COMPOSITION CONTAINING CIRSIUM JAPONICUM EXTRACT AS ACTIVE INGREDIENT FOR STIMULATING MELANOGENESIS

Abstract

The present invention relates to a composition for stimulating melanogenesis, comprising Cirsium japonicum extract as an effective ingredient. The composition has no skin irritation and cytotoxicity and is excellent in human stability and very effective in stimulating melanogenesis. Therefore, the composition can be safely used in cosmetic or pharmaceutical composition for preventing, improving or treating vitiligo, white hair or hypopigmentation.

Claims

1. A composition for stimulating melanogenesis, comprising Cirsium japonicum extract as an effective ingredient.

2. The composition according to claim 1, wherein the composition is a cosmetic composition for preventing or improving vitiligo, white hair or hypopigmentation.

3. The composition according to claim 2, wherein the Cirsium japonicum extract is comprised in an amount of 0.0001 to 15% by weight, based on the total weight of the cosmetic composition.

4. The composition according to claim 2, wherein the composition has a formulation selected form the group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray.

5. The composition according to claim 1, wherein the composition is a pharmaceutic composition for preventing or treating vitiligo, white hair or hypopigmentation.

6. The composition according to claim 4, wherein the Cirsium japonicum extract is comprised in an amount of 0.0001 to 70% by weight, based on the total weight of the cosmetic composition.

Description

DESCRIPTION OF DRAWINGS

[0059] FIG. 1 shows the melanin synthesis promoting effect depending on the concentration of Cirsium japonicum extract in human melanocyte.

[0060] FIG. 2 shows the melanin synthesis promoting effect depending on the concentration of Cirsium japonicum extract in B16 melanoma cell.

[0061] FIG. 3 shows the melanin synthesis promoting effect depending on the concentration of Cirsium japonicum extract in 3D skin.

BEST MODE FOR INVENTION

[0062] In order to achieve the above object, the present invention provides a composition for stimulating melanogenesis comprising Cirsium japonicum extract as an active ingredient.

[0063] In order to achieve the above another object, the present invention provides a cosmetic composition for preventing or improving vitiligo, white hair or hypopigmentation, characterized in that it comprises Cirsium japonicum extract as an active ingredient.

[0064] In order to achieve the above another object, the present invention provides a pharmaceutical composition for preventing or treating vitiligo, white hair or hypopigmentation, characterized in that it comprises Cirsium japonicum extract as an active ingredient.

[0065] In order to achieve the above another object, the present invention provides a method for preventing, improving or treating vitiligo, white hair or hypopigmentation by promoting the melanin production by administering to the subject a composition comprising the Cirsium japonicum extract as an active ingredient.

[0066] Hereinafter, the present invention will be given in detail through Examples. The Examples is given only to more specifically describe the present invention, and it will be self-evident to the ordinary person in the art to which the present invention subject that the scope of the present invention is not limited to such Examples.

Example 1: The Preparation of Cirsium japonicum Extract

[0067] Cirsium japonicum flowers were harvested and washed thoroughly to remove any foreign matter and impurities. It was dried at 20 to 35° C. and then ground to a particle size of 1 mm or less. Thereafter, 1 kg of the Cirsium japonicum flower powder was soaked in 70% ethanol solvent, sonicated for 48 hours, and the obtained extract was filtered using filter paper (Advantes, No. 2). The filtrate was concentrated under reduced pressure to prepare Cirsium japonicum extract.

Example 2: Measurement of the Melanin Promoting Effect of Cirsium japonicum Extract in Human Melanocytes

[0068] Human melanocytes were inoculated into 6-well plate containing M254 medium with HMGS (Human Melanocytes Growth Supplement) at a density 1.5×10.sup.5 cells per well, and then cultured in 5% concentration of CO.sub.2 cultivator under the condition of 37° C. until at least about 80% adhered to the bottom of the well. Thereafter, the medium was removed and the sample of Example 1 was replaced with a medium diluted to an appropriate concentration, and then incubated in 5% concentration of CO.sub.2 under the condition of 37° C. for 5 days. The concentration range of Cirsium japonicum extract according to Example 1 was determined to be 1 ppm, 3 ppm, 5 ppm without cytotoxicity. The cells from which the medium was removed were washed with PBS (phosphated buffer saline), and the cells were obtained. The obtained cells were counted using a hemocytometer and then centrifuged at 10,000 to 13,000 rpm for 10 minutes to remove the supernatant and pellets were obtained. The obtained cell pellet was dried at 60° C., and then 100 μl of 1M sodium hydroxide solution containing 10% DMSO was added to obtain an intracellular melanin solution in a 60° C. thermostat. Using this solution, the absorbance at 450 nm was measured with a microplate reader to determine the amount of melanin per cell. The results are shown in Table 1 and FIG. 1.

TABLE-US-00001 TABLE 1 Treatment Melanin production Sample concentration (ppm) promotion ratio (%) Control — 100 Cirsium japonicum 1 110 extract 3 116 5 122

[0069] As shown in Table 1 and FIG. 1, Cirsium japonicum extract according to the present invention was observed to increase the rate of melanin production in a concentration-dependent compared to the control. Therefore, Cirsium japonicum extract according to the present invention was found to have the effect of promoting the production of melanin.

Example 3: Measurement of the Melanin Promoting Effect of Cirsium japonicum Extract in B16 Melanoma Cells

[0070] B16 melanoma cells were inoculated into 6-well plate containing DMEM medium with 10% FBS (Fetal Bovine Serum) at a density 1.5×10.sup.5 cells per well, and then cultured in 5% concentration of CO.sub.2 cultivator under the condition of 37° C. until at least about 80% adhered to the bottom of the well. Thereafter, the medium was removed and the sample of Example 1 was replaced with a medium diluted to an appropriate concentration, and then incubated in 5% concentration of CO.sub.2 under the condition of 37° C. for 3 days. The concentration range of Cirsium japonicum extract according to Example 1 was determined to be 10 ppm, 50 ppm, 100 ppm without cytotoxicity. The cells from which the medium was removed were washed with PBS (phosphated buffer saline), and the cells were obtained. The obtained cells were counted using a hemocytometer and then centrifuged at 10,000 to 13,000 rpm for 10 minutes to remove the supernatant and pellets were obtained. The obtained cell pellet was dried at 60° C., and then 100 μl of 1M sodium hydroxide solution containing 10% DMSO was added to obtain an intracellular melanin solution in a 60° C. thermostat. Using this solution, the absorbance at 450 nm was measured with a microplate reader to determine the amount of melanin per cell. The results are shown in Table 2 and FIG. 2.

TABLE-US-00002 TABLE 2 Treatment Melanin production Sample concentration (ppm) promotion ratio (%) Control — 100 Cirsium japonicum 10 111 extract 50 159 100 222

[0071] As shown in Table 2 and FIG. 2, Cirsium japonicum extract according to the present invention was observed to increase the rate of melanin production in a concentration-dependent compared to the control. When Cirsium japonicum extract was treated with 100 ppm, it was observed that the melanin production was promoted more than two times compared to the control. Therefore, Cirsium japonicum extract according to the present invention was found to have the effect of promoting the production of melanin.

Example 4: Measurement of the Melanin Promoting Effect of Cirsium japonicum Extract in 3D Skin

[0072] 3D skin were inoculated into 6-well plate containing maintenance medium, and then cultured in 5% concentration of CO.sub.2 cultivator under the condition of 37° C. Thereafter, the medium was removed, and the Cirsium japonicum extracts were treated at ppm and 100 ppm without cytotoxicity. Cirsium japonicum extract was replaced three times with a concentration-treated medium for 10 days and then incubated at 5% CO.sub.2 and 37° C. The cells from which the medium was removed were washed with PBS (phosphated buffer saline), and the cells were obtained. The obtained cells were centrifuged at 10,000 to 13,000 rpm for 10 minutes to remove the supernatant and pellets were obtained. The obtained cell pellet was dried at 60° C., and then 100 μl of 1M sodium hydroxide solution containing 10% DMSO was added to obtain an intracellular melanin solution in a 60° C. thermostat. Using this solution, the absorbance at 450 nm was measured with a microplate reader to determine the amount of melanin per cell. The results are shown in Table 3 and FIG. 3.

TABLE-US-00003 TABLE 3 Treatment Melanin production Sample concentration (ppm) promotion ratio (%) Control — 100 Cirsium japonicum 50 120 extract 100 128

[0073] As shown in Table 3 and FIG. 3, Cirsium japonicum extract according to the present invention was observed to increase the rate of melanin production in a concentration-dependent compared to the control. Therefore, Cirsium japonicum extract according to the present invention was found to have the effect of promoting the production of melanin.

Example 5: Test for Identifying the Safety of Cirsium japonicum Extract to the Human Skin

[0074] In order to identify as to whether the Cirsium japonicum extract is safe or not, a test for identifying the safety to the human skin was performed. For this, a cumulative skin irritation was carried out.

[0075] A nutritious cream containing 0.1%, 0.5% and 1% of Cirsium japonicum extracts of Example 1, respectively, was prepared. Specifically, purified water, triethanolamine, and propylene glycol were heated and dissolved at a temperature of 70° C. (aqueous phase). Beeswax, liquid paraffin, oily ingredients, emulsifiers and preservatives were heated and dissolved at a temperature of 70° C. (oil phase). The oil phase was added to the aqueous phase to prepare an emulsion. After emulsification was completed, the solution was cooled to 45° C., and Cirsium japonicum extracts were added 0.1%, 0.5% and 1% respectively, dispersed and then cooled to 30° C. As the content of the Cirsium japonicum extract was increased to 0.1%, 0.5% and 1%, the nutritional cream was prepared by reducing the content of the liquid paraffin to 9.91%, 9.51% and 9.01%, respectively.

[0076] The nourishing cream prepared as described above was applied as a patch to areas of humerus of 30 adults for accumulated 24 hours on alternate day in total 9 times to test as to whether the Cirsium japonicum extract stimulates the skin.

[0077] The patch used Finn chamber (Epitest Ltd, Filand) and the patch was practiced after dropping with 15 μl of the skin external preparations prepared from the above, respectively. The degree of the reaction represented on the skin in every time was scored by using the below experimental formula 1. The results are represented in Table 4.

Mean reaction degree=[{(reaction index×reaction degree)/(total number of the subject×the highest score (four point))}×100]/times of the test (9 times)  [Experimental Formula 1]

[0078] In regard to the response degree, 1 point was provided for ±, 2 points for +, and 4 points for ++. When the mean reaction degree is less than 3, it is considered as being the safe composition.

TABLE-US-00004 TABLE 4 Number of subjects showing response 1.sup.st week 2.sup.nd week 3.sup.rd week Average Test 1.sup.st 2.sup.nd 3.sup.rd 4.sup.th 5.sup.th 6.sup.th 7.sup.th 8.sup.th 9.sup.th response material ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ degree Control group 1 − − 0 − − − − − − − − − − − − − − − − − − − − − − − 0.09 (Squalene) Cirsium 0 − − 0 − − − − − − − − − − − − − − − − − − − − − − − 0.00 japonicum extract (0.1%) [Test group 1] Cirsium 0 − − 0 − − − − − − − − − − − − − − − − − − − − − − − 0.00 japonicum extract (0.5%) [Test group 2] Cirsium 0 − − 0 − − − − − − − − − − − − − − − − − − − − − − − 0.00 japonicum extract (1%) [Test group 3]

[0079] As shown in the above Table 4, in all of the test groups 1, 2, and 3, all numbers of subjects belonging to ±, +, ++ were 0 and Average response degree was also 0.00. As a result of the above test, test group are all less than 3, and thus, it was determined that the Cirsium japonicum extract is safe for use on human skin which does not show clear cumulative skin irritation.

[0080] The composition of the present invention may be prepared as an example as follows but is not limited thereto.

Preparation Example 1: Preparation of Cosmetics

[0081] 1-1. Preparation of Softening Toner

[0082] Shown in Table 5 below, a softening toner containing the Cirsium japonicum extract as an active ingredient was prepared according to a conventional method.

TABLE-US-00005 TABLE 5 Component Content (wt %) Cirsium japonicum extract 0.01 Glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Carboxyvinyl polymer 0.1 Ethanol 10.0 Triethanolamine 0.1 Preservative, trace amount of pigment, 82.79 trace amount of fragrance, and trace amount of purified water Total 100.0

[0083] 1-2. Preparation of Nourishing Toner

[0084] Shown in Table 6 below, a nourishing toner containing the Cirsium japonicum extract as an active ingredient was prepared according to a conventional method.

TABLE-US-00006 TABLE 6 Component Content (wt %) Cirsium japonicum extract 0.01 Beeswax 4.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Liquid paraffin 5.0 Squalane 5.0 caprylic/capric triglyceride 5.0 Glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Triethanolamine 0.2 Preservative, trace amount of pigment, 69.69 trace amount of fragrance, and trace amount of purified water Total 100.0

[0085] 1-3. Preparation of Nourishing Cream

[0086] Shown in Table 7 below, a nourishing cream containing the Cirsium japonicum extract as an active ingredient was prepared according to a conventional method.

TABLE-US-00007 TABLE 7 Component Content (wt %) Cirsium japonicum extract 0.01 Beeswax 10.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Liquid paraffin 10.0 Squalane 5.0 caprylic/capric triglyceride 5.0 Glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservative, trace amount of pigment, 56.79 trace amount of fragrance, and trace amount of purified water Total 100.0

[0087] 1-4. Preparation of Massage Cream

[0088] Shown in Table 8 below, a massage cream containing the Cirsium japonicum extract as an active ingredient was prepared according to a conventional method.

TABLE-US-00008 TABLE 8 Component Content (wt %) Cirsium japonicum extract 0.01 Beeswax 10.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.8 Liquid paraffin 40.0 Squalane 5.0 caprylic/capric triglyceride 4.0 Glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservative, trace amount of pigment, 27.49 trace amount of fragrance, and trace amount of purified water Total 100.0

[0089] 1-5: Preparation of Pack

[0090] Shown in Table 9 below, a pack containing the Cirsium japonicum extract as an active ingredient was prepared according to a conventional method.

TABLE-US-00009 TABLE 9 Component Content (wt %) Cirsium japonicum extract 0.01 Polyvinyl alcohol 13.0 Sodium carboxymethyl cellulose 0.2 Allantoin 0.1 Ethanol 5.0 Nonyl phenyl ether 0.3 Preservative, trace amount of pigment, Balance trace amount of fragrance, and trace amount of purified water Total 100.0

Preparation Example 2: Preparation of Pharmaceutical Preparation

[0091] 2-1. Preparation of Powder Formulation

TABLE-US-00010 TABLE 10 Component Content (g) Cirsium japonicum extract 2 Lactose 1

[0092] The above components were mixed with each other and then filled in a sealed bag, thereby preparing a powder formulation containing Cirsium japonicum extract as an active ingredient.

[0093] 2-2: Preparation of Tablet Formulation

TABLE-US-00011 TABLE 11 Component Content (mg) Cirsium japonicum extract 100 Corn starch 100 Lactose 100 Magnesium stearate 2

[0094] The above components were mixed with each other and then compressed to a tablet according to a conventional method, thereby preparing a tablet formulation containing Cirsium japonicum extract as an active ingredient.

[0095] 2-3: Preparation of Capsule Formulation

TABLE-US-00012 TABLE 12 Component Content (mg) Cirsium japonicum extract 100 Corn starch 100 Lactose 100 Magnesium stearate 2

[0096] The above components were mixed with each other and then filled into a gelatin capsule according to a conventional method, thereby preparing a capsule formulation containing Cirsium japonicum extract as an active ingredient.

INDUSTRIAL APPLICABILITY

[0097] The composition for stimulating melanogenesis comprising Cirsium japonicum extract as an effective ingredient has no skin irritation and cytotoxicity and is excellent in human stability and very effective in stimulating melanogenesis. Therefore, the composition can be safely used in cosmetic or pharmaceutical composition for preventing, improving or treating vitiligo, white hair or hypopigmentation.