TW201521737A - Skin external composition containing ginsenoside Rh4 - Google Patents

Abstract

The disclosure relates to a skin external composition including Ginsenoside Rh4. To be more specific, the disclosure relates to a composition having Ginsenoside Rh4 which through its antioxidative activities not only provides effects of improving skin problems, such as skin anti-aging effects, skin moisture improvement, anti-inflammatory effects, and acne syndrome relief, and effects of improving overall skin conditions, such as whitening, sebum regulation, pore astringency, and complexion improvement by improving blood circulation, but also provides effects of improving scalp and hair conditions, such as anti-dandruff effects, hair-growth promotion, and grey hair prevention.

Description

Skin external composition containing ginsenoside Rh4

The present invention relates to a composition other than ginsenoside Rh4, and more particularly to a skin anti-aging effect and skin moisturizing improvement by containing ginsenoside Rh4 through its excellent antioxidant power. Anti-inflammatory effect, anti-inflammatory effect, skin problems such as hemorrhoids, whitening effect, sebum regulating effect, pore contraction effect, improvement of skin condition through improvement of blood circulation, etc., can also provide anti-dandruff effect and promotion A composition for improving the scalp and hair condition such as hair growth effect and white hair prevention effect.

The skin of human beings is the first layer of protective film of the human body. It functions to protect organs in the body in the stimulation of external environment such as changes in temperature and humidity, ultraviolet rays, and public nuisance substances. It experiences various internal and external changes with age. It is necessary to change. That is, in terms of internal factors, the secretion of various hormones used to regulate metabolism is reduced, the function of immune cells and the activity of cells are reduced, resulting in a decrease in the biosynthesis of immune proteins and human structural proteins required by the human body; Due to the destruction of the ozone layer, the amount of ultraviolet light reaching the surface of the sun's rays increases, and as the environmental pollution gradually increases, the free radicals and active oxygen increase, resulting in a decrease in skin thickness, an increase in wrinkles, and a decrease in elasticity. Lead to skin The color becomes dull, and skin problems often occur, which increase chloasma and freckles as well as age spots, and the color of the skin becomes darker and darker, resulting in various changes.

In order to prevent changes in the skin state caused by the internal and external causes of the skin and to maintain a healthy skin condition, an attempt is made to add bioactive substances extracted from various known animals, plants, microorganisms, etc. to the cosmetics. Improve skin condition. In particular, research and development of cosmetics using ginseng (Panax ginseng C.A Meyer) ingredients have been carried out many times. The efficacy of ginseng on the skin has long been recognized and widely used in cosmetic compositions. Roots or leaf extracts of ginseng, ginsenoside, aglycon aglycon and ginseng polysaccharides have been used in cosmetics. . However, such an active material containing a ginseng extract, an extract, a polysaccharide, or the like contains a very small amount of active material, and has a disadvantage in comparison with other raw materials: the effect is not remarkably excellent.

In view of this, the present inventors have found that ginseng saponin Rh4 containing ginseng not only provides anti-aging, skin wrinkle, whitening, moisturizing effect, and acne and skin problems, but also provides skin color improving effect, sebum regulation, The skin condition improving effect such as the pore contraction effect can also provide the scalp and hair condition improving effects such as anti-dandruff, hair growth and white hair prevention, and the present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for external use of skin which can improve the overall state of the skin by containing ginsenoside Rh4.

In order to achieve the above object, the present invention provides a skin external preparation composition comprising ginsenoside Rh4 as an active ingredient.

Moreover, the present invention provides a composition for external use of an anti-aging skin which comprises ginsenoside Rh4 as an active ingredient.

Moreover, the present invention provides a composition for external use of a whitening skin containing ginsenoside Rh4 as an active ingredient.

Moreover, the present invention provides a composition for external use of a moisturizing skin comprising ginsenoside Rh4 as an active ingredient.

Moreover, the present invention provides a skin external preparation composition for acne improvement comprising ginsenoside Rh4 as an active ingredient.

Moreover, the present invention provides a composition for external use of skin containing ginsenoside Rh4 as an active ingredient, and a skin color and skin color improving composition thereof.

Further, the present invention provides a composition for external use of a skin containing pores for contracting ginsenoside Rh4 as an active ingredient.

Moreover, the present invention provides a composition for external use of a skin for regulating sebum containing ginsenoside Rh4 as an active ingredient.

Further, the present invention provides a composition for external use of an anti-dandruff skin containing ginsenoside Rh4 as an active ingredient.

Moreover, the present invention provides a composition for external use of skin for hair growth promotion comprising ginsenoside Rh4 as an active ingredient.

Moreover, the present invention provides a composition for external use of skin for preventing white hair comprising ginsenoside Rh4 as an active ingredient.

Further, the present invention provides a composition for external use of skin using ginsenoside Rh4 as a natural preservative.

The composition of the present invention, by containing ginsenoside Rh4, can provide skin anti-aging effect, skin moisturizing effect improving effect, anti-inflammatory effect, acne and other skin problem improving effect, whitening effect, sebum through its excellent antioxidant power. The overall skin condition improvement effect such as the adjustment effect, the pore contraction effect, and the improvement of the color of the blood circulation can be improved, and the scalp and hair state improvement effects such as anti-dandruff effect, hair growth promoting effect, and white hair prevention effect can be provided.

The composition according to the present invention contains ginsenoside Rh4 as an active ingredient.

The ginsenoside Rh4 used in the present invention has the structure of the following Chemical Formula 1.

[Chemical Formula 1]

Ginseng belongs to the genus Panax species of Araliace and is a herbaceous plant that is perennial and semi-inferior. It is produced in Korea, China, Japan and the United States. The main production areas are East Asia; east longitude 85-140 degrees, north latitude 22-49 degrees, North America; west longitude 70-97 degrees, north latitude 34-47 degrees. Thus, ginseng produced in various parts of the world differs in composition, and Korean ginseng contains saponin which is the most important ginseng ginseng (Ginsenoside) which exhibits the most important physiological and pharmacological effects of ginseng. About 34 kinds of ginsenosides have been isolated so far, and each has different pharmacological effects depending on the type of sugar bound to the aglycon or the number of binding saccharides or the binding position. According to structural characteristics, it can be classified into a ginseng diol system, a ginseng triol system, and an oleanane system. Further, the aroma components inherent in ginseng are panacen, polyacetylene, polyphenol, flavonoid, and vitamins.

Ginseng has been recognized for its efficacy in Korea since ancient times and has been used in the treatment of various diseases. Ginseng is known to have physical strength, metabolic improvement, and stress relief in Korean efficacy. In addition to, diabetes treatment, improvement of respiratory diseases, improvement of digestive organs and anti-cancer effects, ginseng has been reported in recent studies to have anti-complement activity, anti-ulcer effect, immune enhancement, anti-cancer effect and hypoglycemic effect. The following studies have been published: on skin effects and anti-inflammatory effects (Korean J. Dermatol. 18(1): 39-42 (1980), Korean J. Dermatol. 14(4): 335-339 (1976)), Hyperkeratosis (Korean J. Dermatol. 28(4): 434-440 (1990)), prevention and treatment of acne (Korean J. Dermatol. 30(1): 27-33 (1992), Korean J. Dermatol. 28(4): 434-440 (1990)), Proceedings of the 2nd international ginseng symposium 13-17 (1978), increased elasticity and hydration (Fitoterapia 57 (1) ): 15-28 (1986), Fitoterapia 57 (4) 217-222 (1986)), wound healing (Brit. J. Pharmacol. 125: 255-262 (1998), Arch. pharm.res. 25 (1) :71-76 (2002)), inhibition of collagen breakdown (Mol. Cells 9 (5): 476-483 (1999)), whitening effect (Journal of the society of cosmetic scientists of korea 27 (2): 45-56 (2001)) and so on.

The ginsenoside Rh4 of the present invention can be extracted from plants, and can also be synthesized according to the methods disclosed in the art, and commercially available ginsenoside Rh4 can also be used. Further, in view of the technical level in the art, it is understood by those skilled in the art that the above-mentioned ginsenoside Rh4 exhibits skin and hair among derivatives obtained by substitution or substitution reaction which is conventionally carried out in the art. Derivatives which improve the effect and the antiseptic effect are also included in the scope of the present invention.

The ginsenoside Rh4 used in the present invention is particularly obtainable from ginseng extract. At this time, the type of ginseng used is not particularly limited, and ginseng, red ginseng, white ginseng, taiji ginseng, and tail ginseng can be used. Further, the above ginseng extract includes all leachate obtained by leaching and decocting from ginseng, by part or all of the leachate a concentrate obtained by concentration again, or a body, a decoction, an elixir, a fluid extract prepared by drying the above-mentioned concentrate, and a chemical substance which is mainly contained in ginseng, and includes a plant thereof In itself, the extract may be taken from all parts of ginseng such as stems, roots, leaves, flowers, fruits, etc., and is not limited to an extract of a specific part. Further, the method of extracting ginsenoside Rh4 from ginseng extract can be carried out by the disclosed method.

Specifically, the above ginsenoside Rh4 can be isolated by extracting the ginseng extract from ginseng 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, diethyl ether, ethyl acetate, chloroform, and a mixed solvent of the organic solvent and water, and preferably 80% ethanol is used. At this time, preferably, the extraction temperature is from 10 ° C to 80 ° C, and extraction is possible for 3 hours to 24 hours. Exceeding the above extraction temperature and extraction time will result in a decrease in extraction efficiency or a change in composition.

Preferably, since the ginsenoside Rh4 is contained in the composition of the present invention in an amount of from 0.001% by weight to 50% by weight based on the total mass of the composition, preferably from 0.01 to 30% by weight, more preferably from 0.1 to 10% by weight. If the content of the above ginsenoside Rh4 is less than 0.001% by weight, the efficacy and effect of the above components are weak; and if the content of the above active ingredient exceeds 50% by weight, problems occur in skin safety or dosage form.

Since ginsenoside Rh4 is a component having excellent antioxidant power, the composition of the present invention containing ginsenoside Rh4 provides excellent antioxidant power and can be used as a composition for external use of skin for anti-aging, which increases skin elasticity, The effect of improving wrinkles is outstanding.

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

The composition of the present invention can be used as a composition for external application for moisturizing skin, which can strengthen the skin barrier function and induce differentiation of skin keratinocytes. Thereby, it can be effectively used as a composition for external preparation of the skin, thereby preventing or improving skin dryness, contact dermatitis or acne due to incomplete differentiation of the epidermis.

The composition of the present invention can be used as a composition for external application of acne for skin improvement, and has excellent antibacterial effect, particularly an antibacterial effect against acne-causing bacteria, and an anti-inflammatory effect.

The composition of the present invention can be used as a composition for external use of skin for improving color and skin color, and is suitable for use in the skin, thereby expanding the capillaries and promoting blood circulation to ensure smooth absorption of nutrients by the skin, thereby suppressing aging, and its color and Excellent skin tone improvement.

The composition of the present invention can be used as a composition for external use of skin for pore contraction, sebum regulation and skin problem improvement, and is suitable for the elimination of active oxygen and collagen by suppressing sebum secreted by excess after application to the skin. Synthetic, thereby shrinking the pores, which has a prominent effect of suppressing the skin problem by reducing the expression of inflammatory factors. Moreover, due to excellent antioxidant power, skin irritation can be prevented.

The composition of the present invention can be used as a composition for external use of anti-dandruff skin, which effectively excretes toxins accumulated in hair and scalp, purifies the scalp, and suppresses the proliferation and growth of dandruff bacteria, thereby preventing scalp inflammatory reaction. Moreover, it has an outstanding anti-oxidation effect against the formation and action of active oxygen, thereby providing an effect of soothing and strengthening the scalp and strengthening the inherent defense ability.

The composition of the present invention can be used as a composition for external use of skin for promoting hair growth, which promotes a hair cycle into a prolonged hair cycle by promoting a rest period Into the growth of hair and prevent hair loss.

The composition of the present invention can be used as a composition for external use of skin for preventing white hair, which can suppress white hair and promote black by significantly enhancing the expression of the small eye deformity-related transcription factor (MITF) of melanocytes. It takes a long time.

Further, the ginsenoside Rh4 used in the composition for external use of the skin of the present invention can provide the effect of a natural preservative.

The skin external preparation composition of the present invention described above can be formulated as a cosmetic composition, and can be formulated by a medium or mechanism which is allowed by cosmetic or dermatological means. It is suitable for all dosage forms suitable for topical application, for example, an emulsion, a suspension, a microemulsion, a microcapsule, a fine particle ball which can be obtained by dissolving a solution, a gel, a solid, a stirred anhydrous product, and dispersing a milk in a water form. Or in the form of ionic (liposomes) and nonionic vesicular dispersing agents, or in the form of creams, lotions, lotions, powders, ointments, sprays or concealers. provide. Alternatively, it can be used in the form of a foam (Foam) or an aerosol composition containing a compressed propellant. These compositions can be prepared in the usual manner in the art.

In particular, the skin external preparation composition of the present invention can be formulated into a composition for scalp and hair as a composition for preventing dandruff, promoting hair growth or preventing white hair, and the dosage form is not particularly limited, for example, It is formulated into a conditioner, hair nutrient water, softener, hair cream, shampoo, hair lotion, hair cream or scalp hair.

Further, the composition according to the present invention may contain a fatty substance, an organic solvent, a solvent, a concentrate, a gel, a softener, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, and a surface. Active agent, water, ionic Or nonionic emulsifiers, fillers, metal ion chelating agents, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipids A vesicular or an adjuvant commonly used in the field of cosmetic or dermatological science, as is commonly used in cosmetics. The amount of the above adjuvant used is based on the amount generally used in the field of cosmetic or dermatological science.

Further, the composition of the present invention may contain a skin absorption promoting substance in order to increase the skin-improving effect.

Hereinafter, the structure and effects of the present invention will be more specifically described by way of test examples and dosage forms. However, the test examples and the formulation examples are provided for the purpose of facilitating the understanding of the present invention, and the scope and scope of the present invention are not limited to the following examples.

[Reference Example 1]

The ginsenoside Rh4 used to test the efficacy of the composition of the present invention was purchased from the AMBO Institute.

[Test Example 1] Reactive oxygen species (ROS) produced an inhibitory effect

Keratinocytes isolated from human epidermal tissue were placed in a 24-well plate at a rate of 5 × 10 ⁴ per well, and allowed to adhere for 24 hours. After 16 hours, ginsenoside Rh4 was treated at 1%. At this time, for comparison, the control group did not treat ginsenoside Rh4. After 2 hours, the culture solution was removed, and 100 μl of phosphate buffer (PBS) was placed in each well. After irradiating ultraviolet rays of 30 mJ/cm ² with ultraviolet B (UVB) lamps (Model: K5T8, UV B 15 W, Sankyo Dennki, Japan) on the keratinocytes, PBS was taken out, and keratinocyte culture solution 200 was added to each well. Ll . The ginsenoside Rh4 is treated again here, and the amount of active oxygen species which is increased by ultraviolet stimuli in each certain time band is quantified. The amount of ROS is quantified by the method of Tan (Tan et al., 1998, J. Cell Biol. Vol. 141, pp 1423-1432), and Tan's method is to measure the fluorescence of DCF-DA (dichlorofluorescin diacetate) oxidized by ROS. The results calculated for the ratio of light to ROS of the control group which only treated the excipients are shown in Table 1.

It can be seen from the results of the above Table 1 that the ginsenoside Rh4 of the present invention can effectively suppress the generation of known ROS which cause skin cell damage by ultraviolet rays, and can not irradiate ultraviolet rays with the amount of ROS after ultraviolet stimulation. The above level inhibits the production of ROS, so that its antioxidant performance is excellent. Therefore, it has been confirmed that the ginsenoside Rh4 of the present invention can prevent pore enlargement by inhibiting oxidation and preventing aging, and can improve skin problems by preventing skin irritation.

[Test Example 2] Elastase activity inhibition potency

The elastase activity inhibiting ability of ginsenoside Rh4 was determined by comparison with epigallocatechin gallate (EGCG). Elastase The substrate was commercially purchased from Sigma-Aldrich (Cat. No. E0127).

The elastase activity inhibition was tested by the following test method.

In a 96-well plate, 200 μl of ginsenoside Rh4 and 50 μL of 20 μg/mL elastase ̇ type III solution were mixed in 10 mg/L Tris-HCl buffer (pH 8.0). . Epigallocatechin gallate (EGCG) 250 μM was used as a positive control group, and distilled water was used as a non-treated group of the negative control group. Then, 100 μL of 0.4514 mg/mL N-SUCCINYL-ALA-ALA-ALA-p-NITROANILIDE formulated with the above buffer was added, and the mixture was reacted at 25 ° C for 15 minutes. After the end of the reaction, the absorbance at a wavelength of 415 nm (Synergy 2, BioTek (VT, USA)) was measured. A blank test was performed by the same method to make corrections.

The calculation method for the inhibition of elastase activity is shown in the following formula 1, and the results are shown in Table 2 below.

[Formula 1] Elastase activity inhibition rate (%) = {1 - (C - D) / (A - B)} × 100

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

B: Absorbance at a wavelength of 415 nm in the absence of addition of the test substance and no addition of the enzyme

C: absorbance at a wavelength of 415 nm in the addition of the test substance, enzyme addition

D: absorbance at a wavelength of 415 nm in the addition of the test substance and no addition of the enzyme

From the results of Table 2 above, it was found that the degree of inhibition of elastase activity of ginsenoside Rh4 was significantly better than that of epigallocatechin gallate (EGCG) which is an inhibitor of elastase activity, thereby confirming this. The ginsenoside Rh4 of the invention has excellent elastase activity inhibitory effect.

[Test Example 3] Collagenase (MMP-1) inhibitory ability

The collagenase production inhibiting ability of the ginsenoside Rh4 of the present invention is determined by comparison with Retinoic acid.

Human fibroblasts were added to a 96-well microtiter plate in a Dulbecco's Modified Eagle's Media (DMEM) medium containing 2.5% fetal bovine serum. In order to reach 5,000 cells/well, it was cultured in a 5% carbon dioxide, 37 ° C incubator until it grew by about 70-80%. After ginsenoside Rh4 or retinoic acid (Retinoic acid) was treated at a concentration of 10 μg/ml for 24 hours, the cell culture solution was extracted.

The degree of collagenase production of the extracted cell culture medium was measured by a commercially available collagenase measuring instrument (American Amway Biotech Co., Inc., Catalog #: RPN 2610). First, the extracted cell culture solution was placed in a 96-well plate, which was uniformly coated with a collagenase antibody, and subjected to an antigen-antibody reaction for 3 hours in a thermostat. After 3 hours, the secondary collagen antibody bound to the chromogenic group was added to a 96-well plate and reacted again for 15 minutes. After 15 minutes, the color-inducing substance (3, 3', 5,5'tetramethylbenzidine, sigma), induced coloration at room temperature for 15 minutes, 1 M sulfuric acid was added again to stop the chromogenic reaction, and the color of the reaction solution was yellow, depending on the extent of the reaction, the degree of yellowness was varied.

The absorbance of a yellow 96-well plate was measured at 405 nm by a light absorption meter, and the degree of synthesis of collagenase was calculated by the following formula 2, and the results are shown in Table 2. At this time, the reaction absorbance of the cell culture solution extracted from the group which did not treat the composition was used as a control group.

[Formula 2] Degree of expression of collagenase (%) = absorbance of substance-treated cell group / absorbance of control group × 100

From the results of the above Table 3, it is known that the degree of collagenase expression of ginsenoside Rh4 is similar to that of the known retroactive acid which is a collagenase inhibitor, similar to the inhibition effect of collagenase. .

From such a result, it is understood that the ginsenoside Rh4 of the present invention has a detrimental effect on the matrix metalloproteinase (MMP-1) and reduces the decomposition of collagen inside the skin, thereby having an anti-aging effect.

[Formulation Example 1 and Comparative Formulation Example 1]

According to the composition of Table 4 below, a nutrient cream (unit: % by weight) was prepared by a usual method.

[Test Example 4] Confirmation of skin elasticity enhancing efficacy

In order to confirm the skin elasticity increasing effect against the human body, the following Formulation Example 1 and Comparative Formulation Example 1 were evaluated as follows.

Twenty healthy women in the age group of 30 to 40 years old were divided into two groups in the group of 10, which corresponded to the two groups of the dosage form example 1 and the comparative dosage form, respectively, and the nutrition cream was applied to the subjects. The skin of the face was measured once a day for 12 weeks, and the skin elasticity was measured by a skin elasticity measuring instrument (Cutometer SEM 575, C+K Electronic Co., Germany). The results are shown in Table 5 below. The results shown in Table 5 are reported as the ΔR8 value of Cutometer SEM 575, and the R8 value indicates the nature of skin viscoelasticity.

From the results of the above Table 5, it was found that the dosage form 1 containing the ginsenoside Rh4 of the present invention further increased the skin elasticity compared to the group of the comparative comparative dosage form 1.

Thus, it was confirmed that the composition containing the ginsenoside Rh4 of the present invention is very effective for increasing skin elasticity.

[Test Example 5] Confirmation of skin wrinkle improvement performance

In order to confirm the wrinkle-improving effect of the composition of the present invention, the above-mentioned Formulation Example 1 and Comparative Formulation Example 1 were used.

In order to confirm the wrinkle improving effect of the above Formulation Example 1 and Comparative Formulation Example 1, evaluation was carried out as follows. Twenty healthy women in the 40-year-old age group were divided into two groups in the group of 10, which corresponded to the two groups of the dosage form example 1 and the comparative dosage form, respectively, and the nutrient cream was applied to the face of the subject. The replica was extracted by gelatin once a day for 12 consecutive weeks, and was measured by a skin measuring instrument (visiometer, SV600, Courage+Khazaka electronic GmbH, Germany) and subjected to image analysis. The results are shown in Table 6 below. The values in Table 6 below represent the average of the values of the parameters before the smearing were removed from the parameter values after 12 weeks of application.

R1: the difference between the highest value and the lowest value of the wrinkle contour line R2: arbitrarily divide the wrinkle contour line into 5 grids, wherein the average value of each R1 value R3: after dividing into 5, the highest value of R1 value R4: in wrinkles In the baseline of the contour line, the average value of the respective peaks and valleys is removed. R5: In the baseline of the wrinkle contour, the average value of the respective wrinkle contours is removed.

From the results of the above Table 6, it is understood that the agent composition of the dosage form of Example 1 has an effect of improving skin wrinkles. excellent.

[Test Example 6] Tyrosinase inhibitory effect

Tyrosinase was extracted from the mushroom using tyrosinase prepared by SIGMA. First, a tyrosine as a substrate was dissolved in distilled water to prepare a solution of 0.3 mg/ml, and the solution was added to a test tube in an amount of 1.0 ml, respectively, and then 1.0 ml of a potassium-phosphate buffer solution (0.1 mol concentration) was added thereto. , pH 6.8) and 0.7 ml of distilled water.

0.2 ml of a sample liquid of ginsenoside Rh4 prepared by mixing at an appropriate concentration was added to the ethanol reaction solution of the present invention, and reacted in a thermostat at 37 ° C for 10 minutes. At this time, 0.2 ml of the solvent was placed in place of each sample solution, and the mixture was placed in a control group, and ascorbic acid was used as a positive control group. To the reaction solution, 2500 units/ml of tyrosinase solution was added in an amount of 0.1 ml each time, and reacted again in a thermostat at 37 ° C for 10 minutes. The test tube to which the reaction solution was added was placed in ice water, rapidly cooled and the reaction was stopped, and the absorbance at a wavelength of 475 nm (Synergy 2, BioTek (VT, USA)) was measured by a photoelectric spectrum analyzer, and the results are shown in the following table. 7. The respective tyrosinase inhibitory effects are calculated by the following formula 3 Out.

[Formula 3] Tyrosinase inhibition rate (%) = 100 - (reaction absorbance of test substance / reaction absorbance of control group) × 100

From the results of the above Table 7, the tyrosinase of the ginsenoside Rh4 according to the present invention has a much higher inhibition rate than the ascorbic acid which is known as a tyrosinase inhibitor, and thus the whitening effect is excellent.

[Test Example 7] Inhibition of melanin production by B16/F10 melanoma cells

A sample containing 0.1% by weight of each of ginsenoside Rh4 and kojic acid was used as a test substance, and this was added to a culture solution of B16/F10 melanoma cells (Korean cell line bank) at a predetermined concentration for 3 days. After the culture, the culture solution was removed, washed with phosphate buffered saline (PBS), and the cells were lysed with 1 N sodium hydroxide (NaOH), and the absorbance was measured at 405 nm (Synergy 2, BioTek (VT, USA)). The cells to which no test substance was added were used as a control group, and the degree of melanin production inhibition of each test substance was measured by comparison with the melanin content in the control group. The melanin production inhibition rate was calculated according to Formula 4, and the results are shown in Table 8.

[Formula 4] Melanin production inhibition rate (%) = 100 - (absorbance of test substance / control group Absorbance) × 100

From the results of the above Table 8, the ginsenoside Rh4 according to the present invention has a melanin production inhibition rate much higher than that of the known Kojic acid as a melanin production inhibitor, and it is found that the whitening effect is excellent.

[Test Example 8] Determination of skin moisturizing effect

In order to determine the effect of ginsenoside Rh4 on the skin moisturizing power, the above Formulation Example 1 and Comparative Formulation Example 1 were used and evaluated as follows.

Twenty adult males and females of the dry skin type of 40 to 50 years old were divided into two groups in the form of 10 in each group, which corresponded to the two groups of the dosage form example 1 and the comparative dosage form example 1, respectively, and the nutrient cream was added. Apply to the subject's face twice a day for 12 weeks. Before the start of application, after 1 week of application, after 2 weeks of application, after 4 weeks of application, and after 2 weeks of smearing (6 weeks in total), under constant temperature and humidity conditions (24 ° C, relative humidity 40%), by The skin moisture content was measured by a skin moisture measuring instrument (Corneometer CM825, C+K Electronic Co., Germany). The results are shown in Table 9 below. The results in Table 9 show the percentage of the increase in the measured value after the treatment for a predetermined period of time, based on the measured value of the skin moisture measured on the eve of the test.

From the above Table 9, when the comparative dosage form example 1 is applied, the fourth week of the application is cut off, which shows a moisture increase rate of about 30%, and the skin moisture content decreases after the application is stopped; on the contrary, When the dosage form 1 containing ginsenoside Rh4 was applied, the skin moisture increase rate of most of 30% or more was also exhibited after the application was stopped. Thereby, it is understood that the composition of the present invention containing ginsenoside Rh4 has excellent skin moisturizing effect.

[Test Example 9] Determination of keratinocyte differentiation promoting effect

In order to understand the differentiation promoting effect of keratinocytes of ginsenoside Rh4, the amount of CE (Cornified Envelop) generated at the time of differentiation of keratinocytes was measured by absorbance in the following manner.

First, the cultured human keratinocytes are placed in a culture flask and attached to the bottom. The keratinocytes are isolated from the epidermis of the newborn, and then the ginsenoside Rh4 is treated to a culture solution at a concentration of 5 ppm. Incubate for 5 days until the cells grow to about 70-80% of the bottom area. At this time, the low calcium (0.03 mM) treatment group and the high calcium (1.2 mM) treatment group were used as a negative control group and a positive control group, respectively. Then, the above-cultured cells were extracted and washed with Phosphate buffered saline, and 1 ml of a 10 mM concentration of 2% SDS (sodium dodecyl sulfate) and 20 mM DTT (Dithiothreitol) was added. Performing sonication, boiling, centrifugation with hydroxymethylaminomethane hydrochloride buffer (Tris-HCl, pH 7.4), resuspending the pellet in 1 ml of phosphate buffer Liquid (PBS), absorbance was measured at 340 nm (Synergy 2, BioTek (VT, USA)). Further, the above-mentioned solution after completion of the ultrasonic treatment was partially extracted, and the protein content thereof was measured, which was used as a reference for evaluating the degree of cell differentiation. The results are shown in Table 10.

From the results of the above Table 10, it was found that when ginsenoside Rh4 was used, the differentiation promoting effect of keratinocytes was excellent.

[Test Example 10] Determination of skin barrier function recovery effect

In order to determine the effect of ginsenoside Rh4 on the recovery of damaged skin barrier function due to skin injury, the following experiment was performed. The skin barrier damage was applied to 10 adult male and female upper arms by the Tape Stripping method, and the two groups of the medicated dosage form 2 and the comparative dosage form 2 were respectively prepared by the composition of the following Table 11, The degree of recovery from the epidermal water loss rate (TWEL) was measured and compared by Vapometer (Delfin, Finland) once a day for 7 consecutive days. Among them, Comparative Formulation Example 2 was used as a negative control group, which was a vehicle. The test results are shown in Table 12 below. The results in Table 12 compare the treatment differences before barrier damage and after barrier damage on a 100% basis.

From the results of Table 12 above, if the comparative dosage form 2 which does not contain ginsenoside Rh4 is treated, the transepidermal water loss rate gradually increases with time; on the contrary, if the dosage form containing ginsenoside Rh4 is treated, Then, the epidermal water loss rate quickly returns to normal, and the barrier damage is restored.

[Test Example 11] Gas color improvement effect

In order to evaluate the skin blood circulation promoting effect of the cosmetic composition according to the present invention, blood circulation of the skin was measured by a laser Doppler Perfusion Imager (Periscan PIM II, Perimed (stochholm, Sweden)). degree. A laser Doppler blood flow imager is a commonly known instrument for measuring blood circulation in the skin, which is currently used to measure not only the speed and amount of blood in the capillaries of the skin, It can also measure the flow in small arteries and venules, which is a very sensitive device.

In a constant temperature and humidity chamber, after washing with a soap, relax for 30 minutes, and the initial value was measured by a laser Doppler blood flow imager. First, the initial blood flow of the lower part of the forehead was measured by a laser Doppler blood flow imager for 30 women who usually had cold hands and feet. Then, the subjects were arranged to use the above-mentioned Formulation Example 1 and Comparative Formulation Example 1 in one week, and the measured blood flow rate and the above-described initial measurement values were compared, and the results (changes in skin blood flow rate) are shown in Table 13 below.

[Table 13]

From the results of the above Table 13, the cosmetic composition according to the present invention significantly increased the skin blood flow rate compared to the comparative dosage form Example 1 which did not contain ginsenoside Rh4, and it was found that the color of the blood was promoted by the promotion of such blood circulation. Improved. In summary, the results represent a cosmetic composition containing ginsenoside Rh4 according to the present invention, which contributes to the effective delivery of nutrients to the skin, inhibits and delays skin aging.

[Test Example 12] Skin color improvement effect

In order to understand the skin color improvement effect of the above Formulation Example 1 and Comparative Formulation Example 1, 30 subjects were arranged to use (1 time/day for 1 week) by Facial Stage DM-3 (Moritex, Japan) equipment. Assess the degree of skin color improvement. Regarding the skin color improvement rate, the skin color improvement rate was measured by measuring the brightness and color measurement value of the skin, and the results are shown in Table 14 below. The greater the brightness and color change value, the higher the skin color improvement.

As is apparent from the results of the above Table 14, the comparative dosage form 1 which does not contain the ginsenoside Rh4 according to the present invention, which does not show significant skin color improving efficacy; on the contrary, the dosage form containing ginsenoside Rh4 as an active ingredient Compared to With the skin color before use, the skin tone after use is greatly improved.

[Test Example 13] Pore contraction effect

1. By promoting the pore shrinkage effect of collagen biosynthesis

The collagen biosynthesis promoting effect of ginsenoside Rh4 according to the present invention was determined by comparison with TGF-β. First, fibroblasts were seeded at 24 wells in a manner of 105 per well, and cultured until the growth was about 90%. After culturing for 24 hours in serum-free Dulbecco's modified Eagle's medium, the ginsenoside Rh4 and TGF-β of the present invention dissolved in serum-free medium were treated at 10 ng/ml, respectively, and cultured in a carbon dioxide medium. 24 hours. The supernatant liquid was removed, and the increase or decrease of collagen procollagen was examined by a collagen prototype (I) ELISA kit (procollagen type (I); #MK101, TAKARA (Shiga, Japan)). The results are shown in Table 15, and the synthesis performance of collagen is shown on the basis of the non-treatment group 100.

As is apparent from the results of the above Table 15, the ginsenoside Rh4 according to the present invention exhibited superiority to the extent of TGF-? as a positive control group. Thus, it is understood that the ginsenoside Rh4 according to the present invention can increase the amount of collagen produced around the pores, thereby reducing the enlarged pores.

2. Pore contraction effect

In order to understand the pore shrinkage effect of Formulation Example 1 and Comparative Formulation Example 1, the following Ways to evaluate. Twenty male and female subjects with enlarged pores were divided into two groups, each of which was divided into two groups. Each group member was applied with facial creaming agent type 1 and comparative dosage form example 1 for 4 weeks. Regarding the effect of the effect of the pore contraction, the naked eye was evaluated by an expert by photographs before the experiment and after 4 weeks of the experiment. The results are shown in Table 15 below (evaluation level: 0 - no reduction; 5 - sharp reduction).

As is apparent from the results of the above Table 16, Comparative Formulation Example 1 did not have a pore-shrinking effect, and Formulation Example 1 exhibited a remarkable pore-shrinking effect which was visible to the naked eye, whereby it was found that the ginsenoside Rh4 according to the present invention The effect of reducing the pore size is excellent.

[Test Example 14] Sebum secretion inhibiting effect

Skin hypersecretion inhibitory effect inhibited by 1.5α-reductase activity

In order to confirm the inhibitory effect of 5α-reductase activity, the ratio of [ ¹⁴ C]testosterone to [ ¹⁴ C]dihydrotestosterone in HEK293-5αR2 cells was measured. P3 x FLAG-CMV-5αR2 was transfected into HEK293 cells and plated in 24-well plates per well/2.5 x 105 cells (Park et al., 2003, JDS. Vol. 31, pp. 191- 98). The next day, the new medium was added to the enzyme matrix and inhibitor. 0.05 μCi of [14C] testosterone (Amersham Pharmacia biotech, UK) was used as a substrate for the medium.

In order to confirm the degree of inhibition of 5α-reductase activity, ginsenoside Rh4 was added and cultured at 37 ° C for 2 hours in a 5% carbon dioxide incubator. At this time, no ginseng soap will be added. The group of glycosides Rh4 was used as a negative control group, and the group of finasteride was added as a positive control group. Then, the medium was taken, the steroid was extracted with 800 μl of ethyl acetate, the upper organic solvent layer was separated and dried, and the remaining residue was again dissolved in 50 μl of ethyl acetate in a plastic sheet of 60 F254 (Silica plastic sheet kieselgel 60). On F254), ethyl acetate-hexane (1:1) was developed as a solvent.

After drying the plastic sample in the air, a bathing system was used to measure the amount of the ectopic element, and the dried plastic sheet and the X-ray film were placed together in a bath cassette, and the testosterone remaining on the film was measured after one week. The amount of the isotopic element of dihydrotestosterone was calculated from the following formula 5 and formula 6, and the conversion ratio and the inhibition ratio were respectively calculated. The results are shown in Table 16 below.

[Formula 5] Conversion rate (%) = radioactivity / total radioactivity in the DHT region × 100

[Formula 6] Inhibition rate (%) = (conversion rate of the control group - conversion rate of the test substance) / conversion ratio of the control group × 100

From the results of Table 17 above, it is known that ginsenoside Rh4 effectively inhibits the activity of 5α-reductase, which converts testosterone into dihydrotestosterone, binds it to the cytoplasmic water-soluble protein, and enters the nucleus. Activates sebaceous gland cells, promotes differentiation, and acts as a sebum secretion in sebaceous glands. Rh4 blockade is converted from testosterone to dihydrotestosterone, which exhibits superior inhibitory effects than Finaster, which is used to inhibit the activity of 5α-reductase. Therefore, it is known that ginsenoside Rh4 inhibits the excessive secretion of sebum by effectively inhibiting the activity of 5α-reductase.

2. Sebum secretion inhibition effect

In order to understand the sebum secretion inhibiting effect of the above Formulation Example 1 and Comparative Formulation Example 1, evaluation was carried out in the following manner. A total of 30 male and female subjects with sebum secretion were selected, and the nutrient cream of the dosage form 1 and the comparative dosage form 1 was applied to the designated site for 4 consecutive weeks. Regarding the determination of the effect of sebum reduction, the average sebum reduction rate (%) after 2 weeks and 4 weeks was measured by a sebum measuring instrument (Sebumeter SM810, C+K Electronic Co., Germany), and the results were shown in Table 18 below.

From the results of the above Table 18, it is known that the dosage form 1 which contains the ginsenoside Rh4 of the present invention as an active ingredient can effectively inhibit excessive secretion of sebum as compared with the comparative dosage form 1 which 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 components and contents (% by weight) shown in Table 19 below. Specifically, in the formulation example 3, ginsenoside Rh4 was blended, and in Comparative Formulation Example 3, the active ingredient for improving acne skin was not contained at all, and the comparative dosage form Example 4 was used as a reference material for antibacterial power, and it was used as a acne treatment. Erythromycin (erythromycin).

Formulation Example 3 and Comparative Formulation Examples 3 to 4 were prepared as follows. The components of A of Table 19 below were completely dissolved, and the component of B was completely dissolved in a separate dissolution tank, and B was added to A and mixed and dissolved. Here, the components of C were added in accordance with the ratio shown in Table 19, uniformly mixed, and then filtered to prepare the composition.

[Test Example 15] Antibacterial test for acne

Each cosmetic composition prepared by the composition of the dosage form Example 3 and the comparative dosage form examples 3 to 4, Propionibacterium acnes (P. acnes) (ATCC 6919: medium-BHI medium (as a source of acne) Broth)) Perform an antimicrobial test.

The test method for acne related antibacterial power is as follows.

(1) Test bacterial preparation

Propionibacterium acnes was cultured using anaerobic culture inoculated in BHI medium.

(2) Preparation of diluted solution

0.15 ml of the above test bacterial solution was added to 15 ml of BHI medium (pH 6.8) or LB medium (pH 4.5), and uniformly mixed, and used as a diluted solution.

(3) Sample preparation

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

(4) Antibacterial test

1) A 96-well cell culture tube (96 well plate) No. 1 was placed, and the sample was placed at a starting concentration, and as a diluted solution, the total amount was added in units of 200 μl.

2) After uniformly mixing the mixture of No. 1 line, 100 μl was taken and placed in line 2, uniformly mixed, 100 μl was again extracted and placed in row 3, and double dilution was performed.

3) After 24 hours and 48 hours of static culture at 32 ° C, the proliferation of the bacteria was judged by the degree of suspension, and the minimum concentration without bacterial growth was taken as the Minimum Inhibitory Concentration (MIC) value. If the mixed solution is opaque and it is difficult to judge whether or not the bacteria are proliferated, it is confirmed by microscopic observation.

The results of the acne-related antibacterial test are shown in Table 20. The minimum inhibitory concentration is labeled in terms of the concentration of the active ingredient contained in the dosage form.

The smaller the ppm concentration of the minimum inhibitory concentration, the more effective the substance can be used as a more effective substance in terms of acne-related antibacterial activity, and the dose level of Example 3 is comparatively higher than that of Comparative Example 4 using erythromycin as a known acne treatment agent. Small, it can be seen that the composition containing ginsenoside Rh4 has remarkably excellent antibacterial activity against the test bacteria.

[Test Example 16] Lipogenesis inhibition test

3T3-L1 cells of the fibroblast cell line of mice were attached to Dürber containing 10% fetal bovine serum (FBS) at 1×10 5 cells/well. A 6-well culture plate of the culture medium of Dulbecco's modified eagle's medium (GIBCO BRL, Life Technologes). After 2 days, the medium was replaced with a new Dulberaceae modified Eagle's medium (containing 10% FBS) and culture was continued for 2 days. Then, for the above-mentioned cells to be cultured, differentiation was carried out with Dulbecco's modified Eagle's medium (containing 10% FBS) containing 1 μg/ml insulin (insulin), 0.5 mM IBMX, and 0.25 μM dexamethasone (dexamethasone). After induction, the ginsenoside Rh4 and the caffeine 50 μM were treated, and after 2 days of treatment, they were replaced with Dulberaceae modified Eagle's medium containing insulin, and culture was continued for 5 days. After 5 days, the medium was again replaced with a normal medium (Durboco modified Eagle's medium containing 10% of FBS), and culture was observed while the cells were changed in morphology to adipocytes.

In order to evaluate the inhibitory potency of ginsenoside Rh4 against fat accumulation in fat cells, Sudan III staining (S4136, sigma-aldrich) was carried out by performing differentiation of 3T3-L1 adipocytes in the above process. The fat cells were fixed in phosphate buffer at 4% polyformaldehyde (pH 7.2) at room temperature, washed with phosphate buffered saline (PBS), stained with Sudan III, and photographed. Compare with the naked eye. In the control group, only the medium to which the test substance or the comparative substance was not added was used, and as the other comparison group, the caffeine was treated at 50 μM. Regarding the degree of suppression of fat accumulation, the degree of dyeing is classified into +++, ++, +, -, and the level is given. In this case, the closer to +++, the higher the degree of dyeing. The results are shown in Table 21 below.

As is apparent from the results of the above Table 21, the ginsenoside Rh4 according to the present invention has not only a small amount of fat accumulated in the fat cells, but also has superior lipid synthesis as compared with caffeine which is known as a lipid synthesis inhibiting substance. Inhibitory effect. Therefore, sebum can be reduced by inhibiting lipid synthesis, thereby suppressing the production of acne.

[Test Example 17] Test for improvement of acne and reduction of sebum secretion and stimulation

Thirty subjects with acne were divided into three groups in the form of 10 persons in each group, and each group of the subjects was arranged to use the cosmetic compositions prepared in the above-mentioned dosage form 3 and comparative dosage forms 3 to 4. The acne improvement scale is set to 1 to 5 points, 1 for "no", 3 for "general", and 5 for "very". Experimental Results The average scores of the 10 subjects in Table 22 below were marked.

The period of disappearance of acne is based on the number of days on which the read acne disappears, and whether the acne recurrence is based on the result one month later. The sebum secretion reduction scale is set to 1 to 5 points, 1 for "no", 3 for "general", and 5 for "very". Experimental Results The average scores of the 10 subjects in Table 22 below were marked. The presence or absence of skin irritation (the number of people who showed irritating response) / (the total number of subjects).

From the results of the above Table 22, it was found that the dosage form of Example 3 had no recurrence of acne as compared with Comparative Formulation Example 3, and as a whole, it had an excellent effect on the improvement of acne. Further, in Comparative Example 4 containing an antibacterial power standard substance, although it exhibited an acne-improving effect, it exhibited strong skin irritation during use, which was not suitable for long-term use, and according to the composition of the present invention, Non-irritating, showing long-term use.

[Test Example 18] IL-8 production inhibitory effect

One day before the start of the experiment, skin human keratinocytes (NHEK, purchased at: Lonza) were dispersed in a 96-well plate at 5x104 cells/well, and a 5% carbon dioxide incubator at 37 °C. Train for 24 hours. After 24 hours, the cells were washed twice with sulfate buffer (PBS) and replaced with serum-free KBM (serum free keratinocyte basement media). To each well, ginsenoside Rh4 was treated according to the concentration shown in Table 23 below, and after reacting for 30 minutes, PGSA (10 μg/ml), PGSA (50 μg/ml), PGSA (50 μg/ml) + LPS (1 μg/ml) were separately treated. . Among them, PGSA (peptidoglycan from S. aureus) is a peptidoglycan extracted from Staphylococcus aureus, which is a major component of the cell wall of Gram-positive (+) bacteria, and the cell membrane component of bacteria can cause inflammation. Further, endotoxin (LPS, lypopolysaccaride) is a major component of the cell membrane of Gram-negative (-) bacteria, which is the main cause of inflammation.

After culturing for 24 hours at 37 ° C in a 5% carbon dioxide (CO 2 ) incubator, the culture solution was extracted and an enzyme-linked immunosorbent assay (ELISA) was performed on Interleukin-8 (IL-8). The results are shown in Table 23. Enzyme-linked immunosorbent assay The test (ELISA) used the experimental method of the manufacturing company (BD science).

As can be seen from the results of Table 23 above, ginsenoside Rh4 significantly reduced and inhibited the secretion of IL-8 which was increased by PGSA and LPS. Therefore, the skin external preparation composition of the present invention can provide an excellent anti-inflammatory effect by significantly reducing the secretion of IL-8 which is increased by PGSA and LPS.

[Test Example 19] Antipruritic evaluation

One day before the start of the experiment, keratinocytes (cell line name: HaCaT source: ATCC) were dispersed in a 96-well plate at 4x104 cells/well, and cultured at 37 ° C in a 5% carbon dioxide incubator for 24 hours. After 24 hours, after washing twice with a Hanks Balanced Salt solution buffer, the reaction buffer (2 μM Fluo-4-AM, 20% pluronic acid, 2.5 mM) was used. Probenecid) cells. The mixture was reacted at 37 ° C in a 5% carbon dioxide incubator for 30 minutes, and further reacted at room temperature for 30 minutes, and then washed twice with Hank's Balanced Salt Buffer solution, respectively, at a concentration (%) of ginsenoside as shown in Table 24 below. Rh4 treatment of cells.

After 10 minutes of reaction, 2 U/ml trypsin (Trypsin) or 5 μM PAR-2 active peptide (SLIGKV) was treated, and the intracellular Ca2+ concentration was measured for 80 seconds. bell. For the measurement of changes in intracellular Ca2+ concentration, a multi-function microplate reader 3 (FlexStation 3: Molecular Device, USA) was used. After treatment of ginsenoside Rh4 and 2 U/ml trypsin (Trypsin) or 5 μM PAR-2 active peptide (SLIGKV), Flex was measured for 80 seconds, and the difference between the obtained minimum and maximum values was determined and compared with 2 U/ The difference between the minimum value and the maximum value of the treatment of ml trypsin or 5 μM PAR-2 active peptide (SLIGKV) was compared, and the intracellular influx inhibition rate (%) of calcium ions is shown in Table 23 below.

As can be seen from the results of Table 24 above, the amount of calcium ions flowing into the cells by trypsin or PAR-2 active peptide (SLIGKV) decreased with the treatment of ginsenoside Rh4, and the concentration of ginsenoside Rh4 was increased. Significantly reduce the influx of calcium ions.

Therefore, the skin external preparation composition containing the ginsenoside Rh4 of the present invention can provide an excellent antipruritic effect by effectively inhibiting the PAR-2 activity which causes itching.

[Formulation Example 4 and Comparative Formulation Example 5]

The shampoo was prepared as follows in the composition of Table 25. Specifically, a surfactant and ethylene glycol distearate are added to purified water, heated to 80 ° C and uniformly dissolved, stirred and gradually cooled to 40 ° C, and added to the above mixture. Active ingredient of the invention and preservative, viscosity regulator, fragrance After the preparation and the hair conditioner, the mixture was stirred and cooled to room temperature to complete the preparation.

[Test Example 20] Anti-dandruff effect test

24 males aged 19-35 who had more dandruff were selected and divided into 2 groups in groups of 12, and each group was treated with the dosage form of Example 4 and the comparative dosage form of Example 5 After the hair was used for one month, the dandruff reduction rate was measured.

Before starting the test, use general shampoo shampoo, collect the dandruff accumulated within 2 days after shampooing, measure the weight of the dandruff, and then use the shampoo of Formulation Example 4 and Comparative Formulation Example 5, respectively. The hair was shampooed once a day, and the dandruff accumulated within 2 days after the completion of the test was collected, and the weight of the dandruff was measured, and the two were compared. At this time, the accumulated dandruff was directly collected from the scalp by a vacuum suction device, and the dandruff reduction rate was determined according to the following formula 7, and the results are shown in Table 26 below.

[Formula 7] dandruff reduction rate (%) = {head dandruff weight (mg) - dandruff weight (mg) after one month} / dandruff weight (mg) before test start × 100

As is apparent from the results of the above Table 26, the dosage form 4 containing ginsenoside Rh4 exhibited an excellent anti-dandruff effect.

[Test Example 21] Test for prevention of scalp pruritus

A total of 24 males and females aged 25 to 45 years old with severe scalp pruritus were selected. Each group was divided into 2 groups in 10 groups. The shampoos of each dosage form 4 and comparative dosage form 5 were arranged for every 3 days. The scalp pruritus prevention effect was evaluated by the following evaluation criteria for one continuous use for two weeks, and the results are shown in Table 27.

[Evaluation Criteria]

Very good -5 points

Excellent -4 points

General -3 points

Failed -2 points

Very unqualified -1 point

[Table 27]

As is apparent from the results of the above Table 27, the dosage form 4 containing ginsenoside Rh4 exhibited a more excellent effect in preventing scalp itching.

[Test Example 22] Proliferation effect of hair follicle dermal papilla cells

The keratin protein constituting the hair is produced by keratinocyte, which forms a cell that differentiates from dermal papilla cells. In order to evaluate the activity of the dermal papilla cells of the present composition, a DP6 (rat immortalized dermal papilla cell) cell strain (Wendy Filsell, Journal of Cell Science 107, 1761-1772 (1994)) was used in the present invention. The dermal papilla cell line is a cell strain obtained by isolating the hair roots of male PVG rat whiskers by microdissection, in DMEM containing 10% FBS (Fetal bovine serum) (Dulbecco's modified Eagle's medium, Gibco BRL). , Gaithersburg, MD, USA), cultured in a 5% carbon dioxide, 37 ° C incubator for 24 hours. The DP6 was placed in a 96-well plate and cultured in an incubator at 37 ° C for 24 hours, and then the ginsenoside Y was treated at a concentration of 5 ppm, 10 ppm, and 20 ppm, respectively. After 24 hours of drug treatment, the cell proliferation ability was measured using a WST-1 kit (Roche). The results are shown in Table 24 below.

As can be seen from the results of Table 28 above, the treatment of ginsenoside Y, hair milk The proliferation of the head cells is increased, and a significant difference in the concentration dependence can be confirmed.

[Test Example 23] Evaluation of the effect of potassium ion channel activity increase

Minoxidil, a hair loss treatment agent, is considered to be a potential mitochondrial potassium channel opener (K _ATP channel opener), which is a representative drug for androgenetic alopecia. In order to evaluate the mechanism of this minoxidil, the following test method was used to treat toluene butyrorea (SIGMA AlDRICH, T0891) for blocking the mitochondrial potassium ion channel in the fibroblasts constituting the dermis of the scalp. Inhibits cell proliferation, reopens potassium channels and restores cell proliferation.

In order to evaluate the function of the present composition as a mitochondrial potassium ion channel opener, a fibroblast cell line NIH3T3 (Mouse embryonic fibroblast cell line) cell line was used in the present invention. This cell line is a natural immortalized cell line isolated from a fibroblast cell line of NIH Swiss mouse embryo under the 3T3 protocol. The above cell strain was cultured in a Dulberaceae modified Eagle's medium (Gibco BRL, Gaithersburg, MD, USA) containing 10% FBS for 24 hours in an incubator maintained at 5% carbon dioxide at 37 °C. NIH3T3 was placed in a 96-well plate, and cultured in a 37 ° C incubator for 24 hours, and then treated with 2.5 mM tolbutamide. After 10 minutes, 10 μM of minoxidil and ginsenoside Rh4 as a positive control group were respectively 2.5 ppm. At a concentration of 5 ppm and 10 ppm, the cell proliferation ability was measured 48 hours after the drug treatment using the WST-1 kit (Roche). The results are shown in Table 29 below.

As shown in the results of Table 29 above, if ginsenoside Rh4 is treated, the proliferation of fibroblasts is restored, and as the concentration of ginsenoside Rh4 treated increases, the cell proliferation ability also increases. If ginsenoside Rh4 is treated at 10 ppm, Then the extent of cell proliferation recovery is reached to the extent that it is treated with minoxidil.

[Test Example 24] Melanin production promoting effect test of ginsenoside Rh4

5% fetal bovine serum, 100 IU of penicillin G, and 0.2 μM of TPA were added to the RPMI medium, and melanocytes (melan-a) were dispersed at 50,000 cells/well in a 24-well microtiter plate. On the next day, ginsenoside Rh4 as a test substance was treated to the dispersed cells at a final concentration of 10 ppm or 50 ppm, 0.1% DMSO was treated as a negative control group, 100 μM IBMX was treated as a positive control group, and cultured at 37 ° C for 3 days. . After the completion of the culture, the wells were washed with phosphate buffered saline (PBS), and 1 N sodium hydroxide (NaOH) was added in an amount of 100 μl each time to dissolve the melanin in the cells. The absorbance of the dissolved melanin was measured by a plate reader using a microplate reader at 405 nm (Synergy 2, BioTek (VT, USA)). The results of the melanin production-promoting effect of ginsenoside Rh4 compared with the control group are shown in Table 30 below.

As is apparent from the results of the above Table 30, the ginsenoside Rh4 promotes the melanin synthesis of melanocytes and increases the amount of melanin production, thereby exhibiting an excellent melanin production-promoting effect.

[Test Example 25] Effect of small eye deformity-related transcription factor (MITF) and tyrosinase expression in melanocytes of ginsenoside Rh4

The cell strain of 501 mel was dispersed to a 6-well microtiter plate at 500,000 cells/well, and for each well, 0.1% of DMSO was treated as a negative control group, and IBMX 100 μM was treated as a positive control group, and as The test group treated ginsenoside Rh4 at 10 ppm, and cultured at 37 ° C for 24 hours, 48 hours, and 72 hours. For the protein thus obtained, immunoblotting was carried out by a small eye deformity-related transcription factor (MITF) and a tyrosinase antibody. Protein extraction and immunoblotting typically use standard methods employed by those skilled in the art. After the immunoblotting was completed, the results were compared with 100 of the negative control group, and the results are shown in Table 31 below.

As is apparent from the results of Table 31 above, ginsenoside Rh4 can enhance the expression of the small eye deformity-related transcription factor (MITF) and tyrosinase protein in melanocytes.

[Test Example 26] Evaluation of antibacterial activity of ginsenoside Rh4

In order to evaluate the antibacterial activity of ginsenoside Rh4, an antibacterial experiment was carried out. The specific method is as follows.

The strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were cultured in tryptic Soy Broth, Candida albicans. Aspergillus niger strain was cultured in Sabouraud Dextrose Broth. A dilution of 1/100 of the culture solution (1/10 of Candida albicans strain) to each medium was used as a test bacterial solution. Aspergillus niger will be used as a test bacterial solution with a spore suspension prepared at 2 x 108 cfu/ml.

0.15 ml of the above test bacterial solution was added to each 15 ml of the medium, uniformly mixed, and used as a diluted solution.

16 μl of each sample was placed in a row of 96 well plates, and each was placed in a diluted solution of 184 μl. To each of the remaining wells, 100 μl of the diluted solution was placed. After uniformly mixing the mixture of No. 1 line, 100 μl was taken and placed in line No. 2, and after uniformly mixing, 100 μl was again extracted and placed in row No. 3, and 2-fold dilution was carried out in this manner.

Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were cultured in a thermostat at 32 ° C, Candida albicans and Aspergillus niger at 25 The culture was carried out in a thermostat of °C.

After 48 hours, the proliferation inhibition and suspension were confirmed by microscopy, and the minimum inhibitory concentration (MIC) value was determined and the results are shown in Table 32 below.

As is apparent from the results of the above Table 32, ginsenoside Rh4 exhibits an antibacterial activity against various strains, whereby it is predicted that ginsenoside Rh4 can function as a natural preservative or an antibacterial agent in the composition.

Claims (23)

A skin external preparation composition comprising: ginsenoside Rh4 as an active ingredient, the ginsenoside Rh4 being represented by the following Chemical Formula 1: The skin external preparation composition according to claim 1, wherein the ginsenoside Rh4 is contained in an amount of 0.001% by weight to 50% by weight based on the total weight of the composition. The skin external preparation composition according to claim 1, wherein the composition is an antioxidant composition. The skin external preparation composition according to claim 1, wherein the composition is an anti-aging composition. The skin external preparation composition according to claim 1, wherein the composition is a composition for increasing skin elasticity. The composition for external use of skin according to item 1 of the patent application, wherein The above composition is a composition for improving skin wrinkles. The skin external preparation composition according to claim 1, wherein the composition is a whitening composition. The skin external preparation composition according to claim 1, wherein the composition is a moisturizing composition. The skin external preparation composition according to the above aspect of the invention, wherein the composition is a composition for strengthening a skin barrier function. The skin external preparation composition according to claim 1, wherein the composition is a skin keratinocyte differentiation-inducing composition. The skin external preparation composition according to claim 1, wherein the composition is a composition for improving acne. The skin external preparation composition according to claim 1, wherein the composition is an antibacterial composition. The skin external preparation composition according to claim 1, wherein the composition is an anti-inflammatory composition. The skin external preparation composition according to claim 1, wherein the composition is a composition for inhibiting lipid formation. The skin external preparation composition according to claim 1, wherein the composition is a gas color and skin color improving composition. The skin external preparation composition according to claim 1, wherein the composition is a composition for pore shrinkage. The skin external preparation composition according to claim 1, wherein the composition is a composition for regulating a sebum. The composition for external use of skin according to item 1 of the patent application, wherein The above composition is a composition for improving skin problems. The skin external preparation composition according to claim 1, wherein the composition is a composition for preventing skin irritation. The skin external preparation composition according to claim 1, wherein the composition is a composition for anti-dandruff. The skin external preparation composition according to claim 1, wherein the composition is a hair growth promoting composition. The skin external preparation composition according to claim 1, wherein the composition is a composition for preventing white hair. The skin external preparation composition according to claim 1, wherein the composition is a composition for a natural preservative.