CN107970251B

CN107970251B - Skin external composition containing ginsenoside Mc

Info

Publication number: CN107970251B
Application number: CN201711171123.1A
Authority: CN
Inventors: 金东泫; 柳权烈; 李沃澯; 廉明勋; 曺濬喆
Original assignee: Amorepacific Corp
Current assignee: Amorepacific Corp
Priority date: 2013-04-24
Filing date: 2014-04-24
Publication date: 2020-07-10
Anticipated expiration: 2034-04-24
Also published as: TWI653042B; CN107970251A; CN105555278A; WO2014175675A1; CN108066170A; TW201832769A; TWI652061B; HK1218077A1; CN105555278B; KR102048709B1; TW201521736A; KR20140126890A

Abstract

The present invention relates to a composition containing ginsenoside Mc as an active ingredient, which can provide not only anti-aging, skin wrinkle improvement, whitening, moisturizing improvement effects, but also anti-inflammatory, acne and skin problem improvement, allergy symptom improvement, skin astringency and pore contraction effects, skin complexion improvement effects, hair growth promotion, white hair improvement, anti-dandruff, and anti-corrosion effects.

Description

Skin external composition containing ginsenoside Mc

This application claims priority to korean patent application No. 10-2013-0045117 filed in the korean intellectual property office at 24.4.2013. The present application is a divisional application of the chinese patent application having the application number of 201480029499.7 entitled "topical skin composition containing ginsenoside Mc".

Technical Field

The present invention relates to a composition containing ginsenoside mc (ginsenoside mc) to provide not only anti-aging, skin wrinkle improvement, whitening, moisturizing improvement effects, but also anti-inflammatory, acne and skin problem improvement, allergy symptom improvement, skin astringency and pore contraction effects, skin blood color improvement, hair growth promotion, white hair improvement, anti-dandruff, and anti-corrosion effects.

Background

The skin serves as a first defense barrier of the human body, and has a function of protecting internal organs from the change of temperature and humidity and the stimulation of external environments such as ultraviolet rays and harmful substances. With age, the skin will change due to a variety of intrinsic and extrinsic factors. That is, in an intrinsic aspect, since secretion of various hormones regulating metabolism is decreased and functions and cellular activities of immune cells are decreased, biosynthesis of immune proteins and organism constituent proteins required for a human body is decreased. Externally, the ultraviolet content reaching the earth surface from the sun increases due to the destruction of the ozone layer, and the free radicals and active oxygen increase with the deepening of environmental pollution, so that not only the thickness of the skin is reduced, wrinkles are increased, elasticity is reduced, but also the skin color is darkened, problems (root) often occur to the skin, and nevi, freckles and age spots are increased, and various changes such as the color deterioration, the darkening of the skin color are caused.

In order to prevent such skin condition changes due to the intrinsic and extrinsic factors of the skin and maintain a healthy skin condition, efforts have been made to improve the skin condition by adding physiologically active substances obtained from various existing animals, plants, microorganisms, etc. to cosmetics and using them.

Ginsenoside Mc is produced by decomposing natural ginsenoside (Rb1, Rg1, Rd, Rb2, Rc, Rf) contained in ginseng with digestive enzymes and intestinal microorganisms. Although ginsenoside Mc has been reported to have a strong anticancer activity, and korean patent No. 164266 discloses a method of using ginsenoside Mc as an anticancer agent, the composition containing ginsenoside Mc as an active ingredient has no reports on the overall effects of a skin external preparation such as an effect of improving the overall skin condition, hair growth, white hair, dandruff resistance, and antisepsis.

Disclosure of Invention

Technical problem to be solved

In contrast, the present inventors have found that ginsenoside Mc contained in ginseng provides not only anti-aging, skin wrinkle improvement, skin whitening, and moisturizing improvement effects, but also anti-inflammatory, acne improvement effects, skin problems, and allergy symptom improvement effects, skin complexion improvement effects, hair growth promotion, white hair improvement, anti-dandruff, and anti-corrosion effects, and have completed the present invention.

Accordingly, an object of the present invention is to provide a composition for external use on skin, which contains ginsenoside Mc and thus can exhibit effects of improving the general state of skin, promoting hair growth, improving white hair, anti-dandruff, and anti-corrosion.

Technical scheme

In order to achieve the above objects, the present invention provides an anti-aging skin external composition containing ginsenoside Mc as an active ingredient.

In addition, the present invention provides a skin external composition for improving wrinkles, comprising ginsenoside Mc as an active ingredient.

In addition, the present invention provides a skin external composition for moisturizing containing ginsenoside Mc as an active ingredient.

In addition, the present invention provides a composition for external use for skin for improving acne, comprising ginsenoside Mc as an effective ingredient.

Further, the present invention provides a skin external composition for improving blood color and skin color, which contains ginsenoside Mc as an active ingredient.

In addition, the present invention provides a skin external composition for shrinking pores, comprising ginsenoside Mc as an active ingredient.

Further, the present invention provides a skin external composition for improving allergic skin, comprising ginsenoside Mc as an active ingredient.

In addition, the present invention provides an anti-inflammatory composition for external use for skin comprising ginsenoside Mc as an active ingredient.

In addition, the present invention provides a skin external composition for whitening skin containing ginsenoside Mc as an active ingredient.

Further, the present invention provides a skin external composition for promoting hair growth, comprising ginsenoside Mc as an active ingredient.

Further, the present invention provides a composition for external application to the skin for preventing white hair, which contains ginsenoside Mc as an active ingredient.

In addition, the present invention provides an anti-dandruff skin external composition comprising ginsenoside Mc as an effective ingredient.

In addition, the invention provides a natural preservative composition containing ginsenoside Mc.

Advantageous effects

The composition of the present invention contains ginsenoside Mc, thereby not only providing anti-aging, skin wrinkle improvement, whitening, moisturizing improvement effects, but also providing anti-inflammatory, acne and skin problem improvement, allergy symptom improvement, skin astringency and pore contraction effects, and skin complexion improvement, hair growth promotion, white hair improvement, anti-dandruff, and anti-corrosion effects.

Detailed Description

The composition for external application to the skin according to the present invention contains ginsenoside Mc as an active ingredient.

Ginsenoside Mc used in the present invention has the following chemical formula 1:

[ chemical formula 1]

Mc。

The ginsenoside Mc of the present invention may be extracted from plants, synthesized and used by a method known in the art, or commercially available ginsenoside Mc may be used. In addition, ginsenoside Mc can be obtained from ginseng extract. The kind of ginseng used in this case is not particularly limited, and ginseng, red ginseng, white ginseng, taiji ginseng, and tailed ginseng, etc. can be used. The ginseng extract may include not only a leachate obtained by leaching or decocting ginseng, but also a concentrate obtained by concentrating a part or the whole of the leachate, or the concentrate may be dried againInfusion, decoction, and tablet prepared by condensing liquid

The fluid extract may be any extract of the whole part of ginseng, including the plant itself, the stem, root, leaf, flower, fruit, etc., and is not limited to a specific part. In addition, known methods can be used to extract ginsenoside Mc from the ginseng extract.

Specifically, the ginsenoside Mc may be obtained by preparing a ginseng extract using water or an organic solvent and then separating it therefrom by a method known in the art. The organic solvent used in the present invention may be selected from ethanol, methanol, butanol, ether, ethyl acetate, chloroform and a mixed solvent of these organic solvents and water, and 80% ethanol is preferably used. At this time, the extraction temperature is preferably 10 to 80 ℃ and the extraction time can be 3 to 24 hours. If the range of the extraction temperature and the extraction time is exceeded, the extraction efficiency may be reduced or a change in composition may occur.

The composition according to the present invention preferably contains 0.001 to 50% by weight of ginsenoside Mc, based on the total weight of the composition. This is because if the content of the ginsenoside Mc is less than 0.001 wt%, the efficacy and effect of the components are weak, and if it exceeds 50 wt%, there is a problem in skin safety or formulation.

The composition of the present invention can be used as an anti-aging skin preparation composition for external use, and is excellent in the effects of improving skin elasticity and improving wrinkles.

The composition of the present invention can be used as a skin external composition for moisturizing, which can strengthen the skin barrier function and induce the differentiation of skin keratinocytes. Therefore, it is useful as a composition for external skin preparations for preventing or improving xeroderma, allergic dermatitis, contact dermatitis, psoriasis, etc., which are caused by incomplete differentiation of the epidermis.

The composition of the present invention can be used as a composition for external skin preparations for improving acne, is excellent in antibacterial effect, particularly excellent in antibacterial effect against acne-causing bacteria, and provides an anti-inflammatory effect.

The composition of the present invention can be used as a composition for external skin preparations for improving the color of blood and the color of skin, and when the composition is applied to the skin, the composition can smoothly supply nutrients to the skin by dilating the capillaries and promoting blood circulation, and can suppress skin aging, thereby having an excellent effect of improving the color of blood and the color of skin.

The composition of the present invention can be used as a composition for external skin preparations for shrinking pores, regulating sebum, and improving skin problems, and when it is applied to the skin, it has an excellent effect of suppressing skin problems by suppressing sebum excessively secreted, promoting elimination of active oxygen and collagen synthesis to shrink pores, and reducing the expression of inflammatory factors.

The composition of the present invention can be used as a composition for improving allergic skin, which not only provides an excellent anti-itch effect by inhibiting the activity of protease-Activated Receptor-2 (PAR-2) inducing itch, but also provides an anti-inflammatory effect by reducing the secretion of Interleukin (Interleukin-8, I L8). accordingly, the ginsenoside Mc of the present invention can be suitably used as an active ingredient of a composition for external skin preparations for stabilizing sensitive, irritant or allergic skin and scalp, and for improving or alleviating sensations of heat and inflammation.

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

The composition of the present invention can be used as a composition for external skin preparation for promoting hair growth, which promotes the transition from the telogen phase to the anagen phase, thereby providing effects including promoting hair growth, promoting the generation of new hair, promoting healthy hair growth, and preventing and suppressing the phenomenon of hair falling off from the scalp or the state of hair thinning or thinning.

The composition of the present invention can be used as a composition for external skin preparations for preventing white hair, which activates melanocytes by increasing the expression of transcription factor (MITF) in melanocytes and promotes the synthesis of melanin, thereby providing the effects of preventing the induction of white hair in advance and promoting the induction of black hair.

The composition of the present invention can be used as a skin external composition for preventing dandruff, which can purify the scalp by effectively discharging toxins accumulated on the hair and scalp, can prevent inflammatory reactions of the scalp by inhibiting the proliferation and growth of dandruff bacteria, and can provide effects of calming and strengthening the scalp and strengthening the inherent defense power because the antioxidant effect of inhibiting the generation and action of active oxygen is prominent.

The composition of the present invention can be used as a natural preservative composition, and is excellent in preservative effect and harmless to the human body because it is a natural component.

The composition of the present invention may be formulated with a cosmetically or dermatologically acceptable carrier or base. It can be provided in all forms suitable for topical use, for example, as a solution, gel, solid, paste, anhydrous product

In the form of emulsions, suspensions, microemulsions, microcapsules, fine-particle spheres or dispersion of ionic (liposomes) and non-ionic vesicles obtained by dispersing the oil phase in the aqueous phase, or in the form of creams, lotions, powders, ointments, sprays or concealer sticks. Also, it may be used in the form of a foam (foam) or an aerosol composition further comprising a compressed propellant. These compositions can be prepared according to methods commonly used in the art.

In particular, when the composition for external application to skin of the present invention is used for preventing dandruff, hair growth, or white hair, the composition can be formulated into a dosage form as a composition for scalp and hair, and the dosage form is not particularly limited, and may be formulated into, for example, hair tonic lotion, scalp care agent, hair care agent, shampoo, hair conditioner, hair lotion, and scalp and hair care agent.

In addition, the composition according to the invention may contain adjuvants commonly used in the field of cosmetics or dermatology, such as fatty substances, organic solvents, solubilizers, concentrates, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, chelating agents, complexing agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics. The adjuvant is introduced in an amount generally used in the field of cosmetics or skin science.

The composition of the present invention may contain a skin absorption-promoting substance for the purpose of enhancing the skin-improving effect.

Examples

Hereinafter, the structure and effects of the present invention will be described more specifically by way of test examples and dosage form examples. However, these test examples and dosage form examples are provided only for the purpose of facilitating understanding of the present invention and are not intended to limit the scope and scope of the present invention to the following examples.

[ reference example 1] preparation of ginsenoside Mc

Ginsenosides Mc used to test the efficacy of the compositions of the invention were purchased from AMBO (AMBO) institute.

[ test example 1] measurement of Elastase Activity inhibitory Effect

The elastase activity inhibitory activity of ginsenoside Mc was measured by comparing with epigallocatechin gallate (EGCG). The elastase and substrate used were elastase commercially available from sigma aldrich, usa (cat. No. eo0127).

The inhibitory effect on elastase activity was tested by the following test method.

In a 96-well plate, 200 μ L0 of ginsenoside Mc and 50 μ L1 of 20 μ g/M L elastase-type III solution were mixed in 10 mg/L of Tris hydrochloride (Tris-HC L) buffer (ph8.0), 250 μ M of EGCG was used as a positive control group, and distilled water was used as a non-treatment group of a negative control group, after which 100 μ L of N-succinyl-alanine-paranitroanilide (N-SUCCINY L-a L a-a L a-a L a-p-NITROANI L) was added at 0.4514mg/M L prepared with the buffer, and after the reaction was finished, absorbance at 415nm was measured (synergistic effect 2, microplate reader (tebio) (us) was performed by the same method as blank test to correct for the reaction at 25 ℃.

The calculation method of the inhibitory effect on elastase activity is shown in the following equation 1, and the results are shown in the following table 1.

Mathematical formula 1

Elastase activity inhibition ratio (%) - (1- (C-D)/(A-B) × 100

A: absorbance at 415nm when no test substance was added and enzyme was added

B: absorbance at 415nm without addition of test substance or enzyme

C: absorbance at 415nm when test substance and enzyme are added

D: absorbance at 415nm with test substance added, but no enzyme added

TABLE 1

Compound (I)	Degree of inhibition (%)
		Non-treatment group	0
EGCG	65
		Ginsenoside Mc	75

As shown in table 1, since ginsenoside Mc exhibits a significantly higher degree of inhibition of elastase activity than EGCG, which is known as an elastase activity inhibitor, it can be confirmed that ginsenoside Mc of the present invention has an excellent elastase activity inhibitory effect.

[ test example 2] collagenase (MMP-1) blocking ability

The collagenase production inhibitory activity of ginsenoside Mc of the present invention is measured by comparing it with retinoic acid.

Human fibroblasts were added in an amount of 5,000 cells/well (well) in a 96-well plate incubator (96-well microtiter plate) containing DMEM (Dulbecco's Modified Eagle's Media) containing 2.5% bovine fetal serum, and cultured in 5% CO₂Culturing is carried out in a 37 ℃ incubator until the growth reaches 70-80%. After 24 hours of treatment with ginsenoside Mc or retinoic acid at a concentration of 10 μ g/ml, a cell culture solution was collected.

The degree of collagenase production in the collected cell culture solution was measured by using a commercially available collagenase measuring apparatus (Amersham pharmacia, Catalog #: RPN 2610). First, the collected cell culture solution was added to a 96-well plate (96-well plate) on which primary collagenase antibody was uniformly applied, and an antigen-antibody reaction was performed in a thermostatic bath for 3 hours. After 3 hours, the secondary collagen antibody bound with the chromogen was added to the 96-well plate and reacted again for 15 minutes. After 15 minutes, 3',5,5' -tetramethylbenzidine (sigma) was added as a color development-inducing substance to induce color development at room temperature for 15 minutes, and 1M sulfuric acid was added again to stop the color development reaction, whereby the reaction solution was yellow in color and the degree of yellow development was different depending on the degree of progress of the reaction.

Absorbance of the 96-well plate in yellow color was measured at 405nm using an absorptiometer, and the degree of synthesis of collagenase was calculated according to the following numerical formula 2, and the results thereof are shown in the following table 2. In this case, the reaction absorbance of the cell culture solution collected from the group not treated with the composition was used as a control group.

Mathematical formula 2

Collagenase expression level (%) - (absorbance of substance-treated cell group/absorbance of control group × 100

TABLE 2

Compound (I)	Degree of expression (%)
		Non-treatment group	100
Retinoic acid	75
		Ginsenoside Mc	73

As shown in the above table 2, it can be known that the collagenase expression level of ginsenoside Mc is similar to that of retinoic acid, which is known as a collagenase expression inhibitor.

From the above results, it was confirmed that ginsenoside Mc of the present invention has an effect of inhibiting matrix metalloproteinase (MMP-1).

Formulation example 1 and comparative formulation example 1

A nourishing cream (unit: wt%) was prepared by a conventional method according to the composition of table 3 below.

TABLE 3

[ test example 3] confirmation of skin elasticity-improving efficacy

In order to confirm the effect of improving skin elasticity of humans, the formulations of the formulation example 1 and comparative formulation example 1 were used, and the following evaluations were made.

For 20 healthy women of 30 to 40 years old, 10 of each group were divided into two groups, and after applying the nutrient creams of the two groups of formulation example 1 and comparative formulation example 1 to the face at a frequency of 1 time per day for 12 weeks, the skin elasticity was measured using a skin elasticity tester (Cutometer SEM575, C + K Electronic Co., ltd., C + K Electronic Co., germany). The results are shown in table 4 below. The results of table 4 are reported as Δ R8 values for a skin elasticity meter (Cutometer SEM 575), where R8 values indicate the viscoelastic (viscoelasticity) properties of the skin.

TABLE 4

Experimental products	Skin elasticity effect
		Dosage form example 1	0.44
Comparative formulation example 1	0.10

As shown in table 4, the formulation example 1 containing ginsenoside Mc of the present invention has more improved skin elasticity than the group coated with the comparative formulation example 1.

Therefore, it was confirmed that the composition containing ginsenoside Mc of the present invention is very effective for improving skin elasticity.

[ test example 4] confirmation of efficacy in improving skin wrinkles

In order to confirm the wrinkle-improving effect of the composition of the present invention on humans, the above-mentioned dosage form example 1 and comparative dosage form example 1 were used.

In order to confirm the wrinkle-improving effects of the formulation example 1 and the comparative formulation example 1, the following evaluations were made. For 20 healthy women of 40 years old, 10 of each group were divided into two groups, and after applying the nourishing cream of both of dosage form example 1 and comparative dosage form example 1 to the face at a frequency of 1 time per day for 12 weeks, the state of wrinkles was measured using a silicon removal replica (replica), and a skin tester (visiometer, SV600, Courage + Khazaka electronic GmbH, germany) and subjected to image analysis. The results are shown in table 5 below. The results in table 5 below show the average values of the parameters before smearing (parameter) subtracted from the parameters after 12 weeks of smearing.

TABLE 5

As shown in table 5 above, it was found that the external preparation composition of dosage form example 1 was very excellent in the effect of improving skin wrinkles.

[ test example 5] tyrosinase-inhibiting effect

Tyrosinase was extracted from Mushroom (Mushroom), and tyrosinase of sigma was used. First, tyrosine as a substrate was dissolved in distilled water to prepare a 0.3mg/ml solution, and after the solution was added to a test tube in an amount of 1.0ml per tube, 1.0ml of a potassium-phosphate buffer solution (0.1mol concentration, pH6.8) and 0.7ml of distilled water were added thereto.

Ginsenoside Mc was mixed in an ethanol solution of the present invention at 10ppm to prepare a sample solution, and 0.2ml of the sample solution was added to the reaction solution, followed by reaction in a 37 ℃ thermostatic bath for 10 minutes. In this case, a control group was prepared by adding only 0.2ml of the solvent instead of each sample solution, and ascorbic acid was used as a positive control group. To the reaction solution, 0.1ml of 2500 cell/ml tyrosinase solution was added, and the reaction was carried out again in a 37 ℃ incubator for 10 minutes. The reaction was stopped by rapidly cooling the reaction solution in ice water, and absorbance at a wavelength of 475nm was measured with a photoelectric spectrophotometer (synergistic effect 2, enzyme labeling instrument (VT, usa), and the results are shown in table 6 below, and the tyrosinase inhibitory effect was calculated by the following equation 3.

Mathematical formula 3

Tyrosinase inhibition (%) 100 — (reaction absorbance of test substance/reaction absorbance of control × 100)

TABLE 6

Test substance	Tyrosinase inhibition ratio (%)
		Control group (without addition)	0
Ascorbic acid	52
		Ginsenoside Mc	63

As can be seen from the results of table 6, the ginsenoside Mc according to the present invention has a much higher inhibition rate of tyrosinase than ascorbic acid, which is a known tyrosinase inhibitor, and thus has very excellent whitening effects.

[ test example 6] effects of inhibiting melanogenesis by B16/F10 melanoma cells

A sample containing ginsenoside Mc and kojic acid in an amount of 0.001 wt% each was added as a test substance to a culture solution of B16/F10 melanoma cells (korean cell line bank) at a certain concentration, and after 3 days of culture, the culture solution was removed, followed by washing with Phosphate Buffered Saline (PBS), and after the cells were lysed with 1N NaOH, absorbance was measured at 405nm (synergistic effect 2, microplate reader (VT, usa) — cells to which no test substance was added were taken as a control group, and compared with the melanin content in the control group, and the degree of inhibition of melanin production by each test substance was measured, and the melanin production inhibition was calculated according to equation 4, and the results are shown in table 7.

Mathematical formula 4

Melanin production inhibition (%) 100 — (test substance absorbance/control group absorbance × 100)

TABLE 7

Test substance	Melanin production inhibition (%)
		Control group (without addition)	0
Kojic acid	53
		Ginsenoside Mc	66

As is clear from the results in table 7, the ginsenoside Mc according to the present invention has a much higher melanin production inhibition rate than kojic acid, which is a known melanin production inhibitor, and thus has very excellent whitening effects.

[ test example 7] measurement of skin moisturizing Effect

In order to measure the effect of ginsenoside Mc on the increase in skin moisturizing ability, the above-mentioned dosage form example 1 and comparative dosage form example 1 were used, and the following evaluations were made.

For 20 adult men and women classified as dry skin in the age range of 40 to 50 years, 10 of each group were divided into two groups, and the nutrition creams of the two groups of formulation example 1 and comparative formulation example 1 were applied to the face at a frequency of 2 times per day for 4 weeks. Skin moisture was measured using a skin moisture tester (Corneometer CM825, C + K Electronic Co., ltd. (C + K Electronic Co.), germany) under constant temperature and humidity conditions (24 ℃, 40% relative humidity) before application was started, after 1 week, after 2 weeks, after 4 weeks, and after application was stopped for 2 weeks (total 6 weeks). The results are shown in table 8 below. The results in Table 8 are expressed as percentages of the increase in the measured values after a certain period of treatment, based on the skin moisture tester values measured before the start of the test.

TABLE 8

As can be seen from the results in table 8, the application of comparative formulation example 1 showed a moisture increase rate of about 30% until 4 weeks after application, but the skin moisture content decreased after application was stopped, whereas the application of formulation example 1 containing ginsenoside Mc showed a skin moisture increase rate of 30% or more mostly after application was stopped. From this, it is found that the composition of the present invention containing ginsenoside Mc has an excellent skin moisturizing effect.

[ test example 8] measurement of keratinocyte differentiation-promoting Effect

As shown below, in order to understand the effect of ginsenoside Mc on the promotion of keratinocyte differentiation, the amount of Cornified Envelope (CE) produced during keratinocyte differentiation was measured by using absorbance.

First, human keratinocytes isolated from the epidermis of an infant and subjected to primary culture were placed in a culture flask and allowed to adhere to the bottom, and then treated with ginsenoside Mc at a concentration of 5ppm in the culture medium, followed by culture for 5 days until the cells grew to 70 to 80% of the area of the bottom. At this time, the low calcium (0.03mM) treated group and the high calcium (1.2mM) treated group were used as a negative control group and a positive control group, respectively. Then, the above cultured cells were taken, washed with phosphate buffered saline, and then 1ml of 10mM Tris-HCl buffer (Tris-HCl, pH7.4) containing 2% Sodium Dodecyl Sulfate (SDS) and 20mM Dithiothreitol (DTT) was added, and subjected to sonication (sonication), boiling (knitting), centrifugal separation, and the pellet was suspended in 1ml of PBS, and absorbance at 340nm was measured (synergy effect 2, microplate reader (VT, USA). additionally, a part of the sonicated solution was taken out, protein content was measured, and this was used as a reference for evaluating the degree of cell differentiation, and the results thereof are shown in Table 9 below.

TABLE 9

Test substance	Differentiation ability in keratinocytes (%)
		Low calcium (0.03mM) solution (negative control group)	100
High calcium (1.2mM) solution (positive control group)	210
		Ginsenoside Mc	285

As shown in table 9, it was confirmed that the differentiation promoting effect of keratinocytes was excellent when ginsenoside Mc was used for the treatment.

[ test example 9] measurement of skin Barrier function recovery Effect

In order to measure the effect of ginsenoside Mc on the recovery of skin barrier function damaged by skin damage, the following experiment was performed, in which the upper arms of 10 adult men and women were peeled off with a Tape (Tape striping), the skin barrier was damaged, two groups of formulation example 2 and comparative formulation example 2 prepared according to the composition of table 10 below were applied, the degree of recovery of the amount of percutaneous water loss (TWE L) was measured 1 time per day with a Vapometer (dolphin, finland), seven days were measured, and comparison was performed, comparative formulation example 2 here was a vehicle (vehicle) of a negative control group, the results of the experiment were shown in table 11 below, and the results of table 11 were compared with the difference before and after barrier damage as a 100% standard.

Watch 10

Ingredient of ingredient	Dosage form example 2	Comparative formulation example 2
			Refined water	69	70
Propylene glycol	30	30
			Ginsenoside Mc	1	-

TABLE 11

As can be seen from table 11, it was confirmed that the amount of transdermal moisture loss gradually increased with the passage of time when the treatment was performed using comparative formulation example 2 containing no ginsenoside Mc, whereas the amount of transdermal moisture loss rapidly recovered to normal and barrier damage was recovered when the treatment was performed using formulation example 2 containing ginsenoside Mc.

[ test example 10] blood color improving effect

In order to evaluate the effect of the cosmetic composition according to the present invention on the promotion of blood circulation in the skin, the degree of blood circulation in the skin was measured using a laser Doppler blood flow Imager (L aser Doppler Perfusion Imager, L DPI; periscan PIM II, miracle (Perimed), in sweden), L DPI is known as an instrument for measuring blood circulation in the skin, and it is an instrument widely used at present, which is a very sensitive instrument that can measure not only the speed and amount of blood in the capillaries of the skin but also the flow in arterioles and venules.

After washing the face with toilet soap in a constant temperature and humidity room, the test piece was adjusted for 30 minutes, and the initial value was measured with L DPI, first, the initial blood flow under the forehead of 30 women whose hands and feet were cold at ordinary times was measured with L DPI, then, the blood flow was measured after the test piece was used for 1 week in the above-mentioned dosage form example 1 and comparative dosage form example 1, and the results (change in skin blood flow) of comparing the measured blood flow with the initial measured value are shown in table 12 below.

TABLE 12

From the results of table 12, it was confirmed that the cosmetic composition according to the present invention significantly increased the skin blood flow and improved the complexion due to the promotion of blood circulation, as compared to comparative formulation example 1 containing no ginsenoside Mc. This finally indicates that the cosmetic composition containing ginsenoside Mc according to the present invention can contribute to effective delivery of skin nutrients, as well as inhibition and delay of skin aging.

[ test example 11] skin color improving effect

In order to understand the skin color improvement effect of the formulation example 1 and the comparative formulation example 1, 30 test subjects were used (1 time/day later, total 1 week of application), and then the skin color improvement degree was evaluated by a Facial Stage DM-3 (jasmonate (Moritex, japan) instrument. The skin color improvement rate was determined using the measured values of the lightness and color of the skin and the values of the change in lightness and color of the skin, and the results are shown in table 13 below. Higher lightness and color variation values indicate improved skin color.

Watch 13

From the results of table 13, it was confirmed that comparative formulation example 1, which did not contain ginsenoside Mc according to the present invention, did not exhibit significant skin color improvement effect, and on the contrary, formulation example 1, which contained ginsenoside Mc as an active ingredient, exhibited much improved skin color after use as compared to before use.

[ test example 12] pore-shrinking Effect

1. Pore-shrinking effect by promoting collagen biosynthesis

The effect of promoting the biosynthesis of collagen by ginsenoside Mc according to the present invention was compared with transforming growth factor- β (TGF- β) and measured first, fibroblasts were cultured at 10 per well⁵The amount of each cell was inoculated into 24 wells (well) and cultured until about 90% of the cells were grown, and after culturing the cells in serum-free cell culture medium (DMEM) for 24 hours, the culture was performed using 10ng/ml of each of ginsenoside Mc and TGF- β of the present invention dissolved in serum-free mediumTo CO₂The culture was carried out in a culture vessel for 24 hours, and the supernatants were removed and the increase and decrease of procollagen (procollagen) were observed using a procollagen type (I) enzyme-linked immunosorbent assay (E L ISA) kit (procollagen type (I); # MK101, TAKARA (Shiga, Japan) and the results are shown in Table 14 below, and the synthesis ability of collagen was compared with the untreated group set to 100.

TABLE 14

Test substance	Collagen synthesizing ability (%)
		Non-treatment group	100
TGF-β	183.5
		Ginsenoside Mc	195.7

From the results of table 14 above, it was confirmed that ginsenoside Mc according to the present invention showed superior collagen synthesis ability at a higher level than that of TGF- β, which is a positive control group.

2. Pore shrinking effect

In order to understand the pore-shrinking effect of the formulation example 1 and the comparative formulation example 1, the following evaluation was performed. 20 male and female test subjects with wide pore sizes are selected, 10 test subjects are divided into two groups according to each group, and the nutrition cream of the preparation example 1 and the nutrition cream of the comparative preparation example 1 are applied to the face every day according to the groups for 4 weeks. The effect of shrinking pores was judged in the following manner. Photographs were taken before and after 4 weeks of the experiment and were evaluated by the expert with the naked eye. The results are shown in Table 15 below (evaluation scale: 0-no shrinkage at all; 5-much shrinkage).

Watch 15

Test substance	Rating of evaluation
		Dosage form example 1	4
Comparative formulation example 1	0

As is clear from the results in table 15, comparative formulation example 1 has no effect of shrinking pores, but formulation example 1 shows a pore-shrinking effect that can be visually confirmed, and thus it is understood that ginsenoside Mc of the present invention has an excellent effect of shrinking pore size.

[ test example 13] sebum secretion-inhibiting Effect

1. Skin hypersecretion-inhibiting effect by inhibiting 5 α -reductase activity

To confirm the inhibitory effect of 5 α -reductase activity, the value of [ 2] was determined in HEK293-5 α R2 cells¹⁴C]Conversion of testosterone to [ alpha ]¹⁴C]Ratio of dihydrotestosterone (DHT: dihydrotestosterone) after transfection of p3x F L AG-CMV-5 α R2 on HEK293 cells and 2.5 × 10 per well⁵The amount of individual cells was seeded in a 24-well plate and cultured (Park et al, 2003, jds. vol.31, pp.191-98). The next day was changed to a new medium supplemented with enzyme substrates and inhibitors. Mixing the particles of 0.05. mu. Ci [ alpha ]¹⁴C]Testosterone (kit (Amersham Pharmaci)a biotech), uk) as culture substrate.

To confirm the degree of inhibition of 5 α -reductase activity, ginsenoside Mc was added and 5% CO was added at 37 deg.C₂The culture was carried out in an incubator for 2 hours. At this time, the group to which ginsenoside Mc was not added was used as a negative control group, and the group to which finasteride (finasteride) was added was used as a positive control group. After recovering the medium and extracting the steroid with 800. mu.l of ethyl acetate, the upper organic solvent layer was separated and dried, and the remaining residue was dissolved with 50. mu.l of ethyl acetate and developed on Silica gel 60F254(Silica plastic sheet kit gel 60F 254) using ethyl acetate-hexane (1:1) as a solvent.

After drying a plastic sample in air, a shower system was used to measure the amount of isotope

Adding the dried plastic sheet and the x-ray film into the bath box

After 1 week, the amounts of isotopes of testosterone and dihydrotestosterone remaining on the film were measured, and then the conversion and inhibition were calculated according to the following numerical formulas 5 and 6, respectively, and the results are shown in table 16 below.

Mathematical formula 5

Conversion (%) — radioactivity/total radioactivity in DHT region × 100

Mathematical formula 6

Inhibition (%) ((conversion of control-conversion of test substance ]/conversion of control × 100

TABLE 16

Test substance	Conversion (%)	Percent inhibition (%)
			Negative control group	48.0	-
Positive control group	27.6	42.5
			Ginsenoside Mc	14.8	67

From the results of the above table 16, it was confirmed that ginsenoside Mc could effectively inhibit the activity of 5 α -reductase, thereby blocking the conversion of testosterone into dihydrotestosterone, and showed more excellent inhibitory effect than finasteride, which is known to inhibit the activity of 5 α -reductase, the 5 α -reductase converts testosterone into dihydrotestosterone, thereby binding to receptor proteins in cytoplasm to enter into nucleus, thereby activating sebaceous gland cells and promoting differentiation, thereby excessively secreting sebum in sebaceous glands.

2. Sebum secretion inhibiting effect

In order to understand the sebum secretion inhibiting effect of the formulation example 1 and the comparative formulation example 1, the following evaluation was performed. 30 male and female test subjects considered to be hyperseborrhea were selected, and the nutrition creams of formulation example 1 and comparative formulation example 1 were applied to the designated area of the skin on the face every day for 4 weeks. The sebum reduction effect was evaluated by measuring the average sebum reduction (%) after 2 weeks and 4 weeks using a sebum amount measuring instrument (Sebumeter SM810, C + K Electronic Co., ltd., C + K Electronic Co., germany), and the results are shown in table 17 below.

TABLE 17

As is clear from the results in table 17, the dosage form example 1 of the present invention containing ginsenoside Mc as an active ingredient can effectively suppress excessive sebum secretion compared to the comparative dosage form example 1 not containing ginsenoside Mc.

[ formulation example 3 and comparative formulation examples 3 to 4]

Dosage form example 3 and comparative dosage form examples 3 to 4 were prepared according to the components and contents (wt%) shown in table 18 below, and are specifically described below. Dosage form example 3 is a substance mixed with ginsenoside Mc, comparative dosage form example 3 is a substance completely not containing an effective ingredient for improving skin containing acne, and comparative dosage form example 4 is a standard substance for antibacterial power, and the substance contains erythromycin (erythromycin) which is often used as an acne therapeutic agent.

The preparation methods of formulation example 3 and comparative formulation examples 3 to 4 are as follows. The ingredients in item A of Table 18 below were completely dissolved, and the ingredients in item B were completely dissolved in another dissolution tank, and then item B was added to item A and mixed and solubilized. The composition was prepared by adding the component of item C to the mixture in the mixing ratio shown in table 18, mixing the mixture uniformly, and then filtering the mixture.

Watch 18

Test example 14 antibacterial efficacy test against acne bacteria

An antibacterial test was conducted on Propionibacterium acnes (ATCC 6919: Medium-brain Heart infusion Broth Medium (BHI broth)) as a causative strain of acne using each of the cosmetic compositions prepared according to the compositions of formulation example 3 and comparative formulation examples 3 to 4.

The test method for the antibacterial ability against acne bacteria is as follows.

(1) Preparation of test bacterial solution

A culture solution obtained by inoculating Propionibacterium acnes in a brain-heart infusion broth culture medium and performing anaerobic culture was used as the test bacterial solution.

(2) Preparation of the diluted solution

To 15ml of brain-heart infusion broth (pH6.8) or L B broth (pH4.5), 0.15ml of the test bacterial suspension was added, and the mixed solution was used as a diluted solution.

(3) Preparation of sample

The cosmetic composition stock solutions prepared in formulation example 3 and comparative formulation examples 3 to 4 were used as samples as they were.

(4) Antibacterial power test

1) A sample was added to the No. 1 row of a 96-well cell culture tube (96well plate) so as to match the initial concentration, and a diluting solution was added so that the total amount was 200. mu.l.

2) After the mixed solution of row 1 was mixed uniformly, 100. mu.l of the mixed solution was taken out and added to row 2, and after mixing uniformly, 100. mu.l of the mixed solution was taken out again and added to row 3. In this way, a secondary dilution (doublydilution) is carried out.

3) After the culture was allowed to stand at 32 ℃ for 24 hours and 48 hours, whether or not the bacteria proliferated was judged by the degree of suspension, and the Minimum Concentration at which the bacteria did not proliferate was defined as the Minimum Inhibitory Concentration (MIC). If the mixture is opaque and it is difficult to determine whether or not the bacteria have proliferated, it is confirmed by observation with a microscope.

The results of the antibacterial activity test for acne bacteria are shown in table 19 below. The minimum inhibitory concentration is marked in terms of the concentration of the active ingredient contained in the dosage form.

Watch 19

Item	pH	Propionibacterium acnes
			Dosage form example 3	5.7	>52ppm
Comparative formulation example 3	5.7	Maximum concentration (no antibacterial power)
			Comparative formulation example 4	5.7	>100ppm

In the results in table 19, it is considered that the substance is effective for the antibacterial activity against acne bacteria as the ppm concentration is smaller in the minimum inhibitory concentration, and it can be confirmed that the composition containing ginsenoside Mc has more excellent antibacterial activity against the test bacteria by showing a significantly lower ppm concentration in the case of using the formulation example 3 than the comparative formulation example 4 using erythromycin which is a known acne therapeutic agent.

[ test example 15] inhibition test of lipid Synthesis (L ipogenensis)

3T 3-L1 cells as a mouse fibroblast line (fibroblast cell line) were cultured in the presence of 1 × 10⁵Cells/well were attached to 6-well culture plates (cultureplates) containing DMEM (Dulbecco's modified eagle's medium, GIBCO BR L, Life technologies) containing 10% Fetal Bovine Serum (FBS). after 2 days, the culture medium was replaced with a new DMEM (containing 10% FBS) and cultured for 2 days, and then, DMEM (containing 10% FBS) containing 1. mu.g/ml insulin (insulin), 0.5mM Isobutylmethylxanthine (IBMX) and 0.25. mu.m dexamethasone (dexamethasone) was usedThe cultured cells were subjected to differentiation induction, treated with 50 μ M of ginsenoside Mc and caffeine, and then, after 2 days, replaced with DMEM containing insulin and cultured for 5 days. After 5 days, the medium was changed to normal medium (DMEM, containing 10% FBS) again, and the cells were observed and cultured until the cells changed from morphological to adipocytes.

In order to evaluate the effect of ginsenoside Mc on the inhibition of fat accumulation in adipocytes, sudan triple staining (S4136, sigma aldrich) was performed using the differentiated 3T 3-L1 adipocytes at room temperature, after fixing the adipocytes in a phosphate buffer with 4% paraformaldehyde (ph7.2), washing with a phosphate buffer, and then staining with sudan triple, photographs were taken and compared with the naked eye, a group using only a medium without a test substance or a comparative substance was used as a control group, and the other comparative groups were treated with 50 μ M caffeine, and the degree of fat accumulation inhibition was assigned to a grade by dividing the degree of staining into +++++, ++, -, at which the closer to +++++++, the greater the degree of staining, and the results are shown in table 20 below.

Watch 20

Sample (I)	Inhibition ratio%
		Control group	+++
Comparison group	+
		Ginsenoside Mc	-

As shown in table 20, it was confirmed that ginsenoside Mc used in the present invention has a small amount of fat accumulated in fat cells and also has an excellent lipid synthesis inhibitory effect compared to caffeine, which is a known lipid synthesis inhibitory substance. Thus, sebum is reduced by inhibiting lipid synthesis, thereby enabling the suppression of the occurrence of acne.

Test example 16 test for improvement of acne and reduction of sebum secretion and Presence of irritation

30 persons suffering from acne were treated as test subjects, 10 persons were divided into three groups, and the cosmetic compositions prepared in the above-mentioned formulation example 3 and comparative formulation examples 3 to 4 were applied to the test subjects corresponding to the respective groups for 1 month. For the acne improvement criteria, a score of 1 to 5 was set, and a score of 1 was "none", 3 was "normal", and 5 was "very good". For the experimental results, 10 average scores are labeled in table 21 below.

The number of days for which elimination was recognized was used as a criterion for the acne elimination period, and the presence or absence of recurrence after 1 month was used as a criterion for the acne recurrence. For the decrease in sebum secretion, 1 to 5 points were set, and the mark was that 1 point was "none", 3 points were "normal", and 5 points were "very good". For the experimental results, 10 average scores are labeled in table 21 below. The presence or absence of skin irritation was judged by (the number of persons who showed irritation reaction)/(the total number of test persons).

TABLE 21

As shown in table 21 above, it can be seen that formulation example 3 had no recurrence of acne and had an excellent effect on acne improvement as a whole, as compared to comparative formulation example 3. In addition, in case of comparative formulation example 4 containing an antibacterial force standard substance, although the acne improvement effect was shown, the use of the substance was not suitable for long-term use because of strong skin irritation, whereas the composition according to the present invention was not irritating and was also shown to be suitable for long-term use.

[ test example 17] inflammation-ameliorating Effect interleukin-8 (I L-8) production-inhibiting Effect

One day before the experiment, skin keratinized epithelial cells (Normal human skin keratinocyte, NHEK, Purchase: L onza) were treated at 5 × 10⁴Cells/well, seeded in 96-well plates, then at 37 ℃ with 5% CO₂After 24 hours in an incubator (incubator), the cells were washed 2 times with phosphate buffer and replaced with serum free Keratinocyte Base Medium (KBM), and after 30 minutes of reaction, the cells were treated with ginsenoside Mc at concentrations of 5ppm, 25ppm and 50ppm, respectively, in each well, and treated with staphylococcus aureus Peptidoglycan (PGSA) (10 μ g/ml), staphylococcus aureus peptidoglycan (50 μ g/ml) and staphylococcus aureus peptidoglycan (50 μ g/ml) + lipopolysaccharide (1 μ g/ml), respectively, wherein staphylococcus aureus peptidoglycan (PGSA: peptidoglycan s. aureus) as a peptidoglycan (peptidoglycan) extracted from staphylococcus is a major constituent of gram-positive bacteria and is known to induce inflammation of cell membrane components of bacteria, particularly, staphylococcus aureus is known to cause secondary bacterial cell membrane infection of about 90% of staphylococcus aureus, and about 90% of staphylococcus is known to be responsible for secondary bacterial cell membrane infection of L. fig..

At 37 ℃ 5% CO₂After 24 hours of culture in the incubator, the culture solution was taken out and subjected to enzyme-linked immunosorbent assay (E L ISA) for Interleukin-8 (Interleukin-8, I L-8), and the results are shown in Table 22 below, for E L ISA, the experimental method of the manufacturing company (BD science) was used.

TABLE 22

Classification	Secretion of Interleukin-8 (pg/ml)
		No treatment control group (control)	935.12
PGSA(10μg/ml)	4812.60
		PGSA(50μg/ml)	5895.08
PGSA(50μg/ml)+LPS(1μg/ml)	6814.91
		Ginsenoside Mc (5ppm)	1573.32
Ginsenoside Mc (25ppm)	1203.54
		Ginsenoside Mc (50ppm)	1001.23

As can be seen from table 22, ginsenoside Mc significantly reduces and inhibits the secretion of interleukin-8 increased by PGSA and lipopolysaccharide L PS, and thus, it is understood that the composition for external skin application of the present invention significantly reduces the secretion of interleukin-8 increased by PGSA and L PS, thereby providing an excellent anti-inflammatory effect.

[ test example 18] evaluation of relief of itching

One day before the experiment, keratinocytes (cell line name: HaCaT, Purchase Notification: American Type Culture Collection (ATCC)) were assigned 4 × 10⁴Cells/well, seeded in 96-well plates, then at 37 ℃ with 5% CO₂The culture was carried out in an incubator for 24 hours. After 24 hours, with hanks' balanced salt solution (After washing (washing) the 96-well plate 2 times with Hanks' Balanced Salt solution, HBSS buffer, reaction buffer (2. mu.M Fluo-4-AM, 20% pluronic acid, 2.5mM probenecid) was put into the cells. At 37 ℃ 5% CO₂After 30 minutes of reaction in the incubator and 30 minutes of reaction at normal temperature, the cells were washed 2 times with HBSS buffer, and treated with ginsenosides Mc at concentrations of 0.05%, 0.1%, 0.5% and 1.0% (wt%).

After 10 minutes of reaction, treatment with 2U/ml trypsin (trypsin) or 5. mu.M PAR-2 active peptide (S L IGKV) was carried out, and intracellular Ca was measured²⁺The concentration was varied for 80 seconds. For intracellular Ca²⁺The concentration change was measured by using a microplate reader 3(FlexStation 3: Molecular Device (USA); after treatment with ginsenoside Mc and 2U/ml trypsin (trypsin) or 5. mu.M PAR-2 active peptide (S L IGKV), the bend (flex) at 80 seconds was measured, the difference between the minimum and maximum values was determined, and the value was compared with the difference between the minimum and maximum values when treated with 2U/ml trypsin or 5. mu.M PAR-2 active peptide (S L IGKV), and the inhibition rate (%) of calcium ion influx into cells was shown in Table 23 below.

TABLE 23

As can be seen from table 23, the influx of calcium ions into cells due to trypsin or PAR-2 active peptide (S L IGKV) decreased with the treatment of ginsenoside Mc, and it was confirmed that the influx of calcium ions into cells was significantly decreased with the increase in the concentration of ginsenoside Mc.

Therefore, the composition for external application to skin containing ginsenoside Mc of the present invention can provide an excellent anti-itch effect by effectively inhibiting PAR-2 activity which induces itch.

Formulation example 4 and comparative formulation example 5

Shampoos were prepared with the compositions of table 24 below. Specifically, a surfactant and ethylene glycol distearate were added to refined water, and heated to 80 ℃ to be uniformly dissolved, and then gradually cooled to 40 ℃ with stirring, and the active ingredient according to the present invention, a preservative, a viscosity modifier, a fragrance, and a hair conditioner were added to the mixture and mixed, and then cooled to room temperature with stirring, to prepare a shampoo.

Watch 24

Composition (I)	Dosage form example 4	Comparative formulation example 5
			Ammonium dodecyl sulfate	10	10
Polyoxyethylene ammonium lauryl sulfate	5	5
			Cocoamidopropyl betaine	2	2
Ethylene glycol distearate	1.5	1.5
			Cocosanoyl monoethanolamide	0.8	0.8
GinsenosideMc	5.0	-
			Polyquaternium-10	0.2	0.2
Blue No. 1	0.0002	0.0002
			Yellow No. 4	0.0001	0.0001
P-hydroxybenzoic acid methyl ester	0.1	0.1
			Perfume	0.8	0.8
Citric acid	0.1	0.1
			Dimethicone	1.0	1.0
Water (W)	To 100	To 100

[ test example 19] dandruff reducing Effect test

After 24 men aged 19 to 35 years, each of which had a high dandruff, were selected and divided into two groups of 12 men, and the shampoos of formulation example 4 and comparative formulation example 5 were used in the following manner, respectively, for 1 month, the dandruff reduction rate was measured.

Before the start of the test, the hair was washed with a conventional shampoo, and then dandruff accumulated for two days after the washing was collected, and the weight of the collected dandruff was compared with the weight of dandruff accumulated for two days after the end of the test in which the hair was washed once every two days with the shampoos of formulation example 4 and comparative formulation example 5, respectively. At this time, the accumulated dandruff was collected directly from the scalp by using a vacuum inhalation device, and the dandruff reduction rate was determined according to the following equation 7, and the results thereof are shown in the following table 25.

Mathematical formula 7

Dandruff reduction (%) as ═ weight of dandruff (mg before the start of test) -weight of dandruff (mg) after one month)/weight of dandruff (mg) before the start of test × 100

TABLE 25

As can be seen from table 25 above, dosage form example 4 containing ginsenoside Mc showed an excellent dandruff preventing effect.

Test example 20 test of the Effect of preventing scalp pruritus

After 24 men and women aged 25 to 45 years who had more severe feeling of scalp itching were selected, 12 of each group were divided into two groups, and the shampoos of each of dosage form example 4 and comparative dosage form example 5 were used at a frequency of once every three days for two weeks, the effect of preventing scalp itching was evaluated by the following evaluation criteria, and the results thereof are shown in table 26 below.

[ evaluation standards ]

Very Excellent-5 points

Excellent-4 point

General score of-3

Bad-2 points

Very poor at 1 point

Watch 26

Classification	Dosage form example 4	Comparative formulation example 5
			Removing effect of scalp pruritus	4.2	2.3

As can be seen from table 26, dosage form example 4 containing ginsenoside Mc showed more excellent effects on the prevention of scalp pruritus.

[ test example 21] evaluation of Effect of increasing Potassium ion channel Activity

Minoxidil as a therapeutic agent for alopecia is known as a potential mitochondrial potassium channel opener (K)_ATPchannel openers) are representative drugs used in the treatment of androgenetic alopecia. To evaluate the mechanism of this minoxidil, the following assay was used. The test method is to use the block K in the fibroblasts constituting the scalp dermis_ATPThe tolbutamide (SIGMAAIDRICH, T0891) of the channel was treated to inhibit cell proliferation and the potassium channel was reopened to restore cell proliferation.

To evaluate K as the present composition_ATPFunction of channel openers, the present invention uses a Mouse embryonic fibroblast line (NIH 3T3) as a fibroblast line. The cell line was obtained by natural immortalization of a fibroblast cell line isolated from NIH Swiss mouse embryo (Swiss mouse embryo) using 3T3 protocol (protocol). The cell line is cultured in the presence of 10% FBSDMEM (Gibco BR L, Gethersburg, Md., USA) maintained at 5% CO₂And cultured in an incubator at 37 ℃ for 24 hours. NIH3T3 was inoculated in a 96-well plate, and after 24 hours of culture in an incubator at 37 ℃, treated with 2.5mM of tolbutamide, and after 10 minutes, treated with 10 μ M of minoxidil and ginsenosides Mc at concentrations of 2.5ppm, 5ppm and 10ppm, respectively, as a positive control group, and after 48 hours of drug treatment, cell proliferation ability was measured using WST-1 kit (Roche). The results are shown in table 27 below.

Watch 27

Classification	Cell proliferation potency (%)
		No treatment control group (control)	100
Minoxidil	132
		Ginsenoside Mc (2.5ppm)	115
Ginsenoside Mc (5ppm)	123
		Ginsenoside Mc (10ppm)	130

As can be seen from table 27 above, when the treatment with ginsenoside Mc was performed, the proliferation of fibroblasts was recovered, and the cell proliferation potency was increased depending on the concentration of ginsenoside Mc that was treated, and it was confirmed that the cell proliferation was recovered to the level when the treatment with ginsenoside Mc of 10ppm was performed.

[ test example 22] test for the melanin production-promoting effect of ginsenoside Mc

Melanocytes were seeded in a 24-well plate at 50,000 cells/well in a serum-free cell cryopreservation medium (RPMI medium) supplemented with 5% fetal bovine serum, 100IU of penicillin G, and 0.2 μ M of terephthalic acid (TPA). The next day, the inoculated cells were treated with ginsenoside Mc as a test substance at a final concentration of 10ppm or 50 ppm. The group treated with 0.1% DMSO was used as a negative control group, the group treated with 100. mu.M Isobutylmethylxanthine (IBMX) was used as a positive control group, and each of the above groups was cultured at 37 ℃ for three days. After incubation, the well plates were washed with Phosphate Buffered Saline (PBS) and melanin in the cells was solubilized after adding 100. mu.l of 1N NaOH to each well. The results of comparing the melanin production-promoting effect of ginsenoside Mc with that of the control group are shown in table 28 below, by measuring the absorbance of the solubilized melanin at 405nm using a plate culture analyzer (synergistic effect 2, microplate reader (VT, usa)).

Watch 28

Test material	Amount of melanin synthesized (%)
		DMSO(0.1％)	100
IBMX(100μM)	120
		Ginsenoside Mc (10ppm)	112
Ginsenoside Mc (50ppm)	121

As is clear from table 28, ginsenoside Mc promotes the synthesis of melanin by melanocytes to increase the production of melanin, and thus can exhibit an excellent melanin production promoting effect.

[ test example 23] Effect of ginsenoside Mc on promoting the expression of transcription factor (MITF) and tyrosinase in melanocytes

Using a 501mel cell line, 500,000 cells/well were seeded in 6-well plates, and in each well, a negative control group treated with 0.1% dimethyl sulfoxide (DMSO), a positive control group treated with 100 μ M IBMX, and an experimental group treated with 10ppm human saponin Mc were used, and after incubation at 37 ℃ for 24 hours, 48 hours, and 72 hours, proteins were obtained. The protein thus obtained was subjected to a western blot (WesternBlot) test using MITF and tyrosinase. Protein extraction and western blotting are performed by standard methods commonly used by those skilled in the art. After western blotting, the negative control group in the results was set to 100, and the results were compared with the values and shown in table 29 below.

Watch 29

As shown in the above table 29, it can be confirmed that ginsenoside Mc increases the expression of MITF and tyrosinase proteins in melanocytes.

[ test example 24] evaluation of antibacterial Activity of ginsenoside Mc

In order to evaluate the antibacterial power of ginsenoside Mc, an antibacterial experiment was performed. Specific test methods are as follows.

Staphylococcus aureus (Staphylococcus aureus) and E.coli used in the experimentEscherichia coli and Pseudomonas aeruginosa were cultured in Tryptic Soy Broth, Candida albicans and Aspergillus niger were cultured in Sabouraud Dextrose Broth, and the culture Broth was used as a test bacterial solution in which 1/100 (Candida albicans strain 1/10) was diluted in each medium, and the culture Broth was prepared as 2 × 10 for Aspergillus niger⁸cfu/ml spore suspension was used as the test bacterial liquid.

A mixed solution obtained by adding 0.15ml of the test bacterial suspension to 15ml of each culture medium and mixing the mixture was used as a diluted solution.

In the No. 1 row of the 96-well plate, 10ppm of ginsenoside Mc was added in an amount of 16 μ l per well, respectively, and 184 μ l of the diluted solution was added, respectively. To the other wells, 100. mu.l of each diluted solution was added. After the mixed liquid of row 1 is mixed uniformly, 100 mul of the mixed liquid is taken out and added into row 2, and after the mixed liquid is mixed uniformly, 100 mul of the mixed liquid is taken out again and added into row 3. In this way, a double dilution was performed.

The staphylococcus aureus, the escherichia coli and the pseudomonas aeruginosa are cultured in a constant temperature tank at 32 ℃; candida albicans and Aspergillus niger were cultured in a thermostatic bath at 25 ℃.

After 48 hours, the Minimum Inhibitory Concentration (MIC) value was determined by confirming proliferation of the bacteria using a suspension ratio and a microscope, and the results are shown in table 30 below.

Watch 30

As shown in the above table 30, it can be confirmed that ginsenoside Mc exhibits antibacterial power against various strains, and from these, it can be predicted that ginsenoside Mc can function as a natural preservative or antibacterial agent in the composition.

Claims

1. Use of a skin external composition comprising ginsenoside Mc as an active ingredient in the preparation of a cosmetic composition for moisturizing skin.

2. Use of ginsenoside Mc as the only active ingredient in preparing cosmetic composition for moisturizing skin is provided.

3. Use of a skin external composition comprising ginsenoside Mc as an effective ingredient in the preparation of a cosmetic composition for enhancing skin barrier function.

4. Use of ginsenoside Mc as the only active ingredient in preparing cosmetic composition for enhancing skin barrier function is provided.

5. Use of a skin external composition comprising ginsenoside Mc as an active ingredient in the preparation of a cosmetic composition for inducing differentiation of skin keratinocytes.

6. Use of ginsenoside Mc as the only active ingredient in the preparation of cosmetic composition for inducing the differentiation of skin keratinocyte.