Description

COMPOSITION FOR SKIN WHITENING CONTAINING

DIOSGENIN

Technical Field

[1] The present invention relates to a composition for skin whitening, and more particularly to a composition for skin whitening comprising diosgenin that has excellent stability without side effects on skin, and has a superior effect to inhibit pigmentation on skin by restraining melanin generation.

[2]

Background Art

[3] Human skin color is determined by the concentration and distribution of melanin in the skin, and affected by environmental or physiological factors such as ultraviolet rays of the sun, fatigue, or stress as well as hereditary factors. Melanin synthesized in the epidermal melanocytes is a polymer of polyphenols existing in the form of complexes of dark pigment and protein. It is responsible for protecting the skin against UV radiation. It is known that tyrosinase present in melanocytes is the greatest contributor in melanin biosynthesis. Tyrosinase is a key enzyme in skin pigmentation, and catalyzes the conversion of tyrosine to DOPA and dopaquinone, which are intermediate products formed during melanin biosynthesis. However, the mechanism inducing melanin synthesis before tyrosinase works is not clarified yet in detail while the process through which melanin is made is disclosed.

[4] Overproduction of the melanin may induce discoloration, melasma, freckles or the like. Accordingly, the inhibition of melanin overproduction improves skin hyperpig- mentation such as melasma and freckles due to UV, hormone, and hereditary factors, as well as skin brightening and whitening effects.

[5] In general, conventional materials having tyrosinase inhibiting activities such as hy droquinone, ascorbic acid, kojic acid, and glutathione have been used for improving skin whitening effect or hyperpigmentation. However, hydroquinone has problems in that only an extremely restricted amount should be used due to the severe skin irritation, even though it exhibits skin whitening effects. Ascorbic add is easily oxidized, so that cosmetics blended with it have problems of color and odor changes, and kojic add is unstable in a solution, which requires a complicated preparation process. In addition, thiol based compounds such as glutathione and cysteine have unique unpleasant odors as well as problems in transdermal absorption, and glycosides

and derivatives thereof have problems in that they cannot be appropriately used as mixed ingredients of cosmetics due to their high polarities. Vitamin C is disadvantageous in that it is easily oxidized in an aqueous solution, so as not to continuously exhibit its effect. In recent years, skin- whitening compositions containing extracts of natural medicinal herbs have been developed. However, since most of them are colored, there are limitations in blending. Further, since their effective ingredients are not identified, consistent effects cannot be expected in products.

[6] On the other hand, diosgenin is a plant-derived sapogenin, and reported to reduce arterial pressure and show preventive and therapeutic effect on cholesterin steatosis. In US patent No. 6,878,367, disclosed is diosgenin as being usable in slimming compositions, on account of its ability to inhibit the storage of biochemical lipid material in adipose tissue. In WO 97/2504?, disclosed is a composition containing diosgenin for the prevention or treatment of osteoporosis. As mentioned above, many studies have been made on the efficacy of a natural substance, diosgenin.

[7]

Disclosure of Invention Technical Problem

[8] In order to solve the problems, the present inventors have made an effort to develop a single substance, which has excellent whitening effect and stability without side effect on skin, thereby completing the present invention. [9]

Technical Solution [10] It is an object of the present invention to provide a composition for whitening skin, comprising diosgenin. [11] It is another object of the present invention to provide a cosmetic composition for whitening skin, comprising the composition. [12] It is still another object of the present invention to provide a pharmaceutical composition for whitening skin, comprising the composition. [13]

Best Mode for Carrying out the Invention [14] In accordance with one aspect, the present invention relates to a composition for whitening skin, comprising diosgenin. [15] Diosgenin {(3U,25R)-spirost-5-en-3-όf, of the present invention is a plant-derived sapogenin, represented by the following Ibrmula 1.

[16] ChemistryRgure 1 [Chem.l]

[17] Diosgenin of the present invention may be extracted and isolated from natural sources (e.g., D. japonica, D. tokoro, D. septemloba), prepared by chemical modification of saponins obtained from natural sources, or synthesized by chemical methods. Alternatively, a commercially available diosgenin (e.g., Diosgenin available from Sabinsa) may be used. R>r example, diosgenin of the present invention may be extracted and isolated from plants such as D. japonica and D. tokoro using a solvent such as water, alcohol and acetone at 0 to 5O ⁰ C, which is well known to those skilled in the art.

[18] In order to confirm the whitening effect of diosgenin of the present invention, its efficacy of suppressing melanin synthesis and tyrosinase activity was measured, which is a general screening method of whitening substances, and compared to that of a known skin whitening cosmetic material, vitamin C. As a result, diosgenin of the present invention exhibited excellent effects of suppressing melanin synthesis and tyrosinase activity, which was about 10% more than that of vitamin C at the same concentration. Thus, diosgenin of the present invention can be used for whitening skin.

[19] It is apparent to those skilled in the art that diosgenin of Ibrmula 1 of the present invention includes derivatives that suppress melanin synthesis and/or tyrosinase activity to exhibit the effect of whitening skin, among its derivatives produced by addition or substitution of substituent.

[20] In the skin- whitening composition of the present invention, the content of diosgenin may be preferably 0.0001 to 15 wt%, based on the total weight of the composition. If the content is less than 0.0001 wt%, the whitening effect is not sufficient, and if the content is more than 15 wt%, the whitening effect is slightly increased, whereas there is a problem in the stability upon formulation. More preferably, diosgenin is contained in an amount of 0.0001 to 10 wt%, based on the total weight of the composition.

[21]

[22] In accordance with another aspect, the present invention relates to a cosmetic composition for whitening skin, comprising the composition.

[23] The cosmetic composition may include additives oommonly used in cosmetic formulations, for example, conventional auxiliary agents such as an antioxidant, a stabilizer, a solubilizing agent, a vitamin, a pigment, and a scent, and/or a carrier, in addition to diosgenin as an effective ingredient. The cosmetic composition may further include a skin absorption enhancer to improve skin- whitening effect.

[24] The cosmetic composition of the present invention may be prepared as any formulation commonly prepared in the art. The cosmetic composition may be formulated as, for example, 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 powdered foundation, an emulsion foundation, a wax foundation, a spray, or the like, but not limited thereto. More specifically, the cosmetic composition may be prepared as a formulation such as a softening toner, a nutrient toner, a nutrient cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, and a powder.

[25] If the formulation of the cosmetic composition of the present invention is a paste, a cream or a gel, an animal oil, a vegetable oil, a wax, paraffin, a starch, traganth, a cellulose derivative, a polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or the like may be used as the carrier ingredient.

[26] If the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talς silica, aluminum hydroxide, calcium silicate, or polyamide powders may be used as the carrier ingredient, and in particular, if the formulation is a spray, a propellent such as chlorofluorohydrocarbon, propane/butane and dimethyl ether may be used.

[27] If the formulation of the oosmetic composition of the present invention is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifier may be used as the carrier ingredient, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan fatty acid esters.

[28] If the formulation of the cosmetic composition of the present invention is a suspension, a liquid diluent such as water, ethanol and propylene glycol; a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and poly-

oxyethylene sorbitan ester; macrocrystalline cellulose; aluminum metahydroxide; bentonite; agar, traganth, or the like may be used as the carrier ingredient.

[29] If the formulation of the cosmetic composition of the present invention is a surfactant-containing cleanser, aliphatic aloohol sulfate, aliphatic aloohol ether sulfate, sulphosucdnic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetain, aliphatic alcohol, fatty add glyceride, fatty acid diethanolamide, vegetable oils, a lanolin derivative or ethoxylated glycerol fatty add ester, or the like may be used as the carrier ingredient.

[30] In the oosmetic composition of the present invention, the content of diosgenin may be 0.0001 to 15 wt%, and preferably 0.0001 to 10 wt%, based on the total weight of the cosmetic composition.

[31]

[32] In accordance with still another aspect, the present invention relates to a pharmaceutical composition for whitening skin, comprising the composition.

[33] In the pharmaceutical composition of the present invention, the content of diosgenin may be 0.0001 to 15 wt%, and preferably 0.0001 to 10 wt%, based on the total weight of the pharmaceutical composition.

[34] The pharmaceutical composition of the present invention may further include suitable carriers, exdpients and diluents oommonly used in the preparation of pharmaceutical compositions.

[35] According to the conventional methods, the pharmaceutical composition of the present invention may be formulated into any suitable formulation including oral formulations such as powder, granule, tablet, capsule, suspension, emulsion, syrup and aerosol; external preparations such as ointment and cream; suppository, and sterilized solution for injection.

[36] The pharmaceutical composition according to the present invention may be administered to mammals such as rat, mouse, livestock, and human via various routes such as oral and parenteral routes (e.g., oral, rectal or intravenous, intramuscular, subcutaneous, intraepidural or intracerebrovascular injection), preferably transcutaneous route among parenteral routes, and more preferably topical application.

[37] An effective dosage of the present composition may be determined depending on the patient's age, gender, body weight, health state, and administration mode, lor topical administration, diosgenin may be preferably administered at a daily dosage of 1.0 to 3.0 mH one time or five times for 1 month or more.

[38] Further, for oral administration, diosgenin may be administered at a daily dosage of

0.1 to 100 mg/kg one time or several times, depending on the patient's age, gender, and body weight. The dosage may be adjusted depending on administration route, severity of the disease, the patient's gender, body weight, and age. However, the scope of the present invention is not limited to the dosage.

[39]

[40] On the other hand, as a result of cumulative irritation test in a specific Example of the present invention, diosgenin was found to be safe for human skin as a natural substance. Accordingly, diosgenin of the present invention has no toxicity and side effects, thereby being safely used for a long period of time, in particular, applied to cosmetic and pharmaceutical compositions.

[41]

Mode for the Invention

[42] Hereinbelow, the present invention will be described in more detail with reference to

Examples. However, the present invention is not limited to these Examples.

[43]

[44] Example 1 : Evaluation of inhibitory effect of diosgenin on melanin synthesis

[45] Murine B 16 melanoma cells were used to measure the inhibitory effect of diosgenin

(Sigma) on melanin synthesis, which was compared with the inhibitory effect of vitamin C on melanin synthesis.

[46] Murine melanoma (B- 16 FlO) cells were inoculated on 6- well plates containing

DMEM media supplemented with 10% FBS (fetal bovine serum) (1 x 10 cells per well), and cultured in a 5% CO incubator at 37 ⁰ C until reaching about 80% confluency. Then, media was removed from the cells, replaced with diluted fresh media, and cultured in a 5% CO incubator at 37 ⁰ C for 3 days. The treatment concentration of diosgenin was determined over a range of 1 uM, 10 uM and 50 uM, which show no cytotoxicity. Media was removed from the cells, and the cells were washed with PBS (phosphate buffered saline), followed by trypsin treatment. The cells were recovered, and counted using a hemocytometer. Then, the cells were centrifuged at 5,000 to 10,000 rpm for 10 min, and the supernatant was removed to obtain a pellet. The pellet was dried at 6O ⁰ C, and suspended in 100 μJl of 1 M sodium hydroxide solution containing 10% DMSO in a 60 ⁰ C water bath to obtain melanin in the cells. Absorbance was determined at 4)0 nm using a microplate reader to assess the melanin content per cell. The results are shown in the following Table 1.

[47]

[48] Table 1 [Table 1] [Table ]

[49] [50] As shown Table 1, diosgenin exhibited about 7%, 14% and 11% higher inhibition rate of melanin synthesis than vitamin C at the same treatment concentration.

[51] [52] Example 2 : Evaluation of inhibitory effect of diosgenin on tyrosinase activity

[53] Murine B 16 melanoma cells were used to measure the inhibitory effect of diosgenin on tyrosinase activity, which was compared with the inhibitory effect of vitamin C on tyrosinase activity.

[54] Murine melanoma (B- 16 Fl) cells were inoculated on 6- well plates containing DMEM media supplemented with 10% FBS (fetal bovine serum) (1 x 10 cells per well), and cultured in a 5% CO incubator at 37 ⁰ C until reaching about 80% confluency. Then, media was removed from the cells, replaced with diluted fresh media, and cultured in a 5% CO incubator at 37 ⁰ C for 3 days. The treatment concentration of diosgenin was determined over a range of 1 uM, 10 uM and 50 uM, which show no cytotoxicity. Media was removed from the cells, and the cells were washed with PBS (phosphate buffered saline), followed by trypsin treatment. The cells were recovered, and counted using a hemocytometer. Then, the cells were centrifuged at 5,000 to 10,000 rpm for 10 min, and the supernatant was removed to obtain a pellet. The pellet was resuspended using a lysis buffer, and centrifuged at 12,000 rpm for 10 min to collect the supernatant. Absorbance was determined at 492 nm using a microplate reader to assess the tyrosinase activity per cell. The results are shown in the

following Table 2.

[55] [56] Table 2 [Table 2] [Table ]

[57] [58] As shown Table 2, diosgenin exhibited about 11%, 12% and 11% higher inhibition rate of tyrosinase activity than vitamin C at the same treatment concentration.

[59] [60] Example 3 : Evaluation of skin whitening effect in animal [61] Brown guinea pigs (Tortoiseshell guinea pigs), which are known to increase pigmentation upon exposure to UV like human, were used to measure the whitening effect of diosgenin.

[62] To cause pigmentation in the brown guinea pig by UV, light- shielding aluminum foil

2 with windows of 3 x 3 cm was adhered to hair-removed abdominal skin of brown guinea pig, and then UV light was irradiated thereon with a SE lamp (wavelength

2

290-320 nm, Toshiba) (total irradiation energy = 1350 mJ/cm ). After UV irradiation, the aluminum foil was removed, and then the samples (diosgenin and vitamin C) were applied as follows. Increased pigmentation was observed at 2 or 3 days after UV irradiation, and reached a maximum after about 2 weeks. From the maximum, each sample was applied.

[63] Applications were performed once or twice a day for 50 days. The samples were dissolved and diluted in a certain solvent (a mixed solvent of propylene glycol: ethanol: water=5 : 3: 2) and applied with a swab. Another region was applied with the solvent only as a control. Occurrence of cumulative irritation also was examined.

[64] The degree of skin pigmentation was determined using a chromameter (CR2002 chromameter, Minolta, Japan) to evaluate the effects. The results are shown in the following Table 3. L*A*B* colorimetric system is used to classify color, and an L* value was used as standard in the present invention. The L* value was corrected using white board standard, and measured more than five times at one region, repeatedly. Pigmentation was evenly distributed. Skin oolor differences (δL*) between initial application point and terminal application point were determined, and then using these values, their effects of the applied samples were evaluated. The results are shown in the following Table 3.

[65] [66] [Mathematical Equation 1] [67] δL* = L* value at 00 days after application - L* value at initial application day [68] [69] δL* values were obtained at sample application region and control application region and compared. From the result, the skin whitening effects of the samples can be evaluated.

[70] [71] Table 3 [Table 3] [Table ]

[72] [73] As shown Table 3, it was found that diosgenin exhibited better skin whitening effect than vitamin C. Further, in the cumulative irritation test, diosgenin causes no cumulative irritation. Therefore, it can be seen that diosgenin is safe for skin.

[74] [75] Example 4 : Safety test of diosgenin on human skin [76] 4-1. External preparation containing diosgenin

[77] In order to confirm the safety of diosgenin on human skin, external preparations containing diosgenin and vitamin C were prepared, and safety test of the external preparations was performed.

[78] Skin external preparations containing diosgenin and vitamin C were prepared using the ingredients shown in Table 4 below. As a control group, a skin external preparation was prepared without a tyrosinase inhibitor. The experimental groups 1 and 2 are skin external preparations containing diosgenin and vitamin C, respectively.

[79] To prepare the skin external preparations, purified water, glycerin, and butylene glycol were mixed and dissolved at 7O ⁰ C (aqueous phase). The remaining components except for the above three components and trimethanolamine were dissolved at 7O ⁰ C (oil phase). The oil phase was added to the aqueous phase, and stirred with a Homomixer (Tokushu Kika company, Japan) to primarily emulsify. Finally, trimethanolamine was added thereto. loam produced in the mixed solution was removed, and then cooled to room temperature to prepare skin external preparations.

[80]

[81] Table 4

[Table 4] [Table ]

[82] [83] 4-2. Cumulative irritation test [84] Each skin external preparation prepared in Example 4- 1 was applied every other day to the forearms of 30 healthy adults, and left for 24 hours, and this was repeated so that each subject was treated with 9 fresh patches in total, so as to confirm whether skin irritation is caused by diosgenin.

[85] The patch test was performed using a Hnn chamber (Epitest Ltd, Finland). The external preparations were loaded drop wise in an amount of 15 ul per patch on the chamber. At every round of the patch application, the response of the skin was scored using the following Mathematical Equation. The results are shown in the following Table 5.

[86] [87] [Mathematical Equation 2]

[88] Average response degree = [89] [[Response index x Response degree / Total number of subjects x Highest score (4 points)] x 100] í Times of examination (9 rounds)

[90] [91] In regard to the response degree, 1 point was provided for ±, 2 points for +, 4 points for ++. When the average response degree was less than 3, the composition was determined to be safe for the skin.

[92] [93] Table 5 [Table 5] [Table ]

[94] [95] As shown in Table 5, in Experimental group 1, the subjects corresponding to +, +, and ++ numbered 4, 0 and 0, respectively, while the others showed no response. Further, the average response degree was calculated to be 0.37 ([(4xl)/(20x4)]x 100/9=0.37), which is less than 3, demonstrating that the composition of the present invention is safe for human skin.

[96] As shown in Table 5, the external preparation containing diosgenin (Experimental group 1) causes no noticeable cumulative irritation, like the control group and the external preparation containing vitamin C. Therefore, diosgenin according to the present invention was found to be safe for human skin.

[97]

Industrial Applicability

[98] According to the present invention, diosgenin exhibited higher inhibitory effects on tyrosinase activity and melanin synthesis than the known skin whitening agent, vitamin C. Further, diosgenin exhibits no cytotoxicity and skin complications, thereby being safely applied to cosmetic, pharmaceutical and food compositions.