation Examples 10 to 12: Preparation of a mixed solvent extract of bitter grass, western red pepper and Haneultari

After mixing (100 g) of dried bitter grass, red pepper herb outpost, and Haneultari root in the weight ratio shown in Table 1 below, 90% by weight of water, 5% by weight of Betain, and 5% by weight of Saccharose for extraction by the NADES method as an extraction solvent with 10 times the weight. % solvent at 45° C. for 3 hours.

After extraction by the above method, the mixture was left at room temperature for 3 days, and the precipitate was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. Then, using a vacuum concentrator (Coolace CCA-1100, EYELA), it was concentrated at a temperature of 40°C to 50°C, and then, using a spray dryer (B-290, BUCHI), the inlet temperature was 180°C, and the inhaler (Aspirator) Drying under the conditions of 100% efficiency (35㎤/hour), 25% pump efficiency (7.5ml/min), Nozzle Cleaner 4 and Rotameter: 30mm (357 liters/hour) to obtain the title extract powder.

weight ratio Preparation 10 Preparation 11 Preparation 12 bitter grass One One One western red pepper One 2 3 sky One 3 2

Preparation Examples 13 to 15: Preparation of mixed subcritical extract of bitter grass, western red pepper and Haneultari

After mixing (100 g) of dried bitter grass, red pepper herb outpost, and Haneultari root in the weight ratio shown in Table 2 below, 10 times the weight of purified water as an extraction solvent was added, followed by extraction under subcritical water conditions of 121° C. and pressure of 1 MPa for 15 minutes.

After extraction by the above method, the mixture was left at room temperature for 3 days, and the precipitate was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. Then, using a vacuum concentrator (Coolace CCA-1100, EYELA), it was concentrated at a temperature of 40°C to 50°C, and then, using a spray dryer (B-290, BUCHI), the inlet temperature was 180°C, and the inhaler (Aspirator) Drying under the conditions of 100% efficiency (35㎤/hour), 25% pump efficiency (7.5ml/min), Nozzle Cleaner 4 and Rotameter: 30mm (357 liters/hour) to obtain the title extract powder.

weight ratio Preparation 13 Preparation 14 Preparation 15 bitter grass One One One western red pepper One 2 3 sky One 3 2

Examples 1 to 3: Preparation of mixed extracts of bitter grass, western red pepper and Haneultari

After mixing (100 g) dried bitter grass, red pepper herb outpost, and Haneultari root in the weight ratio shown in Table 3 below, 10 times the weight of purified water as an extraction solvent to the eutectic solvent extract obtained by extraction in the same manner as in Preparation Examples 4 to 6 and extracted for 15 minutes under subcritical water conditions of 121°C and pressure of 1 MPa.

After extraction by the above method, the mixture was left at room temperature for 3 days, and the precipitate was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. Then, using a reduced pressure concentrator (Coolace CCA-1100, EYELA), it was concentrated at a temperature of 40 ℃ to 50 ℃, and then, using a spray dryer (B-290, BUCHI), the inlet temperature was 180 ℃, the inhaler (Aspirator) Drying under the conditions of 100% efficiency (35㎤/hour), 25% pump efficiency (7.5ml/min), Nozzle Cleaner 4 and Rotameter: 30mm (357 liters/hour) to obtain the title extract powder

weight ratio Example 1 Example 2 Example 3 bitter grass One One One western red pepper One 2 3 sky One 3 2

Test Example 1: Confirmation of cytotoxicity

In order to check whether cytotoxicity, fibroblasts were inoculated in IMDM medium supplemented with 10% FBS at ^{a cell concentration of 5×10 5} , and cultured at 37° C. 5% CO _{2 in an} incubator for 24 hours. After culturing, the medium was removed, and the extracts prepared in Preparation Examples and Examples were treated with a diluted solution prepared by diluting the extracts prepared in Preparation Examples and Examples with dimethyl sulfoxide so that the sample concentrations were 50, 100, 500, and 1000 μg/ml, and cultured for 24 hours. After that, 100 ml of MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazoliumboramide, Sigma, USA) solution was added to each well (3 mg/ml) and incubated for 4 hours more. Then, the supernatant was removed, 150 μl of dimethyl sulfoxide was added, and the resulting formazan was dissolved by shaking for 30 minutes, and absorbance was measured at 540 nm using a multimicroplate reader (Molecular device Spectra max190). Cell viability was calculated according to the following formula, and the results are shown in Table 4 below.

Cell viability (%) = Absorbance of the sample added group / Absorbance of the control group × 100

Cell viability (%) Sample concentration (%) 0 0.1 0.5 1.0 Preparation Example 1 100 100 100 100 Preparation 2 100 100 100 100 Preparation 3 100 100 100 100 Preparation 4 100 100 100 100 Preparation 5 100 100 100 100 Preparation 6 100 100 100 100 Preparation 7 100 100 100 100 Preparation 8 100 100 100 100 Preparation 9 100 100 100 100 Preparation 10 100 100 100 100 Preparation 11 100 100 100 100 Preparation 12 100 100 100 100 Preparation 13 100 100 100 100 Preparation 14 100 100 100 100 Preparation 15 100 100 100 100 Example 1 100 100 100 100 Example 2 100 100 100 100 Example 3 100 100 100 100

As shown in the results of Table 4, it was confirmed that the cytotoxicity was not affected as the cell viability was confirmed at the concentration of 500 μg/ml in both the samples to which the extracts of the Preparation Examples and Examples were applied, and there is no problem in safety. confirmed that.

Test Example 2: Confirmation of reduction in tyrosinase expression

Cells were seeded on 24-well plates at 1×10 ⁵ cells/mL and cultured for 24 hours. After 24 hours, samples were prepared by concentration in a medium not added with FBS and antibiotics, treated with cells, and further cultured for 24 hours. After removing the sample treatment medium, it was washed with phosphate-buffer saline (PBS) of pH 6.8, and PBS containing 1% Triton X-100 was added to the wells, and then the cells attached to the wells were removed with a cell scraper and a 1.5 mL tube It was collected and thawed after being rapidly frozen at -70°C, and this method was repeated 3 times to destroy the cell membrane. After centrifugation, the supernatant was used as an enzyme source for tyrosinase activity measurement and as a sample for protein measurement. Protein was quantified with Bio-Rad protein kit, and tyrosinase activity was measured after adding 200 μl of 10 mM L-dopa, 500 μl of 0.1 M PBS (pH 6.8), and 300 μl of tyrosinase enzyme source (supernatant from cells). Absorbance was measured at 475 nm after incubation at ℃ for 1 hour. The tyrosinase expression inhibition rate was calculated by the following formula, and the results are shown in Table 5 below.

Tyrosinase expression inhibition rate (%) = [(A-B)/A]X100

A: Amount of melanin in wells without sample added

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

Tyrosinase expression inhibition rate (%) Sample concentration (%) 0.05 0.1 0.2 Control Arbutin (2%) 80.4 Preparation Example 1 4.7 11.7 16.7 Preparation 2 6.7 12.7 18.4 Preparation 3 5.1 12.4 17.6 Preparation 4 14.8 20.6 27.9 Preparation 5 15.1 22.7 29.1 Preparation 6 15.2 21.4 28.1 Preparation 7 18.4 25.3 31.7 Preparation 8 20.1 27.4 34.2 Preparation 9 19.8 27.1 33.3 Preparation 10 22.4 28.4 35.5 Preparation 11 25.8 31.7 38.4 Preparation 12 24.2 30.7 36.9 Preparation 13 25.6 32.4 40.5 Preparation 14 29.5 37.7 48.2 Preparation 15 28.4 35.9 40.5 Example 1 30.1 56.4 59.8 Example 2 30.2 57.9 60.3 Example 3 59.4 68.7 79.5

As can be seen in Table 5, after the extraction of the natural eutectic solvent, the mixed extract of Examples 1 to 3 prepared by subcritical extraction is each extract (Preparation Examples 1 to 9) or , showed an excellent tyrosinase expression inhibitory effect compared to the natural eutectic solvent mixed extract (Preparation Examples 10 to 12) or the subcritical mixed extract (Preparation Examples 13 to 15).

Test Example 3: Confirmation of a decrease in melanin synthesis

Based on the degree of inhibition of melanin production for B16F1 melanocytes, their whitening effects were judged. The B16F1 melanocyte used in this test example is a mouse-derived cell line, and is a cell secreting a melanin called melanin. The degree of melanin melanin decreased by processing the sample during artificial culture of these cells was evaluated. B16F1 melanosite used in this test example was used after receiving from ATCC (American Type Culture Collection, Accession No. 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocytes was measured as follows. B16F1 melanocytes were dispensed in a 6-well plate at a ^{concentration of 2×10 6} cells/mL per well, and after attaching the cells, the sample was treated at a concentration of 0.1% (wt/v) and 5% carbon dioxide was released at 37°C for 72 hours. It was cultured in an incubator containing Here, the solvent used to adjust the concentration of the sample was PBS. After culturing for 72 hours, the number of cells in liver culture was treated with trypsin-EDTA 500 μl to measure the number of cells removed, and then centrifuged to recover the cells. Quantification of intracellular melanin was carried out by modifying the method of Lotan (Lotan: Cancer Res., 40: 3345-3350, 1980). After washing the cell pellet once with PBS, 1 ml of a homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Tripton X-100, 2 mM PMSF) was added and vortexed for 5 minutes to destroy the cells. After dissolving the extracted melanin by adding 120 μl of 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), the absorbance of melanin was measured at 405 nm with a microplate reader, and then melanin was quantified. Melanin production inhibition rate (%) of the sample was measured. The inhibition rate (%) of melanin production of B16F1 melanocytes was calculated by the following formula, and the results are shown in Table 6.

Melanogenesis inhibition rate (%) = [(A-B)/A]X100

A: Amount of melanin in wells without sample added

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

Melanin production inhibition rate (%) Sample concentration (%) 0.05 0.1 0.2 Control Arbutin (2%) 77.1 Preparation Example 1 7.4 12.4 17.4 Preparation 2 6.9 12.7 18.1 Preparation 3 6.1 13.2 16.8 Preparation 4 15.4 20.1 29.1 Preparation 5 14.8 21.4 29.7 Preparation 6 15.9 20.8 29.4 Preparation 7 19.4 24.3 30.4 Preparation 8 20.5 25.4 32.2 Preparation 9 20.1 26.1 31.2 Preparation 10 23.4 27.4 34.5 Preparation 11 26.1 32.9 36.7 Preparation 12 25.1 31.7 35.8 Preparation 13 26.2 34.4 41.1 Preparation 14 28.1 38.1 44.2 Preparation 15 27.9 36.7 43.7 Example 1 29.1 53.1 67.8 Example 2 30.7 55.1 68.7 Example 3 55.5 68.4 75.7

As confirmed in Table 6 above, as a result of testing the melanogenesis inhibitory effect of B16F1 melanocytes, the extract samples of Examples 1 to 3 showed that there was a better melanogenesis inhibitory effect than Comparative Examples 1 to 15, and the Among them, the extract of Example 3 showed the highest inhibition rate.

Test Example 4: Confirmation of inhibition of tyrosinase activity

Tyrosinase (SigmaAldrich Inc.) isolated from mushrooms was diluted in a buffer solution (50 mM sodium phosphate, pH 6.8) to a concentration of 250 U/μl. The experiment was carried out at room temperature, and was performed in a transparent 96-well plate. Each well was prepared with 80 μl of mushroom tyrosinase, 80 μl of tyrosine and 40 μl of a sample to make a final volume of 200 μl. The reaction was carried out in a dark room at 37 °C for 10 minutes, and absorbance was measured at 475 nm using an ELISA reader. The tyrosinase inhibition rate (%) of the sample was calculated by the following formula, and the results are shown in Table 7.

Inhibition rate (%) = [(A-B)/A]X100

A: Absorbance of wells to which no sample was added

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

Inhibition rate (%) Sample concentration (%) 0.05 0.1 0.2 Control Arbutin (2%) 89.1 Preparation Example 1 6.5 12.7 16.4 Preparation 2 7.2 13.4 17.9 Preparation 3 7.1 13.0 17.4 Preparation 4 14.9 22.1 28.8 Preparation 5 15.1 23.2 30.1 Preparation 6 15.2 23.8 29.9 Preparation 7 16.1 24.5 31.4 Preparation 8 17.5 25.1 31.3 Preparation 9 18.1 25.9 31.0 Preparation 10 20.1 25.4 32.5 Preparation 11 21.1 27.9 34.9 Preparation 12 21.5 29.1 35.2 Preparation 13 25.2 32.6 41.2 Preparation 14 26.4 34.4 43.8 Preparation 15 25.9 35.1 43.1 Example 1 50.1 61.1 70.4 Example 2 52.3 61.9 71.5 Example 3 60.5 79.5 84.5

As can be seen in Table 7, the extract samples of Examples 1 to 3 showed a better tyrosinase inhibitory effect than Comparative Examples 1 to 15, and among them, the extract of Example 3 showed the highest inhibition rate It was.

Test Example 5: Confirmation of inhibition of L-DOPA oxidative activity

3-(3,4-Dihydroxyphenyl)-L-alanine (L-DOPA) was purchased from TOKYO CHEMICAL INDUSTRY CO., LTD. L-DOPA was diluted to a concentration of 10 mM in a buffer solution (50 mM sodium phosphate, pH 6.8). The experiment was carried out in a transparent 96-well plate, and the final volume of each well was 200 μl. The experimental solution was 80 μl of mushroom tyrosinase, 80 μl of L-DOPA and 40 μl of the sample, and the reaction was carried out in a dark room at 37° C. for 10 minutes. Absorbance was measured at 475 nm using an ELISA reader. The L-DOPA oxidation activity inhibition rate (%) of the sample was calculated by the following formula, and the results are shown in Table 8.

Inhibition rate (%) = [(A-B)/A]X100

A: Absorbance of wells to which no sample was added

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

Inhibition rate (%) Sample concentration (%) 0.05 0.1 0.2 Control Arbutin (2%) 79.5 Preparation Example 1 3.5 11.7 15.7 Preparation 2 4.2 11.9 17.1 Preparation 3 3.9 11.1 16.3 Preparation 4 11.7 20.1 27.8 Preparation 5 13.1 23.4 29.1 Preparation 6 12.9 22.8 28.9 Preparation 7 16.7 25.3 31.4 Preparation 8 18.1 27.1 34.2 Preparation 9 17.5 25.9 32.9 Preparation 10 19.7 23.9 33.4 Preparation 11 21.5 26.4 35.1 Preparation 12 20.9 24.9 34.7 Preparation 13 24.1 30.4 42.4 Preparation 14 26.7 36.2 43.4 Preparation 15 26.1 35.1 42.9 Example 1 40.9 51.7 75.9 Example 2 42.1 52.1 76.7 Example 3 45.6 54.5 78.9

As can be seen in Table 8, the extract samples of Examples 1 to 3 showed a better L-DOPA oxidative activity inhibitory effect than Comparative Examples 1 to 15, and among them, the highest inhibition rate in the extract of Example 3 was shown.

Test Example 6: Whitening-related gene analysis

Gene analysis to determine the effect of the samples of Preparation Examples and Examples on the expression of Tyrosinase (TYR), Melanocyte inducing transcription factor (MITF), Tyrosine relative protein 1 (TYRP1) and Dopachrome Tautomerase (DCT) involved in melanin synthesis proceeded.

B16F10 cells were inoculated in a 24 well plate at ^{a cell concentration of 4×10 5} , respectively, and cultured at 37° C. 5% CO _{2 in an} incubator for 24 hours. The samples of Preparation Examples and Examples were added to the medium for each concentration and treated with cells for 24 hours, followed by induction of melanin production with α-MSH (100 nM) and cultured for 24 hours, washed twice with PBS, and then cells was harvested.

Then, 100 μl of Trizol reagent was dispensed into each well to lyse the cells, and 200 μl of chloroform was dispensed and shaken up and down for 3 minutes. Then, centrifuged at 13,200 rpm for 20 minutes, and the supernatant was transferred to a tube containing 200 μl of isopropanol and mixed. After centrifugation again at 13,200 rpm for 20 minutes, after removing the supernatant, 500 μl of 70% EtOH-diethylpyrocarbonate water was dispensed into each tube, centrifuged at 13,200 rpm for 5 minutes, and then the supernatant was removed and dried at room temperature. . After dissolving 20 μl of DEPC, 5 μl of RNA and 195 μl of sterile water were added to a 96-well plate, and absorbance was measured at 260 nm and 280 nm, respectively, to measure the total RNA amount. The extracted RNA was synthesized as cDNA using the High-Capacity RNA-to-cDNA™ Kit cDNA (Applied Biosystems™). The experiment was performed according to the manufacturer's manual.

Then, using the primer sequences shown in Table 9, Tyrosinase (TYR), Melanocyte inducing transcription factor (MITF), Tyrosine relative protein 1 (TYRP1) and Dopachrome Tautomerase (DCT) genes were amplified and then electrophoresed to confirm their expression levels. The results are shown in Table 10, Table 11, Table 12, and Table 13, respectively.

TYR Forward 5'- GGC CAG CTT TCA GGC AGA GGT ?? 3' Reverse 5'- TGG TGC TTC ATG GGC AAA ATC - 3 MITF Forward 5'-CTA GAG CGC ATG GAC TTT CC - 3' Reverse 5'-AAG TTG GAG CCC ATC TTC CT - 3' TYRP1 Forward 5'- GCT GCA GGA GCC TTC TTT CTC - 3' Reverse 5'- AAG ACG CTG GTC T - 3' DCT Forward 5'-TAC CAT CTG TTG TGG CTG GA-3' Reverse 5'-TGG CAC TGC TGG TCA TCT TGT CTT GCT G - 3'

TYR expression rate (%) Sample concentration (%) 0.05 0.1 0.2 Control Arbutin (2%) 18.2 Preparation Example 1 55.2 53.2 48.2 Preparation 2 49.2 45.2 44.2 Preparation 3 45.2 46.2 43.2 Preparation 4 51.2 49.2 42.2 Preparation 5 55.3 44.2 35.2 Preparation 6 52.3 48.2 35.2 Preparation 7 49.2 45.2 32.5 Preparation 8 48.2 38.2 35.2 Preparation 9 50.2 48.2 40.2 Preparation 10 51.2 46.2 40.2 Preparation 11 52.3 46.2 42.3 Preparation 12 49.2 47.2 39.2 Preparation 13 48.2 40.2 35.7 Preparation 14 47.2 40.5 35.2 Preparation 15 46.2 42.3 32.2 Example 1 42.3 38.5 30.2 Example 2 41.2 35.2 31.2 Example 3 25.3 20.2 18.8

MITF expression rate (%) Sample concentration (%) 0.05 0.1 0.2 Control Arbutin (2%) 19.2 Preparation Example 1 60.2 55.2 51.2 Preparation 2 52.3 49.5 44.2 Preparation 3 48.2 45.2 40.1 Preparation 4 47.2 42.2 35.6 Preparation 5 53.6 50.1 49.2 Preparation 6 49.3 41.2 35.2 Preparation 7 43.5 34.5 33.2 Preparation 8 48.2 38.2 35.2 Preparation 9 51.2 46.5 38.7 Preparation 10 49.6 47.2 39.5 Preparation 11 52.3 46.2 42.3 Preparation 12 45.6 41.8 35.6 Preparation 13 48.2 40.2 35.7 Preparation 14 47.2 40.5 35.2 Preparation 15 43.5 36.7 31.2 Example 1 49.5 41.2 35.2 Example 2 39.5 32.5 29.8 Example 3 30.2 25.2 20.1

TYRP1 expression rate (%) Sample concentration (%) 0.05 0.1 0.2 Control Arbutin (2%) 17.6 Preparation Example 1 59.2 50.2 45.2 Preparation 2 55.3 45.5 41.2 Preparation 3 45.2 41.2 35.1 Preparation 4 49.2 40.2 38.6 Preparation 5 56.6 51.1 47.2 Preparation 6 46.3 42.2 37.2 Preparation 7 49.5 43.5 33.2 Preparation 8 48.2 37.2 32.2 Preparation 9 56.2 49.5 42.7 Preparation 10 49.6 48.2 34.5 Preparation 11 55.3 47.2 44.3 Preparation 12 46.6 40.8 34.6 Preparation 13 46.2 41.2 35.7 Preparation 14 48.2 41.5 39.2 Preparation 15 44.5 39.7 31.2 Example 1 49.7 44.5 37.2 Example 2 36.5 32.5 29.8 Example 3 34.5 27.7 18.5

DCT expression rate (%) Sample concentration (%) 0.05 0.1 0.2 Control Arbutin (2%) 19.8 Preparation Example 1 53.2 50.2 49.2 Preparation 2 45.2 42.2 40.2 Preparation 3 49.2 46.2 43.2 Preparation 4 56.2 44.2 45.2 Preparation 5 59.3 45.2 39.2 Preparation 6 55.3 48.2 36.2 Preparation 7 49.2 44.2 35.5 Preparation 8 47.2 35.2 34.2 Preparation 9 50.2 47.2 48.2 Preparation 10 54.2 47.2 42.2 Preparation 11 56.3 46.2 48.3 Preparation 12 48.2 45.2 42.2 Preparation 13 49.2 40.2 35.7 Preparation 14 42.2 38.5 35.2 Preparation 15 49.2 42.3 39.2 Example 1 49.3 37.5 35.2 Example 2 45.2 36.2 32.2 Example 3 35.9 29.8 21.4

As a result of observation after performing RT-PCR, the samples of the above Examples were superior to the samples of Preparation Example for gene expression of Tyrosinase (TYR), Melanocyte inducing transcription factor (MITF), Tyrosine relative protein 1 (TYRP1) and Dopachrome Tautomerase (DCT). It was confirmed that a reduction effect was exhibited.

Formulation Example 1: Preparation of emulsion base containing bitter grass, western red pepper and Haneultari mixed solvent and subcritical extract

An emulsion base containing bitter grass, a mixed co-solvent of western red pepper and Haneultari and a subcritical extract (Example 3) was prepared according to the composition of Table 14 below by a conventional method.

Raw material content (wt%) Formulation Example 1 Example 3 0.2 beeswax 1.0 Polysorbate 60 1.5 Sorbitan Sesquiolate 0.5 mineral oil 8.0 Sorbitan sesquioleate 0.4 lipophilic monostearate glycerin 0.5 stearic acid 1.0 Glyceryl Stearate/PEG-400 Stearate 1.0 propylene glycol 5.0 carboxy polymer 0.1 triethanolamine 0.16 1,2-Hexanediol a very small amount Spices a very small amount Purified water remaining amount

Formulation Example 2: Serum containing bitter grass, western red pepper and Haneultari mixed solvent and subcritical extract

A serum containing a mixed co-solvent of bitter grass, western red pepper sprouts and Haneultari and a subcritical extract (Example 3) was prepared in a conventional manner according to the composition of Table 15 below.

Raw material content (wt%) Formulation Example 2 Example 3 0.2 xanthan gum 1.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Caprylic/Capric Triglycerides 8.0 Sorbitan Stearate 0.4 lipophilic monostearate glycerin 0.5 stearic acid 1.0 Glyceryl Stearate/PEG-400 Stearate 1.0 butalene glycol 5.0 carboxy polymer 0.1 triethanolamine 0.16 Phenoxyethanol a very small amount Spices a very small amount Purified water remaining amount

Test Example 7: Formulation stability confirmation

For the formulations of Formulation Example 1 and Formulation Example 2, stored in an opaque glass container in an opaque glass container in a room (25°C), refrigeration (4°C) and constant temperature (50°C) constantly maintained at a constant temperature (50°C) and stored for 12 weeks Observation (discoloration, discoloration, and separation) was performed, and stability was confirmed. The results are shown in Table 16.

temperature condition Stability check (discoloration, odor and separation) Formulation Example 1 Formulation Example 2 Indoor (25℃) 0 0 Refrigeration (4℃) 0 0 constant temperature (50℃) 0 0

<Formulation stability grade>

0: no change, 1: small change, 2: change, 3: extreme change

As shown in Table 16, Formulation Example 1 and Formulation Example 2 were confirmed to be stable without discoloration, discoloration and separation under 25°C, 4°C and 50°C temperature conditions.

Claims (6)

A mixture of bitter grass, red pepper greens and Hanultari mixed in a weight ratio of 1:3:2, respectively, is used as a natural eutectic solvent consisting of betaine and saccharose and water as a solvent. Bitter grass, red pepper greens and Hanultari mixed extract prepared by subcritical extraction under subcritical conditions of 120~200℃ and pressure of 0.1~15MPa for 10~30 minutes as active ingredients by adding an extraction solvent to the total weight of the composition A cosmetic composition for skin whitening containing 0.1 to 10% by weight with respect to. delete The cosmetic composition for skin whitening according to claim 1, wherein the natural eutectic solvent is a mixture of Betaine and Saccharose in a weight ratio of 1:1. delete delete delete