KR20140131027A - Skin external composition containing ginsenoside Rh4 - Google Patents

Abstract

The present invention relates to a composition containing ginsenoside Rh4 and, more specifically, to a composition containing ginsenoside Rh4 which has effects of improving overall skin condition, such as anti-aging, alleviating skin wrinkles, whitening, moisturizing effect, anti-inflammation, alleviating acne and skin troubles, alleviating atopic symptoms, skin astringency, shrinking pores, improving the blood circulation to improve skin complexion; and effects of improving scalp and hair condition such as anti-dandruff effect, promoting hair growth, and alleviating grey hair, via the excellent antioxidative ability.

Description

[0001] The present invention relates to a skin external composition containing ginsenoside Rh4,

The present invention relates to a composition for external application for skin containing ginsenoside Rh4, and more particularly to a composition for external application for skin comprising ginsenoside Rh4, Provides effects to improve scalp and hair condition such as anti-dandruff effect, hair growth effect and anti-whiplash effect as well as overall skin condition improvement such as trouble improvement effect, whitening effect, sebum control effect, pore contraction effect, ≪ / RTI >

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

In order to prevent the change of the skin condition due to the intrinsic and extrinsic factors, and to maintain a healthy skin condition, physiologically active substances obtained from various known animals, plants, microorganisms and the like are added to cosmetics to improve the skin condition There have been many efforts to develop cosmetics using ginseng (Panax ginseng C. A Meyer) ingredients. Ginseng has been widely used in cosmetic compositions since its effectiveness in skin has been proven early. Ginseng root or leaf extract, ginseng saponin, ginseng aglycon and ginseng polysaccharides have been used. However, the cosmetic compositions containing such ginseng extract, extract, polysaccharide, etc. have disadvantages in that their effects are not remarkably superior to those of other raw materials because they contain a very small amount of the active substance.

Accordingly, the present inventors have found that ginsenoside Rh4 contained in ginseng can provide an anti-aging effect, an improvement in skin wrinkles, a whitening effect, a moisturizing effect, as well as an effect of improving acne and skin troubles, It is possible to provide a skin condition improving effect such as a shrinking effect and also to provide an effect of improving scalp and hair condition such as prevention of dandruff, hair growth and hair loss, and completed the present invention.

Accordingly, an object of the present invention is to provide a composition for external application for skin, which can contain the ginsenoside Rh4 and improve the overall condition of the skin.

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

The present invention also provides a composition for external application for skin for anti-aging comprising ginsenoside Rh4 as an active ingredient.

In addition, the present invention provides a whitening skin external application composition containing ginsenoside Rh4.

The present invention also provides a composition for external application for skin moisturizing comprising ginsenoside Rh4 as an active ingredient.

The present invention also provides a composition for external application for skin for acne improvement comprising ginsenoside Rh4 as an active ingredient.

The present invention also provides a composition for external application for skin color and skin tone, which comprises ginsenoside Rh4 as an active ingredient.

The present invention also provides a composition for external application for skin pore reduction comprising ginsenoside Rh4 as an active ingredient.

The present invention also provides a composition for external application for sebum control comprising ginsenoside Rh4 as an active ingredient.

The present invention also provides a composition for external application for skin for preventing dandruff comprising ginsenoside Rh4 as an active ingredient.

The present invention also provides a composition for external application for hair growth comprising ginsenoside Rh4 as an active ingredient.

The present invention also provides a composition for external application for skin whitening prevention comprising ginsenoside Rh4 as an active ingredient.

The present invention also provides a composition for external application for skin using ginsenoside Rh4 as a natural preservative.

The composition of the present invention contains an excellent antioxidant ability by containing the ginsenoside Rh4. The antioxidant activity of the composition of the present invention is superior to the antioxidant effect of the skin, such as skin aging prevention effect, skin moisturizing power improvement effect, antiinflammatory effect, skin troubles such as acne, whitening effect, sebum control effect, Improvement of the complexion through improvement, improvement of the scalp and hair condition such as anti-dandruff effect, hair growth effect and anti-whiplash effect.

The composition for external application for skin according to the present invention contains ginsenoside Rh4 as an active ingredient.

The ginsenoside Rh4 used in the present invention has a structure represented by the following formula (1).

Ginseng belongs to the genus Ginseng of Araliace, a herbaceous perennial plant. It is produced in various places in the world such as Korea, China, Japan and the United States. It is cultivated mainly in East Asia 85-140 degrees, North latitude 22-49 degrees, North America 70-97 degrees in the west and 34-47 degrees in the North. Ginseng saponin, which is the most important ginsenoside, is most abundant in Korean ginseng. It is the most important ginseng saponin produced in many parts of the world. It has various physiological and pharmacological effects. To date, about 34 species of ginsenosides have been isolated and their pharmacological effects differ depending on the type of sugar bound to the aglycon, the number of sugars attached or the binding site. Depending on the structural characteristics, it is classified into diol type, triol type and oleanane type. In addition, it contains panacen, polyacetylene compound, polyphenol compound, flavonoid and vitamins, which are the fragrance ingredients of ginseng.

Ginseng has been used for the treatment of various diseases because its efficacy has been acknowledged as a one - sided treatment. Recent studies have shown that ginseng has antioxidant activity, anti - ulcer activity, immunity enhancement, and anti - inflammatory effect. Have been reported to have anti-cancer and hypoglycemic effects. Skin-related effects include anti-inflammatory effects (Korean J. Dermatol. 18 (1): 39-42 (1980), Korean J. Dermatol.14 (4): 335-339 (1976)). 28 (4): 434-440 (1990)), acne prevention and treatment (Korean J. Dermatol. 30 (1): 27-33 (1992), Korean J. Dermatol. (1990)), antioxidative effects (Fitoterapia 57 (1): 15-28 (1986), Fitoterapia 57 (4) 217-7, increase in elasticity and hydratability (Proceedings of the 2nd international ginseng symposium 13-17 222 (1986)), wound healing (Brit. J. Pharmacol. 125: 255-262 (1998), Arch Pharm Res. 25 (1): 71-76 (2002)), collagen degradation inhibition 9 (5): 476-483 (1999)), whitening effect (Journal of the Society of Cosmetic Scientists of Korea 27 (2): 45-56 (2001)).

The ginsenoside Rh4 of the present invention may be extracted from a plant, synthesized according to a method known in the art, or commercially available one may be used. It is also within the scope of the present invention that a derivative which is obtained by subjecting the above-mentioned ginsenoside Rh4 to substitution or substitution reaction with substituents which are conventionally practiced in the art is also included in the scope of the present invention. As will be apparent to those skilled in the art.

The ginsenoside Rh4 used in the present invention can be obtained especially from ginseng extract. The type of ginseng to be used at this time is not particularly limited, and ginseng, red ginseng, white ginseng, taegeuk ginseng and ginseng can be used. In addition, the ginseng extract is not only contained in the leached liquid obtained by leaching and transferring from ginseng but also contained in the concentrate obtained by partially or completely concentrating the leach solution or in the slump, premix, regular season, liquid extract and ginseng prepared by drying the concentrate again It contains all of the plant itself as well as chemicals that exert the main effect. Extracts from all parts of ginseng such as stem, root, leaf, flower, and fruit are available and are not limited to any particular part of the extract. Also, a known method can be used for extracting ginsenoside Rh4 from ginseng extract.

Specifically, the ginsenoside Rh4 can be isolated from ginseng by preparing ginseng extract from 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, preferably 80% ethanol. At this time, the extraction temperature is preferably 10 to 80 ° C, and the extraction can be performed for 3 to 24 hours. If the extraction temperature and the extraction time are exceeded, the extraction efficiency may be deteriorated or the component may be changed.

The composition according to the present invention may contain ginsenoside Rh4 in an amount of 0.001 to 50% by weight, preferably 0.01 to 30% by weight, more preferably 0.1 to 10% by weight based on the total weight of the composition. If the content of ginsenoside Rh4 is less than 0.001% by weight, the effect and effect of the ingredient are insignificant. If the content is more than 50% by weight, skin safety or shape problems may occur.

Since ginsenoside Rh4 is a component having excellent antioxidant ability, the composition of the present invention containing ginsenoside Rh4 can be used as an anti-aging external composition for skin by providing excellent antioxidative power, which can improve skin elasticity and improve wrinkles The effect is excellent.

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

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

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

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

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

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

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

The composition of the present invention can be used as a composition for external application for skin preventing anti-whiplash, which can significantly increase the expression of MITF in melanocytes, thereby inhibiting white moth and promoting gray hair induction.

In addition, the ginsenoside Rh4 used in the composition for external application for skin of the present invention can provide an effect as a natural preservative.

The above composition for external application for skin of the present invention can be formulated as a cosmetic composition, and can be formulated containing a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example as a solution, a gel, a solid, a paste anhydrous product, an emulsion obtained by dispersing an oil phase in water, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic form (liposome) In the form of ionic fibrin dispersions, or in the form of creams, skins, lotions, powders, ointments, sprays or conical sticks. It can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. These compositions may be prepared according to conventional methods in the art.

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

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

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

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

[Referential Example 1]

Ginsenoside Rh4 was used from Ambo Laboratories to test the efficacy of the composition of the present invention.

[Test Example 1] Inhibitory effect on reactive oxygen species (ROS) production

Keratinocytes isolated from human epidermal tissue were added to each well of a 24-well plate at 5 × 10 ⁴ for 24 hours. After 16 hours, ginsenoside Rh4 was treated with 1%. For comparison, the control group did not treat ginsenoside Rh4. After 2 hours, the culture solution was removed, and 100 μl of phosphate buffered saline (PBS) was added to each well. This keratinocyte was irradiated with ultraviolet rays of 30 mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: K5T8, UVB 15 W, Sankyo Dennki, Japan), and then PBS was removed. The keratinocyte culture medium 200 ≪ / RTI > The amount of reactive oxygen species increased by ultraviolet stimulation was determined at a certain time interval by treating the ginsenoside Y again. The amount of ROS was determined by referring to Tan's method of measuring the fluorescence of DCF-DA (dichlorofluorescin diacetate) oxidized by ROS (Tan et al., 1998, J. Cell Biol. Vol. 141, pp1423-1432) The ratios of the vehicle-treated control to the ROS were calculated. The results are shown in Table 1 below.

Test substance UVB 30 mJ / cm ² Time elapsed after investigation 0hr 2 hr 3hr Vehicle 100 244 287 UVB + vehicle 100 325 381 UVB + ginsenoside Rh4 100 235 279

In the results shown in Table 1, the ginsenoside Rh4 according to the present invention effectively inhibits the production of ROS, which is known to cause skin cell damage by ultraviolet rays, and the amount of ROS after ultraviolet stimulation is higher than that when no ultraviolet light is irradiated. And the antioxidant effect is excellent. Thus, it has been confirmed that the ginsenoside Rh4 according to the present invention can prevent the pores from being widened by inhibiting oxidation and preventing aging, and can prevent skin irritation, thereby improving skin troubles.

[Test Example 2] Elastase activity inhibition assay

The inhibitory activity on the elastase activity of ginsenoside Rh4 was measured in comparison with EGCG. The elastase and substrate used were commercially purchased from Sigma Aldrich, USA (Cat. No. E0127).

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

(200 μL) of ginsenoside Rh4 and 50 μL of 20 μg / mL elastase type III solution were mixed in a 96-well plate prepared with 10 mg / L Tris-HCl buffer (pH 8.0) EGCG 250 μM was used as a positive control, and negative control group, distilled water, was used. Then, 100 μL of 0.4514 mg / mL N-SUCCINYL-ALA-ALA-ALA-p-NITROANILIDE prepared as the buffer solution 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 carried out in the same manner and corrected.

The method for calculating the activity of inhibiting the activity of the elastase was as shown in the following formula (1), and the results are shown in Table 2 below.

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

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

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

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

compound % Inhibition Untreated group 0 EGCG 65 Ginsenoside Rh4 77

As shown in Table 2, the degree of inhibition of elastase activity of ginsenoside Rh4 was significantly superior to that of EGCG, which is known as an inhibitor of elastase activity. Thus, the inhibitory effect of ginsenoside Rh4 of the present invention on elastase activity It can be confirmed that it is excellent.

[Test Example 3] Collagenase (MMP-1)

The ability of the ginsenoside Rh4 of the present invention to inhibit collagenase production was measured in comparison with retinoic acid.

Human fibroblasts were plated at 5,000 cells / well in a 96-well microtiter plate containing 2.5% fetal bovine serum-containing DMEM (Dulbecco's Modified Eagle? Media) And incubated at 37 ° C in an incubator at 5% CO ₂ until the cells grew to 70-80%. Ginsenoside Rh4 or retinoic acid was treated at a concentration of 10 占 퐂 / ml for 24 hours, and then the cell culture solution was collected.

The degree of collagenase production in the cell culture was measured using a commercially available collagenase assay device (Amersham Pharmacia, USA, Catalog #: RPN 2610). First, the cell culture solution collected on a 96-well plate uniformly coated with primary collagenase antibody was added and the antigen-antibody reaction was performed in a thermostatic chamber for 3 hours. After 3 hours, the second collagen antibody bound to the chromophore was placed in a 96-well plate and reacted for another 15 minutes. After 15 minutes, 3,3 ', 5,5'-tetramethylbenzidine (Sigma), which is a color inducing substance, was added and color development was induced at room temperature for 15 minutes. Then, 1M sulfuric acid When the color reaction was stopped, the color of the reaction solution became yellow, and the degree of yellow was different according to the progress of the reaction.

The absorbance of a yellow 96-well plate was measured at 405 nm using a spectrophotometer and the degree of synthesis of collagenase was calculated by the following equation (2) Respectively. At this time, the reaction absorbance of the cell culture fluid collected from the non-treated group was used as a control.

compound Expression level (%) Untreated group 100 Retinoic acid 75 Ginsenoside Rh4 73

As shown in Table 3, it can be seen that the collagenase expression inhibition effect is similar to that of retinoic acid, in which the degree of collagenase expression of ginsenoside Rh4 is known as a collagenase expression inhibitor.

From these results, it can be confirmed that the ginsenoside Rh4 of the present invention inhibits the substrate metalloprotease (MMP-1) and has anti-aging effect by reducing collagen decomposition in the skin.

[Formulation Example 1 and Comparative Formulation Example 1]

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

Compounding ingredient Formulation Example 1 Comparative Formulation Example 1 Purified water To 100 To 100 Ginsenoside Rh4 0.1 - Vegetable hydrogenated oil 1.50 1.50 Stearic acid 0.60 0.60 Glycerol stearate 1.00 1.00 Stearyl alcohol 2.00 2.00 Polyglyceryl-10pentastearate and behenyl alcohol and sodium stearoyl lactylate 1.00 1.00 Arachidyl behenyl alcohol and arachidyl glucoside 1.00 1.00 Cetylaryl Alcohol and Cetearyl Glucoside 2.00 2.00 PEG-100 Stearate & Glycerololate & Propylene Glycol 1.50 1.50 Caprylic / carboxy triglyceride 11.00 11.00 Cyclomedicone 6.00 6.00 Preservative, incense Suitable amount Suitable amount Triethanolamine 0.1 0.1

[Test Example 4] Confirmation of skin elasticity improving effect

In order to confirm the skin elasticity improvement effect in human, the formulations of Formulation Example 1 and Comparative Formulation Example 1 were evaluated as follows.

Twenty healthy women in the 30s to 40s were divided into two groups of 10 each for the two groups of Formulation Example 1 and Comparative Formulation Example 1, and the nutritional cream was applied to the face for one week for 12 weeks. Then, the skin elasticity was measured with a cut- 575, C + K Electronic Co., Germany). The results are shown in Table 5 below. The results in Table 5 are reported as ΔR8 values of Cutometer SEM 575, where the value of R8 indicates the nature of viscoelasticity.

Experimental product Skin elasticity effect Formulation Example 1 0.38 Comparative Formulation Example 1 0.10

As shown in Table 5, Formulation Example 1 containing the ginsenoside Rh4 of the present invention showed a greater skin elasticity than the group to which Comparative Formulation Example 1 was applied.

Therefore, it can be confirmed that the composition containing ginsenoside Rh4 of the present invention is very effective for improving skin elasticity.

[Test Example 5] Confirmation of skin wrinkle improving effect

Formulation Example 1 and Comparative Formulation Example 1 were used in order to confirm the wrinkle-reducing effect of the composition of the present invention in humans.

In order to confirm the effect of improving the wrinkles of Formulation Example 1 and Comparative Formulation Example 1, the following evaluation was made. Twenty healthy women in their 40s were divided into two groups of 10 each for Formulation Example 1 and Comparative Formulation Example 1 to apply the nutritional cream once a day for 12 weeks to the face, Were visually analyzed by viscometer (SV600, Courage + Khazaka electronic GmbH, Germany). The results are shown in Table 6 below. The values in Table 6 below are the average values obtained by subtracting the pre-application parameter values from the respective parameter values after 12 weeks of application.

After 8 weeks of use
Clinical outcome R1 R2 R3 R4 R5 Formulation Example 1 0.14 0.13 0.09 0.02 0.01 Comparative Formulation Example 1 0.27 0.26 0.21 0.03 0.03

R1: Difference between the maximum value and the minimum value of the wrinkle contour line

R2: The wrinkle contour line is arbitrarily divided into 5 squares, and the average of R1 values

R3: the highest value of R1 divided by 5

R4: Average value obtained by subtracting the value of each apex and valley from the baseline of the wrinkle contour line

R5: Difference between the baseline of the wrinkle contour line minus the contour of each wrinkle

As shown in Table 6, the external preparation composition of Formulation Example 1 showed excellent skin wrinkle-reducing effect.

Test Example 6: Effect of tyrosinase inhibition

The tyrosinase enzyme was extracted from Mushroom and was obtained from Sigma (SIGMA). First, the substrate, tyrosine, is dissolved in distilled water to make a 0.3 mg / ml solution. The solution is put into a test tube in an amount of 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 .

0.2 ml of the sample solution prepared by mixing the ginsenoside Rh4 of the present invention at an appropriate concentration into the ethanol solution was added to the reaction solution, and the mixture was allowed to react for 10 minutes in a 37 ° C thermostat. At this time, the control group was prepared by adding 0.2 ml of a solvent instead of each sample solution, and ascorbic acid was used as a positive control group. 0.1 ml of tyrosinase solution of 2500 units / ml was added to the reaction solution, followed by reaction at 37 ° C for 10 minutes. The reaction tube was immersed in ice water to quench the reaction, and absorbance at a wavelength of 475 nm was measured by a photoelectric spectrometer. The results are shown in Table 7 below. Each tyrosinase inhibitory effect was calculated by the following formula (3).

Test substance Inhibition rate of tyrosinase (%) Control group (no addition) 0 Ascorbic acid 52 Ginsenoside Rh4 63

From the results of Table 7, it can be seen that the inhibition of tyrosinase of ginsenoside Rh4 according to the present invention is much higher than that of ascorbic acid, which is a known tyrosinase inhibitor, and thus the whitening effect is excellent.

[Test Example 7] Inhibitory effect on melanin formation by B16 / F10 melanoma cells

A sample containing 0.1% by weight of ginsenoside Rh4 and 0.1% by weight of kojic acid was added to the culture medium of B16 / F10 melanoma cells (Korean Cell Line Bank) at a constant concentration for 3 days, And the cells were dissolved with 1N NaOH, and absorbance was measured at 405 nm. Cells to which the test substance was not added were used as a control group, and the melanin formation inhibition degree of each test substance was measured by comparing with the melanin content in the control group. The inhibition rate of melanin production was calculated according to Equation (4) and the results are shown in Table 8. < tb > < TABLE >

Test substance Melanin inhibition rate (%) Control group (no addition) 0 Kojic acid 53 Ginsenoside Rh4 68

From the results of Table 8, it can be seen that the inhibitory rate of melanin formation of ginsenoside Rh4 according to the present invention is much higher than that of kojic acid, which is a known melanin formation inhibitor, and thus the whitening effect is excellent.

[Test 8] Measurement of skin moisturizing effect

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

20 men and women in the 40s and 50s classified as dry skin were divided into two groups of 10 persons for each of Formulation Example 1 and Comparative Formulation Example 1 to apply the nutritional cream twice daily for 4 weeks to the face. (24 ° C, relative humidity 40%) after 1 week, 2 weeks, and 4 weeks after application, and after 2 weeks (total 6 weeks) (Corneometer CM825, C + K Electronic Co., Germany). The results are shown in Table 9 below. The results in Table 9 are based on the skin moisture meter measured immediately before the start of the test, and the percent increase in the measured value after treatment for a certain period of time.

Test group Moisture growth rate (%) 1 week Two weeks 4 weeks 6 weeks Formulation Example 1 32 33 37 34 Comparative Formulation Example 1 30 32 32 15

The results of Table 9 indicate that when Comparative Formulation Example 1 is applied, the moisture increase rate is about 30% until 4 weeks of application, but the amount of skin water decreases after stopping application, while the amount of ginsenoside Rh4 It can be confirmed that the skin moisture increase rate is 30% or more even after the application is stopped. Thus, it can be seen that the composition of the present invention containing ginsenoside Rh4 has excellent skin moisturizing effect.

[Test Example 9] Measurement of promoting effect of keratinocyte differentiation

In order to examine the differentiation promoting effect of kinsenoside Rh4 on keratinocytes, the amount of cornified envelope (CE) generated upon differentiation of keratinocytes was measured by absorbance as described below.

First, keratinocytes isolated from the epidermis of newborns and primary cultured human keratinocytes were placed in a culture flask and adhered to the bottom. After treating ginsenoside Rh4 with a concentration of 5 ppm in the culture medium, the cells were treated with 70% to 80% The cells were cultured for 5 days until they were grown. At this time, low calcium (0.03 mM) and high calcium (1.2 mM) treatment groups were negative control and positive control, respectively. Then, the cultured cells were harvested and washed with PBS (phosphate buffered saline). Then, 10 mM Tris-HCl buffer (Tris-HCl, pH 7.0) containing 2% SDS (sodium dodecyl sulfate) and 20 mM DTT (Dithiothreitol) pH 7.4) was added, sonication, boiling, and centrifugation. The precipitate was suspended again in 1 ml of PBS and the absorbance at 340 nm was measured. Separately, a part of the solution after the sonication was taken to measure the protein content and used as a standard in evaluating the degree of cell differentiation. The results are shown in Table 10 below.

Test substance The ability to differentiate in keratinocytes (%) Low calcium (0.03 mM) solution
(Negative control) 100 High calcium (1.2 mM) solution
(Positive control group) 210 Ginsenoside Rh4 285

As shown in Table 10, when ginsenoside Rh4 was treated, it was confirmed that the effect of accelerating the differentiation of keratinocytes was excellent.

[Test Example 10] Measurement of recovery effect on skin barrier function

In order to measure the effect of ginsenoside Rh4 on the recovery of damaged skin barrier function due to skin damage, the following experiment was conducted. Ten adult male and female adult rats were injured on the skin barrier using a tape stripping method, and applied to the two groups of Formulation Example 2 and Comparative Formulation Example 2 prepared in the following Table 11 for 7 days The recovery rate of TWEL was measured and compared with Vapometer (Delfin, Finland) once a day. Here, Comparative Formulation Example 2 is a vehicle as a negative control. The experimental results are shown in Table 12 below. The results in Table 12 were compared before treatment before barrier damage and after treatment before barrier damage on the basis of 100%.

Compounding ingredient Formulation Example 2 Comparative Formulation Example 2 Purified water 69 70 Propylene glycol 30 30 Ginsenoside Rh4 One -

Test group TWEL Change (%) Before processing 1 day 2 days 3 days 4 days 5 days 6 days Formulation Example 2 100 123.5 126.4 121.8 114.9 111.0 107.4 Comparative Formulation Example 2 100 121.4 112.7 98.3 70.5 62.3 43.5

As can be seen from the above Table 12, when Comparative Formulation Example 2 containing no ginsenoside Rh4 was treated, the transdermal water loss was gradually increased over time, whereas the dosage form containing ginsenoside Rh4 2 treatment, the rate of transdermal water loss returned to normal and the barrier damage was recovered at a rapid rate.

[Test Example 11]

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

The face was soaked in a constant temperature and humidity chamber, and then adapted for 30 minutes. Initial values were measured using LDPI. First, the initial blood flow in the lower part of the forehead of 30 women with cold hands and feet was measured by LDPI. The results of the blood flow measured after using Formulation Example 1 and Comparative Formulation Example 1 for one week to the subject and the initial measured value (skin blood flow change) are shown in Table 13 below.

LDPI results before and after using cosmetics - skin blood flow Test substance Percent change of skin blood flow after 1 week use (%) Formulation Example 1 11 Comparative Formulation Example 1 5

From the results shown in Table 13, it was confirmed that the cosmetic composition according to the present invention significantly increased skin blood flow compared to Comparative Formulation Example 1, which did not contain ginsenoside Rh4, and that blood circulation was improved by promoting blood circulation . This ultimately suggests that the cosmetic composition containing ginsenoside Rh4 according to the present invention can contribute to effectively transmit nutrients of the skin and inhibit and retard skin aging.

[Test Example 12] Skin tone improvement effect

In order to examine the skin tone improvement effect of Formulation Example 1 and Comparative Formulation Example 1, 30 subjects were used (one evening / one day application for a total of one week) and then facial stage DM-3 (Moritex, Japan) To evaluate the degree of skin tone improvement. The improvement rate of the skin tone was determined by the brightness and color change value of the skin and the brightness and color change value of the skin. The results are shown in Table 14 below. The larger the change in brightness and color, the better the skin tone.

Test substance Skin tone improvement rate (%) Brightness (mean ± standard deviation) Color (mean ± standard deviation) Formulation Example 1 13 ± 2.37 13 ± 3.04 Comparative Formulation Example 1 5 ± 2.34 5 ± 2.05

In the results of Table 14, Comparative Formulation Example 1 which does not contain ginsenoside Rh4 according to the present invention shows no significant skin tone improving effect, whereas Formulation Example 1 containing ginsenoside Rh4 as an active ingredient has a higher effect It was confirmed that the skin tone after the treatment was greatly improved.

[Test Example 13] Pore reduction effect

1. Collagen biosynthesis promotes pore reduction effect

The collagen biosynthesis promoting effect of ginsenoside Rh4 according to the present invention was measured in comparison with TGF-beta. First, fibroblasts were seeded at 10 ⁵ per well in 24 wells and cultured until they grew to about 90%. After culturing for 24 hours in serum-free DMEM medium, the ginsenosides Rh4 and TGF-β of the present invention dissolved in serum-free medium were treated with 10 ng / ml each, and CO ₂ And cultured in an incubator for 24 hours. The supernatant was removed and the procolagen (I) ELISA kit (procollagen type (I)) was used to determine whether procollagen was increased or decreased. The results are shown in Table 15, and the combined performance of the collagen was set at 100 for the non-treated group.

Test substance Collagen Total Performance (%) Untreated group 100 TGF-beta 183.5 Ginsenoside Rh4 196.1

In the results shown in Table 15, it was confirmed that the ginsenoside Rh4 according to the present invention exhibits a superior collagen combining performance higher than that of the positive control TGF-beta. Therefore, it was confirmed that the ginsenoside Rh4 according to the present invention can increase the collagen production around the pore, thereby reducing the enlarged pores.

2. Pore reduction effect

In order to examine the pore reduction effect of Formulation Example 1 and Comparative Formulation Example 1, the following evaluation was made. Twenty male and female subjects with large pore sizes were selected and divided into two groups of 10 persons. The nutritional creams of Formulation Example 1 and Comparative Formulation Example 1 were applied to the faces for each group for 4 weeks each day. The evaluation of the effectiveness of the pore reduction was made by a visual evaluation of experts by taking pictures before and after the experiment. The results are shown in Table 16 below (rating scale: 0 - no reduction at all; 5 - very shrunk).

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

From the results shown in Table 16, Comparative Formulation Example 1 has no effect of reducing the pore size, but in the case of Formulation Example 1, the effect of reducing pores is visible to the naked eye. Ginsenoside Rh4 according to the present invention reduces the size of pores And the effect was excellent.

Test Example 14 Effect of suppressing sebum secretion

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

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

To confirm the inhibition of 5α-reductase activity, ginsenoside Rh4 was added and cultured in a 5% CO ₂ incubator at 37 ° C. for 2 hours. At this time, no ginsenoside Rh4 was used as a negative control and finasteride was used as a positive control. Then, the culture medium was collected, and the steroid was extracted with 800 쨉 l of ethyl acetate. The upper organic solvent layer was separated, dried, and the remaining residue was again dissolved in 50 쨉 l of ethyl acetate to obtain a silica plastic sheet kiesel gel 60 F254 ) Using ethyl acetate-hexane (1: 1) as the solvent.

After drying the plastic sample in air, the bath system was used to measure the amount of isotope. The dried plastic sheet and x-ray film were put together in a bath cassette, and after 1 week, the testosterone remaining in the film and isotopes of dihydrotestosterone The conversion and inhibition rates were calculated according to the following equations (5) and (6), respectively, and the results are shown in Table 17 below.

Test substance Conversion Rate (%) Inhibition rate (%) Negative control group 48.0 - Positive control group 27.6 42.5 Ginsenoside Rh4 14.9 61.3

In the results shown in Table 17, testosterone is converted into dihydrotestosterone, which binds to the receptor protein in the cytoplasm to enter the nucleus, activates the sebaceous gland, accelerates differentiation, and induces 5α-reductase Was effectively inhibited by ginsenoside Rh4 to block the conversion of testosterone to dihydrotestosterone and showed an inhibitory effect superior to that of finasteride, which is known to inhibit 5α-reductase activity. Thus, it was confirmed that ginsenoside Rh4 effectively inhibited the activity of 5? -Redoxytase, thereby suppressing sebaceous hyperpermeability.

2. Inhibitory effect of sebum secretion

To evaluate the inhibitory effect of sebum secretion of Formulation Example 1 and Comparative Formulation Example 1, the following evaluation was made. 30 men and women who felt that sebum secretion was high were selected and nutritional creams of Formulation Example 1 and Comparative Formulation Example 1 were made daily for four weeks in designated areas. The results are shown in Table 18 below. The results are shown in Table 18 below. The results are shown in Table 18 below. Respectively.

Test substance Sebum reduction rate (%) After 2 weeks After 4 weeks Formulation Example 1 32 40 Comparative Formulation Example 1 5 5

From the results of Table 18, it can be seen that Formulation Example 1 containing ginsenoside Rh4 according to the present invention as an active ingredient can effectively inhibit excess sebum secreted from Comparative Formulation Example 1 which does not contain ginsenoside Rh4 as an active ingredient.

[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 19 below. More specifically, Formulation Example 3 was prepared by blending ginsenoside Rh4, Comparative Formulation Example 3 contained no active ingredient for acne skin improvement, Comparative Formulation Example 4 was prepared by using standard material Which contains erythromycin, which is widely used as an acne remedy.

The manufacturing method of Formulation Example 3 and Comparative Formulation Examples 3 to 4 is as follows. The components of phase A in Table 19 were completely dissolved and the components of phase B were completely dissolved in a separate melting tank, and the phase B was added to the phase A to be mixed and solubilized. The components of the C phase were added thereto in accordance with the mixing ratios shown in Table 19, mixed and homogenized and then filtered to prepare the compositions.

division Formulation Example 3 Comparative Formulation Example 3 Comparative Formulation Example 4 A Deionized Water To 100 To 100 To 100 EDTA-2Na 0.02 0.02 0.02 glycerin 5.0 5.0 5.0 B ethanol 2.0 2.0 2.0 PEG-60 hardened castor oil
(hydrogenated castor oil) 0.4 0.4 0.4 Perfume 0.04 0.04 0.04 C Ginsenoside Rh4 5.0 - - Erythromycin - - 5.0

[Test Example 15] Test for antibacterial activity against acne bacteria

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

The test method for the antimicrobial activity against acne bacteria was as follows.

(1) Preparation of test strain

Propionibacterium acnes were cultured in an anaerobic culture inoculated with BHI broth.

(2) Dilution solution preparation

0.15 ml of the above test solution was added to 15 ml of BHI broth (pH 6.8) or LB broth (pH 4.5), and the mixture was used as a diluting solution.

(3) Preparation of sample

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

(4) Antimicrobial activity test

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

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

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

The results of the antimicrobial activity test against acne bacteria are shown in Table 20 below. The MIC was expressed in terms of the concentration of the active ingredient contained in the formulation.

Item pH Propionibacterium acnes Formulation Example 3 5.7 > 30 ppm Comparative Formulation Example 3 5.7 Maximum concentration (no antimicrobial activity) Comparative Formulation Example 4 5.7 > 100ppm

In the case of Formulation Example 3, the concentration of ppm was remarkably smaller than that of Comparative Formulation Example 4 using erythromycin, which is a known acne treatment agent, It can be confirmed that the composition containing the side Rh4 has much better antibacterial activity against the test bacteria.

[Test Example 16] Lipogenesis inhibition test

3T3-L1 cells, a fibroblast cell line of mice, were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Dulbecco's modified eagles medium, GIBCO BRL, Life Technologies) Lt; ⁵ > cells / well in a well culture plate. After 2 days, the cells were replaced with fresh DMEM (containing 10% FBS) medium and cultured for 2 days. Then, the cultured cells were again induced to differentiate into DMEM (containing 10% FBS) containing 1 μg / ml insulin, 0.5 mM IBMX and 0.25 μM dexamethasone, and ginsenoside Rh4 and caffeine After 2 days of treatment, the cells were replaced with insulin-containing DMEM and cultured for 5 days. After 5 days, the cells were replaced with normal medium (DMEM, containing 10% FBS) and cultured while observing until the cells morphologically changed into adipocytes.

In order to evaluate the inhibitory effect of ginsenoside Rh4 on adipocyte accumulation in adipocytes, 3T3-L1 adipocytes differentiated as above were stained with S-III (S4136, sigma-aldrich). The adipocytes were fixed in 4% paraformaldehyde (pH 7.2) in phosphate buffer at room temperature, washed with PBS (phosphate buffered saline), stained with means III and then photographed and compared visually. The control group was fed with the test substance or the test substance without the reference substance, and 50 μM of caffeine was treated with another comparative group. The degree of inhibition of fat accumulation was graded by dividing the degree of staining by +++, +, +, -, which means that the degree of staining is greater toward +++. The results are shown in Table 21 below.

sample % Inhibition Control group +++ Comparative group + Ginsenoside Rh4 -

As shown in Table 21, it was found that the ginsenoside Rh4 used in the present invention had an effect of inhibiting lipid synthesis which is superior to caffeine, which is a known lipid synthesis inhibitor, in addition to a small amount of fat accumulated in adipocytes have. Therefore, lipid synthesis is inhibited, and sebum is reduced, so that occurrence of acne can be suppressed.

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

30 persons who had acne were divided into 3 groups of 10 persons, and subjects corresponding to each group were allowed to use the cosmetic composition prepared in Formulation Example 3 and Comparative Formulation Examples 3 to 4 for one month. Acne improvement scale is from 1 point to 5 points, 1 point is 'No', 3 points are 'Normal' and 5 points are 'Very agree'. The experimental results are shown in Table 22 below with an average score of 10 persons.

The acne extermination period was based on the number of days of disappearance readings, and the results were based on the results after 1 month with or without recurrence of acne. The reduction of sebaceous secretion was from 1 point to 5 points, 1 point was 'No', 3 points were 'Normal' and 5 points were 'Very agree'. The experimental results are shown in Table 22 below with an average score of 10 persons. The presence or absence of skin irritation was evaluated as (number of stimuli) / (total number of test subjects).

Inflammatory acne improvement Acne-bloating period Recurrence of acne Decrease sebaceous glands Presence or absence of stimulation Formulation Example 3 4.2 3 days radish 4.1 0/10 Comparative Formulation Example 3 2.1 13th U 2.0 0/10 Comparative Formulation Example 4 4.2 2 days radish 4.1 9/10

As shown in Table 22, it can be seen that Formulation Example 3 has no recurrence of acne compared to Comparative Formulation Example 3, and has an excellent effect on improvement of acne as a whole. On the other hand, Comparative Formulation Example 4 containing the antibacterial activity reference material shows the effect of improving acne but it is not suitable for long-term use due to strong skin irritation in use. However, since the composition according to the present invention has no irritation, .

[Test Example 18] IL-8 production inhibitory effect

The day before the experiment, normal human skin keratinocyte (NHEK, available from Lonza) was dispensed into a 96 well plate at 5 × 10 ⁴ cells / well and incubated at 37 ° C. in a 5% CO ₂ incubator And cultured for 24 hours. Twenty-four hours later, the cells were washed twice with PBS and replaced with serum free keratinocyte basement media (KBM). (50 μg / ml), PGSA (50 μg / ml) and LPS (1 μg / ml) were added to each well, Mu] g / ml), respectively. Here, peptidoglycan from S. aureus (PGSA ) is a peptidoglycan extracted from Staphylococcus aureus , which is a major constituent of the cell wall of Gram-positive (+) bacteria, and the cell membrane components of bacteria are known to cause inflammation. In addition, LPS (lypopolysaccaride) is a major constituent of Gram negative (-) bacterial cell membrane and is known to be the main cause of inflammation.

After culturing in a 5% CO ₂ incubator at 37 ° C for 24 hours, the culture was taken and ELISA for interleukin-8 (IL-8) was performed. The results are shown in Table 23 below. The ELISA used the experimental method of BD science.

division IL-8 secretion (pg / ml) Untreated Control (Control) 935.12 PGSA (10 [mu] g / ml) 4812.60 PGSA (50 占 퐂 / ml) 5895.08 PGSA (50 占 퐂 / ml) + LPS (1 占 퐂 / ml) 6814.91 Ginsenoside Rh4 (5 ppm) (1398.33) Ginsenoside Rh4 (25 ppm) (1212.41) Ginsenoside Rh4 (50 ppm) (1010.37)

In Table 23, it was confirmed that ginsenoside Rh4 significantly reduced and suppressed the secretion of IL-8 increased by PGSA and LPS. Therefore, it can be seen that the composition for external application for skin of the present invention can provide a superior anti-inflammatory effect by significantly reducing the secretion of IL-8 increased by PGSA and LPS.

[Test Example 19] Evaluation of itching relief

The day before the experiment, keratinocytes (cell line name: HaCaT available from ATCC) were dispensed in a 96 well plate at 4x10 ⁴ cells / well and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours Respectively. Twenty-four hours later, a 96-well plate was washed twice with HBSS (Hanks' Balanced Salt Solution) buffer, and the reaction buffer (2 μM Fluo-4-AM, 20% pluronic acid, 2.5 mM probenecid) gave. After incubation at 37 ° C in a 5% CO ₂ incubator for 30 minutes and at room temperature for 30 minutes, the cells were treated with ginsenoside Rh4 at the concentration (%) shown in Table 24 below by washing twice with HBSS buffer.

After reaction for 10 minutes to process the 2U / ml trypsin (Trypsin) or 5μM PAR-2 activation peptide (SLIGKV) and was measured in Ca ^{2 +} concentration in the cell 80 seconds. Measurement of intracellular Ca ²⁺ concentration was performed using FlexStation 3 (Molecular Device, USA). The difference between the minimum value and the maximum value obtained by measuring the flex for 80 seconds after the treatment with ginsenoside Rh4 and 2U / ml trypsin or 5 μM PAR-2 active peptide (SLIGKV) was obtained, The percent inhibition of intracellular entry of calcium ions compared to the difference between the minimum and maximum values in the treatment of 2 U / ml trypsin or 5 μM PAR-2 active peptide (SLIGKV) is shown in Table 24 below.

Ginsenoside Rh4 concentration % Inhibition of intracellular entry of calcium ions Trypsin (2 U / ml) PAR-2 active peptide (5 [mu] M) 0.05% 27 29 0.1% 32 33 0.5% 39 43 1.0% 44 52

As can be seen in Table 24 above, the influx of intracellular calcium ions by trypsin or PAR-2 activating peptide (SLIGKV) decreases with the treatment of ginsenoside Rh4, and as the concentration of ginsenoside Rh4 increases, It was confirmed that the inflow of calcium ions was remarkably reduced.

Accordingly, the composition for external application for skin containing ginsenoside Rh4 of the present invention can effectively provide an excellent antinociceptive effect by effectively inhibiting the itching-inducing PAR-2 activity.

[Formulation Example 4 and Comparative Formulation Example 5]

A shampoo was prepared with the composition shown in Table 25 below. Specifically, the surfactant and ethylene glycol distearate were added to purified water, heated to 80 DEG C to dissolve uniformly, and then gradually cooled to 40 DEG C with stirring. To the mixture were added the active ingredient according to the present invention and a preservative, A viscosity adjusting agent, a perfume, and a hair conditioning agent were mixed and stirred, followed by cooling to room temperature.

ingredient Formulation Example 4 Comparative Formulation Example 5 Ammonium lauryl sulfate 10 10 Polyoxyethylene lauryl ammonium sulfate 5 5 Cocoamidopropyl betaine 2 2 Ethylene glycol distearate 1.5 1.5 Cocoyl monoethanolamide 0.8 0.8 Ginsenoside Rh4 5.0 - Polyquaternium-10 0.2 0.2 Blue No. 1 0.0002 0.0002 Yellow No. 4 0.0001 0.0001 Methyl paraben 0.1 0.1 Spices 0.8 0.8 Citric acid 0.1 0.1 Dimethicone 1.0 1.0 water to 100 to 100

[Test Example 20] Dandruff reduction test

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

Before the start of the test, the dandruff was smeared with ordinary shampoo, and the dandruff accumulated for 2 days after shedding was collected. The dandruff weight was sampled once every two days with the shampoo of each of Formulation Example 4 and Comparative Example 5, The weight of accumulated dandruff was compared and evaluated. The accumulated dandruff was collected directly from the scalp using a vacuum suction device, and the dandruff reduction rate was calculated according to the following equation (7). The results are shown in Table 26 below.

Formulation Example 4 Comparative Formulation Example 5 Dandruff reduction rate (%) 62.8
55.9
73.1
65.3
48.1
59.3
69.6
72.6
58.6
71.6
59.6
68.4 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 63.7 2.5 SD 7.72 2.2

As shown in Table 26, it can be seen that Formulation Example 4 containing ginsenoside Rh4 exhibits excellent dandruff prevention effect.

[Test Example 21] Test for preventing the itchiness of the scalp

Twenty-four male and female twenty-four to twenty-five to fifty-year-olds who felt severe scalp itching were selected and divided into two groups of twelve persons. Each shampoo of Formulation Example 4 and Comparative Example 5 was used once every three days for two weeks. Then, scalp itching The preventive effect was evaluated through the following evaluation criteria, and the results are shown in Table 27 below.

[Evaluation standard]

Very good - 5 points

Excellent - 4 points

Usually - 3 points

Poor - 2 points

Very bad - 1 point

division Formulation Example 4 Comparative Formulation Example 5 The itching effect of scalp 4.2 2.3

As can be seen from the above Table 27, it can be seen that Formulation Example 4 containing ginsenoside Rh4 exhibits a more excellent effect in preventing scalp itching.

[Test Example 22] Effect of proliferation of dermal papilla cells

The keratin proteins that make up the hair are produced in hair follicle keratinocyte, which keratinocytes differentiate from the dermal papilla cells. In order to evaluate the activity of the dermal papilla cells of the present composition, DP6 (rat immortalized dermal papilla cell) cell line was used in the present invention (Wendy Filsell, Journal of Cell Science 107, 1761-1772 (1994)). This dermal papilla cell line was obtained by microdissection in the hair roots of male PVG rat beard and cultured in DMEM (Dulbecco's modified Eagle's medium, Gibco BRL, Gaithersburg, MD, USA) containing 10% FBS (Fetal bovine serum) USA) for 24 hours in an incubator maintained at 37 ° C in 5% CO ₂ . DP6 was added to a 96-well plate and cultured in a 37 ° C incubator for 24 hours. Then, the main ginsenoside Rh4 was treated at concentrations of 5 ppm, 10 ppm and 20 ppm, respectively. After 24 hours of drug treatment, cell proliferation was measured using WST-1 kit (Roche). The results are shown in Table 28 below.

division Cell proliferation (%) Untreated Control (Control) 100 Ginsenoside Rh4 (5 ppm) 131 Ginsenoside Rh4 (10 ppm) 139 Ginsenoside Rh4 (20 ppm) 148

As can be seen from Table 28, when the ginsenoside Rh4 was treated, the proliferation of the dermal papilla cells was increased, which was significantly increased in a concentration-dependent manner.

[Test Example 23] Evaluation of Potassium Ion Channel Activity Increase Effect

Minoxidil, a remedy for depilation, is known as a potential mitochondrial potassium ion channel opener (K _ATP channel opener) and is a representative drug used for the treatment of androgenetic alopecia. In order to evaluate the mechanism of minoxidil, cell proliferation was inhibited by treatment with tolbutamide (SIGMA AlDRICH, T0891) blocking K _ATP channel in fibroblasts constituting the dermis of the scalp, The restored test method was used.

To evaluate the function of this composition as a K _ATP channel opener, the fibroblast cell line NIH3T3 (mouse embryonic fibroblast cell line) cell line was used in the present invention. This cell line is a cell line obtained by natural immortalization of fibroblasts isolated from NIH Swiss mouse embryo by the 3T3 protocol. The cells were cultured in DMEM (Gibco BRL, Gaithersburg, MD, USA) containing 10% FBS for 24 hours in an incubator maintained at 37 ° C in 5% CO ₂ . NIH3T3 was added to a 96-well plate, cultured in a 37 ° C incubator for 24 hours, treated with 2.5 mM tolbutamide, and 10 minutes after the positive control, minoxidil 10 μM and ginsenoside Rh 4 were added at concentrations of 2.5 ppm, 5 ppm and 10 ppm And 48 hours after drug treatment, cell proliferation was measured using WST-1 kit (Roche). The results are shown in Table 29 below.

division Cell proliferation (%) Untreated Control (Control) 100 Minoxidil 132 Ginsenoside Rh4 (2.5 ppm) 111 Ginsenoside Rh4 (5 ppm) 129 Ginsenoside Rh4 (10 ppm) 136

As can be seen in Table 29, when ginsenoside Rh4 was treated, the proliferation of fibroblasts was restored and the cell proliferative capacity was increased depending on the concentration of the treated ginsenoside Rh4. The ginsenoside Rh4 Is treated with 10 ppm, it can be confirmed that cell proliferation is restored to the level when minoxidil is treated.

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

Melanocytes (melan-a) were added to a 24-well microtiter plate at 50,000 cells / well in RPMI medium supplemented with 5% fetal bovine serum, 100 IU penicillin G and 0.2 μM TPA Respectively. Dissociated cells were treated with ginsenoside Rh4 as a test substance at a final concentration of 10 ppm or 50 ppm, treated with 0.1% DMSO as a negative control, 100 袖 M IBMX as a positive control, and then incubated at 37 째 C for 3 days. After incubation, the wells were washed with PBS, and 100 μl of 1N NaOH was added thereto, and melanin in the cells was dissolved. The absorbance of dissolved melanin was measured at 405 nm using a microplate reader. The results of comparing melanin production promoting effect of ginsenoside Rh4 with the control group are shown in Table 30 below.

sample Amount of melanin synthesis (%) DMSO (0.1%) 100 IBMX (100 [mu] M) 120 Ginsenoside Rh4 (10 ppm) 109 Ginsenoside Rh4 (50 ppm) 123

Table 30 shows that ginsenoside Rh4 promotes melanin synthesis of melanocyte to increase melanin production and exhibit excellent melanin production promoting effect.

[Test Example 25] Promotion of MITF and tyrosinase expression promotion in melanocytes of ginsenoside Rh4

The cells were divided into 6-well microtiter plates at a density of 500,000 cells / well using a 501mel cell line. DMSO 0.1% as a negative control, IBMX 100 μM as a positive control group, and ginsenoside Rh4 was treated at 10 ppm and cultured at 37 DEG C for 24 hours, 48 hours, and 72 hours to obtain proteins. The proteins thus obtained were subjected to western blotting using MITF and tyrosinase antibodies. Protein extraction and Western blotting were routinely performed by standard methods used by those skilled in the art. After Western blotting, the result was compared with the negative control group of 100, and the results are shown in Table 31 below.

MITF Tyrosinase IBMX Ginsenoside Rh4 IBMX Ginsenoside Rh4 24hr 121 98 160 155 48hr 98 115 149 156 72hr 224 123 298 179

From Table 31, it can be seen that ginsenoside Rh4 elevates MITF and tyrosinase protein expression in melanocytes.

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

Antimicrobial experiments were conducted to evaluate the antimicrobial activity of ginsenoside Rh4. Specific experimental methods are as follows.

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

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

In a 96-well 96-well plate, add 16 μl of each sample to the first line, and add 184 μl of the diluted solution. In the remaining wells, 100 μl of the dilution solution was added. 100 μl of the mixed solution of No. 1 line was mixed well and 100 μl of the mixture was added to the No. 2 line. After mixing well, 100 μl of the mixture was added to the No. 3 line and diluted 2-fold.

Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa are 32? In a thermostat, Candida albicans and Aspergillus niger were cultured in a 25 ° C incubator.

After 48 hours, the microbial proliferation was determined by suspension and microscope to determine the minimum inhibitory concentration (MIC) value. The results are shown in Table 32 below.

Sample Minimum inhibitory concentration (MIC), unit% Sedona Monas
Aruzinosa Staphylococcus aureus Escherichia coli Candida Albikans Aspergillus niger Ginsenoside Rh4 0.21 0.04 0.124 > 3 > 1

As shown in Table 32, it can be confirmed that ginsenoside Rh4 exhibits antibacterial activity against various strains, and it can be predicted that ginsenoside Rh4 can act as a natural preservative or an antimicrobial agent in the composition.

Claims (23)

A composition for external application for skin comprising ginsenoside Rh4 represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]
The composition for external application for skin according to claim 1, wherein the ginsenoside Rh4 is contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. The composition for external application for skin according to claim 1, wherein the composition is for antioxidation. The composition for external application for skin according to claim 1, wherein the composition is anti-aging. The composition for external application for skin according to claim 1, wherein the composition is for improving skin elasticity. The composition for external application for skin according to claim 1, wherein the composition is for improving skin wrinkles. The composition for external application for skin according to claim 1, wherein the composition is for whitening. The composition for external application for skin according to claim 1, wherein the composition is for moisturizing. The composition for external application for skin according to claim 1, wherein the composition is for enhancing skin barrier function. The composition for external application for skin according to claim 1, wherein the composition is for inducing differentiation of dermal keratinocytes. The composition for external application for skin according to claim 1, wherein the composition is for improving acne. The composition for external application for skin according to claim 1, wherein the composition is antibacterial. The composition for external application for skin according to claim 1, wherein the composition is for anti-inflammation. The composition for external application for skin according to claim 1, wherein the composition inhibits lipid production. The composition for external application for skin according to claim 1, wherein the composition is for improving color and skin tone. The composition for external application for skin according to claim 1, wherein the composition is for shrinking pores. The composition for external application for skin according to claim 1, wherein the composition is for sebum control. The composition for external application for skin according to claim 1, wherein the composition is for improving skin troubles. The composition for external application for skin according to claim 1, wherein the composition is for skin irritation. The composition for external application for skin according to claim 1, wherein the composition is for preventing dandruff. The composition for external application for skin according to claim 1, wherein the composition is for hair growth. The composition for external application for skin according to claim 1, wherein the composition is for prevention of white hair. The composition for external application for skin according to claim 1, wherein the ginsenoside Rh4 is used as a natural preservative.