TECHNICAL FIELD The present invention relates to composition for skin whitening, and more particularly, to composition for skin whitening that has excellent stability and gives no side effects on skin, and have a superior effect to inhibit pigmentation on skin by restraining melanin from being generated.

BACKGROUND ART Most people want to have white and fine skin. The color of human skin is determined by the density and the distribution of melanin inside skin and affected by the environmental or physiological factor such as ultraviolet rays of the sun, fatigue, or stress as well as the genetic factor. Melanin is made through the following steps: first, enzyme tyrosinase affects on a kind of amino acid, tyrosine to change the same into DOPA or dopaquinone and then the same goes through non-enzymatic oxidation reaction. However, the mechanism that derives melanin synthesis, which is a step before tyrosinase affects, is not clarified in detail though the process through which melanin is made is disclosed.

When the melanin synthesis is excessively performed inside skin, the tone of skin darkens and chloasma and frekles can be generated. Accordingly, when the melanin synthesis inside skin is inhibited, the skin whitening is possible, plus hyperpigmentation such as chloasma, freckles, etc. due to ultraviolet rays or hormonic and genetic factors can be improved.

Conventionally, the skin whitening is tried by mixing material having inhibition

function against tyrosinase such as hydroquinone, ascorbic acid, kojic acid, or glutathione with cosmetic such as essence or ointment for external use. However, although hydroquinone shows a prescribed effect of whitening, the mixture amount of the same should be restricted to minimum since the same seriously irritates skin, in case of ascorbic acid, since the same is easy to be oxidized, cosmetic mixed with the same has problems of discoloration and change of scent, and in case of kojic acid, the same is restricted to be used since the same is unstable. Further, thiol compound such as glutathione or cysteine has a peculiar bad smell and low absorptiveness to skin.

DISCLOSURE OF THE INVENTION Accordingly, an object of the present invention is to overcome the above- mentioned and to provide composition for skin whitening that has excellent stability and gives no side effects on skin, and has a superior effect to inhibit pigmentation on skin by restraining melanin from being generated.

The detailed description about the composition according to the present is provided hereinafter.

The present invention provides composition for skin whitening containing one selected from the group consisting of veratramine (3, 23-14, 15,16, 17- tetradehydroveratramine-3, 23-diol), verazine, ( (3S, 20S, 25S)-22, 26-iminocholesta- 5,22 (N)-dien-3-ol), epi-verazine ( (3S, 20R, 25S)-22, 26-iminocholesta-5, 22 (N)-dien-3-ol) and mixtures thereof as an effective component, which are represented by the following chemical formula 1 to 3, respectively.

[Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3]

It was reported that veratramine shows antibacterial effect (Wakan lyakugaku Zasshi, 1999,16 (5/6), 196-200) ), and it was also reported that verazine and epi- verazine have antifungal effect (Chem. Pharam. Bull, 35 (12), 1987,4862-7) and inhibitory effect on formation of DNA (Lanzhou Daxue Xuebao, Ziran Kexueban, 21 (2), 1985,86-90), but, skin-whitening effect of these materials was not known yet.

The present inventors have found that veratramine, verazine and epi-verazine show very strong inhibitory effect on formation of melanin and show excellent skin whitening effect as a result from the experiment on B16 mouse melanoma cell which can even screen the material which restrain induction of melanin synthesis itself.

Therefore, if one or mixtures of these materials are added and mixed into the composition for skin whitening such as skin lotion, cream, foundation, essence, gel, pack, foam cleansing, soup and ointment for external use according to the present invention, the high-potency of skin whitening without any special side effects can be obtained.

Veratramine, verazine and epi-verazine can be obtained synthetically, or can be easily extracted from the herbal medicines that can be purchased from the traditional market. Veratrum nigrum, Veratrum album, Veratrum maackii, Veratrum patulum, Eclipta alba, Veratrum taliense, Veratrum loblianum, Zygadenus sibiricus, Veratrum dahuricum, Veratrum oxysepalum, Solanum nigrum, Solanum hostmannii, etc. , are the examples for such raw material of the herbal medicines.

Hereinafter, extraction method of Veratramine, verazine and epi-verazine from the root of Veratrum nigrum or Veratrum album will be explained as follows, but should not be restricted within this.

At first, after pulverizing the dried Veratrum nigrum or Veratrum album into small pieces, the same is heat extracted with water or organic solvent such as dried or aqueous alcohol of low molecular weight having 1 to 4 carbon atoms, ethylacetate and acetone, and then, separated and purified by solvent-fractionation using hexane, chloroform, butanol, etc. , to obtain high purity of Veratramine, verazine and epi-verazine.

Veratramine, verazine and epi-verazine obtained by the above mentioned method can be used by adding one or mixtures thereof into various skin whitening composition such as ointment for external use or cosmetic, and the content is preferably 0.000001 to 10 weight% based on total weight of the composition in consideration of whitening effect and economic efficiency.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The detailed description of the present invention referring to the embodiments is provided hereinafter. However, the embodiments according to the present invention can be modified in various ways and should not be understood to be restricted to the embodiments described below. The embodiments of the present invention are provided to explain the present invention more completely to a person who has standard knowledge in the art.

Extraction Examples of Veratramine Extraction Example 1 After 1Kg of dried root of Veratrum nigrum purchased at traditional market was pulverized into small pieces, the same was extracted heating for 3 hours using extractor wherein reflux condenser was equipped. Filtering the output with 300 mesh filter and extracting the residual by the same method more than one time, all the extract was concentrated with vacuum and freeze-dried to obtain 52g of dried extract. By solvent fractionation, hexane and chloroform fractions were eliminated from this dried extract, and the obtained fraction was fractionated 3 times with water-saturated butanol. And after 24g of the obtained butanol fraction was purified with silica gel chromatography and high-performance liquid chromatography (HPLC), then the structure of Veratramine

obtained by recrystallization method was validated using mass-spectroscopy and NMR spectroscopy.

Extraction Example 2 To 20g of the extract obtained by extracting 1Kg of dried root of Veratrum nigrum with chloroform, 5% of aqueous tartaric acid was added, and the dissolved fraction was neutralized with NaOH and solvent-fractionated with chloroform to obtain 1g fraction. And the structure of Veratramine obtained by recrystallization was validated using mass-spectroscopy and NMR spectroscopy.

Extraction Example 3 After 1Kg of dried root of Veratrum album purchased at traditional market was pulverized into small pieces, the same was extracted heating for 3 hours using extractor wherein reflux condenser was equipped. Filtering 43g of the output with filter and extracting the residual by the same method more than one time, all the extract was concentrated with vacuum and freeze-dried to obtain dried extract. By solvent fractionation, hexane and chloroform fractions were eliminated from this dried extract, and the obtained fraction was fractionated 3 times with water-saturated butanol. And after 18g of the obtained butanol fraction was purified with silica gel chromatography and high-performance liquid chromatography (HPLC) to obtain 0.4g fraction containing Veratramine, the structure of Veratramine obtained by using preparative HPLC and recrystallization of the fration was validated using mass-spectroscopy and NMR spectroscopy.

Extraction Examples of Verazine and Epiverazine Extraction Example 4 After the dried root of Veratrum nigrum purchased at traditional market was

pulverized into small pieces, the same was put into 1000g of methanol, and was extracted by heating using reflux extractor wherein cooling condenser was equipped.

The output was filtered with 100 mesh filter and the residual was extracted by the same method once again. After all of the summed extract was filtered with Whatman filter paper No. 2 at room temperature to remove insoluble material, and was concentrated with vacuum using distillator wherein cooling condenser is equipped, 52g of dried extract by freeze-drying. This extract was put into butanol, hexane, chloroform, and hexane and chloroform fractions were eliminated from this dried extract by solvent fractionation, and the obtained fraction was fractionated 3 times with water-saturated butanol to obtain 24g of butanol fraction. Subsequently, the obtained butanol fraction was purified by silica column chromatography for several times to obtain 1g fraction containing Verazine and Epi-verazine. Next, Verazine and Epi-verazine was obtained by preparative thin layer chromatography (Preparative TLC). The content of obtained verazine and epi-verazine were validated to be more than 99.9% each of their purity from the result of the analysis using mass spectroscopy and NMR spectroscopy.

Extraction Example 5 The same method of extraction example 4 except using the root of Veratrum album instead of Veratrum nigrum was performed, and the content of obtained verazine and epi-verazine were validated to be more than 99.9% each of their purity from the result of the analysis using mass spectroscopy and NMR spectroscopy.

Extraction Example 6 After the dried root of Veratrum nigrum purchased at traditional market was pulverized into small pieces, the same was put into 1000g of methanol, and was extracted by heating using reflux extractor wherein cooling condenser was equipped.

The output was filtered with 100 mesh filter and the residual was extracted by the same method once again. After all of the summed extract was filtered with Whatman filter paper No. 2 at room temperature to remove insoluble material, and was concentrated with vacuum using distillator wherein cooling condenser is equipped, 52g of dried extract by freeze-drying. After dissolving this extract with chloroform, the same was solvent fractionated with 5% aqueous chloric acid several times to obtain acidic material fraction. Subsequently, after sodium hydroxide was added to control pH of the aqueous layer to basic, and solvent-fractionation with chloroform was performed to transmit the basic material to chloroform, then the basic material fraction was obtained by concentration with vacuum. Next, the obtained basic material fraction was purified by silica column chromatography treated with sodium hydroxide to obtain 1g fraction containing verazine and epi-verazine. Next, verazine and epi-verazine was obtained by preparative thin layer chromatography (Preparative TLC). The content of obtained verazine and epi-verazine were validated to be more than 99.9% each of their purity from the result of the analysis using mass spectroscopy and NMR spectroscopy.

Experimental Example The whitening effect in a cellular level is tested by adding aqueous solutions of Veratramine, Verazine and Epi-verazine obtained according to above extraction examples from 1 to 3 and aqueous albutin solution to a culture medium of B-16 mouse melanoma cell (Lotan R. , Lotan D. Cancer Res. 40: 3345-3350,1980). The final concentrations of Veratramine, Verazine and Epi-verazine and albutin were prepared as described at table 1 respectively, and each was added to the culture medium of B-16 mouse melanoma cell, and then cultured for 3 days. Next, the cultured cells were

treated with trypsin, and separated from the culture plate, and then centrifuged to extract melanin.

1ml of sodium hydroxide solution (1N of concentration) was added to each of above obtained extracts and the mixtures were boiled for 10 minutes for melanin to melt.

Then, the light absorbancy at 400mn was measured by spectrophotometer and the amount of generated melanin was indicated as the light absorbancy per unit number of cell (106 cell). Then, the inhibition ratio (%) was obtained by calculating the amount of generated melanin relative to the comparative group, and the results are described at table 1.

[Table 1] Testing material Final concentration Amount of generated Inhibition (gg/rn) melanin ratio (%) Comparative group 0 0. 0540. 003- 0. 1 0.031~0.001 43 Veratramine 1 0. 028~0. 001 48 5 0. 0160. 001 70 20 0. 0060. 001 89 0. 1 0. 0310. 001 43 1 0.026~0.001 52 Verazine 5 0.019~0.001 65 0.009~0.001 83 20 0.1 0.032~0.001 41 Epi-verazine 1 0. 0270. 001 50 5 0. 019f0. 001 65 200. 0080. 001 85 Albutin 200 0. 029~0. 001 46

Referring to the tables 1, Veratramine, Verazine and Epi-verazine have a very high-potency of restraining the generation of melanin of the cultured mouse melanoma cells by comparison with that of the comparative group. Especially, considering that the concentrations of added Veratramine, Verazine and Epi-verazine are small, the potency

of restraining the generation of melanin of Veratramine, Verazine and Epi-verazine at same concentration are much more excellent than that of albutin, the conventional skin- whitening material.

Hereinafter, preparing composition for skin whitening by adding Veratramine, Verazine, Epi-verazine and mixtures thereof to the cosmetics such as ointment for external use, cream, softening lotion, essence, pack, and nutritious lotion, the effect of inhibiting pigmentation and occurrence of side-effect were tested by applying them to the testees.

Embodiment 1 and comparative example 1 Ointments for external use were manufactured with the components and contents as described at table 2.

[Table 2] Embodiment 1 Comparative example 1 a b C d Veratramine 2 - - - Verazine - 2 - 1 - Epi-verazine--2 1- Diethyl sebacate 8 8 8 8 8 Spermaceti 5 5 5 5 Polyoxyethylene oleylether 6 6 6 6 6 phosphate Sodium benzoic acid q. s q. s q. s q. s q. s Vaseline to 100 To 100 to 100 to 100 to 100

Embodiment 2 and comparative example 2 Creams were manufactured with components and contents as described at table 3.

[Table 3] Name of material (wt%) Embodiment 2 Comparative example 2 a b C d Veratramine 0. 5 Verazine-0. 5-0. 25 Epi-verazine--0. 5 0. 25- Stearic acid 15. 0 1.0 1.0 1. 0 1. 0 Cetanol 1.0 2.0 2.0 2. 0 2. 0 Potassium hydroxide 0.7 1.0 1.0 1. 0 1. 0 Glycerin 5. 0 1.0 1.0 1. 0 1. 0 Propylene glycol 3.0 10.0 10.0 10. 0 10. 0 Preservative q.s q.s q.s q.s q.s Aromatic material q.s q.s q.s q.s q.s Water to 100 to 100 to 100 to 100 to 100

Embodiment 3 and comparative example 3 Softening lotions were manufactured with components and contents as described in table 4.

[Table 4] Name of material (wt%) Embodiment 3 Comparative example 3 a b c d Veratramine 0. 2 - - - - Verazine-0. 2-0. 1 Epi-verazine--0. 2 0. 1- Ethanol 10.0 10.0 10.0 10. 0 10. 0 Polyoxyethylene sorbitan 1.0 1.0 1.0 1.0 1.0 polylaurate Paraoxymethylbensoate 0.2 0. 2 0.2 0.2 0. 2 Glycerin 5. 0 5.0 5.0 5.0 5. 0 1, 3-buthylene glycol 6.0 6. 0 6. 0 6.0 6. 0 Aromatic material q.s q.s q.s q.s q.s Pigments q.s q.s q.s q.s q.s Water to 100 to 100 to 100 to 100 to 100

Embodiment 4 and comparative example 4 Nutritious lotions were manufactured with components and contents as described in table 5.

[Table 5] Name of material (wt%) Embodiment 6 Comparative example 6 a b C d Veratramine 0. 1 Verazine-0. 1-0. 05 Epi-verazine--0. 1 0. 05 Vaseline 2. 0 2. 0 2.0 2.0 2. 0 Sorbitan sesquioleate 0.8 0.8 0.8 0.8 0. 8 Polyoxyethylene oleylether 1.2 1.2 1.2 1.2 1.2 phosphate Paraoxymethylbensoate q. s q. s q. s q. s. s Propylene glycol 5. 0 5.0 5.0 5.0 5. 0 Ethanol 3. 2 3.2 3.2 3.2 3. 2 Carboxyvinyl polymer 18.0 18. 0 18. 0 18. 0 18. 0 Potassium hydroxide 0.1 0.1 0.1 0. 1 0. 1 Pigments q. s q. s q. s q. s q. s Aromatic material q.s q.s q.s q.s q.s Water to 100 To 100 to 100 to 100 to 100

Embodiment 5 and comparative example 5 Packs were manufactured with components and contents as described in table 6.

[Table 6] Name of material (wt%) Embodiment 5 Comparative example 5 a B zu Veratramine 0. 2 Verazine-0. 2-0. 1- Epi-verazine - - 0.2 0. 1 Glycerin 5. 0 5. 0 5.0 5.0 5. 0 Propylene glycol 4. 0 4.0 4.0 4.0 4. 0 Polyvinyl alcohol 15.0 15.0 15.0 15. 0 15. 0 Ethanol 8. 0 8.0 8.0 8.0 8. 0 Polyoxyethylenen oleylether 1.0 1.0 1.0 1.0 1.0 phosphate Methyl I paraben 0.2 0.2 0.2 0. 2 0. 2 Aromatic material q.s q.s q.s q.s q.s Pigments. s q. s q. s q. s. s Water to 100 to 100 to 100 to 100 to 100

Embodiment 6 and comparative example 6 Essences were manufactured with components and contents as described in table 7.

[Table 7] Name of material (wt%) Embodiment 4 Comparative example 4 a b u a Veratramine 1. ne1. 0- Verazine-1. 0-0. 5 Epi-verazine--1. 0 0. 5- Propylene glycol 10.0 10.0 10.0 10. 0 10. 0 Glycerin 10. 0 10.0 10.0 10. 0 10. 0 Sodium hyaluronic acid 5. 0 5. 0 5. 0 5. 0 5. 0 aquous solution (1%) Ethanol 5. 0 5.0 5.0 5.0 5. 0 Polyoxyethylene hardened 1.0 1.0 1.0 1.0 1.0 caster-oil Methyl paraben 0. 1 0. 1 0.1 0.1 0. 1 Aromatic materiall. s. s. s. s. s Water to 100 to 100 to 100 To 100 To 100

The process for testing the pigmentation inhibitory effect by the ointment for external use, cream, softening lotion, essence, pack, and nutritious lotion manufactured as described above is as follows.

First, an aluminium foil having two rows of six holes of 7mm diameter was adhered to each forearm of twenty healthy men and women and 60mJ/cm2 of light was irradiated at 10cm distance from arm by ORIEL solar simulator 1000W. Before irradiation, the portion to be irradiated is washed with 70% of aqueous ethanol solution.

From 3 days before irradiation to 3 weeks after irradiation, each pair of the cosmetic composition according to the embodiments 1 to 6 and the comparative examples 1 to 6 respectively was applied to a same row twice a day. The packs of the embodiment 5 and the comparative examples 5 were removed 15 minutes after application.

After applying the cosmetics according to each embodiments and comparative examples to the testees as described above, the pigmentation degree is determined with naked eye, and comparing the degrees of restraining pigmentation of the cosmetics according to each embodiments with those of comparative examples respectively, the result was evaluated into two stages, i. e. , effective or no-difference, and was depicted along with the result of side effect occurrence as following Table 8.

[Table 8] Testing t'ou Effective No-difference Side effect occurrence a 17 3 0 Embodiment 1 b 16 4 0 c 17 3 0 d 16 4 0 a 14 6 0 Embodiment 2 b 14 6 0 c 16 4 0 d 15 5 0 a 14 6 0 Embodiment 3 b 14 6 0 c 14 6 0 a 16 4 0 a 13 7 0 Embodiment 4 b 13 7 0 c 14 6 0 d 14 6 0 a 14 6 0 Embodiment 5 b 14 6 0 c 15 5 0 d 15 5 0 a 16 4 0 Embodiment 6 b 16 4 0 c 15 5 0 d 17 3 0

As shown in table 8, at least 13 persons of 20 testees applied the cosmetics containing Veratramine, Verazine, Epi-verazine and mixtures thereof according to the embodiments 1 to 6 show superior skin whitening effect and show no side effect.

INDUSTRIAL APPLICABILITY As described above, Veratramine, Verazine, and Epi-verazine according to the present invention show no side effects on skin, and have a superior effect to inhibit pigmentation on skin by restraining melanin from being generated. Thus, the composition containing the same is effectively used for skin whitening.