KR102048709B1 - External composition for skin containing Ginsenoside Mc - Google Patents

Abstract

The composition of the present invention can provide anti-aging, skin wrinkle improvement, whitening, moisturizing effect as well as anti-inflammatory, acne and skin troubles, atopic symptoms, skin convergence and pore contraction effect by containing ginsenoside Mc. The present invention relates to a composition capable of providing skin color improvement, hair growth, white hair improvement, antidandruff and antiseptic effect.

Description

External composition for skin containing Ginsenoside Mc

The present invention can provide anti-aging, skin wrinkle improvement, whitening, moisturizing effect as well as anti-inflammatory, acne and skin troubles, atopic symptoms improvement, skin convergence and pore contraction effect by containing ginsenoside Mc and skin color It relates to a composition that can provide the effect of improvement, hair growth, white hair improvement, antidandruff and antiseptic effect.

Human skin is the body's primary protective barrier, which protects the body's organs from changes in temperature and humidity, and from external environmental stimuli such as ultraviolet rays and pollutants. Undergo a change. That is, internally, the secretion of various hormones that regulate metabolism decreases, and the function of immune cells and the activity of cells decreases, thereby reducing the biosynthesis of immune proteins and constituent proteins necessary for living organisms. Due to the increase in the amount of ultraviolet rays that reach the surface of the sun's rays and intensifying environmental pollution, free radicals and free radicals increase, thereby reducing the thickness of the skin, increasing wrinkles, reducing elasticity, Skin color becomes dull, skin problems frequently occur, blemishes, freckles and blotch also increase, the color becomes worse and the skin tone becomes darker.

In order to prevent changes in the skin condition caused by these internal and external factors and to maintain a healthy skin condition, the skin condition is improved by adding bioactive substances obtained from various known animals, plants, and microorganisms to cosmetics. Efforts have been made.

Ginsenoside Mc (ginsenoside Mc) is produced by the decomposition of ginsenosides (Rb1, Rg1, Rd, Rb2, Rc, Rf) in the form of natural products contained in ginseng by digestive enzymes and intestinal microorganisms. Although the point is reported, Korean Patent No. 164266 describes a method of using it as an anticancer agent, but the overall skin condition improvement effect, hair growth and white hair improvement, antidandruff and antiseptic effect of the composition comprising ginsenoside Mc as an active ingredient. The overall effect as an external skin preparation such as has not been reported yet.

Registered Patent Publication No. 10-164266 (January 15, 1999)

Therefore, the present inventors can provide ginsenoside Mc anti-aging, skin wrinkle improvement, whitening, moisturizing effect as well as anti-inflammatory, acne and skin troubles, atopic symptoms, and improves skin color and hair growth and white hair. The present invention has been found to be able to provide improvements, antidandruff and antiseptic effects.

Accordingly, it is an object of the present invention to provide a composition for external application for skin containing ginsenoside Mc, which improves the overall condition of the skin and promotes hair growth, white hair improvement, antidandruff and antiseptic effect.

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

The present invention also provides a skin external composition for improving wrinkles containing ginsenoside Mc as an active ingredient.

The present invention also provides a moisturizing skin external composition containing ginsenoside Mc as an active ingredient.

In addition, the present invention provides a composition for external application for improving acne containing ginsenoside Mc as an active ingredient.

The present invention also provides a skin external preparation composition for improving color and skin tone containing ginsenoside Mc as an active ingredient.

The present invention also provides a topical skin composition for reducing pores containing ginsenoside Mc as an active ingredient.

In addition, the present invention provides a skin external preparation composition for improving atopic skin containing ginsenoside Mc.

The present invention also provides an anti-inflammatory external composition for skin containing ginsenoside Mc.

The present invention also provides a whitening skin external preparation composition containing ginsenoside Mc.

The present invention also provides a composition for promoting hair growth containing ginsenoside Mc.

The present invention also provides a composition for preventing white hair containing ginsenoside Mc.

The present invention also provides a composition for anti-dandruff containing ginsenoside Mc.

The present invention also provides a natural preservative composition containing ginsenoside Mc.

The composition of the present invention can provide anti-aging, skin wrinkle improvement, whitening, moisturizing effect as well as anti-inflammatory, acne and skin troubles, atopic symptoms, skin convergence and pore contraction effect by containing ginsenoside Mc. It can provide the effect of skin complexion improvement, hair growth and white hair improvement, antidandruff and antiseptic effect.

The composition according to the present invention contains ginsenoside Mc (ginsenoside Mc) as an active ingredient.

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Ginsenoside Mc of the present invention can be extracted from plants, can be used synthesized according to methods known in the art, and may be commercially available. Ginsenoside Mc can also be obtained from ginseng extract. The type of ginseng used at this time is not particularly limited, and ginseng, red ginseng, white ginseng, taeguksam, misam and the like can be used. In addition, the ginseng extract is contained not only in the leachate obtained by leaching and transferring from ginseng, but also in the concentrate obtained by partially or fully concentrating the leachate, or in stagnation, whole, regular, liquid extract and ginseng prepared by drying the concentrate again. The chemicals that exert the main effect, of course, include all of the plant itself, and extracts from all parts of ginseng, such as stems, roots, leaves, flowers, fruits, can be used and are not limited to extracts of any particular part. In addition, a method for extracting ginsenoside Mc from ginseng extract may use a known method.

Specifically, the ginsenoside Mc may be separated from the ginseng extract after preparing ginseng extract with water or an organic solvent by 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 of the present invention preferably contains the ginsenoside Mc in an amount of 0.001 to 50% by weight based on the total weight of the composition. This is because when the content of the active ingredient is less than 0.001% by weight, the efficacy and effect by the above ingredients are weak, and when the amount of the active ingredient exceeds 50% by weight, there is a problem of skin safety or formulation.

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

The composition of the present invention may be used as a moisturizing skin external preparation composition, which may enhance skin barrier function and induce differentiation of skin keratinocytes. Therefore, it can be usefully used as an external composition for skin to prevent or improve skin dryness, atopic dermatitis, contact dermatitis, or psoriasis caused by incomplete epidermal differentiation.

The composition of the present invention can be used as an external preparation composition for improving acne, which is excellent in antibacterial effect, in particular, antibacterial effect against acne causing bacteria, and also provides an anti-inflammatory effect.

The composition of the present invention can be used as an external skin composition for improving color and skin tone, and when applied to the skin, it smoothly supplies nutrients to the skin by promoting capillaries and promotes blood circulation and inhibits skin aging to improve color and skin tone. The effect is excellent.

The composition of the present invention can be used as a skin external preparation composition for pore reduction, sebum control and skin trouble improvement, which suppresses excessive secretion of sebum when applied to the skin, promotes free radical removal and collagen synthesis to shrink pores. In addition, the effect of suppressing skin problems is excellent by reducing the expression of inflammatory factors.

The composition of the present invention can be used as a composition for improving atopic dermatitis, which inhibits the activity of protease-activated receptor-2 (PAR-2), which causes itching, to provide an excellent anti-pruritic effect. In addition to providing anti-inflammatory effects, the secretion of Interleukin-8 (IL-8) can be reduced. Therefore, the ginsenoside Mc of the present invention can be suitably used as an active ingredient of the skin external preparation composition for stabilizing sensitive, irritant or atopic skin and scalp, and to improve or alleviate itching, heat and inflammation.

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

The composition of the present invention can be used as a composition for promoting hair growth, which promotes hair growth by promoting the transition of the resting hair cycle to the growing hair cycle, and also promotes the production of new hair, as well as promoting healthy hair. It includes growing, and provides the effect of preventing and suppressing the phenomenon of hair falling off from the scalp or the condition of hair growth or thinning.

The composition of the present invention can be used as a composition for preventing white hair, and increases the MITF expression of melanocytes to activate melanocytes and promote melanin synthesis, thereby preventing the induction of white hair and providing an effect of promoting hair loss.

The composition of the present invention can be used as an anti-dandruff skin external composition, which effectively discharges toxins accumulated in the hair and scalp to cleanse the scalp, inhibit the growth and growth of dandruff bacteria, and prevent the scalp inflammatory reaction, and also active It has an excellent antioxidant effect that inhibits the production and action of oxygen, so it can provide the effect of calming and strengthening the scalp and strengthening its natural defense.

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

The composition according to the invention may 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-obtained by dispersing an oil phase in solution, gels, solids, pasty anhydrous products, aqueous phases. 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 may also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. These compositions can be prepared according to conventional methods in the art.

In particular, when the topical skin composition of the present invention is used for the prevention of dandruff, hair growth or white hair, it may be formulated as a composition for scalp and hair, and the formulation is not particularly limited, for example, hair tonic, hair nourishing cosmetics, scalp It can be formulated as a treatment, hair treatment, hair shampoo, hair rinse, hair lotion or scalp hair combination treatment and the like.

In addition, the composition according to the present invention is a fatty substance, an organic solvent, a dissolving agent, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, an ionic or nonionic. Such as type emulsifiers, fillers, metal ion sequestrants, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredients commonly used in cosmetics. It may contain adjuvants conventionally used in the cosmetic or dermatology field. Such adjuvants are introduced in amounts generally used in the cosmetic or dermatological arts.

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

Hereinafter, the configuration 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 only for the purpose of illustration to help the understanding of the present invention is not limited to the scope and scope of the present invention by the following examples.

Reference Example 1 Preparation of Ginsenoside Mc

Ginsenoside Mc for testing the efficacy of the composition of the present invention was purchased from Ambo laboratory.

Test Example 1 Measurement of Elastase Activity Inhibition Efficacy

The inhibition of elastase activity of ginsenoside Mc was measured in comparison with EGCG. The elastase and substrate used were purchased commercially from Sigma-Aldrich, USA (Cat. No. E0127).

Elastase activity inhibitory activity was tested by the following test method.

200 μg of ginsenoside Mc and 50 μL of 20 μg / mL elastase type III solution were mixed in a 96-well plate with 10 mg / L Tris-HCL buffer (pH 8.0). 250 μM of EGCG was used as a positive control and distilled water was used as a non-treated group as a negative control. Thereafter, 100 µL of 0.4514 mg / mL N-SUCCINYL-ALA-ALA-ALA-p-NITROANILIDE prepared with the above buffer was added and reacted at 25 ° C for 15 minutes. After completion of the reaction, the absorbance at a wavelength of 415 nm was measured. A blank test was performed in the same manner to correct.

Calculation method of the elastase activity inhibitory activity is shown in Equation 1 below, the results are shown in Table 1 below.

A: Absorption at wavelength 415 nm in the absence of test substance and addition of enzyme

B: Absorbance at a wavelength of 415 nm with no test substance and no enzyme

C: absorbance at wavelength 415 nm in test substance addition and enzyme addition

D: absorbance at wavelength 415 nm with test substance added and without enzyme

compound Suppression degree (%) Untreated group 0 EGCG 65 Ginsenoside Mc 75

In the results of Table 1, the degree of suppressing the elastase activity of ginsenoside Mc is significantly superior to the EGCG known as an elastase activity inhibitor, it is excellent in the effect of inhibiting the elastase activity of ginsenoside Mc of the present invention. can confirm.

Test Example 2 Collagenase (MMP-1) Inhibitory Activity

Inhibition of collagenase production of ginsenoside Mc of the present invention was measured in comparison with retinoic acid.

Place human fibroblasts at 5,000 cells / well in a 96-well microtiter plate containing Dulbecco® Modified Eagle® Media (DMEM) medium containing 2.5% fetal calf serum. The culture was incubated in an incubator at 37 ° C., 5% CO ₂ until 70-80% growth. Ginsenoside Mc or retinoic acid was treated at a concentration of 10 μg / ml for 24 hours, followed by cell culture.

The degree of collagenase production of cell cultures was measured using a commercially available collagenase measuring instrument (Amersham Pharmacia, USA, Catalog #: RPN 2610). First, the cell culture solution collected in a 96-well plate uniformly coated with primary collagenase antibody was placed in an incubator for 3 hours. After 3 hours, the chromophore-bound secondary collagen antibody was placed in a 96-well plate and reacted for another 15 minutes. After 15 minutes, color-causing substances (3,3 ', 5,5'tetramethylbenzidine, sigma) were added to induce color development at room temperature for 15 minutes, and when 1M sulfuric acid was added to stop the color reaction, the color of the reaction solution became yellow. The degree of yellow color was different according to the progress of reaction.

The absorbance of the yellow 96-well plate was measured at 405 nm using an absorbance meter, and the degree of synthesis of collagenase was calculated by Equation 2 below, and the results are shown in Table 2 below. Indicated. At this time, the reaction absorbance of the cell culture liquid collected from the group not treated with the composition was used as a control.

compound Expression degree (%) Untreated group 100 Retinoic acid 75 Ginsenoside Mc 73

In the results of Table 2, it can be seen that the degree of collagenase expression of ginsenoside Mc is similar to the collagenase expression inhibitory effect compared to retinoic acid known as a collagenase expression inhibitor.

Through these results, it can be confirmed that the ginsenoside Mc of the present invention has an effect of inhibiting the substrate metalloprotease (MMP-1).

[Formulation Example 1 and Comparative Formulation Example 1]

Nutritional cream was prepared in a conventional manner according to the composition of Table 3 (unit: wt%).

Ingredient Formulation Example 1 Comparative Formulation Example 1 Purified water To 100 To 100 Ginsenoside Mc 0.1 - Vegetable Cured 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 Arachidil Behenyl Alcohol & Arachidil 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 Cyclomedicon 6.00 6.00 Preservative, incense Quantity Appropriate Triethanol amine 0.1 0.1

 Test Example 3 Confirmation of Skin Elasticity Improvement Efficacy

In order to confirm the effect of improving skin elasticity in humans, the formulation of Formulation Example 1 and Comparative Formulation Example 1 were evaluated as follows.

Twenty healthy women in their 30s to 40s were divided into two groups of 10 people in two groups of Formulation Example 1 and Comparative Formulation Example 1, and applied nutrition cream to the face once a day for 12 weeks. 575, C + K Electronic Co., Germany) was used to measure skin elasticity. The results are shown in Table 4 below. The results in Table 4 are described as ΔR8 values of Cutometer SEM 575, where R8 values indicate the nature of skin viscoelasticity.

Experimental product Skin elasticity effect Formulation Example 1 0.44 Comparative Formulation Example 1 0.10

In the results of Table 4, Formulation Example 1 containing the ginsenoside Mc of the present invention was more skin elasticity compared to the group to which Comparative Example 1 was applied.

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

Test Example 4 Confirmation of Skin Wrinkle Improvement Effect

Formulation Example 1 and Comparative Formulation Example 1 were used to confirm the effect of improving wrinkles in humans by the composition of the present invention.

In order to confirm the wrinkle improvement effect of the Formulation Example 1 and Comparative Formulation Example 1 was evaluated as follows. Twenty healthy women in their 40s were divided into two groups of 10 people for each of two groups of Formulation Example 1 and Comparative Formulation Example 1, and the nourishing cream was applied to the face once a day for 12 weeks. The state of the image was measured by a skin meter (visiometer, SV600, Courage + Khazaka electronic GmbH, Germany) and analyzed. The results are shown in Table 5 below. The values in Table 5 below represent the average of each parameter value after 12 weeks of application minus the parameter value before application.

8 weeks after use
Clinical results R1 R2 R3 R4 R5 Formulation Example 1 -0.22 -0.20 -1.2 -0.04 -0.03 Comparative Formulation Example 1 0.27 0.26 0.21 0.03 0.03 R1: difference between the highest and lowest of the wrinkle contour
R2: Divide the wrinkle contours by 5 spaces, and then average the R1 values
R3: The highest value among the R1 values divided by five
R4: The mean value of the baseline of the fold contour minus the top and valley values of each angle
R5: Difference value of the baseline of the wrinkle contour line minus each wrinkle outline

In the results of Table 5, it was found that the external preparation composition of Formulation Example 1 has a very good skin wrinkle improvement effect.

Test Example 5 Tyrosinase Inhibitory Effect

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.

0.2 ml of a sample solution prepared by mixing 10 g of ginsenosides Mc in the ethanol solution of the present invention was added to the reaction solution and reacted for 10 minutes in a 37 ° C thermostat. In this case, the control group was prepared by adding only 0.2 ml of 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 test 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 6 below. Each tyrosinase inhibitory effect was calculated by the following equation.

Test substance Tyrosinase inhibition rate (%) Control group (no addition) 0 Ascorbic acid 52 Ginsenoside Mc 63

From the results of Table 6, it can be seen that the ginsenoside Mc tyrosinase inhibition rate of the present invention is much higher than the known tyrosinase inhibitor ascorbic acid, the whitening effect is very excellent.

Test Example 6 Effect of Inhibiting Melanin Production Using B16 / F10 Melanoma Cells

A sample containing 0.001% by weight of ginsenoside Mc and kojic acid, respectively, was added as a test substance to the culture medium of B16 / F10 melanoma cells (Korea Cell Line Bank) at a constant concentration for 3 days, after which the culture solution was removed and PBS was removed. The cells were washed with 1N NaOH and absorbed 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. According to Equation 4 to calculate the melanin inhibition rate is shown in Table 7 the results.

Test substance Melanogenesis inhibition rate (%) Control group (no addition) 0 Kojisan 53 Ginsenoside Mc 66

From the results of Table 7, it can be seen that the ginsenoside Mc melanin production inhibition rate of the present invention is much higher than the known melanin production inhibitor kojic acid is very excellent whitening effect.

Test Example 7 Measurement of Increased Skin Moisturizing Effect

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

Twenty male and female adults in their 40s to 50s, classified as dry skin, were divided into two groups of 10 people for each of the two groups, Formulation Example 1 and Comparative Formulation Example 1, and applied to the face twice daily for four weeks. Skin moisture meter at constant temperature and constant humidity (24 ° C, 40% relative humidity) before start of application, after 1 week, 2 weeks, 4 weeks after application, and after 2 weeks (total 6 weeks) after application was stopped. Skin moisture content was measured by (Corneometer CM825, C + K Electronic Co., Germany). The results are shown in Table 8 below. The results in Table 8 show the percentage increase of the measured value after a certain period of treatment based on the skin moisture meter value measured immediately before the start of the test.

Test group Moisture Growth Rate (%) 1 week past 2 weeks past 4 weeks past After 6 weeks Formulation Example 1 32 35 36 32 Comparative Formulation Example 1 30 32 32 15

In the results of Table 8, when the Comparative Formulation Example 1 was applied, it showed a water increase rate of about 30% up to 4 weeks after the application, but after stopping the application, the moisture content of the skin decreased, whereas ginsenoside Mc In the case of coating containing Formulation Example 1, it can be confirmed that even after the application was stopped, the skin moisture increase rate was more than 30%. It can be seen that the composition of the present invention containing ginsenoside Mc has excellent skin moisturizing effect.

Test Example 8 Measurement of Keratinocyte Differentiation Promotion Effect

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

First, the keratinocytes of primary cultures isolated from the epidermis of newborns were put in a culture flask and adhered to the bottom. After treatment with Ginsenoside Mc at 5 ppm concentration in the culture medium, the cells were approximately 70-80% of the floor area. Incubate for 5 days until growth. At this time, the low calcium (0.03mM) treated group and the high calcium (1.2mM) treated group were used as negative and positive controls, respectively. Then, the cultured cells were harvested and washed with PBS (Phosphate buffered saline), followed by 10 mM Tris-HCl buffer (Tris-HCl, containing 2% SDS (sodium dodecyl sulfate) and 20 mM Dithiothreitol (DTT). pH 7.4) 1 ml was added to sonication, boiling and centrifugation, and the precipitate was suspended in 1 ml of PBS again to measure absorbance at 340 nm. Separately, a portion of the solution after the sonication was taken to measure the protein content and used as a reference when evaluating the degree of cell differentiation. The results are shown in Table 9 below.

Test substance Differentiation capacity in keratinocytes (%) Low Calcium (0.03 mM) Solution
(Negative control) 100 High Calcium (1.2mM) Solution
(Positive control) 210 Ginsenoside Mc 285

In the results of Table 9, it was confirmed that the treatment of ginsenoside Mc is excellent in promoting the differentiation of keratinocytes.

Test Example 9 Measurement of Skin Barrier Function Restoration Effect

In order to measure the effect of ginsenoside Mc on the recovery of damaged skin barrier function due to skin damage, the following experiment was performed. Ten adult men and women's upper and lower limbs were damaged by a tape stripping method for 7 days while applying two groups of Formulation Example 2 and Comparative Formulation Example 2, each prepared by the composition of Table 10 below. The recovery of transdermal moisture loss (TWEL) was measured once a day using a Vapometer (Delfin, Finland). Comparative Formulation Example 2 is a vehicle as the negative control group. The experimental results are shown in Table 11 below. The results in Table 11 were compared on the basis of the 100% difference before treatment and after barrier damage.

Compounding Ingredients (wt%) Formulation Example 2 Comparative Formulation Example 2 Purified water 69 70 Propylene glycol 30 30 Ginsenoside Mc One -

Test group TWEL change (%) Before treatment 1 day 2 days 3 days 4 days 5 days 6 days Formulation Example 2 100 125.6 127.3 122.3 117.5 112.1 105.3 Comparative Formulation Example 2 100 121.4 112.7 98.3 70.5 62.3 43.5

In the results of Table 11, when the Comparative Formulation Example 2 containing no ginsenoside Mc is treated, the transdermal moisture loss increases more and more over time, whereas the Formulation Example 2 containing ginsenoside Mc is treated. In this case, the loss of transdermal moisture returns to normal and the barrier damage is recovered.

Test Example 10 Hemoglobin Improvement Effect

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 a LDPI (Laser Doppler Perfusion Imager). LDPI is a well-known and currently used device for measuring blood circulation in the skin, and is a very sensitive device that can measure not only the speed and amount of blood in the capillaries of the skin but also the flow in the small arteries and the venous veins.

After washing the face with soap in a thermo-hygrostat room for 30 minutes, the initial value was measured using LDPI. Initially, initial blood flow in the lower forehead of 30 women with cold hands and feet was measured by LDPI. Then, after the formulation Example 1 and Comparative Formulation Example 1 were used in the test subjects for one week, the result of comparing the blood flow rate measured with the initial measurement value (skin blood flow change) is shown in Table 12 below.

LDPI Results-Skin Blood Flow Before and After Cosmetic Use Test substance % Change in skin blood flow after 1 week of use Formulation Example 1 13 Comparative Formulation Example 1 5

In the results of Table 12, the cosmetic composition according to the present invention significantly increased the skin blood flow than Comparative Formulation Example 1 containing no ginsenoside Mc, it was confirmed that the blood color is improved through the blood circulation. . This ultimately suggests that the cosmetic composition containing ginsenoside Mc according to the present invention can contribute effectively to the skin's nutrient delivery, inhibit skin aging and delay.

Test Example 11 Skin Tone Improvement Effect

In order to find out the skin tone improvement effect of Formulation Example 1 and Comparative Formulation Example 1, each of 30 subjects were used (once evening / daily application, for a total of 1 week), and then, a Facial Stage DM-3 (Moritex, Japan) device The improvement of skin tone was evaluated using. Skin tone improvement rate was determined by the lightness and color measurement value of the skin as the measured value of the brightness and color of the skin, the results are shown in Table 13 below. The higher 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 14 ± 2.85 12 ± 3.31 Comparative Formulation Example 1 5 ± 2.34 5 ± 2.05

In the results of Table 13, Comparative Formulation Example 1 containing no ginsenoside Mc according to the present invention showed no significant skin tone improvement effect, while Formulation Example 1 containing ginsenoside Mc as an active ingredient was used before use. It was confirmed that a lot of later skin tone is improved.

Test Example 12 Pore Reduction Effect

1. Pore reduction effect through promoting collagen biosynthesis

Ginsenoside Mc according to the present invention was measured by comparing collagen biosynthesis promoting effect with TGF-β. First, fibroblasts were seeded by 10 ⁵ per hole in 24 wells and cultured until 90% growth. This was incubated with serum-free DMEM medium for 24 hours and then treated with 10 ng / ml of ginsenoside Mc and TGF-β of the present invention dissolved in serum-free medium, respectively, and CO ₂ The incubator was incubated for 24 hours. These supernatants were removed and procollagen increased or decreased using procollagen type I ELISA kit (procollagen type (I)). The results are shown in Table 14, and the collagen synthesis ability was compared with the non-treated group as 100.

Test substance Collagen Synthesis (%) Untreated group 100 TGF-β 183.5 Ginsenoside Mc 195.7

In the results of Table 14, ginsenoside Mc 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 ginsenoside Mc 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 determine the pore reduction effect of Formulation Example 1 and Comparative Formulation Example 1 was evaluated as follows. Twenty male and female subjects with large pore sizes were selected and divided into two groups of ten, and the nutritional creams of Formulation Example 1 and Comparative Formulation Example 1 were applied to the face each day for 4 weeks. Determination of the effect of pore reduction was made by visual assessment of experts by taking pictures before and after 4 weeks of the experiment. The results are shown in Table 15 below (rating rating: 0-not scaled down at all; 5-scaled down).

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

In the results of Table 15, 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, ginsenoside Mc according to the present invention reduces the size of the pores It was found that the effect was excellent.

Test Example 13 Sebum Secretion Inhibitory Effect

1. Inhibition of skin hypersecretion through inhibition of 5α-reductase activity

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

In order to confirm the degree of inhibition of 5α-reductase activity, ginsenoside Mc was added and incubated for 2 hours at 37 ° C and 5% CO ₂ incubator. In this case, the ginsenoside Mc was not used 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 in 50 μl ethyl acetate. The silica plastic sheet kieselgel 60 F254 ) 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 isotope amount. The isotope of testosterone and dihydrotestosterone remaining in the film after one week by placing the dried plastic sheet and the x-ray film together in the bath cassette. After the amount was measured, the conversion and inhibition rates were calculated according to the following Equations 5 and 6, and the results are shown in Table 16 below.

Test substance % Conversion % Inhibition Negative Control 48.0 - Positive control group 27.6 42.5 Ginsenoside Mc 14.8 67

In the results of Table 16, 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. It was confirmed that Ginsenoside Mc effectively inhibits the activity of blocking the conversion of testosterone to dihydrotestosterone, and it was shown to have a superior inhibitory effect than finasteride known to inhibit the activity of 5α-reductase. Therefore, it was confirmed that ginsenoside Mc can suppress sebum hypersecretion by effectively inhibiting the activity of 5α-reductase.

2. Sebum secretion inhibitory effect

In order to determine the sebum secretion inhibitory effect of Formulation Example 1 and Comparative Formulation Example 1 was evaluated as follows. Thirty male and female subjects who felt sebum secretion were selected and the nutritional creams of Formulation Example 1 and Comparative Formulation Example 1 were applied to the designated areas of the facial skin every day for 4 weeks. The determination of the effect of sebum reduction was measured using the sebum meter (Sebumeter SM810, C + K Electronic Co., Germany) and the average sebum reduction rate (%) after 2 weeks and 4 weeks, respectively, and the results are shown in Table 17 Shown in

Test substance Sebum reduction rate (%) After 2 weeks After 4 weeks Formulation Example 1 29 39 Comparative Formulation Example 1 5 5

From the results of Table 17, it can be seen that Formulation Example 1 containing ginsenoside Mc according to the present invention as an active ingredient can effectively inhibit the sebum secreted in excess than Comparative Formulation Example 1 does not contain 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 18 below. Specifically, Formulation Example 3 is a formulation of ginsenoside Mc, Comparative Formulation Example 3 does not contain any active ingredients of acne skin improvement, Comparative Formulation Example 4 is a standard to be used as a reference for the antimicrobial activity It contains erythromycin, which is widely used as an acne treatment.

The preparation method of Formulation Example 3 and Comparative Formulation Examples 3-4 is as follows. To complete the dissolution of the components of phase A in Table 18 and completely dissolve the components of phase B in a separate dissolution bath, phase B was added to phase A for solubilization. The components of phase C were added thereto according to the blending ratios shown in Table 18, homogenized, mixed and filtered to prepare the present 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 Cured Castor Oil
(Hydrogenated castor oil) 0.4 0.4 0.4 Perfume 0.04 0.04 0.04 C Ginsenoside Mc 5.0 - - Erythromycin - - 5.0

Test Example 14 Antibacterial Activity Test against Acne Bacteria

Antibacterial activity was tested against propionibacterium acnes (ATCC 6919: Medium-BHI broth), which is an acne-causing strain, with each cosmetic composition prepared in the formulation of Formulation Example 3 and Comparative Formulation Examples 3-4. .

The antibacterial test method for acne bacteria was as follows.

(1) Test bacteria preparation

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) sample preparation

The cosmetic composition stock solutions prepared from Formulation Example 3 and Comparative Formulation Examples 3 to 4 were used as samples.

(4) antimicrobial activity test

1) Insert the sample to the starting concentration in the first well of 96-well cell culture tube (96 well plate) and add 200 μl of the total amount into the dilution solution.

2) Mix the mixed solution in the first row well, take 100μl into the second row, mix well, and then add 100μl to the third row and perform double dilution.

3) After incubation for 24 hours and 48 hours at 32 ℃, determine the growth of bacteria to the extent of suspension and determine the minimum concentration without growth of bacteria as 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 antibacterial activity test results for acne bacteria are shown in Table 19 below. MIC is expressed in terms of the concentration of the active ingredient contained in the formulation.

Item pH Propionibacterium Acnes Formulation Example 3 5.7 > 52 ppm Comparative Formulation Example 3 5.7 Highest concentration (no antimicrobial activity) Comparative Formulation Example 4 5.7 > 100 ppm

In the results of Table 19, it can be said that the smaller the ppm concentration in the MIC is effective for the antimicrobial activity against acne bacteria, in the case of Formulation Example 3 ppm concentration than Comparative Formulation Example 4 using the known acne treatment erythromycin It can be seen that the composition containing the ginsenoside Mc has significantly superior antimicrobial activity against the test bacteria.

Experimental Example 15 Lipogenesis Inhibition Test

3T3-L1 cells, a mouse fibroblast cell line, were loaded with DMEM (Dulbecos modified eagles medium, GIBCO BRL, Life Technologes) medium containing 10% fetal bovine serum (FBS). The well culture plate was attached at 1 × 10 ⁵ cells / well. After 2 days, it was again exchanged with fresh DMEM (containing 10% FBS) medium and incubated 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 ginsenoside Mc and caffeine. After 2 days of treatment 50μM was exchanged for DMEM containing insulin and incubated for 5 days. After 5 days, the cells were exchanged with normal medium (DMEM, containing 10% FBS) and cultured while observing until the cells changed to fat cells.

Sudan III staining (S4136, sigma-aldrich) was performed using the differentiated 3T3-L1 adipocytes in order to evaluate the inhibitory effect of ginsenoside Mc in adipocyte fat accumulation. Adipose cells were fixed at room temperature with 4% paraformaldehyde (pH 7.2) in phosphate buffer, washed with PBS (phosphate buffered saline), stained with Sudan III, and photographed for visual comparison. As a control group, only the medium without the test substance or the comparative substance was used, and 50 μM of caffeine was treated with another control group. The degree of inhibition of fat accumulation was given by dividing the degree of staining into +++, ++, +, and-, which means that the degree of staining is increased toward +++. The results are shown in Table 20 below.

sample % Inhibition Control +++ Comparison + Ginsenoside Mc -

From the results of Table 20, ginsenoside Mc used in the present invention can be seen that not only the amount of fat accumulated in adipocytes, but also has a superior lipid synthesis inhibitory effect than caffeine, a known lipid synthesis inhibitor. . Therefore, sebum is reduced by inhibiting lipid synthesis, thereby suppressing the occurrence of acne.

[Test Example 16] Test for acne improvement, sebum secretion and irritation

Thirty people with acne were divided into three groups of 10 people, 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. The acne improvement scale ranged from 1 to 5 points, with 1 being ‘no’, 3 being ‘normal’ and 5 being ‘very yes’. Experimental results are shown in the average score of 10 people in Table 21 below.

Acne disappearance was based on the number of days the extinction was read, acne recurrence was based on the results after one month. Sebum secretion is reduced from 1 to 5 points, with 1 being ‘no’, 3 being ‘normal’ and 5 being ‘very yes’. Experimental results are shown in the average score of 10 people in Table 21 below. The presence or absence of skin irritation was determined as (number of irritants) / (total number of test subjects).

Inflammatory Acne Improvement Cotton acne extinction Acne recurrence Reduce sebum secretion Irritation Formulation Example 3 4.1 3 days radish 4.4 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

In the results of Table 21, Formulation Example 3 did not recur acne compared to Comparative Formulation Example 3, it can be seen that there is an excellent effect on the overall acne improvement. On the other hand, Comparative Formulation Example 4 containing an antimicrobial activity standard shows an acne-improving effect, but due to the strong skin irritation in use, it may not be suitable for long-term use, but the composition according to the present invention has no irritation for long-term use. Even appeared to be appropriate.

[Test Example 17] Inflammation improving effect-IL-8 production inhibitory effect

One day before the experiment, normal human skin keratinocytes (NHEK, obtained from Lonza) were dispensed in 96-well plates at 5x10 ⁴ cells / well, and then in a 37 ° C, 5% CO ₂ incubator. Incubated for 24 hours. After 24 hours, the cells were washed twice with PBS and changed to serum-free keratinocyte basement media (KBM). Each well was treated with ginsenoside Mc at concentrations of 5 ppm, 25 ppm and 50 ppm, 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) was treated respectively. Here, PGSA (peptidoglycan from S. aureus ) is a peptidoglycan (peptidoglycan) extracted from Staphylococcus aureus, a major component of the Gram-positive (+) cell wall and bacterial membrane components are known to cause inflammation, In particular, in staphylococci, about 90% of patients with atopic dermatitis have been reported to cause secondary infection. Lypopolysaccaride (LPS) is a major component of the cell membrane of Gram-negative bacteria and is known to be a major 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 22 below. ELISA used the experimental method of the manufacturer (BD science).

division IL-8 secretion (pg / ml) Untreated Control 935.12 PGSA (10 μg / ml) 4812.60 PGSA (50 μg / ml) 5895.08 PGSA (50 μg / ml) + LPS (1 μg / ml) 6814.91 Ginsenoside Mc (5 ppm) 1573.32 Ginsenoside Mc (25 ppm) 1203.54 Ginsenoside Mc (50 ppm) 1001.23

In the results of Table 22, it was confirmed that ginsenoside Mc significantly reduces and inhibits the secretion of IL-8 increased by PGSA and LPS. Therefore, it can be seen that the topical skin composition of the present invention can provide an excellent anti-inflammatory effect by significantly reducing the secretion of IL-8 increased by PGSA and LPS.

[Test Example 18] Itching relief evaluation

One day before the experiment, the keratinocytes (Cell name: HaCaT obtained from ATCC) were dispensed into 96 well plates at 4x10 ⁴ cells / well, followed by incubation for 24 hours in a 37 ° C, 5% CO ₂ incubator. It was. After 24 hours, wash 96 well plates twice with Hanks'Balanced Salt solution (HBSS) buffer, and then add reaction buffer (2 μM Fluo-4-AM, 20% pluronic acid, 2.5 mM probenecid) to the cells. gave. After reaction at 37 ° C., 5% CO ₂ incubator for 30 minutes, at room temperature for 30 minutes, wash twice with HBSS buffer and with Ginsenoside Mc at 0.05%, 0.1%, 0.5% and 1.0% concentrations (wt%), respectively. Cells were treated.

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. Intracellular Ca ²⁺ concentration change was measured using FlexStation3 (Molecular Device, USA). After the ginsenoside Mc and 2U / ml Trypsin or 5 μM PAR-2 active peptide (SLIGKV) treatment, the flex was measured for 80 seconds to determine the difference between the minimum and maximum values. The percent inhibition of intracellular influx of calcium ions compared to the difference between the minimum and maximum values treated with 2U / ml trypsin or 5 μM PAR-2 active peptide (SLIGKV) is shown in Table 23 below.

Ginsenoside Mc Concentration Inhibition rate on the cellular influx of calcium ions (%) Trypsin (2U / ml) PAR-2 active peptide (5 μM) 0.05% 25 27 0.1% 30 33 0.5% 41 41 1.0% 45 49

In the results of Table 23, the influx of intracellular calcium ions by trypsin or PAR-2 active peptide (SLIGKV) is reduced by the treatment of ginsenoside Mc, increasing the concentration of ginsenoside Mc, the intracellular calcium It can be seen that the influx of ions is significantly reduced.

Therefore, the external preparation composition for skin containing ginsenoside Mc 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 24. 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 (% by weight) 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 Ginsenoside Mc 5.0 - Polyquaternium-10 0.2 0.2 Blue No. 1 0.0002 0.0002 Yellow No. 4 0.0001 0.0001 Methylparaben 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 19 Dandruff Reduction Effect Test

Twenty-four males aged 19 to 35 years with relatively high dandruff were selected to use 12 groups of shampoos of Formulation Example 4 and Comparative Formulation Example 5 for 1 month in the following manner, and then the rate of dandruff reduction was measured. .

Triturate with a normal shampoo before the start of the test, and collect the dandruff accumulated for 2 days after the shampoo, triturate once every 2 days with the weight of the collected dandruff and the shampoo of Formulation Example 4 and Comparative Formulation Example 5, 2 days after the end of the test. The weight of accumulated dandruff was evaluated. At this time, the accumulated dandruff was collected directly from the scalp with a vacuum suction device, to obtain a dandruff reduction rate based on Equation 7 below and the results are shown in Table 25 below.

Formulation Example 4 Comparative Formulation Example 5 Dandruff reduction rate (%) 63.7
55.2
73.1
63.2
45.9
54.8
66.8
73.5
55.2
62.3
66.7
55.8 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 61.35 2.5 SD 8.21 2.2

In the result of Table 25, it can be seen that the formulation example 4 containing ginsenoside Mc exhibits an excellent anti-dandruff effect.

Test Example 20 Scalp Itching Prevention Test

Twenty-four men and women aged 25 to 45 who feel severely itchy scalp were selected and divided into two groups of 12 people each. The shampoos of Formulation Example 4 and Comparative Formulation Example 5 were used once every three days for two weeks. The prevention effect was evaluated through the following evaluation criteria and the results are shown in Table 26 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 4.2 2.3

From the results of Table 26, it can be seen that the case of Formulation Example 4 containing ginsenoside Mc has a better effect in preventing scalp itching.

Test Example 21 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 ℃. NIH3T3 was placed in a 96-well plate and incubated in a 37 ° C. incubator for 24 hours, treated with 2.5 mM tolbutamide, and 10 minutes later, 2.5 ppm, 5 ppm and 10 ppm of 10 μM of minoxidil and ginsenoside Mc, respectively, were positive controls. The cell proliferation was measured using the WST-1 kit (Roche) after 48 hours of drug treatment. The results are shown in Table 27 below.

division Cell proliferation (%) Untreated Control 100 Minoxidil 132 Ginsenoside Mc (2.5 ppm) 115 Ginsenoside Mc (5 ppm) 123 Ginsenoside Mc (10 ppm) 130

In the results of Table 27, it can be seen that the treatment of ginsenoside Mc, the proliferation of fibroblasts, the cell proliferation capacity is increased depending on the concentration of the treated ginsenoside Mc, 10ppm ginsenoside Mc In the case of treatment with, it can be confirmed that the proliferation of cells is restored to the level when minoxidil is treated.

[Test Example 22] Ginsenoside Mc melanin production promoting effect test

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 ginsenoside Mc at a final concentration of 10 ppm or 50 ppm as a test substance, 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 dissolved. 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 ginsenoside Mc with the control is shown in Table 28 below.

sample Melanin Synthesis (%) DMSO (0.1%) 100 IBMX (100 μM) 120 Ginsenoside Mc (10 ppm) 112 Ginsenoside Mc (50 ppm) 121

In the results of Table 28, it can be seen that ginsenoside Mc promotes melanin synthesis of melanocytes, melanin production is increased, showing an excellent melanin production promoting effect.

Test Example 23 Effect of Ginsenoside Mc on Melanocite Promotes MITF and Tyrosinase Expression

Dispense 500,000 cells / well in a 6-well microtiter plate using a 501mel cell line, in each hole 0.1% DMSO for negative control, 100 μM for positive control, and ginsenoside for test. Mc was treated with 10 ppm to obtain proteins after incubation for 24 hours, 48 hours, 72 hours at 37 ℃ temperature. The protein thus obtained was subjected to western blot using MITF and tyrosinase antibody. Protein extraction and western blot were typically performed by standard methods used by those skilled in the art. The result after Western blot is shown in Table 29 below by comparing the negative control to 100.

MITF Tyrosinase IBMX Ginsenoside Mc IBMX Ginsenoside Mc 24hr 121 98 160 150 48hr 98 113 149 153 72hr 224 121 298 188

In the results of Table 29, it can be seen that ginsenoside Mc increases the expression of MITF and tyrosinase protein in melanocytes.

 Test Example 24 Evaluation of Antibacterial Activity of Ginsenoside Mc

Antimicrobial experiments were conducted to evaluate the antimicrobial activity of ginsenoside Mc. The specific experimental method is as follows.

Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa strains used in the experiments were tested on Tryptic Soy Broth. Candida albicans and Aspergillus niger strains were cultured in Sabouraud Dextrose Broth. Incubate 1/100 (Candida albicans) in each medium 1/10) of the strain was used as the test bacteria. Aspergillus niger used a spore suspension prepared to be 2 × 10 ⁸ cfu / ml as the test cell solution.

0.15 ml of the test bacteria was added to 15 ml of each medium, and a well mixed solution was used as a dilution solution.

16 μl of ginsenoside Mc 10 ppm was added to the first row of a 96 deep well plate, and 184 μl of the diluted solution was added thereto. The remaining wells were added with 100 μl of dilution solution. The mixture was well mixed in the first row, and then 100 μl of the mixed solution was added to the second row and mixed well. Then, 100 μl was again taken and placed in the third row.

Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, in a 32 ° C. thermostat. niger) was incubated in a 25 ℃ thermostat.

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 30 below.

Sample Minimum Inhibitory Concentration (MIC),% Sedonas
Aruginosa Staphylococcus aureus Escherichia Coli Candida Albicans Aspergillus Niger Ginsenoside Mc 0.3 0.04 0.148 > 3 > 1

In the results of Table 30, it can be confirmed that ginsenoside Mc exhibits antimicrobial activity against various strains, through which it can be predicted that ginsenoside Mc can act as a natural preservative, or antimicrobial agent in the composition.

Claims (22)

delete delete delete Cosmetic composition for external application for skin comprising ginsenoside Mc as an active ingredient, wherein the composition is for anti-aging cosmetic composition. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is a skin external preparation cosmetic composition for enhancing skin elasticity. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is a skin external preparation cosmetic composition, characterized in that for improving skin wrinkles. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is a moisturizing cosmetic composition for external use. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is a skin external preparation cosmetic composition for enhancing skin barrier function. A composition for external application for skin comprising ginsenoside Mc as an active ingredient, wherein the composition is for inducing skin differentiation of keratinocytes. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is an external skin cosmetic composition for improving acne. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is an external preparation cosmetic composition for antibacterial use. Cosmetic composition for external application for skin comprising ginsenoside Mc as an active ingredient, wherein the composition is for anti-inflammatory skin composition. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is an external skin cosmetic composition for improving atopic skin. Cosmetic composition for external application for skin comprising ginsenoside Mc as an active ingredient, wherein the composition is for improving color and skin tone. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is a skin external preparation cosmetic composition, characterized in that for reducing pores. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is a skin external preparation cosmetic composition, characterized in that it is for sebum suppression. Cosmetic composition for external application for skin comprising ginsenoside Mc as an active ingredient, wherein the composition is for improving skin troubles. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is a skin external preparation cosmetic composition for whitening. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is a skin external preparation cosmetic composition for promoting hair growth. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is a skin external preparation cosmetic composition, characterized in that for preventing white hair. A skin external preparation cosmetic composition comprising ginsenoside Mc as an active ingredient, wherein the composition is an external anti-dandruff cosmetic composition. Cosmetic composition for external application for skin comprising ginsenoside Mc as an active ingredient, wherein the composition is for natural preservatives.