KR20140003198A - Composition of skin external application containing compound k - Google Patents

Abstract

The present invention relates to a composition of an skin external application containing compound K, more specifically a composition containing compound K for providing excellent antioxidative activity, a skin moisturizing improvement effect, an anti-inflammatory effect, a skin trouble treatment effect for acne and atopy, a skin whitening effect, a sebum controlling effect, a pore contraction effect, a complexion improvement effect by improving blood circulation, an overall skin condition improvement effect, and hair condition improvement effects including a dandruff prevention effect, a hair growing effect, and a white hair growing preventing effect. [Reference numerals] (AA) Untreated group; (BB) Comparison formulation 6; (CC) Formulation 5

Description

Composition of skin external application containing compound K}

The present invention relates to an external composition for skin containing compound K, and more particularly, by containing compound K, having excellent antioxidant power, improving skin moisturizing effect, anti-inflammatory effect, skin trouble improvement effect such as acne and atopy, and whitening. In the composition that can provide the effects of improving the overall skin condition such as the effects, sebum control effect, pore contraction effect, improving the complexion by improving blood circulation, as well as improving the scalp and hair condition, such as the anti-dandruff effect, the hair growth effect, and the white hair prevention effect. It is about.

Human skin is the primary protective membrane of the human body. It functions to protect the internal organs from external environmental stimuli such as changes in temperature and humidity, ultraviolet rays, and pollutants. Depending on various internal and external factors, It undergoes change. In other words, internally, secretion of various hormones that regulate metabolism is reduced, the function of immune cells and the activity of cells are lowered, and the biosynthesis of immune proteins and biocompatible proteins necessary for the living body is reduced, and ozone layer destruction As the content of ultraviolet rays reaching the surface of the sunlight increases and environmental pollution is further intensified, free radicals and active noxious oxygen are increased, so that the thickness of the skin decreases, the wrinkles increase, the elasticity decreases Skin color is grayish, skin troubles are frequent, spots, freckles and black spots are increased, bleeding is poor, skin tone is getting dark, and so on.

In order to prevent the change of the skin condition due to the intrinsic and extrinsic factors, and to maintain a healthy skin condition, physiologically active substances obtained from various known animals, plants, microorganisms and the like are added to cosmetics to improve the skin condition There has been effort for.

Accordingly, the present inventors can provide a compound K to improve skin condition such as skin whitening, sebum control, pore contraction, as well as whitening and moisturizing, as well as improving acne and skin troubles and atopic symptoms. It has been found that the present invention can also provide effects of improving scalp and hair condition such as anti-dandruff, hair growth and white hair prevention, and have completed the present invention.

Accordingly, it is an object of the present invention to provide a composition for external application of the skin which contains Compound K and which can improve the overall condition of the skin.

In order to achieve the above object, in order to achieve the above object, the present invention provides an external composition for skin containing Compound K as an active ingredient.

The present invention also provides a whitening skin external composition comprising compound K as an active ingredient.

The present invention also provides a moisturizing skin external composition comprising compound K as an active ingredient.

In addition, the present invention provides an external composition for improving acne containing the compound K as an active ingredient.

The present invention also provides a topical skin composition for improving atopy containing compound K as an active ingredient.

The present invention also provides a composition for external application for complexion and skin tone, which contains Compound K as an active ingredient.

The present invention also provides a skin external preparation composition for reducing pores, containing compound K as an active ingredient.

The present invention also provides a skin external composition for controlling sebum containing compound K as an active ingredient.

The present invention also provides a topical anti-dandruff composition containing Compound K as an active ingredient.

The present invention also provides a composition for external application for hair growth comprising a compound K as an active ingredient.

The present invention also provides an external composition for preventing white hair containing Compound K as an active ingredient.

The present invention also provides a topical skin composition using compound K as a natural preservative.

The composition of the present invention has an excellent antioxidant power by containing the compound K, while improving skin moisturizing effect, anti-inflammatory effect, acne and atopy skin trouble improvement effect, whitening effect, sebum control effect, pore contraction effect, blood circulation improvement In addition to improving the overall skin condition, such as improving the complexion, it is possible to provide the effects of improving the scalp and hair condition, such as the anti-dandruff effect, the hair growth effect, and the white hair prevention effect.

Figure 1 shows the hair growth effect after the application of Formulation Example 5 and Comparative Formulation Example 6.

The external preparation composition for skin according to the present invention contains compound K as an active ingredient.

Compound K used in the present invention has a structure of formula (1).

Compound K of the present invention may be extracted from plants, synthesized according to methods known in the art, and may be commercially available. Compound K may also be obtained from ginseng extracts. The type of ginseng to be used at this time is not particularly limited, and ginseng, red ginseng, white ginseng, taegeuk ginseng and ginseng can be used. In addition, the ginseng extract is not only contained in the leached liquid obtained by leaching and transferring from ginseng but also contained in the concentrate obtained by partially or completely concentrating the leach solution or in the slump, premix, regular season, liquid extract and ginseng prepared by drying the concentrate again It contains all of the plant itself as well as chemicals that exert the main effect. Extracts from all parts of ginseng such as stem, root, leaf, flower, and fruit are available and are not limited to any particular part of the extract. In addition, a method of extracting compound K from ginseng extract can use a known method.

Specifically, the compound K may be separated from the ginseng extract after preparing the ginseng extract with water or an organic solvent in a method well known in the art. The organic solvent used in the present invention may be selected from the group consisting of ethanol, methanol, butanol, ether, ethyl acetate, chloroform and a mixed solvent of these organic solvents and water, and preferably 80% ethanol is used. At this time, the extraction temperature is preferably 10 ~ 80 ℃, it can be extracted for 3 to 24 hours. If the extraction temperature and extraction time is out of the extraction efficiency may decrease or change of components may occur.

The composition according to the present invention may contain ginsenoside K in an amount of 0.001 to 50% by weight, preferably 0.01 to 30% by weight, more preferably 0.1 to 10% by weight, based on the total weight of the composition. If the content of the compound K is less than 0.001% by weight, the efficacy and effect by the above components are insignificant, and when the content of the compound K exceeds 50% by weight, there is a problem of skin safety or formulation.

Since compound K is a component having excellent antioxidant power, the composition of the present invention containing compound K provides excellent antioxidant power to the skin.

The composition of the present invention can be used as a whitening composition, which can provide an excellent whitening effect by inhibiting tyrosinase activity and inhibiting the production of melanin.

The composition of the present invention can be used as a composition for external application for moisturizing skin, which can enhance the skin barrier function and induce differentiation of dermal keratinocytes. Therefore, it can be effectively used as a composition for external application for skin, which prevents or improves skin dryness, atopic dermatitis, contact dermatitis, psoriasis or the like caused by incompleteness of epidermal differentiation.

The composition of the present invention can be used as a composition for external application for skin for improving acne, and has excellent antimicrobial effect, especially antimicrobial effect against acne causing bacteria, and also provides anti-inflammatory effect.

The composition of the present invention can be used as a composition for external application of skin for improving color and skin tone. It expands capillary blood vessels when applied to skin and promotes blood circulation, thereby smoothly supplying nutrients to the skin and suppressing skin aging, The effect is excellent.

The composition of the present invention can be used as a composition for external application for skin for reducing pores, controlling sebum, and improving skin troubles. It reduces sebum secreted by the skin when applied to the skin, reduces active oxygen and promotes collagen synthesis, , And suppression of skin troubles due to decreased expression of inflammatory factors. In addition, excellent antioxidant power can prevent the generation of skin irritation.

The composition of the present invention can be used as a composition for external application for skin prevention of dandruff. It can purify the scalp by effectively discharging toxins accumulated in hair and scalp, inhibit proliferation and growth of dandruff to prevent scalp inflammation reaction, It has excellent antioxidant effect which inhibits the production and action of oxygen, so it can calm and strengthen the scalp and provide the effect of strengthening the original defense force.

The composition of the present invention can be used as a composition for external application for hair growth, which promotes the transition of the resting hair cycle to the growing hair cycle, thereby promoting hair growth and preventing hair loss.

The composition of the present invention can be used as a topical anti-skin composition for white hair, which can significantly increase the expression of MITF in melanocytes, inhibit white hair and promote the induction of black hair.

In addition, the compound K used in the external preparation composition for skin of the present invention can provide an effect as a natural preservative.

The above composition for external application for skin of the present invention can be formulated as a cosmetic composition, and can be formulated containing a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) and non- It may be provided in the form of an ionic vesicle dispersant or in the form of a cream, skin, lotion, powder, ointment, spray or cone stick. It can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. These compositions may be prepared according to conventional methods in the art.

In particular, when the composition for external application for skin of the present invention is used for prevention of dandruff, hair growth or hair loss prevention, it may be formulated as a composition for scalp and hair, and the formulation is not particularly limited, but, for example, hair tonic, A hair treatment, a hair treatment, a hair shampoo, a hair rinse, a hair lotion, or a scalp hair combined treatment.

In addition, the composition according to the present invention may further comprise at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening agents, gelling agents, softening agents, antioxidants, suspending agents, stabilizing agents, Such as fillers, emulsifiers, emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics May contain adjuvants commonly used in the field of cosmetics or dermatology. Such adjuvants are introduced in amounts commonly used in the cosmetics or dermatological fields.

In addition, the composition of the present invention may contain a skin absorption promoting substance to increase the skin improving effect.

Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to test examples and formulation examples. However, these test examples and formulation examples are provided for illustrative purposes only for the sake of understanding of the present invention, and the scope and scope of the present invention are not limited by the following examples.

[Referential Example 1]

Compound K for testing the efficacy of the composition of the present invention was purchased from Ambo laboratory.

Test Example 1 Inhibitory Effect of Reactive Oxygen Species Production

Keratinocytes isolated from human epidermal tissue were added to each well of a 24-well plate at 5 × 10 ⁴ for 24 hours. After 16 hours, compound K was treated with 1%. At this time, the control group did not process Compound K for comparison. After 2 hours, the culture solution was removed, and 100 μl of phosphate buffered saline (PBS) was added to each well. This keratinocyte was irradiated with ultraviolet rays of 30 mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: K5T8, UVB 15 W, Sankyo Dennki, Japan), and then PBS was removed. The keratinocyte culture medium 200 ≪ / RTI > Compound K was again treated and the amount of reactive oxygen species increased by UV stimulation at certain time points was quantified. The amount of ROS was quantified by referring to Tan's method for measuring the fluorescence of DCF-DA (dichlorofluorescin diacetate) oxidized by ROS (Tan et al., 1998, J. Cell Biol. Vol. 141, pp1423-1432). Table 1 shows the results of calculating the ratio of the vehicle-treated control group ROS.

Test substance UVB 30 mJ / cm ² Time elapsed after investigation 0hr 2 hr 3hr Vehicle 100 244 287 UVB + vehicle 100 325 381 UVB + Compound K 100 245 289

In the results of Table 1, the compound K according to the present invention effectively inhibits the generation of ROS known to cause skin cell damage by ultraviolet rays, and the amount of ROS after ultraviolet stimulation is almost the same as that of the case where no ultraviolet rays are irradiated. It can be seen that the antioxidant effect is very good to inhibit the production of. Therefore, it was confirmed that compound K according to the present invention can prevent pores from widening by inhibiting oxidation and preventing aging, and can improve skin trouble by defending the generation of skin irritation.

Test Example 2: Effect of tyrosinase inhibition

Tyrosinase enzyme was extracted from the mushroom (Mushroom) was used as the Sigma (SIGMA). First, the substrate tyrosine was dissolved in distilled water to make a solution of 0.3 mg / ml, and the solution was added to the test tube by 1.0 ml. Then, 1.0 ml of potassium-phosphate buffer solution (0.1 mol concentration, pH 6.8) and 0.7 ml of distilled water were added thereto. Added.

Compound K of the present invention was mixed with an appropriate concentration of ethanol solution 0.2ml of the sample solution prepared in the reaction solution and then reacted for 10 minutes in a 37 ℃ thermostat. At this time, the control group was prepared by adding 0.2 ml of a solvent instead of each sample solution, and ascorbic acid was used as a positive control group. 0.1 ml of a tyrosinase solution of 2500 units / ml was added to the reaction solution and reacted for 10 minutes in a 37 ° C thermostat. The reaction tube containing the reaction solution was placed in iced water, quenched to stop the reaction, and the absorbance at 475 nm was measured with a photospectrometer. The results are shown in Table 2 below. Each tyrosinase inhibitory effect was calculated by the following equation.

Test substance Tyrosinase inhibition rate (%) Control group (no addition) 0 Ascorbic acid 52 Compound K 61

From the results of Table 2, it can be seen that the tyrosinase inhibition rate of compound K according to the present invention is much higher than the known tyrosinase inhibitor ascorbic acid, which is very excellent in the whitening effect.

[Test Example 3] Inhibitory effect on melanin formation by B16 / F10 melanoma cells

A sample containing 0.1 wt% of compound K and kojic acid, respectively, was added as a test substance to a culture solution of B16 / F10 melanoma cells (Korea Cell Line Bank) at a constant concentration for 3 days, followed by removing the culture solution and washing with PBS. The cells were dissolved with 1N NaOH and absorbance was measured at 405 nm. The cells without test substance were used as a control group, and the degree of inhibition of melanin production of each test substance was measured by comparing with the melanin content of the control group. The inhibition rate of melanin production was calculated according to Equation (2), and the results are shown in Table 3.

Test substance Melanogenesis inhibition rate (%) Control group (no addition) 0 Kojic acid 53 Compound K 67

From the results of Table 3, it can be seen that the rate of inhibition of melanin production of Compound K according to the present invention is much higher than that of kojic acid, a known melanin production inhibitor.

[Formulation Example 1 and Comparative Formulation Example 1]

Nutritive creams were prepared according to the composition of Table 4 below in a conventional manner (unit: wt%).

Compounding ingredient Formulation Example 1 Comparative Formulation Example 1 Purified water To 100 To 100 Compound K 0.1 - Vegetable hydrogenated oil 1.50 1.50 Stearic acid 0.60 0.60 Glycerol stearate 1.00 1.00 Stearyl alcohol 2.00 2.00 Polyglyceryl-10 Pentastearate & Behenyl Alcohol & Sodium Stearoyl Lactylate 1.00 1.00 Arakidil Behenyl Alcohol & Arakidil Glucoside 1.00 1.00 Cetylaryl Alcohol & Cetearyl Glucoside 2.00 2.00 PEG-100 Stearate & Glycerolate & Propylene Glycol 1.50 1.50 Caprylic / Carric Triglycerides 11.00 11.00 Cyclomedicone 6.00 6.00 Preservative, incense Suitable amount Suitable amount Triethanolamine 0.1 0.1

[Test Example 4] Measurement of skin moisturizing effect

In order to measure the effect of compound K on increasing skin moisturizing power, Formulation Example 1 and Comparative Formulation Example 1 were used and evaluated as follows.

20 men and women in the 40s and 50s classified as dry skin were divided into two groups of 10 persons for each of Formulation Example 1 and Comparative Formulation Example 1 to apply the nutritional cream twice daily for 4 weeks to the face. (24 ° C, relative humidity 40%) after 1 week, 2 weeks, and 4 weeks after application, and after 2 weeks (total 6 weeks) (Corneometer CM825, C + K Electronic Co., Germany). The results are shown in Table 5 below. The results in Table 5 are based on the skin moisture meter measured just before the start of the test, and the increase in the measured value after a certain period of treatment is expressed as a percentage.

Test group Moisture growth rate (%) 1 week Two weeks 4 weeks 6 weeks Formulation Example 1 31 33 36 33 Comparative Formulation Example 1 30 32 32 15

In the results of Table 5, when the Comparative Formulation Example 1 was applied, it showed a water increase rate of about 30% up to 4 weeks after the application was made, but after stopping the application, the moisture content of the skin decreased, but it contained the compound K. In the case of application of Formulation Example 1, it can be confirmed that even after the application was stopped, the skin moisture increase rate was more than 30%. Through this, the composition of the present invention containing the compound K can be seen that the skin moisturizing effect and excellent.

[Test Example 5] Measurement of promoting effect of keratinocyte differentiation

In order to examine the differentiation promoting effect of keratinocytes of Compound K, the amount of Cornified Envelop (CE) generated during the differentiation of keratinocytes was measured using absorbance.

First, the keratinocytes of the primary cultured humans isolated from the epidermis of the newborn are put in a culture flask and attached to the bottom. After the compound K is treated at 5 ppm in the culture medium, the cells grow about 70 to 80% of the floor area. Incubated for 5 days. At this time, low calcium (0.03 mM) and high calcium (1.2 mM) treatment groups were negative control and positive control, respectively. Then, the cultured cells were harvested and washed with PBS (phosphate buffered saline). Then, 10 mM Tris-HCl buffer (Tris-HCl, pH 7.0) containing 2% SDS (sodium dodecyl sulfate) and 20 mM DTT (Dithiothreitol) pH 7.4) was added, sonication, boiling, and centrifugation. The precipitate was suspended again in 1 ml of PBS and the absorbance at 340 nm was measured. Separately, a part of the solution after the sonication was taken to measure the protein content and used as a standard in evaluating the degree of cell differentiation. The results are shown in Table 6 below.

Test substance The ability to differentiate in keratinocytes (%) Low calcium (0.03 mM) solution
(Negative control) 100 High calcium (1.2 mM) solution
(Positive control group) 210 Compound K. 295

As shown in Table 6, when compound K was treated, it was confirmed that the effect of promoting the differentiation of keratinocytes was excellent.

[Test Example 6] Measurement of recovery effect on skin barrier function

In order to measure the effect of compound K on the recovery of damaged skin barrier function due to skin damage, the following experiment was performed. Ten adult male and female adult rats were treated with a tape stripping method to damage the skin barrier, and the two groups of Formulation Example 2 and Comparative Formulation Example 2, which were prepared in the composition shown in Table 7 below, were applied for 7 days The recovery rate of TWEL was measured and compared with Vapometer (Delfin, Finland) once a day. Here, Comparative Formulation Example 2 is a vehicle as a negative control. The experimental results are shown in Table 8 below. The results in Table 8 were compared before treatment before barrier damage and after treatment before barrier damage on the basis of 100%.

Compounding ingredient Formulation Example 2 Comparative Formulation Example 2 Purified water 69 70 Propylene glycol 30 30 Compound K One -

Test group TWEL Change (%) Before processing 1 day 2 days 3 days 4 days 5 days 6 days Formulation Example 2 100 126.8 128.5 122.8 116.8 111.2 108.5 Comparative Formulation Example 2 100 121.4 112.7 98.3 70.5 62.3 43.5

As can be seen in Table 8, when the Comparative Formulation Example 2 containing no compound K was treated, the amount of transdermal water loss gradually increased over time, whereas Formulation Example 2 containing Compound K was treated. In this case, the loss of transdermal moisture returns to normal and the barrier damage is recovered.

[Test Example 7]

In order to evaluate the skin blood circulation promoting effect of the cosmetic composition according to the present invention, the degree of blood circulation in the skin was measured using LDPI (Laser Doppler Perfusion Imager). LDPI is a widely used and widely used device for measuring blood circulation in skin. It is a highly sensitive device that can measure not only blood velocity and quantity in the capillaries of the skin, but also flow in the small artery and the parenchyma.

The face was soaked in a constant temperature and humidity chamber, and then adapted for 30 minutes. Initial values were measured using LDPI. First, the initial blood flow in the lower part of the forehead of 30 women with cold hands and feet was measured by LDPI. Table 9 below shows the result of comparing the blood flow measured with the initial measurement value (skin blood flow change) measured after using Formulation Example 1 and Comparative Formulation Example 1 for one week to the subject.

LDPI results before and after using cosmetics - skin blood flow Test substance Percent change of skin blood flow after 1 week use (%) Formulation Example 1 12 Comparative Formulation Example 1 5

In the results of Table 9, the cosmetic composition according to the present invention significantly increased the skin blood flow than Comparative Formulation Example 1 containing no compound K, it was confirmed that the blood color is improved through the blood circulation. This ultimately suggests that the cosmetic composition containing the compound K according to the present invention can contribute effectively to the skin's nutrient delivery, inhibit skin aging and delay.

[Test 8] Effect of improving skin tone

In order to examine the skin tone improvement effect of Formulation Example 1 and Comparative Formulation Example 1, 30 subjects were used (one evening / one day application for a total of one week) and then facial stage DM-3 (Moritex, Japan) To evaluate the degree of skin tone improvement. The improvement rate of the skin tone was determined by the brightness and the color change value of the skin and the brightness and the color change value of the skin. The results are shown in Table 10 below. The greater the brightness and color change, the better the skin tone.

Test substance Skin tone improvement rate (%) Brightness (mean ± standard deviation) Color (mean ± standard deviation) Formulation Example 1 15 ± 3.24 12 ± 2.34 Comparative Formulation Example 1 5 ± 2.34 5 ± 2.05

In the results of Table 10, Comparative Formulation Example 1, which does not contain Compound K according to the present invention, did not show significant skin tone improvement effect, whereas Formulation Example 1, which contained Compound K as an active ingredient, had more skin tones after use than before use. It was confirmed that the improvement.

[Test Example 9] Pore reduction effect

1. Collagen biosynthesis promotes pore reduction effect

Compound K according to the present invention was measured by comparing collagen biosynthesis promoting effect with TGF-β. First, fibroblasts were seeded at 10 ⁵ per well in 24 wells and cultured until they grew to about 90%. After incubation with serum-free DMEM medium for 24 hours, the compound K and TGF-β of the present invention dissolved in serum-free medium were treated with 10ng / ml, respectively, and CO ₂ And cultured in an incubator for 24 hours. The supernatant was removed and the procolagen (I) ELISA kit (procollagen type (I)) was used to determine whether procollagen was increased or decreased. The results are shown in the following Table 11, and the combined performance of the collagen was set to 100 in the non-treatment group.

Test substance Collagen Total Performance (%) Untreated group 100 TGF-beta 183.5 Compound K 196.2

In the results of Table 11, the compound K according to the present invention was confirmed to exhibit a higher level of collagen synthesis than the positive control group TGF-β. Therefore, it was confirmed that the compound K according to the present invention can reduce the enlarged pores by increasing the amount of collagen production around the pores.

2. Pore reduction effect

In order to examine the pore reduction effect of Formulation Example 1 and Comparative Formulation Example 1, the following evaluation was made. Twenty male and female subjects with large pore sizes were selected and divided into two groups of 10 persons. The nutritional creams of Formulation Example 1 and Comparative Formulation Example 1 were applied to the faces for each group for 4 weeks each day. The evaluation of the effectiveness of the pore reduction was made by a visual evaluation of experts by taking pictures before and after the experiment. The results are shown in Table 12 below (rating rating: 0-not scaled down at all; 5-scaled down).

Test substance Rating Rating Formulation Example 1 4 Comparative Formulation Example 1 0

From the results of Table 12, Comparative Formulation Example 1 does not have a pore reduction effect, but in the case of Formulation Example 1 shows a pore reduction effect that can be visually confirmed, Compound K according to the present invention has an effect of reducing the size of the pores It was found to be excellent.

Test Example 10 Effect of suppressing sebum secretion

1. Suppression of skin hypersecretion by 5α-reductase inhibition

In order to confirm 5α-reductase inhibitory effect, the ratio of [ ¹⁴ C] testosterone to [ ¹⁴ C] dihydrotestosterone (DHT) in HEK293-5αR2 cells was measured. HEK293 cells were transfected with p3 x FLAG-CMV-5αR2, and cultured in 2.5 × 10 ⁵ cells / well in a 24-well plate (Park et al., 2003, JDS Vol. 31, pp. 191-98). The next day, the medium was changed to a new medium supplemented with an enzyme substrate and an inhibitor. 0.05 [mu] Ci [ ¹⁴ C] testosterone (Amersham Pharmacia biotech, UK) was used as the substrate of the medium.

To confirm the degree of inhibition of 5α-reductase activity, compound K was added and incubated for 2 hours at 37 ° C. in a 5% CO ₂ incubator. In this case, the compound K was not added as a negative control group, and the finasteride was used as a positive control group. Thereafter, the culture medium was collected, the steroid was extracted with 800 μl ethyl acetate, the organic solvent layer was separated, dried, and the remaining residue was dissolved again with 50 μl ethyl acetate to make silica plastic sheet kieselgel 60 K54. ) Was developed using ethyl acetate-hexane (1: 1) as a solvent.

After drying the plastic sample in air, the bath system was used to measure the amount of isotope. The dried plastic sheet and x-ray film were put together in a bath cassette, and after 1 week, the testosterone and dihydrootestosterone isotope The conversion and inhibition rates were calculated according to the following equations (3) and (4), respectively, and the results are shown in Table 13 below.

Test substance Conversion Rate (%) Inhibition rate (%) Negative control group 48.0 - Positive control group 27.6 42.5 Compound K 15.9 60.8

In the results of Table 13, 5α-reductase, which converts testosterone to dihydrotestosterone, binds to a receptor protein in the cytoplasm, enters the nucleus, activates sebaceous gland cells, promotes differentiation, and hypersecretes sebum in sebaceous glands. Compound K effectively inhibited the activity of and inhibited the conversion of testosterone to dihydrotestosterone and was found to have a superior inhibitory effect than finasteride, which is known to inhibit the activity of 5α-reductase. Therefore, it was confirmed that compound K can suppress sebum hypersecretion by effectively inhibiting the activity of 5α-reductase.

2. Inhibitory effect of sebum secretion

To evaluate the inhibitory effect of sebum secretion of Formulation Example 1 and Comparative Formulation Example 1, the following evaluation was made. 30 men and women who felt that sebum secretion was high were selected and nutritional creams of Formulation Example 1 and Comparative Formulation Example 1 were made daily for four weeks in designated areas. The results of the evaluation of the effect of reducing sebum were measured by using a sebum level meter (Sebumeter SM810, C + K Electronic Co., Germany) after 2 weeks and 4 weeks, Respectively.

Test substance Sebum reduction rate (%) After 2 weeks After 4 weeks Formulation Example 1 33 42 Comparative Formulation Example 1 5 5

From the results in Table 14, it was found that Formulation Example 1 containing Compound K according to the present invention as an active ingredient can effectively inhibit sebum secreted in excess than Comparative Formulation Example 1 without containing it.

[Formulation Example 3 and Comparative Formulation Examples 3 to 4]

Formulation Example 3 and Comparative Formulation Examples 3 to 4 were prepared according to the ingredients and the contents (% by weight) shown in Table 15 below. Specifically, Formulation Example 3 is compounded with Compound K, Comparative Formulation Example 3 does not contain any active ingredients for improving acne skin, Comparative Formulation Example 4 is a standard to be used as a reference for antimicrobial activity, It contains erythromycin, a popular acne treatment.

The manufacturing method of Formulation Example 3 and Comparative Formulation Examples 3 to 4 is as follows. The components of phase A in Table 15 were completely dissolved, and the components of phase B were completely dissolved in a separate dissolution tank, and then mixed and solubilized by adding phase B to phase A. The ingredients of the C phase were added thereto in accordance with the mixing ratios shown in Table 15, mixed and homogenized and then filtered to prepare the compositions.

division Formulation Example 3 Comparative Formulation Example 3 Comparative Formulation Example 4 A Deionized Water To 100 To 100 To 100 EDTA-2Na 0.02 0.02 0.02 glycerin 5.0 5.0 5.0 B ethanol 2.0 2.0 2.0 PEG-60 hardened castor oil
(hydrogenated castor oil) 0.4 0.4 0.4 Perfume 0.04 0.04 0.04 C Ginsenoside K 5.0 - - Erythromycin - - 5.0

[Test Example 11] Test for antibacterial activity against acne bacteria

Each of the cosmetic compositions prepared in the formulations of Formulation Example 3 and Comparative Formulations 3 to 4 was tested for the antibacterial activity against Propionibacterium acnes (ATCC 6919: medium-BHI broth), a causative strain of acne .

The antibacterial test method for acne bacteria was as follows.

(1) Preparation of test strain

Propionibacterium acnes was used as a culture broth inoculated in BHI broth and anaerobic culture.

(2) Dilution solution preparation

0.15 ml of the test bacteria was added to 15 ml of BHI broth (pH 6.8) or LB broth (pH 4.5) and mixed well as a dilution solution.

(3) Preparation of sample

The undiluted solution of the cosmetic composition prepared from Formulation Example 3 and Comparative Formulation Examples 3 to 4 was used as a sample.

(4) Antimicrobial activity test

1) In a 96-well 96-well plate, place the sample in line 1 and add 200 μl of diluted solution.

2) Thoroughly mix the mixture of line 1, and then take 100 μl of the mixture, place it in the second row, mix well, and then repeat the double dilution by adding 100 μl to the third row.

3) After culturing the cells at 32 ° C for 24 hours and 48 hours, determine whether the bacteria are proliferating to the extent of suspension, and determine the minimum concentration without bacterial growth as the MIC (Minimum Inhibitory Concentration) value. If the mixed solution is opaque and it is difficult to determine the growth of bacteria, check through a microscope.

The results of the antimicrobial activity test against acne bacteria are shown in Table 16 below. The MIC was expressed in terms of the concentration of the active ingredient contained in the formulation.

Item pH Propionibacterium Acnes Formulation Example 3 5.7 > 40 ppm Comparative Formulation Example 3 5.7 Maximum concentration (no antimicrobial activity) Comparative Formulation Example 4 5.7 > 100 ppm

The smaller the ppm concentration in MIC, the more effective the antimicrobial activity against acne bacteria. In the case of Formulation Example 3, the concentration of ppm was significantly lower than that of Comparative Formulation Example 4 using erythromycin, which is a known acne treatment. It can be confirmed that the composition containing has much superior antibacterial activity against test bacteria.

[Test Example 12] Lipogenesis inhibition test

3T3-L1 cells, a fibroblast cell line of mice, were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Dulbecco's modified eagles medium, GIBCO BRL, Life Technologies) Lt; ⁵ > cells / well in a well culture plate. After 2 days, the cells were replaced with fresh DMEM (containing 10% FBS) medium and cultured for 2 days. The cultured cells were then induced to differentiate into DMEM (containing 10% FBS) containing 1 μg / ml insulin, 0.5 mM IBMX and 0.25 μM dexamethasone, and 50 μM of compound K and caffeine. After 2 days of treatment, the cells were replaced with DMEM containing insulin and cultured for 5 days. After 5 days, the cells were replaced with normal medium (DMEM, containing 10% FBS) and cultured while observing until the cells morphologically changed into adipocytes.

Sudan III staining (S4136, sigma-aldrich) was performed using the differentiated 3T3-L1 adipocytes to evaluate the effect of compound K on adipocyte inhibition of adipocyte accumulation. The adipocytes were fixed in 4% paraformaldehyde (pH 7.2) in phosphate buffer at room temperature, washed with PBS (phosphate buffered saline), stained with means III and then photographed and compared visually. The control group was fed with the test substance or the test substance without the reference substance, and 50 μM of caffeine was treated with another comparative group. The level of inhibition of fat accumulation was graded by dividing the degree of staining by +++, ++, +, -. At this time, it means that the degree of dyeing is larger toward +++. The results are shown in Table 17 below.

sample % Inhibition Control group +++ Comparative group + Compound K -

As shown in Table 17, the compound K used in the present invention is not only a small amount of fat accumulated in the adipocytes, it can be seen that there is an excellent lipid synthesis inhibitory effect than caffeine, a known lipid synthesis inhibitor. Therefore, lipid synthesis is inhibited, and sebum is reduced, so that occurrence of acne can be suppressed.

[Test Example 13] Test for improvement of acne and reduction of sebum secretion and stimulation

30 persons who had acne were divided into 3 groups of 10 persons, and subjects corresponding to each group were allowed to use the cosmetic composition prepared in Formulation Example 3 and Comparative Formulation Examples 3 to 4 for one month. Acne improvement scale is from 1 point to 5 points, 1 point is 'No', 3 points are 'Normal' and 5 points are 'Very agree'. The experimental results are shown in Table 18 below with an average score of 10 persons.

Acne disappearance was based on the number of days the extinction was read, acne recurrence was based on the results after one month. The reduction of sebaceous secretion was from 1 point to 5 points, 1 point was 'No', 3 points were 'Normal' and 5 points were 'Very agree'. Experimental results are shown in the average score of 10 people in Table 18 below. The presence or absence of skin irritation was determined as (number of irritants) / (total number of test subjects).

Inflammatory Acne Improvement Acne-bloating period Acne recurrence Decrease sebaceous glands Irritation Formulation Example 3 4.5 3 days radish 4.6 0/10 Comparative Formulation Example 3 2.1 13th U 2.0 0/10 Comparative Formulation Example 4 4.2 2 days radish 4.1 9/10

As shown in Table 18, it can be seen that Formulation Example 3 has no recurrence of acne compared to Comparative Formulation Example 3, and has excellent effects on improvement of acne as a whole. On the other hand, Comparative Formulation Example 4 containing the antibacterial activity reference material shows the effect of improving acne but it is not suitable for long-term use due to strong skin irritation in use. However, since the composition according to the present invention has no irritation, .

Test Example 14 Inflammation Improvement Effect

1. Inhibitory effect of prostaglandins

The anti - inflammatory effect was evaluated by the inhibitory effect of prostaglandin production. Compound K was used to measure the effect on macrophages. First, aspirin was added to macrophages taken from the abdominal cavity of mice to a final concentration of 500 M, thereby irreversibly inhibiting cyclooxygenase (COX) activity remaining in the cells. Then, 100 μl of the suspension was added to each well of the 96 well cell culture tube and incubated for 2 hours in an incubator at 37 ° C. with 5% CO ₂ to attach macrophages to the container surface. Then, the adhered macrophages were washed three times with PBS and used for the inflammation improving effect test. After incubating for 12 hours by adding RPMI medium containing 1% (w / v) of LPS to the cultured macrophages 5x10 ⁴ cells / ml, prostaglandin production was induced and compound K was treated with 100 μl to release free prostaglandins. Quantification was performed using enzyme immunoassay (ELISA).

In this case, the production inhibitory activity of the compound K prostaglandin was set to 100% of the difference between the prostaglandins produced in the LPS-treated and untreated groups, and the LPS and the sample were treated together to reduce the percentage of prostaglandins. It was compared with, and determined, and the results (the effect of inhibiting the production of prostaglandins) are shown in Table 19 below.

Blank 100% Control group (Aspirin treated group) 25.0% Compound K 20.8%

As shown in Table 19, it can be seen that even when compound K is treated like the control group treated with aspirin, the effect of inhibiting the production of prostaglandins is very high.

It can be seen that Compound K of the present invention can provide an excellent inflammation improving effect.

In addition, it can be seen that Compound K prevents and improves skin trouble by inhibiting expression of prostaglandin, a skin inflammatory factor.

2. IL-8 production inhibitory effect

The day before the experiment, normal human skin keratinocyte (NHEK, available from Lonza) was dispensed into a 96 well plate at 5 × 10 ⁴ cells / well and incubated at 37 ° C. in a 5% CO ₂ incubator And cultured for 24 hours. Twenty-four hours later, the cells were washed twice with PBS and replaced with serum free keratinocyte basement media (KBM). Compound K was treated in each well by the concentrations of Table 20, followed by reaction for 30 minutes, followed by PGSA (10 µg / ml), PGSA (50 µg / ml), and PGSA (50 µg / ml) + LPS (1 µg / ml). Ml) were treated respectively. Here, peptidoglycan from S. aureus (PGSA ) is a peptidoglycan extracted from Staphylococcus aureus , which is a major constituent of the cell wall of Gram-positive (+) bacteria, and the cell membrane components of bacteria are known to cause inflammation, Especially in staphylococcus, about 90% of atopic patients are reported to cause secondary infection by this bacterium. LPS (lypopolysaccaride) is a major constituent of Gram negative (-) bacterial cell membrane and is known to be the main cause of inflammation.

After incubation for 24 hours at 37 ℃, 5% CO ₂ incubator, the culture medium was taken and subjected to ELISA for Interleukin-8 (IL-8), the results are shown in Table 20 below. The ELISA used the experimental method of BD science.

division IL-8 secretion (pg / ml) Untreated Control 935.12 PGSA (10 [mu] g / ml) 4812.60 PGSA (50 占 퐂 / ml) 5895.08 PGSA (50 占 퐂 / ml) + LPS (1 占 퐂 / ml) 6814.91 Compound K (5 ppm) 1563.25 Compound K (25 ppm) 1103.29 Compound K (50 ppm) 1003.57

In Table 20, Compound K was found to significantly reduce and inhibit the secretion of IL-8 increased by PGSA and LPS. Therefore, it can be seen that the composition for external application for skin of the present invention can provide a superior anti-inflammatory effect by significantly reducing the secretion of IL-8 increased by PGSA and LPS.

[Test Example 15] Evaluation of itching relief

The day before the experiment, keratinocytes (cell line name: HaCaT available from ATCC) were dispensed in a 96 well plate at 4x10 ⁴ cells / well and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours Respectively. After 24 hours, the 96-well plate was washed twice with HBSS (Hanks' Balanced Salt Solution) buffer, and the reaction buffer (2 μM Fluo-4-AM, 20% pluronic acid, 2.5 mM probenecid) gave. After reaction at 37 ° C., 5% CO ₂ incubator for 30 minutes, and at room temperature for 30 minutes, the cells were washed twice with HBSS buffer and treated with compound K at a concentration (%) as shown in Table 25 below.

After reaction for 10 minutes to process the 2U / ml trypsin (Trypsin) or 5μM PAR-2 activation peptide (SLIGKV) and was measured in Ca ^{2 +} concentration in the cell 80 seconds. Measurement of intracellular Ca ²⁺ concentration was performed using FlexStation 3 (Molecular Device, USA). The difference between the minimum and maximum values obtained by measuring flex for 80 seconds after treatment with Compound K and 2U / ml Trypsin or 5μM PAR-2 active peptide (SLIGKV) was determined. The percent inhibition of calcium ions into the cellular influx compared to the difference between the minimum and maximum values when treated with ml trypsin or 5 μM PAR-2 active peptide (SLIGKV) is shown in Table 21 below.

Compound K Concentration % Inhibition of intracellular entry of calcium ions Trypsin (2 U / ml) PAR-2 active peptide (5 [mu] M) 0.05% 26 28 0.1% 31 34 0.5% 40 42 1.0% 46 50

As can be seen in Table 21, the influx of intracellular calcium ions by trypsin or PAR-2 active peptide (SLIGKV) is reduced by the treatment of compound K, the influx of intracellular calcium ions by increasing the concentration of compound K It was confirmed that this is significantly reduced.

Therefore, the external composition for skin containing the compound K of the present invention can provide an excellent antipruritic effect by effectively inhibiting PAR-2 activity causing itching.

[Formulation Example 4 and Comparative Formulation Example 5]

To prepare a shampoo in the composition of Table 22. Specifically, the surfactant and ethylene glycol distearate are added to purified water, heated to 80 ° C., uniformly dissolved, and then slowly cooled to 40 ° C. under stirring, and the active ingredient and preservative according to the present invention are added to the mixture. A viscosity modifier, a fragrance, and a hair conditioner were added and mixed, followed by cooling to room temperature under stirring.

ingredient Formulation Example 4 Comparative Formulation Example 5 Ammonium Lauryl Sulfate 10 10 Polyoxyethylene lauryl ammonium sulfate 5 5 Cocoamidopropylbetaine 2 2 Ethylene Glycol Distearate 1.5 1.5 Cocoyl Monoethanolamide 0.8 0.8 Compound K 5.0 - Polyquaternium-10 0.2 0.2 Blue No. 1 0.0002 0.0002 Yellow No. 4 0.0001 0.0001 Methyl paraben 0.1 0.1 Spices 0.8 0.8 Citric acid 0.1 0.1 Dimethicone 1.0 1.0 water to 100 to 100

Test Example 16 Anti-dandruff force evaluation

The antidandruff force of Formulation Example 4 and Comparative Formulation Example 5 were measured and compared. At this time, the test strain is dandruff Pytirosporum Ovale ( Pityrosporum) Obalae (ATCC 12078)).

Antibacterial activity was measured by the skin disk diffusion method. The skin disc diffusion method (SDDM) is a method that is closest to a consumer's habit of use, and is a method of measuring antimicrobial activity on guinea pig skin similar to human skin.

Specifically, after peeling off the guinea pig skin (70% ethanol treatment) to uniformly dry by using a ethanol, the cut skin disk was used to cut to a certain size. The sterilized skin discs were soaked in dilute shampoo solution for 3 minutes and then washed with running water. Then, the skin disk was placed on a solid medium inoculated with the test bacteria and then cultured. After incubation, the relative antimicrobial activity was measured by measuring the size of the clear zone in which the growth of bacteria was suppressed. The experimental results are shown in Table 23 below as average values of the results of three measurements.

Anti-dandruff division Inhibitor size (mm) Formulation Example 4 Comparative Formulation Example 5 Dandruff inhibitor ring size 3.4 0

Looking at the Table 23, in the case of Comparative Formulation Example 5 containing no compound K, there is no dandruff reduction effect, but in the case of Formulation Example 4 containing compound K effectively reduce dandruff bacteria, it was confirmed that there is an excellent anti-dandruff effect Can be.

Test Example 17 Dandruff Reduction Effect Test

Twenty-four men aged 19 to 35 years with a relatively large number of dandruff were selected and the dandruff reduction rate was measured after using the shampoo of each of Formulation Example 4 and Comparative Example 5 for 2 months in the following manner for 12 months in the following manner .

Before the start of the test, the dandruff was smeared with ordinary shampoo, and the dandruff accumulated for 2 days after shedding was collected. The dandruff weight was sampled once every two days with the shampoo of each of Formulation Example 4 and Comparative Example 5, The weight of accumulated dandruff was compared and evaluated. At this time, the accumulated dandruff was collected directly from the scalp with a vacuum suction device, to obtain a rate of dandruff reduction based on Equation 5 below and the results are shown in Table 24 below.

Formulation Example 4 Comparative Formulation Example 5 Dandruff reduction rate (%) 64.8
56.2
72.6
63.1
46.9
55.9
68.5
72.3
51.0
69.7
66.7
53.2 0.7
-0.5
-1.1
1.5
2.2
1.3
6.3
3.6
3.3
4.6
3.7
4.2 medium 61.7 2.5 SD 8.4 2.2

As can be seen in Table 24 above, in the case of Formulation Example 4 containing Compound K it can be seen that the excellent anti-dandruff effect.

Test Example 18 Scalp Itching Prevention Test

Twenty-four men and women aged 25 to 45 who feel severely itching scalp were divided into two groups of 12 people, each of whom used the shampoos of Formulation Example 4 and Comparative Formulation Example 5 once every three days for two weeks. The prevention effect was evaluated through the following evaluation criteria and the results are shown in Table 25 below.

[Evaluation standard]

Very good-5 points

Excellent-4 points

Normal-3 points

Poor-2 points

Very bad-1 point

division Formulation Example 4 Comparative Formulation Example 5 Itching effect of scalp 4.4 2.3

As can be seen in Table 25, it can be seen that the case of Formulation Example 4 containing Compound K has a better effect in preventing scalp itching.

[Test Example 19] hair follicle dermal papilla cell proliferation effect

The keratin proteins that make up hair are produced in hair root keratinocytes, which are differentiated from dermal papilla cells. In order to evaluate the activity of the dermal papilla cells of the composition, a rat immortalized dermal papilla cell (DP6) cell line was used in the present invention (Wendy Filsell, Journal of Cell Science 107, 1761-1772 (1994)). This dermal papilla cell line was cultured by microdissection from the hair roots of male PVG rat whiskers and cultured with DMEM (Dulbecco's modified Eagle's medium, Gibco BRL, Gaithersburg, MD, USA) was incubated for 24 hours in an incubator maintained at 5% CO ₂ , 37 ℃. DP6 was placed in a 96 well plate and incubated in a 37 ° C. incubator for 24 hours, and then treated with Compound K at concentrations of 5 ppm, 10 ppm and 20 ppm, respectively. After 24 hours of drug treatment, cell proliferation was measured using the WST-1 kit (Roche). The results are shown in Table 26 below.

division Cell proliferation (%) Untreated Control 100 Compound K (5 ppm) 121 Compound K (10 ppm) 129 Compound K (20 ppm) 145

As can be seen in Table 26, when compound K is treated, the proliferation of dermal papilla cells increases, which can be confirmed to increase significantly in a concentration-dependent manner.

Test Example 20 Evaluation of Potassium Ion Channel Activity Increase Effect

Hair loss treatment of minoxidil is known as mitochondrial potential potassium ion channel openers (K _ATP channel opener), a representative drug used in the treatment of androgenetic alopecia. To evaluate the mechanism of minoxidil, the treatment of toltamide (SIGMA AlDRICH, T0891), which blocks the K _ATP channel in fibroblasts constituting the dermis of the scalp, inhibits cell proliferation, and then opens potassium ion channels to allow cell proliferation. A recovering test method was used.

In order to evaluate the function of the composition as a K _ATP channel opener, in the present invention, a mouse embryonic fibroblast cell line (NIH3T3) cell line was used. The cell line is a cell line in which the fibroblast cell line isolated from NIH Swiss mouse embryo was naturally immortalized by 3T3 protocol. The cell line was incubated in DMEM (Gibco BRL, Gaithersburg, MD, USA) containing 10% FBS for 24 hours in an incubator maintained at 5% CO ₂ , 37 ℃. After the NIH3T3 was placed in a 96-well plate and incubated in a 37 ° C. incubator for 24 hours, 2.5 mM tolbutamide was treated, and after 10 minutes, 10 μM of minoxidil and compound K were treated at concentrations of 2.5 ppm, 5 ppm, and 10 ppm, respectively. After 48 hours of drug treatment, cell proliferation was measured using the WST-1 kit (Roche). The results are shown in Table 27 below.

division Cell proliferation (%) Untreated Control 100 Minoxidil 132 Compound K (2.5 ppm) 116 Compound K (5 ppm) 127 Compound K (10 ppm) 131

As can be seen in Table 27, when the compound K is treated, the proliferation of fibroblasts is restored, and the cell proliferation ability is increased by the concentration-dependent increase of the treated compound K, and the compound K is treated at 10 ppm. It can be seen that the proliferation of fibroblasts is restored to the minoxidil level.

Formulation Example 5 and Comparative Formulation Example 6

To the cream-based composition was prepared in a conventional manner according to the composition of Table 28 (unit: wt%).

Compounding ingredient Formulation Example 5 Comparative Formulation Example 6 Labrafac 23 23 Tween 80 10.5 10.5 Glycerol monostearate 4.0 4.0 Stearic acid 7.0 7.0 Cetyl alcohol 2.0 2.0 Propylene glycol 9.0 9.0 Glycerol Monooleate 3.5 3.5 Compound K One - water 40 41

Test Example 21 Hair Growth Effect of Compound K

After the hair on the back of the ICR mouse was depilated with a hair remover, hair was completely removed using a hair removal cream (beet cream). The skin inflammation was induced for 3 days by applying 200 μl once daily to the skin with 1% DNCB for the other 2 groups except the control group (normal group). After 3 days, the cream base of Formulation Example 5 and Comparative Formulation Example 6 was applied to the group except the control group once a day, and the degree of hair growth of each group was observed, and the results are shown in FIG. 1.

Referring to FIG. 1, the control group did not show the hair growth phenomenon in the depilated portion during the observation for 15 days, but the group treated only with the cream base containing no compound K showed a slight hair growth effect, and treated with the cream containing the compound K. In the group, hair growth was remarkable in the entire depilated area.

Test Example 22 Wool Effect of Compound K

By treating the hair of the back hair by the method of Test Example 21 by treating dinitrochlorobenzene (DNCB) to induce skin irritation by setting the stress conditions in the skin to maximize the growth of the hair, the present invention In order to evaluate the hair regeneration efficacy of the composition, the creams of Formulation Example 5 and Comparative Formulation Example 6 containing Compound K were treated, and the length of the hair grown according to the treatment days was measured and compared with the control group. The results are shown in Table 29 below.

Days of Treatment (Days) Hair length (cm) Formulation Example 5 Treatment Comparative Formulation 6 Treatment 0 0.1 0.1 6 0.2 0.1 9 0.5 0.1 12 0.9 0.1

Referring to Table 29, the length of the hair of the group treated with the cream base of Formulation Example 5 showed a statistically significant difference (p <0.01) compared to the group treated with the cream base of Comparative Formulation Example 6, The length of the hair of the group treated with the cream base of Formulation Example 5 compared to the group treated with the cream base of Comparative Example 6 containing no compound K was increased to about 0.9 cm on the 12th day. You can see that the length is longer.

It is determined that compound K promotes hair regeneration even under the condition of inhibiting hair regeneration formed under skin stress, and it can be seen that compound K has a regeneration function for hair that has been bald under stress.

Test Example 23 Test of Melanin Promoting Effect of Compound K

Add melan-a to 50,000 cells / well in a 24-well microtiter plate in medium containing 5% fetal bovine serum, RPMI medium, 100 IU penicillin G and 0.2 μM TPA in RPMI medium. Busy. The next day, the divided cells were treated with Compound K as a test substance at a final concentration of 10 ppm or 50 ppm, treated with 0.1% DMSO as a negative control, and 100 μM IBMX as a positive control, followed by incubation at 37 ° C. for 3 days. After incubation, the wells were washed with PBS, 100 μl of 1N NaOH was added, and the melanin in the cells was lysed. The absorbance of the dissolved melanin was measured at 405 nm using a microplate reader. The result of comparing the melanin production promoting effect of the compound K with the control is shown in Table 30 below.

sample Melanin Synthesis (%) DMSO (0.1%) 100 IBMX (100 μM) 120 Compound K (10 ppm) 111 Compound K (50 ppm) 122

Looking at the Table 30, it can be seen that compound K promotes melanin synthesis of melanocytes to increase melanin production, showing an excellent melanin production promoting effect.

Experimental Example 24 Effect of Compound K on Melanosite-induced Expression of MITF and Tyrosinase

Dispense 500,000 cells / well in a 6-well microtiter plate using a 501mel cell line, and in each hole, DMSO 0.1% for negative control, IBMX 100μM for positive control, and compound K for test group. Treatment with 10 ppm, incubated for 24 hours, 48 hours, 72 hours at 37 ℃ temperature to obtain a protein. The protein thus obtained was subjected to western blot using MITF and tyrosinase antibody. Protein extraction and Western blot were usually performed by standard methods used by those skilled in the art. After Western blotting, the result was compared with the negative control group of 100, and the results are shown in Table 31 below.

MITF Tyrosinase IBMX Compound K IBMX Compound K 24hr 121 96 160 151 48hr 98 112 149 154 72hr 224 120 298 185

Looking at Table 31, it can be seen that Compound K increases the expression of MITF and tyrosinase protein in melanocytes.

[Test Example 25] Evaluation of Efficacy of Anti-Blank Hair and Induction of Black Hair in Vivo Compound K in Vivo Using Leukemia-Induced Leukemia Mice

Vitiligo mice (C57bl / 6- Mitf ^{mi - vit} ) were purchased from The Jackson Lab, USA. Experimental method for the effect of white hair prevention material using the mouse to promote the development of white hair is as follows. Dorsal hairs of 12 week old mice were removed by depilation. However, the area of the hair removal site was adjusted in the same manner for each individual. From the day after the hair was removed, the anti-hair white matter was applied to the area where the hair was removed twice a day. As a vehicle for the anti-harvest material, a mixture of EtOH: 1,3-BG: DW = 3: 2: 5 (volume ratio) was used, and the carrier was used as a negative control, and the solution added with IBMX 50 mM was positive. As a control group, a solution to which 2.5% of compound K was added was used as a test group. After about three weeks, when the difference in anti-white hair efficacy between each substance was visually identified, newly grown hairs were collected and the amount of melanin in the hair was measured. The amount of melanin in the hair was measured using a proteinase enzyme, Esperase (Novozyme). A reaction buffer was prepared by dissolving esperase at a concentration of 1 NPU / ml in a buffer (50 mM Tris-HCl, 5 mM DTT, pH 9.3). 5 mg of mouse hair was added to 1 ml of the reaction buffer, followed by reaction for 13 hours while shaking at a speed of 1,000 rpm at 37 ° C., and the hair and the reaction solution were separated by instant centrifugation. The reaction solution thus obtained was placed in a 96 well plate, and the absorbance at 405 nm was measured to determine the amount of melanin in the reaction solution. When the negative control group, the positive control group, and the test group material were treated in the leukemia-promoting vitiligo mouse model, the results of visual observation and measurement of melanin in the hair were shown in Table 32 below.

Negative control group IMBX Compound K % Against control 100 105.9 108.2

According to Table 32, it can be seen that the compound K can promote the induction of black hair by inhibiting white hair and increasing the amount of melanin in the hair in mice in which white hair development is promoted.

Test Example 26 Evaluation of Antibacterial Activity of Compound K

Antimicrobial experiments were conducted to evaluate the antimicrobial activity of Compound K. Specific experimental methods are as follows.

Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa strains used in the experiment were obtained from Tryptic Soy Broth, Candida albicans and Aspergillus niger strains were cultured in Sabouraud Dextrose broth. The culture was added to each medium at a ratio of 1/100 (Candida albicans 1/10 diluted diluted solution was used as a test solution. Aspergillus niger, a spore suspension prepared to have a concentration of 2 × 10 ⁸ cfu / ml was used as the test strain.

0.15 ml of the above-mentioned test solution was added to 15 ml of each medium, and well-mixed solution was used as a diluting solution.

In a 96-well 96-well plate, add 16 μl of each sample to the first line, and add 184 μl of the diluted solution. In the remaining wells, 100 μl of the dilution solution was added. After mixing well the mixture of the first row, 100 μl was taken in the second row, mixed well, and then diluted 100 times by taking 100 μl again in the third row.

Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were incubated in a 32 ° C thermostat with Candida albicans, Aspergillus niger (Aspergillus niger), Staphylococcus aureus niger) were incubated in a 25 ° C incubator.

After 48 hours, the microbial growth was checked by suspension and microscope to determine the minimum inhibitory concentration (MIC) value. The results are shown in Table 33 below.

Sample Minimum inhibitory concentration (MIC), unit% Sedona Monas
Aruzinosa Staphylococcus aureus Escherichia coli Candida Albicans Aspergillus niger
Compound K 0.2 0.05 0.134 > 3 > 1

As shown in Table 33, it can be seen that compound K exhibits antimicrobial activity against various strains, through which it can be predicted that compound K may act as a natural preservative or antimicrobial agent in the composition.

Claims (20)

An external preparation composition for skin containing compound K represented by the following Chemical Formula 1 as an active ingredient.
[Chemical Formula 1]

The composition for applying the external of the skin according to claim 1, wherein the compound K is contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. The composition for external application for skin according to claim 1, wherein the composition is for whitening. The composition for external application for skin according to claim 1, wherein the composition is for moisturizing. The composition for external application for skin according to claim 1, wherein the composition is for enhancing skin barrier function. The composition for external application for skin according to claim 1, wherein the composition is for inducing differentiation of dermal keratinocytes. The composition for external application for skin according to claim 1, wherein the composition is for improving acne. The composition for external application for skin according to claim 1, wherein the composition is antibacterial. The composition for external application for skin according to claim 1, wherein the composition is for anti-inflammation. The composition for external application for skin according to claim 1, wherein the composition inhibits lipid production. The external preparation of the skin of claim 1, wherein the composition is for improving atopic dermatitis. The composition for external application for skin according to claim 1, wherein the composition is for improving color and skin tone. The composition for external application for skin according to claim 1, wherein the composition is for shrinking pores. The composition for external application for skin according to claim 1, wherein the composition is for sebum control. The composition for external application for skin according to claim 1, wherein the composition is for improving skin troubles. The composition for external application for skin according to claim 1, wherein the composition is for skin irritation. According to claim 1, wherein the composition is an external topical composition, characterized in that for preventing dandruff. According to claim 1, wherein the composition for external application to the skin, characterized in that for hair growth. According to claim 1, wherein the composition for external application to the skin, characterized in that for preventing white hair. The composition for external application of skin according to claim 1, wherein the compound K is used as a natural preservative.

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