KR102021764B1 - Skin external composition containing ginsenoside Rh4 - Google Patents

Abstract

The present invention relates to an external composition for skin containing ginsenoside Rh4, and more particularly, by containing ginsenoside Rh4, an anti-aging effect, skin moisturizing effect, anti-inflammatory effect, acne, and the like through excellent antioxidant power. It improves the scalp and hair condition such as anti-dandruff, hair growth and white hair as well as improvement of overall skin condition such as skin trouble improvement, whitening effect, sebum control effect, pore contraction effect and improvement of complexion through blood circulation. It relates to a composition that can be provided.

Description

Skin external composition containing ginsenoside Rh4

The present invention relates to an external composition for skin containing ginsenoside Rh4, and more particularly, by containing ginsenoside Rh4, skin anti-aging effect, skin moisturizing effect, anti-inflammatory effect, acne, etc. In addition to improving the overall skin condition such as trouble improvement, whitening effect, sebum control effect, pore contraction effect, and improvement of complexion through blood circulation improvement, it provides scalp and hair condition improvement effects such as anti-dandruff effect, hair growth effect and anti-white hair effect. It relates to a composition which can be done.

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

In order to prevent changes in the skin condition caused by these internal and external factors and to maintain a healthy skin condition, the skin condition is improved by adding bioactive substances obtained from various known animals, plants, and microorganisms to cosmetics. Efforts have been made, and research and development of cosmetics using ginseng (Panax ginseng C. A Meyer) in particular has been conducted. Ginseng has been widely used in cosmetic compositions since its efficacy in the skin has been proven early, and ginseng root or leaf extract, ginseng saponin (ginsenoside), ginseng aglycon and ginseng polysaccharides have been used. However, the cosmetics containing such ginseng extract, extract, polysaccharides, etc. contain a very small amount of the active substance has a disadvantage that the effect is not significantly superior to other raw materials.

Ginsenoside Rh4, a genuine dammarane glycoside from Korean red ginseng (Baek NI et al., Planta Med. 1996 Feb; 62 (1): 86-7.)

Therefore, the present inventors can provide ginsenoside Rh4 contained in ginseng to provide anti-aging, skin wrinkle improvement, whitening, and moisturizing effect as well as to improve acne and skin troubles, and to improve skin color, sebum control, and pores. It has been found that the present invention can provide a skin condition improvement effect such as a contraction effect, and can also provide a scalp and hair condition improvement effect such as anti-dandruff, hair growth, and white hair prevention.

Accordingly, it is an object of the present invention to provide an external preparation composition for skin containing ginsenoside Rh4 which can improve the overall condition of the skin.

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

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

The present invention also provides a whitening skin external composition containing ginsenoside Rh4.

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

In addition, the present invention provides a topical composition for improving acne containing ginsenoside Rh4 as an active ingredient.

The present invention also provides a skin external composition for improving color and skin tone containing ginsenoside Rh4 as an active ingredient.

The present invention also provides a topical skin composition for reducing pores containing ginsenoside Rh4 as an active ingredient.

The present invention also provides a skin external composition for controlling sebum containing ginsenoside Rh4 as an active ingredient.

The present invention also provides a topical anti-dandruff composition containing 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.

In another aspect, the present invention provides a composition for preventing skin whitening containing ginsenoside Rh4 as an active ingredient.

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

The composition of the present invention, by containing ginsenoside Rh4, through the excellent antioxidant power, anti-aging effect, skin moisturizing effect, anti-inflammatory effect, skin trouble improvement effect such as acne, whitening effect, sebum control effect, pore contraction effect, blood circulation In addition to improving the overall skin condition such as improving the complexion through improvement, it is possible to provide an effect of improving the scalp and hair condition such as an anti-dandruff effect, a hair growth effect and a white hair prevention effect.

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

Ginsenoside Rh4 used in the present invention has a structure of Formula 1 below.

Ginseng belongs to the genus Ginseng of Araliace and is a herbaceous plant as a perennial ring-negative root. It is produced in many parts of the world, including Korea, China, Japan, and the United States, and is mainly cultivated at 85-140 degrees east, 22-49 degrees north latitude, 70-97 degrees west latitude, and 34-47 degrees north latitude in North America. As described above, ginseng produced in different parts of the world is different in its composition, and ginseng saponin, which is the most important ginsenoside, is used in Korean ginseng to represent various physiological and pharmacological effects of ginseng. About 34 kinds of ginsenosides have been isolated so far, and the pharmacological efficacy differs depending on the type of sugars bound to the aglycone, the number of the sugars bound thereto, or the binding sites thereof. According to the structural characteristics, it is divided into diols, triols and oleanes. In addition, it contains panacen, a polyacetylene compound, a polyphenol compound, flavonoids and vitamins, which are inherent fragrance components of ginseng.

Ginseng has long been recognized for its efficacy and has been used for the treatment of various diseases. The herbal efficacy of ginseng is known to improve stamina, improve metabolism, relieve stress, treat diabetes, improve respiratory diseases, improve digestive system, and anti-cancer activity.In recent research, ginseng has anti-complement activity, anti-ulcer activity, immune boosting effect, It has been reported to have anticancer action and hypoglycemic action. Skin-related effects include anti-inflammatory activity (Korean J. Dermatol. 18 (1): 39-42 (1980), Korean J. Dermatol. 14 (4): 335-339 (1976)), prevention of hyperkeratosis (Korean J. Dermatol. 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)), antioxidant effects (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 degradation (Mol. Cells 9 (5): 476-483 (1999)) and the whitening effect (Journal of the society of cosmetic scientists of Korea 27 (2): 45-56 (2001)).

Ginsenoside Rh4 of the present invention may be extracted from plants, synthesized according to methods known in the art, and may be commercially available. In addition, among the derivatives obtained by addition or substitution reaction of substituents commonly performed in the art with the ginsenoside Rh4, derivatives showing skin, hair improvement effect and antiseptic effect are included in the scope of the present invention. It is clear to those skilled in the art in consideration of this.

Ginsenoside Rh4 used in the present invention can be obtained especially from ginseng extract. The type of ginseng used at this time is not particularly limited, and ginseng, red ginseng, white ginseng, taeguksam, misam and the like can be used. In addition, the ginseng extract is contained not only in the leachate obtained by leaching and transferring from ginseng, but also in the concentrate obtained by partially or fully concentrating the leachate, or in stagnation, whole, regular, liquid extract and ginseng prepared by drying the concentrate again. The chemicals that exert the main effect, of course, include all of the plant itself, and extracts from all parts of ginseng, such as stems, roots, leaves, flowers, fruits, can be used and are not limited to extracts of any particular part. In addition, a method for extracting ginsenoside Rh4 from ginseng extract may use a known method.

Specifically, the ginsenoside Rh4 may be isolated from ginseng extract prepared by water or an organic solvent by a method well known in the art in ginseng. The organic solvent used in the present invention may be selected from the group consisting of ethanol, methanol, butanol, ether, ethyl acetate, chloroform and a mixed solvent of these organic solvents and water, and preferably 80% ethanol is used. At this time, the extraction temperature is preferably 10 ~ 80 ℃, it can be extracted for 3 to 24 hours. If the extraction temperature and extraction time is out of the extraction efficiency may decrease or change of components may occur.

The composition according to the present invention may contain ginsenoside 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 the ginsenoside Rh4 is less than 0.001% by weight, the efficacy and effect by the above components are insignificant. If the content of the ginsenoside Rh4 is more than 50% by weight, there is a problem of skin safety or formulation.

Since ginsenoside Rh4 is a component having excellent antioxidant power, the composition of the present invention containing ginsenoside Rh4 can be used as an anti-aging skin external preparation composition by providing excellent antioxidant power, which enhances skin elasticity and improves wrinkles. Excellent effect to let

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

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

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

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

The composition of the present invention can be used as a skin external preparation composition for pore reduction, sebum control and skin trouble improvement, which suppresses excessive secretion of sebum when applied to the skin, and reduces pores by promoting free radical removal and collagen synthesis. In addition, the effect of suppressing skin problems is excellent by reducing the expression of inflammatory factors. In addition, it is possible to defend against the generation of skin irritation due to its excellent antioxidant power.

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

The composition of the present invention can be used as a skin external preparation composition for hair growth, which can promote the growth of hair and prevent hair loss by promoting the transition from the resting hair cycle to the growing hair cycle.

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

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

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

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

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

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

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to test examples and formulation examples. However, these test examples and formulation examples are provided only for the purpose of illustration to help the understanding of the present invention is not limited to the scope and scope of the present invention by the following examples.

Reference Example 1

Ginsenoside Rh4 for testing the efficacy of the composition of the present invention was purchased from Ambo laboratory.

Test Example 1 Inhibitory Effect of Reactive Oxygen Species Production

The keratinocytes (keratinocyte) isolated from the skin tissue of the person into a 5 × 10 ⁴ are in each well of a 24-well plate was attached for 24 hours. After 16 hours, ginsenoside Rh4 was treated with 1%. At this time, the control group was not treated with ginsenoside Rh4 for comparison. After 2 hours, the culture solution was removed, and 100 µl of phosphate buffered saline (PBS) was added to each well. The keratinocytes were irradiated with UV 30mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: K5T8, UV B 15 W, Sankyo Dennki, Japan), and then PBS was removed and each cell was treated with keratinocyte culture solution 200. Μl was added. Ginsenoside Y was again treated and the amount of reactive oxygen species increased by UV stimulation at certain time points was quantified. The amount of ROS was quantified by referring to Tan's method for measuring the fluorescence of DCF-DA (dichlorofluorescin diacetate) oxidized by ROS (Tan et al., 1998, J. Cell Biol. Vol. 141, pp1423-1432). Table 1 shows the results of calculating the ratio of the vehicle-treated control group ROS.

Test substance UVB 30 mJ / cm ² Time since survey 0hr 2hr 3hr Vehicle 100 244 287 UVB + Vehicle 100 325 381 UVB + Ginsenoside Rh4 100 235 279

In the results of Table 1, ginsenoside Rh4 according to the present invention effectively inhibits the production of ROS known to cause skin cell damage by ultraviolet rays and ROS to a level higher than that when the amount of ROS after ultraviolet stimulation is not irradiated with ultraviolet rays. It can be seen that the antioxidant effect is very excellent by inhibiting the production of. Therefore, it was confirmed that ginsenoside Rh4 according to the present invention can prevent pores from widening by inhibiting oxidation and preventing aging, and can improve skin trouble by defending generation of skin irritation.

Test Example 2 Measurement of Elastase Inhibition Effect

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

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

Ginsenoside Rh4 (200 μL) and 50 μL of 20 μg / mL elastase type III solution were mixed in a 96 mg plate prepared with 10 mg / L Tris-HCL buffer (pH 8.0). 250 μM of EGCG was used as a positive control and distilled water was used as a non-treated group as a negative control. Thereafter, 100 µL of 0.4514 mg / mL N-SUCCINYL-ALA-ALA-ALA-p-NITROANILIDE prepared with the above buffer was added and reacted at 25 ° C for 15 minutes. After completion of the reaction, the absorbance at a wavelength of 415 nm was measured. A blank test was performed in the same manner to correct.

Calculation method of the elastase activity inhibitory activity is shown in Equation 1 below, the results are shown in Table 2.

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

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

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

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

compound Suppression degree (%) Untreated group 0 EGCG 65 Ginsenoside Rh4 77

As shown in Table 2, the degree of suppressing the elastase activity of ginsenoside Rh4 was significantly higher than that of EGCG, which is known as an elastase inhibitor, and the effect of inhibiting the elastase activity of ginsenoside Rh4 of the present invention was It can be confirmed that excellent.

Test Example 3 Collagenase (MMP-1) Inhibitory Activity

Inhibition of collagenase production of ginsenoside Rh4 of the present invention was measured in comparison with retinoic acid.

Place human fibroblasts at 5,000 cells / well in a 96-well microtiter plate containing Dulbecco® Modified Eagle® Media (DMEM) medium containing 2.5% fetal calf serum. The culture was incubated in an incubator at 37 ° C., 5% CO ₂ until 70-80% growth. Ginsenoside Rh4 or retinoic acid was treated at a concentration of 10 µg / ml for 24 hours, followed by cell culture.

The degree of collagenase production of cell cultures was measured using a commercially available collagenase measuring instrument (Amersham Pharmacia, USA, Catalog #: RPN 2610). First, the cell culture solution collected in a 96-well plate uniformly coated with primary collagenase antibody was placed in an incubator for 3 hours. After 3 hours, the chromophore-bound secondary collagen antibody was placed in a 96-well plate and reacted for another 15 minutes. After 15 minutes, 3,3 ', 5,5'-tetramethylbenzidine (sigma), a color-causing substance, was added to induce color development for 15 minutes at room temperature. 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 the yellow 96-well plate was measured at 405 nm using an absorbance meter, and the degree of synthesis of collagenase was calculated by Equation 2 below, and the results are shown in Table 3 below. Indicated. At this time, the reaction absorbance of the cell culture liquid collected from the group not treated with the composition was used as a control.

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

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

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

[Formulation Example 1 and Comparative Formulation Example 1]

Nutritional cream was prepared in a conventional manner according to the composition of Table 4 (unit: wt%).

Ingredient Formulation Example 1 Comparative Formulation Example 1 Purified water To 100 To 100 Ginsenoside Rh4 0.1 - Vegetable Cured Oil 1.50 1.50 Stearic acid 0.60 0.60 Glycerol Stearate 1.00 1.00 Stearyl alcohol 2.00 2.00 Polyglyceryl-10 Pentastearate & Behenyl Alcohol & Sodium Stearoyl Lactylate 1.00 1.00 Arakidil Behenyl Alcohol & Arakidil Glucoside 1.00 1.00 Cetylaryl Alcohol & Cetearyl Glucoside 2.00 2.00 PEG-100 Stearate & Glycerolate & Propylene Glycol 1.50 1.50 Caprylic / Carric Triglycerides 11.00 11.00 Cyclomedicon 6.00 6.00 Preservative, incense Quantity Quantity Triethanol amine 0.1 0.1

Test Example 4 Confirmation of Skin Elasticity Improvement Efficacy

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

Twenty healthy women in their 30s to 40s were divided into two groups of 10 people in two groups of Formulation Example 1 and Comparative Formulation Example 1, and the nutrition cream was applied to the face once a day for 12 weeks. 575, C + K Electronic Co., Germany) was used to measure skin elasticity. The results are shown in Table 5 below. The results in Table 5 are described as ΔR8 values of Cutometer SEM 575, where R8 values indicate the nature of skin viscoelasticity.

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

As shown in Table 5, Formulation Example 1 containing the ginsenoside Rh4 of the present invention was further increased skin elasticity compared to 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 Improvement Efficacy

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

In order to confirm the wrinkle improvement effect of the Formulation Example 1 and Comparative Formulation Example 1 was evaluated as follows. Twenty healthy women in their 40s were divided into two groups of 10 people for each of two groups of Formulation Example 1 and Comparative Formulation Example 1, and the nourishing cream was applied to the face once a day for 12 weeks. The condition of the skin was measured by a skin meter (visiometer, SV600, Courage + Khazaka electronic GmbH, Germany) and analyzed. The results are shown in Table 6 below. The values in Table 6 below represent the average of each parameter value after 12 weeks of application minus the parameter value before application.

8 weeks after use
Clinical results R1 R2 R3 R4 R5 Formulation Example 1 0.14 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 highest and lowest of the wrinkle contour

R2: Divide the wrinkle contours by 5 spaces, and then average the R1 values

R3: The highest value among the R1 values divided by five

R4: The mean value of the baseline of the fold contour minus the top and valley values of each angle

R5: Difference value of the baseline of the wrinkle contour line minus each wrinkle outline

As shown in Table 6, it was found that the external preparation composition of Formulation Example 1 has a very good skin wrinkle improvement.

Test Example 6 Tyrosinase Inhibitory Effect

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

0.2 ml of the sample solution prepared by mixing ginsenoside Rh4 of the present invention in an appropriate concentration in an ethanol solution was added to the reaction solution and reacted for 10 minutes in a 37 ° C thermostat. In this case, the control group was prepared by adding only 0.2 ml of solvent instead of each sample solution, and ascorbic acid was used as a positive control group. 0.1 ml of a tyrosinase solution of 2500 units / ml was added to the reaction solution and reacted for 10 minutes in a 37 ° C thermostat. The reaction tube containing the reaction solution was quenched in ice water to stop the reaction, and the absorbance at wavelength 475 nm was measured with a photospectrometer. The results are shown in Table 7 below. Each tyrosinase inhibitory effect was calculated by the following equation.

Test substance Tyrosinase inhibition rate (%) Control group (no addition) 0 Ascorbic acid 52 Ginsenoside Rh4 63

In the results of Table 7, the tyrosinase inhibition rate of ginsenoside Rh4 according to the present invention is much higher than the known tyrosinase inhibitor ascorbic acid, which shows that the whitening effect is very excellent.

[Test Example 7] inhibitory effect of melanin production using B16 / F10 melanoma cells

A sample containing 0.1% by weight of ginsenoside Rh4 and kojic acid, respectively, was added as a test substance to a culture medium of B16 / F10 melanoma cells (Korea Cell Line Bank) at a constant concentration for 3 days, after which the culture solution was removed and PBS was removed. The cells were washed with 1N NaOH and absorbed at 405 nm. The cells without test substance were used as a control group, and the degree of inhibition of melanin production of each test substance was measured by comparing with the melanin content of the control group. According to Equation 4, the melanin production inhibition rate is calculated and the results are shown in Table 8.

Test substance Melanogenesis inhibition rate (%) Control group (no addition) 0 Kojisan 53 Ginsenoside Rh4 68

In the results of Table 8, it can be seen that the ginsenoside Rh4 melanin production inhibition rate of the present invention is much higher than the known melanogenesis inhibitor kojic acid is very excellent whitening effect.

Test Example 8 Measurement of Increased Skin Moisturizing Effect

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

Twenty male and female adults in their 40s to 50s, classified as dry skin, were divided into two groups of 10 people for each of the two groups, Formulation Example 1 and Comparative Formulation Example 1, and applied to the face twice daily for four weeks. Skin moisture meter at constant temperature and constant humidity (24 ° C, 40% relative humidity) before start of application, after 1 week, 2 weeks, 4 weeks after application, and after 2 weeks (total 6 weeks) after application was stopped. Skin moisture content was measured by (Corneometer CM825, C + K Electronic Co., Germany). The results are shown in Table 9 below. The results in Table 9 show the percentage increase of the measured value after treatment for a period of time based on the skin moisture meter value measured immediately before the start of the test.

Test group Moisture Growth Rate (%) 1 week past 2 weeks past 4 weeks past After 6 weeks Formulation Example 1 32 33 37 34 Comparative Formulation Example 1 30 32 32 15

In the results of Table 9, when the Comparative Formulation Example 1 was applied, it showed a water increase rate of about 30% up to 4 weeks after the application, but after stopping the application, the moisture content of the skin decreased, whereas ginsenoside Rh4 In the case of application of Formulation Example 1 containing, it can be seen that the skin moisture increase rate is more than 30% even after the application is stopped. It can be seen that the composition of the present invention containing ginsenoside Rh4 has an excellent skin moisturizing effect.

Test Example 9 Measurement of Keratinocyte Differentiation Promotion Effect

In order to determine the differentiation promoting effect of keratinocytes of ginsenoside Rh4, the amount of CE (Cornified Envelop) generated during the differentiation of keratinocytes was measured using absorbance.

First, the keratinocytes of humans isolated from the epidermis of the newborn were cultured and attached to the bottom of the culture flask, and then treated with 5 ppm concentration of ginsenoside Rh4 in the culture medium. Incubate for 5 days until growth. At this time, the low calcium (0.03mM) treated group and the high calcium (1.2mM) treated group were used as negative and positive controls, respectively. Then, the cultured cells were harvested and washed with PBS (Phosphate buffered saline), followed by 10 mM Tris-HCl buffer solution containing 2% SDS (sodium dodecyl sulfate) and 20 mM Dithiothreitol (DTT). pH 7.4) 1 ml was added to sonication, boiling and centrifugation, and the precipitate was suspended in 1 ml of PBS again to measure absorbance at 340 nm. Separately, a portion of the solution after the sonication was taken to measure the protein content and used as a reference when evaluating the degree of cell differentiation. The results are shown in Table 10 below.

Test substance Differentiation capacity in keratinocytes (%) Low Calcium (0.03 mM) Solution
(Negative control) 100 High Calcium (1.2mM) Solution
(Positive control) 210 Ginsenoside Rh4 285

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

Test Example 10 Measurement of Skin Barrier Function Restoration Effect

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 performed. Ten adult men and women's upper and lower limbs were damaged by a tape stripping method for 7 days while applying two groups of Formulation Example 2 and Comparative Formulation Example 2, each prepared by the composition of Table 11 below. The recovery of transdermal moisture loss (TWEL) was measured once a day using a Vapometer (Delfin, Finland). Comparative Formulation Example 2 is a vehicle as the negative control group. The experimental results are shown in Table 12 below. The results in Table 12 were compared on the basis of the 100% difference before treatment and after barrier damage.

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

Test group TWEL change (%) Before treatment 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 in Table 12, when the Comparative Formulation Example 2 containing no ginsenoside Rh4 is treated, the transdermal moisture loss increases over time, whereas the ginsenoside Rh4 contains a formulation example. In case of treatment of 2, transdermal moisture loss returns to normal and barrier damage is recovered.

Test Example 11 Hemoglobin Improvement Effect

In order to evaluate the skin blood circulation promoting effect of the cosmetic composition according to the present invention, the degree of blood circulation in the skin was measured using a Laser Doppler Perfusion Imager (LDPI). LDPI is a well-known and currently used device for measuring blood circulation in the skin, and is a very sensitive device that can measure not only the speed and amount of blood in the capillaries of the skin but also the flow in the small arteries and the venous veins.

After washing the face with soap in a thermo-hygrostat room for 30 minutes, the initial value was measured using LDPI. Initially, initial blood flow in the lower forehead of 30 women with cold hands and feet was measured by LDPI. Then, after the formulation Example 1 and Comparative Formulation Example 1 were used in the test subjects for one week, the blood flow rate measured after the initial measurement value (skin blood flow change) is shown in Table 13 below.

LDPI Results-Skin Blood Flow Before and After Cosmetic Use Test substance % Change in skin blood flow after 1 week of use Formulation Example 1 11 Comparative Formulation Example 1 5

In the results of Table 13, the cosmetic composition according to the present invention significantly increased the skin blood flow than Comparative Formulation Example 1 containing no ginsenoside Rh4, it was confirmed that the blood color is improved through the blood circulation. . This ultimately suggests that the cosmetic composition containing ginsenoside Rh4 according to the present invention can contribute effectively to the skin's nutrient delivery, inhibit skin aging and delay.

Test Example 12 Skin Tone Improvement Effect

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

Test substance Skin tone improvement rate (%) Brightness (mean ± standard deviation) Color (mean ± standard deviation) Formulation Example 1 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 containing no ginsenoside Rh4 according to the present invention showed no significant skin tone improvement, whereas Formulation Example 1 containing ginsenoside Rh4 as an active ingredient was used more than before use. It was confirmed that a lot of later skin tone was improved.

Test Example 13 Pore Reduction Effect

1. Pore reduction effect through promoting collagen biosynthesis

Ginsenoside Rh4 according to the present invention was measured by comparing collagen biosynthesis promoting effect with TGF-β. First, fibroblasts were seeded by 10 ⁵ per hole in 24 wells and cultured until 90% growth. This treatment serum and then cultured in DMEM medium free Ginsenoside Rh4 and TGF-β of the present invention was dissolved in serum-free medium by each of 10ng / ml for 24 hours, CO ₂ The incubator was incubated for 24 hours. These supernatants were removed and procollagen increased or decreased using procollagen type I ELISA kit (procollagen type (I)). The results are shown in Table 15, and the synthesis ability of collagen was compared with the non-treated group as 100.

Test substance Collagen Synthesis (%) Untreated group 100 TGF-β 183.5 Ginsenoside Rh4 196.1

In the results of Table 15, ginsenoside Rh4 according to the present invention was confirmed to exhibit a higher level of collagen synthesis than the positive control group TGF-β. Therefore, it was confirmed that ginsenoside Rh4 according to the present invention can reduce the enlarged pores by increasing the amount of collagen production around the pores.

2. Pore Reduction Effect

In order to determine the pore reduction effect of Formulation Example 1 and Comparative Formulation Example 1 was evaluated as follows. Twenty male and female subjects with large pore sizes were selected and divided into two groups of ten, and the nutritional creams of Formulation Example 1 and Comparative Formulation Example 1 were applied to the face each day for 4 weeks. Determination of the effect of pore reduction was made by visual assessment of experts by taking pictures before and after 4 weeks of the experiment. The results are shown in Table 16 below (rating rating: 0-not reduced at all; 5-very reduced).

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

From the results of Table 16, Comparative Formulation Example 1 does not have a pore reduction effect, but in the case of Formulation Example 1 shows a pore reduction effect that can be visually confirmed, ginsenoside Rh4 according to the present invention reduces the size of the pores It was found that the effect was excellent.

Test Example 14 Sebum Secretion Inhibitory Effect

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

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

In order to confirm the degree of inhibition of 5α-reductase activity, ginsenoside Rh4 was added and incubated for 2 hours at 37 ° C and 5% CO ₂ incubator. At this time, the ginsenoside Rh4 was used as a negative control group, the pinasteride was used as a positive control group. Thereafter, the culture medium was collected, the steroid was extracted with 800 μl ethyl acetate, the organic solvent layer was separated, dried, and the remaining residue was dissolved in 50 μl ethyl acetate. The silica plastic sheet kieselgel 60 F254 ) Was developed using ethyl acetate-hexane (1: 1) as a solvent.

After drying the plastic sample in air, the bath system was used to measure the isotope amount. The isotopic isomer of testosterone and dihydrotestosterone remaining in the film after one week by placing the dried plastic sheet and the x-ray film together in the bath cassette. After the amount was measured, 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 % Inhibition Negative Control 48.0 - Positive control group 27.6 42.5 Ginsenoside Rh4 14.9 61.3

In the results of Table 17, 5α-reductase, which converts testosterone into dihydrotestosterone, binds to a receptor protein in the cytoplasm, enters the nucleus, activates sebaceous gland cells, promotes differentiation, and hypersecretes sebum in sebaceous glands. It was confirmed that ginsenoside Rh4 effectively inhibits the activity of blocking the conversion of testosterone to dihydrotestosterone, and it was shown to have a superior inhibitory effect than finasteride known to inhibit the activity of 5α-reductase. Accordingly, it was confirmed that ginsenoside Rh4 can suppress sebum hypersecretion by effectively inhibiting the activity of 5α-reductase.

2. Sebum secretion inhibitory effect

In order to determine the sebum secretion inhibitory effect of Formulation Example 1 and Comparative Formulation Example 1 was evaluated as follows. Thirty male and female subjects who felt sebum secretion were selected and the nutritional creams of Formulation Example 1 and Comparative Formulation Example 1 were applied to the designated sites every day for 4 weeks. Determination of the effect of sebum reduction was measured using the sebum meter (Sebumeter SM810, C + K Electronic Co., Germany) to measure the average sebum reduction rate (%) after 2 weeks and 4 weeks, respectively, the results are shown in Table 18 Shown in

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 sebum secreted in excess than Comparative Formulation Example 1 not containing it.

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

Formulation Example 3 and Comparative Formulation Examples 3 to 4 were prepared according to the ingredients and the contents (% by weight) shown in Table 19 below. Specifically, Formulation Example 3 is a formulation of ginsenoside Rh4, Comparative Formulation Example 3 does not contain any active ingredients for improving acne skin, Comparative Formulation Example 4 is a standard to be used as a reference for antimicrobial activity It contains erythromycin, which is widely used as an acne treatment.

The preparation method of Formulation Example 3 and Comparative Formulation Examples 3-4 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 dissolution bath, followed by solubilization by adding phase B to phase A. The components of phase C were added thereto according to the blending ratios described in Table 19 to homogenize the mixture 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 Cured 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 Antibacterial Activity Test against Acne Bacteria

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

The antibacterial test method for acne bacteria was as follows.

(1) Test bacteria preparation

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

(2) dilution solution preparation

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

(3) sample preparation

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

(4) antimicrobial activity test

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

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

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

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

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

The smaller the ppm concentration in MIC, the more effective the antimicrobial activity against acne bacteria. In the case of Formulation Example 3, the concentration of ppm was significantly lower than that of Comparative Formulation Example 4 using erythromycin, a known acne treatment agent. The composition containing the side Rh4 can be confirmed to have a much better antimicrobial activity against the test bacteria.

Test Example 16 Lipogenesis Inhibition Test

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

Sudan III staining (S4136, sigma-aldrich) was performed on the differentiated 3T3-L1 adipocytes to evaluate the inhibitory effect of ginsenoside Rh4 on adipocyte accumulation in adipocytes. Adipose cells were fixed at room temperature with 4% paraformaldehyde (pH 7.2) in phosphate buffer, washed with PBS (phosphate buffered saline), stained with Sudan III, and photographed for visual comparison. As a control group, only the medium without the test substance or the comparative substance was used, and 50 μM of caffeine was treated with another control group. The degree of inhibition of fat accumulation was given by dividing the degree of staining into +++, ++, +, and-, which means that the degree of staining is increased toward +++. The results are shown in Table 21 below.

sample % Inhibition Control +++ Comparison + Ginsenoside Rh4 -

As shown in Table 21, ginsenoside Rh4 used in the present invention is not only a small amount of fat accumulated in adipocytes, but also has a superior lipid synthesis inhibitory effect than caffeine, a known lipid synthesis inhibitor. have. Therefore, sebum is reduced by inhibiting lipid synthesis, thereby suppressing the occurrence of acne.

[Test Example 17] Test of acne improvement, sebum secretion and irritation

Thirty people with acne were divided into three groups of 10 people, and subjects corresponding to each group were allowed to use the cosmetic composition prepared in Formulation Example 3 and Comparative Formulation Examples 3 to 4 for one month. The acne improvement scale ranged from 1 to 5 points, with 1 being 'no', 3 being 'normal' and 5 being 'very so'. Experimental results are shown in the average score of 10 people in Table 22 below.

Acne disappearance was based on the number of days the extinction was read, acne recurrence was based on the results after one month. Sebum secretion is reduced from 1 to 5 points, 1 point is 'No', 3 points are 'Normal' and 5 points are 'Yes.' The experimental results are expressed in the average score of 10 people in Table 22 below. The presence or absence of skin irritation was determined as (number of irritants) / (total number of test subjects).

Inflammatory Acne Improvement Cotton acne extinction Acne recurrence Reduce sebum secretion Irritation Formulation Example 3 4.2 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, Formulation Example 3 did not recur acne compared to Comparative Formulation Example 3, it can be seen that there is an excellent effect on the overall acne improvement. On the other hand, Comparative Formulation Example 4 containing an antimicrobial activity standard shows an acne improvement effect, but in use, the skin irritation seems to be unsuitable for long-term use due to the strong skin irritation, the composition according to the present invention does not have a stimulus for long-term use Even appeared to be appropriate.

Test Example 18 Effect of Inhibiting IL-8 Production

One day before the experiment, normal human skin keratinocytes (NHEK, obtained from Lonza) were dispensed in 96-well plates at 5x10 ⁴ cells / well, and then in a 37 ° C, 5% CO ₂ incubator. Incubated for 24 hours. After 24 hours, the cells were washed twice with PBS and changed to serum-free keratinocyte basement media (KBM). Each well was treated with ginsenoside Rh4 according to the concentration of Table 23 for 30 minutes, followed by PGSA (10 µg / ml), PGSA (50 µg / ml), and PGSA (50 µg / ml) + LPS (1). [Mu] g / ml) was treated respectively. Here, PGSA (peptidoglycan from S. aureus ) is a peptidoglycan (peptidoglycan) extracted from Staphylococcus aureus, a major component of the Gram-positive (+) cell wall and bacterial cell membrane components are known to cause inflammation. In addition, LPS (lypopolysaccaride) is a major component of the cell membrane of Gram-negative (-) bacteria and is known to be a major cause of inflammation.

After incubation for 24 hours at 37 ℃, 5% CO ₂ incubator, the culture medium was taken and subjected to ELISA for Interleukin-8 (Interleukin-8, IL-8), the results are shown in Table 23 below. ELISA used the experimental method of the manufacturer (BD science).

division IL-8 secretion (pg / ml) Untreated Control 935.12 PGSA (10 μg / ml) 4812.60 PGSA (50 μg / ml) 5895.08 PGSA (50 μg / ml) + LPS (1 μg / ml) 6814.91 Ginsenoside 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 inhibited the secretion of IL-8 increased by PGSA and LPS. Therefore, it can be seen that the topical skin composition of the present invention can provide an excellent anti-inflammatory effect by significantly reducing the secretion of IL-8 increased by PGSA and LPS.

[Test Example 19] Itching relief evaluation

One day before the experiment, the keratinocytes (Cell name: HaCaT obtained from ATCC) were dispensed into 96 well plates at 4x10 ⁴ cells / well, followed by incubation for 24 hours in a 37 ° C, 5% CO ₂ incubator. It was. After 24 hours, wash 96 well plates twice with Hanks'Balanced Salt solution (HBSS) buffer, and then add reaction buffer (2 μM Fluo-4-AM, 20% pluronic acid, 2.5 mM probenecid) to the cells. gave. After reaction at 37 ° C., 5% CO ₂ incubator for 30 minutes, and at room temperature for 30 minutes, the cells were washed twice with HBSS buffer and treated with ginsenoside Rh4 at a concentration (%) as shown in Table 24 below.

After reaction for 10 minutes to process the 2U / ml trypsin (Trypsin) or 5μM PAR-2 activation peptide (SLIGKV) and was measured in Ca ^{2 +} concentration in the cell 80 seconds. Intracellular Ca ²⁺ concentration change was measured using FlexStation3 (Molecular Device, USA). After the ginsenoside Rh4 and 2U / ml Trypsin or 5μM PAR-2 active peptide (SLIGKV) treatment, the flex was measured for 80 seconds to determine the difference between the minimum and maximum values. Inhibition rate (%) for the cellular influx of calcium ions compared to the difference between the minimum value and the maximum value when treated with 2 U / ml trypsin or 5 μM PAR-2 active peptide (SLIGKV) is shown in Table 24 below.

Ginsenoside Rh4 Concentration Inhibition rate on the cellular influx of calcium ions (%) Trypsin (2U / ml) PAR-2 active peptide (5 μM) 0.05% 27 29 0.1% 32 33 0.5% 39 43 1.0% 44 52

As can be seen in Table 24, the influx of intracellular calcium ions by trypsin or PAR-2 active peptide (SLIGKV) is reduced by the treatment of ginsenoside Rh4, and the concentration of ginsenoside Rh4 is increased. It was confirmed that the influx of calcium ions was significantly reduced.

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

Formulation Example 4 and Comparative Formulation Example 5

To prepare a shampoo in the composition of Table 25. Specifically, the surfactant and ethylene glycol distearate are added to purified water, heated to 80 ° C., uniformly dissolved, and then slowly cooled to 40 ° C. under stirring, and the active ingredient and preservative according to the present invention are added to the mixture. A viscosity modifier, a perfume, and a hair conditioner were added and mixed, followed by cooling to room temperature under stirring.

ingredient Formulation Example 4 Comparative Formulation Example 5 Ammonium Lauryl Sulfate 10 10 Polyoxyethylene lauryl ammonium sulfate 5 5 Cocoamidopropylbetaine 2 2 Ethylene Glycol Distearate 1.5 1.5 Cocoyl Monoethanolamide 0.8 0.8 Ginsenoside Rh4 5.0 - Polyquaternium-10 0.2 0.2 Blue No. 1 0.0002 0.0002 Yellow No. 4 0.0001 0.0001 Methylparaben 0.1 0.1 Spices 0.8 0.8 Citric acid 0.1 0.1 Dimethicone 1.0 1.0 water to 100 to 100

Test Example 20 Dandruff Reduction Effect Test

Twenty-four males aged 19 to 35 years with relatively high dandruff were selected and used in two groups of 12 shampoos for each of Formulation Example 4 and Comparative Formulation Example 5 for one month as follows. .

Before the start of the test, triturate with a normal shampoo, and collect the dandruff accumulated for 2 days after the shampoo, and the weight of the collected dandruff and trituration once every 2 days with the shampoos of Formulation Examples 4 and Comparative Formulation Example 5, 2 days after the end of the test. The weight of accumulated dandruff was evaluated. At this time, the accumulated dandruff was collected directly from the scalp with a vacuum suction device, to obtain a rate of dandruff reduction based on Equation 7 below and 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 can be seen in Table 26, it can be seen that the formulation example 4 containing ginsenoside Rh4 exhibits an excellent anti-dandruff effect.

Test Example 21 Scalp Itching Prevention Test

Twenty-four men and women aged 25 to 45 who feel severely itching scalp were divided into two groups of 12 people, each of whom used the shampoos of Formulation Example 4 and Comparative Formulation Example 5 once every three days for two weeks. The prevention effect was evaluated through the following evaluation criteria and the results are shown in Table 27 below.

[Evaluation standard]

Very good-5 points

Excellent-4 points

Normal-3 points

Poor-2 points

Very bad-1 point

division Formulation Example 4 Comparative Formulation Example 5 Itching effect of scalp 4.2 2.3

As can be seen in Table 27, it can be seen that the formulation of Example 4 containing ginsenoside Rh4 shows a better effect in preventing scalp itch.

Test Example 22 Hair Follicle Hair Papillary Cell Proliferation Effect

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

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

As can be seen in Table 28, the treatment of ginsenoside Rh4 increases the growth of dermal papilla cells, which can be confirmed to increase significantly in a concentration-dependent manner.

Test Example 23 Evaluation of Potassium Ion Channel Activity Increase Effect

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

In order to evaluate the function of the composition as a K _ATP channel opener, in the present invention, a mouse embryonic fibroblast cell line (NIH3T3) cell line was used. The cell line is a cell line in which the fibroblast cell line isolated from NIH Swiss mouse embryo was naturally immortalized by 3T3 protocol. The cell line was incubated in DMEM (Gibco BRL, Gaithersburg, MD, USA) containing 10% FBS for 24 hours in an incubator maintained at 5% CO ₂ , 37 ℃. NIH3T3 was placed in a 96-well plate and incubated in a 37 ° C. incubator for 24 hours, treated with 2.5 mM tolbutamide, and after 10 minutes, 10 μM of minoxidil and ginsenoside Rh4 at concentrations of 2.5 ppm, 5 ppm, and 10 ppm, respectively, were tested. After 48 hours of drug treatment, cell proliferation was measured using the WST-1 kit (Roche). The results are shown in Table 29 below.

division Cell proliferation (%) Untreated 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 the ginsenoside Rh4 is treated, the proliferation of fibroblasts is recovered, and the cell proliferation ability is increased depending on the concentration of the treated ginsenoside Rh4. When treated with 10ppm it can be confirmed that the proliferation of the cells is restored to the level when the minoxidil treatment.

TEST EXAMPLE 24 Melanin Promoting Effect Test of Ginsenoside Rh4

Add melan-a to 50,000 cells / well in a 24-well microtiter plate in medium containing 5% fetal bovine serum, RPMI medium, 100 IU penicillin G and 0.2 μM TPA in RPMI medium. Busy. The next day the cells were treated with ginsenoside Rh4 at a final concentration of 10 ppm or 50 ppm as a test substance, 0.1% DMSO as a negative control and 100 μM IBMX as a positive control, followed by incubation at 37 ° C for 3 days. After incubation, the wells were washed with PBS, 100 μl of 1N NaOH was added, and the melanin in the cells was lysed. The absorbance of the dissolved melanin was measured at 405 nm using a microplate reader. The result of comparing the melanin production promoting effect of ginsenoside Rh4 with the control is shown in Table 30 below.

sample Melanin Synthesis (%) DMSO (0.1%) 100 IBMX (100 μM) 120 Ginsenoside Rh4 (10 ppm) 109 Ginsenoside Rh4 (50 ppm) 123

Looking at Table 30, ginsenoside Rh4 promotes melanin synthesis of melanocytes, the melanin production is increased, showing an excellent melanin production promoting effect.

Experimental Example 25 Effect of Ginsenoside Rh4 on Melanosite in Promoting MITF and Tyrosinase Expression

Dispense 500,000 cells / well in a 6-well microtiter plate using a 501mel cell line, in each hole 0.1% DMSO as a negative control, 100 μM as a positive control, and ginsenoside as a test group. Rh4 was treated with 10 ppm, and the protein was obtained after incubating at 37 ° C. for 24 hours, 48 hours, and 72 hours. The protein thus obtained was subjected to western blot using MITF and tyrosinase antibody. Protein extraction and Western blot were usually performed by standard methods used by those skilled in the art. After Western blot the results are shown in Table 31 below by comparing the negative control to 100.

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

Looking at Table 31, it can be seen that ginsenoside Rh4 increases the expression of MITF and tyrosinase protein in melanocytes.

Test Example 26 Evaluation of Antimicrobial Activity of Ginsenoside Rh4

Antimicrobial experiments were conducted to evaluate the antimicrobial activity of ginsenoside Rh4. The specific experimental method is as follows.

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

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

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

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

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

Sample Minimum Inhibitory Concentration (MIC),% Sedonas
Aruginosa Staphylococcus aureus Escherichia Coli Candida Albicans Aspergillus Niger Ginsenoside Rh4 0.21 0.04 0.124 > 3 > 1

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

Claims (23)

A whitening cosmetic composition containing ginsenoside Rh4 as an active ingredient. The cosmetic composition for whitening 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. Moisturizing cosmetic composition containing ginsenoside Rh4 as an active ingredient. The moisturizing cosmetic composition according to claim 3, wherein the ginsenoside Rh4 is contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. Cosmetic composition for enhancing skin barrier function containing ginsenoside Rh4 as an active ingredient. The cosmetic composition for enhancing skin barrier function according to claim 5, wherein the ginsenoside Rh4 is contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. Cosmetic composition for inducing skin keratinocyte differentiation containing ginsenoside Rh4 as an active ingredient. The cosmetic composition for inducing skin keratinocyte differentiation according to claim 7, wherein the ginsenoside Rh4 is contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. Acne improvement cosmetic composition containing ginsenoside Rh4 as an active ingredient. The cosmetic composition for improving acne according to claim 9, wherein the ginsenoside Rh4 is contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. Antibacterial cosmetic composition containing ginsenoside Rh4 as an active ingredient. The antimicrobial cosmetic composition according to claim 11, wherein the ginsenoside Rh4 is contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. A cosmetic composition for inhibiting lipid formation, comprising ginsenoside Rh4 as an active ingredient. The cosmetic composition for inhibiting lipid production according to claim 13, wherein the ginsenoside Rh4 is contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. A cosmetic composition for improving color and skin tone, comprising ginsenoside Rh4 as an active ingredient. A cosmetic composition for shrinking pores containing ginsenoside Rh4 as an active ingredient. Cosmetic composition for sebum secretion containing ginsenoside Rh4 as an active ingredient. Cosmetic composition for improving skin problems containing ginsenoside Rh4 as an active ingredient. A cosmetic composition for defense against skin stimulation, comprising ginsenoside Rh4 as an active ingredient. Anti-dandruff cosmetic composition containing ginsenoside Rh4 as an active ingredient. Hair growth cosmetic composition containing ginsenoside Rh4 as an active ingredient. White hair prevention cosmetic composition containing ginsenoside Rh4 as an active ingredient. A cosmetic composition comprising ginsenoside Rh4 as an active ingredient, wherein the ginsenoside Rh4 is used as a natural preservative.