Whitening cosmetic composition comprising <i>Caragana sinica </i>root extract

Abstract

The present invention relates to a cosmetic composition for whitening comprising a Caragana sinica root extract, and more specifically relates to a cosmetic composition having an outstanding skin-whitening effect, the composition comprising a Caragana sinica root extract, a fraction thereof or α-viniferin isolated therefrom. In the present invention, the Caragana sinica root extract, fraction thereof or α-viniferin isolated therefrom inhibits tyrosinase activity and suppresses melanin production and is therefore effective in skin whitening.

Claims

1. A skin whitening method, comprising applying a cosmetic composition to skin, wherein the cosmetic composition comprises: (a) an ethyl acetate fraction of an ethanol extract of Caragana Sinica root, wherein the ethyl acetate fraction comprises alpha-viniferin at a concentration of 4 μM to 8 μM as an active ingredient, and (b) a carrier, wherein the ethyl acetate fraction is present in an amount of 0.01 wt % to 10 wt % based on the total weight of the cosmetic composition, and wherein the alpha-viniferin has an IC50 of 7 uM on melanin production.

2. The method of claim 1, wherein the ethyl acetate fraction contains 0.1 wt % to 1 wt % of α-viniferin based on the total weight of the fraction.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows results of analyzing an ingredient of an ethyl acetate fraction of an ethanol extract of Caragana Sinica root through a high-performance liquid chromatography (HPLC).

(2) FIG. 2 shows results of analyzing an ingredient of α-viniferin isolated from the ethanol extract of Caragana Sinica root through the HPLC.

(3) FIG. 3a shows results of analyzing the α-viniferin isolated from the ethyl acetate fraction of the ethanol extract of Caragana Sinica root through the 1H-nuclear magnetic resonance (1H-NMR).

(4) FIG. 3b shows results of analyzing the α-viniferin isolated from the ethyl acetate fraction of the ethanol extract of Caragana Sinica root through the 13C-NMR.

(5) FIG. 4 shows a melanin production inhibitory effect of the α-viniferin isolated from the ethyl acetate fraction of the ethanol extract of Caragana Sinica root.

(6) FIG. 5 shows results of a clinical evaluation on skin brightness changes according to an application of a cosmetic composition comprising a fraction of Caragana Sinica root extract.

(7) FIG. 6 shows results of a clinical evaluation on melanin content changes of skin according to the application of the cosmetic composition comprising the fraction of Caragana Sinica root extract.

(8) FIG. 7 shows results of a visual evaluation on skin color changes according to the application of the cosmetic composition comprising the fraction of Caragana Sinica root extract.

MODE FOR INVENTION

(9) Hereinafter, the present invention will be described in detail through examples, experimental examples and formulation examples.

(10) However, the following examples, experimental examples and formulation examples are set forth just as the illustrative description of the present invention, but are not to be construed to limit the content of the present invention.

Example 1: Preparing of a Caragana Sinica Root Extract

(11) 500 g of Caragana Sinica root, which was finely cut and dried in the shade, was pulverized to prepare a pulverized Caragana Sinica root. 5 l of 70% ethanol was added to the pulverized one, after which an extract was obtained by being reflux extracted for 5 hours and macerated. The extract was filtered with a 400-mesh filtering cloth, followed by filtered again with a filtering paper No. 2 (Whatman). After that, a final resulting filtrate was concentrated under reduced pressure to obtain 24 g of ethanol extract of Caragana Sinica root.

Examples 2 to 5: Preparing of a Fraction of Caragana Sinica Root Extract

(12) 10 g of the ethanol extract of Caragana Sinica root obtained from the Example 1 was solvent fractionated by means of a conventional method using hexane, chloroform, ethyl acetate and butanol, followed by concentrated under reduced pressure, such that 2.12 g of an hexane fraction of Example 2, 1.32 g of a chloroform fraction of Example 3, 1.24 g of an ethyl acetate fraction of Example 4 and 0.82 g of a butanol fraction of Example 5 were obtained, respectively.

Experimental Example 1: Isolation and Analysis of the Active Ingredient of the Fraction of Caragana Sinica Root Extract

(13) 0.1 g of each fraction of Caragana Sinica root extract obtained from the Examples 2 to 5 was dissolved in methanol to obtain 10 ml of test liquid. Also, 10 mg of a standard α-viniferin (C42H30O9:678.68) was dissolved in methanol so as to obtain a 100 ml solution, out of which 0.5 ml was dissolved again in methanol so as to obtain a 100 ml solution, and then this solution was diluted to 1/2 and 1/4 solutions to obtain standard solution. 20 μl of each of the test liquid and the standard solution was analyzed through an HPLC to measure a content of α-viniferin.

(14) This HPLC analysis was made by means of Gilson HPLC System (Gilson Inc., Middleton, Wis., USA) equipped with an UV/VIS 151 detector. A 5 μm Hector-M C18 column (4.6 mm×250 mm; RS tech co., Daejeon, Korea) was used for a fixed phase. A mixed solution of 0.5% formic acid (A) and acetonitrile (B) was used for a mobile phase, wherein a content of B was increased from 30% to 100% with a certain gradient for 50 minutes. A flow rate was 0.8 ml per minute, and a detection wavelength of an UV detector was set to 280 nm. Results of HPLC analysis were shown in FIGS. 1 and 2. As a result, contents of α-viniferin of the Examples 2 to 5 were indicated in the following Table 1.

(15) TABLE-US-00001 TABLE 1 Sample Content of α-viniferin (wt %) Example 2 — Example 3 0.12% Example 4 0.52% Example 5 0.17%

(16) Also, the α-viniferin comprised in the ethyl acetate fraction of the ethanol extract of Caragana Sinica root obtained from the above Example 4 was identified through an NMR analysis.

(17) Specifically, 15 g of the ethyl acetate fraction of the ethanol extract of Caragana Sinica root obtained from the above Example 4 was divided into 8 fractions (CE1 to CE8) by carrying out a silica gel column chromatography (2.5×28 an, EtOAc:MeOH=10:1.fwdarw.100% MeOH). With regard to a fraction CE7, the silica gel column chromatography (2.5×28 cm, CH2Cl2: MeOH=50:1.fwdarw.100% MeOH) and a sephadex LH-20 column chromatography (70% MeOH 100% MeOH) were performed to isolate 0.1 g of α-viniferin from the fraction.

(18) For NMR device, 1H-NMR of Varian (GEMINI, 500 MHz) and a 13C-NMR of Varian (GEMINI, 200 MHz) were used to analyze a structure of the α-viniferin, wherein a solvent used therefor was acetone-d6, purchased from Aldrich, and an internal standard substance used therefor was trimethylsilane so as to indicate results as a δ value (ppm), wherein NMR results were shown in FIGS. 3a and 3b.

(19) 1H-NMR (500 MHz, Acetone-d): δppm 4.11 (s, 1H, H-3(I)), 4.77 (d, 1H, H-3(II)), 4.85 (d, 1H, H-3(II)), 5.05 (d, 1H, H-2(III)), 6.09 (d, 1H, H-2(II)), 6.14 (d, 1H, H-5(I)), 6.22 (s, 1H, H-2(I)), 6.38 (m, 3H, H-7(I, II, III)), 6.74 (d, 1H, H-5(III)), 6.87 (m, 3H, H-3′, 5′(I), H-5(II)), 6.92 (m, 4H, H-3′, 5′(II, III)), 7.18 (m, 4H, H-2′, 6′(I, III)), 7.36 (m, 2H, H-2′, 6′ (II))

(20) 13C-NMR (125 MHz, Acetone-d): δppm 46.4 (C-3(I)), 52.8 (C-3(II)), 55.7 (C-3(III)), 86.4 (C-2(I)), 90.0 (C-2(II)), 95.6 (C-2(III)), 96.6 (C-7(II, III)), 98.0 (C-7(I)), 105.8 (C-5(III)), 106.2 (C-5(II)), 108.5 (C-5(I)), 115.7 (C-3′, 5′ (I)), 116.1 (C-3′, 5′ (III,II)), 118.8 (C-3a(I)), 119.7 (C-3a(III)), 120.9 (C-3a(II)), 128.1 (C-2′, 6′ (I)), 128.2 (C-2′, 6′ (II)), 128.6 (C-2′, 6′ (III)), 132.0 (C-1′ (I)), 132.3 (C-1′ (II)), 132.5 (C-1′ (III)), 138.7 (C-4(III)), 159.4 (C-6(II,I)), 160.7 (C-7a(II)), 160.8 (C-6(III)), 161.6 (C-7a(I)), 161.8 C-7a(III))

Experimental Example 2: Confirming Whitening Effect of the Caragana Sinica Root Extract and the Fraction Thereof

Experimental Example 2-1: Confirming Tyrosinase Activity Inhibitory Effect of the Caragana Sinica Root Extract and the Fraction Thereof

(21) A tyrosinase is an enzyme that promotes an oxidization process of a substance called tyrosine in vivo to produce a melanin. Thus, it was confirmed that the ethanol extract of Caragana Sinica root and the fraction thereof prepared from the above Examples 1 to 5 had an inhibitory effect on an activity of tyrosinase.

(22) Specifically, tyrosinase, isolated and refined from a mushroom, was bought from Sigma L-tyrosine, as a substrate, was dissolved in 0.05 M sodium phosphate buffer solution (pH 6.8) to obtain a 0.1 mg/ml solution, after which the ethanol extract of Caragana Sinica root and the fraction thereof obtained from the above Examples 1 to 5 were dissolved in 0.05 M sodium phosphate buffer solution (pH 6.8) at a concentration of 0.05%, 0.1% and 0.2%. After that, 0.5 ml of a resulting L-tyrosine solution was inserted into a test tube, after which 0.5 ml of a sample fluid of the above Caragana Sinica root extract and the fraction thereof was added thereto, such that the test tube of a resulting mixture was left alone in a 37° C. constant temperature chamber for 10 minutes. After that, 0.5 ml of 200 unit/ml tyrosinase was added, followed by reacted at 37° C. for 10 minutes. The reaction was stopped by placing the test tube containing that reaction solution on ice to be rapidly cooled down, after which an absorbance of the resulting product was measured with a spectrophotometer at a wavelength of 475 nm. Control group was one containing 0.5 ml of a buffer solution instead of the Caragana Sinica root extract, and the comparison target was arbutin, well-known as a skin whitening agent. At that time, an inhibition rate of tyrosinase activity (%) was calculated according to the following Mathematical formula 1, wherein results thereof were shown in the following Table 2.

(23) $\left[\mathrm{Mathematical}\mathrm{formula}1\right]$$\mathrm{Inhibition}\mathrm{rate}\mathrm{of}\mathrm{tyrosinase}\mathrm{activity}\left(\%\right)=100-\left[\frac{\mathrm{Absorbance}\mathrm{of}\mathrm{test}\mathrm{group}}{\mathrm{Absorbance}\mathrm{of}\mathrm{control}\mathrm{group}}\right]\times 100$

(24) TABLE-US-00002 TABLE 2 Inhibition rate of Sample Concentration tyrosinase activity (%) Example 1 0.2% 67.2 0.1% 45.8 0.05% 32.2 Example 2 0.2% 45.5 0.1% 23.2 0.05% 13.2 Example 3 0.2% 53.1 0.1% 31.2 0.05% 16.4 Example 4 0.2% 79.2 0.1% 55.8 0.05% 36.2 Example 5 0.2% 49.7 0.1% 29.2 0.05% 12.4 Arbutin 0.2% 78.7 0.1% 38.4 0.05% 21.2

(25) As a result, as shown in the Table 2, the Caragana Sinica root extract and the fraction thereof obtained from the Examples 1 to 5 inhibited an activity of tyrosinase in a concentration-dependent manner. In particular, when the ethyl acetate fraction of Caragana Sinica root extract of the Example 4 was compared with arbutin as a positive control group, the former showed an inhibitory effect similar to the latter one. Thus, it was confirmed that the ethyl acetate fraction of Caragana Sinica root extract had an excellent effect on inhibiting an activity of tyrosinase.

Experimental Example 2-2: Confirming Melanin Production Inhibitory Effect of the Caragana Sinica Root Extract and the Fraction Thereof

(26) In order to confirm a whitening effect of the ethanol extract of Caragana Sinica root and the fraction thereof prepared in the above Examples 1 to 5, its effect on inhibiting a production of melanin was confirmed with regard to a B16F1 melanin-forming cell.

(27) Specifically, the B16F1 melanin-forming cells, as a mouse-derived cell line, were obtained from American Type Culture Collection (ATCC) to use. The B16F1 melanin-forming cells were divided into a 6-well plate at a concentration of 2×10.sup.5 per well, after which the cells were attached thereto, thus being divided at such a concentration as not to cause toxicity. After that, the ethanol extract of Caragana Sinica root and the fraction thereof obtained from the above Examples 1 to 5 were treated at each concentration, and then being cultured for 72 hours. After this 72-hour cultivation, the cells were taken off by trypsin-EDTA, after which the number of cells was measured, and then cells were collected by centrifugation. A quantification of melanin in cells was performed in such a way that modifying method of Lotan (Cancer Res., 40: 3345-3350, 1980). A cell pallet was washed once with PBS, after which 1 ml of a homogenization buffer solution (50 mM sodium phosphate, pH6.8, 1% Triton X-100, 2 mM PMSF) was added thereto and swirled for 5 minutes to crush the cells. 1N NaOH (10% DMSO) was added to a cell lysate obtained through centrifugation (3,000 rpm, 10 minutes) so as to dissolve the extracted melanin, after which an absorbance of the melanin was measured by microplate reader at 405 nm, and the melanin was quantified to measure an inhibition rate of melanin production (%) of a sample. The inhibition rate of melanin production (%) of the B16F1 melanin-forming cell was calculated according to the following [Mathematical formula 2], wherein results thereof were indicated at Table 3.

(28) $\left[\mathrm{Mathematical}\mathrm{formula}2\right]$$\mathrm{Inhibition}\mathrm{rate}\mathrm{of}\mathrm{melanin}\mathrm{production}\left(\%\right)=\left[\frac{A-B}{A}\right]\times 100$$A\text{:}\mathrm{Amount}\mathrm{of}\mathrm{melanin}\mathrm{in}a\mathrm{well}\mathrm{without}a\mathrm{sample}\mathrm{added}$$B\text{:}\mathrm{Amount}\mathrm{of}\mathrm{melanin}\mathrm{in}a\mathrm{well}\mathrm{with}a\mathrm{sample}\mathrm{added}$

(29) TABLE-US-00003 TABLE 3 Treatment concentration Inhibition rate of Sample (μg/ml) melanin production (%) Untreated group 0.2 — Example 1 200 75.3 100 60.5 Example 2 200 69.4 100 51.9 Example 3 200 71.7 100 58.9 Example 4 200 82.1 100 69.6 Example 5 200 34.7 100 24.9 Arbutin 200 75.7 100 63.5

(30) As a result, as shown in the Table 3, it was confirmed that the Caragana Sinica root extract and the fraction thereof obtained from the Examples 1 to 5 inhibited a production of melanin in a concentration-dependent manner. In particular, it was confirmed that the ethyl acetate fraction of the Caragana Sinica root extract of the Example 4 showed more excellent inhibitory effect on the production of melanin than arbutin known as a whitening agent.

Experimental Example 2-3: Confirming Inhibitory Effect of α-Viniferin on Melanin Production

(31) In order to confirm whitening effect of α-viniferin isolated from the fraction of Caragana Sinica root of the above Example 4, its effect on inhibiting a production of melanin was confirmed with regard to a B16F1 melanin-forming cell.

(32) Specifically, the α-viniferin isolated from the ethyl acetate fraction of the ethanol extract of Caragana Sinica root obtained from the above Example 4 or arbutin, and α-MSH, a stimulant of melanin production, were treated at the same time, after which an amount of melanin was measured in 70 to 74 hours later as describe in the above Example 2-2.

(33) As a result, as shown in FIG. 4, an inhibitory effect of α-viniferin on melanin production, which was induced by α-MSH, was indicated as inhibition rate of 8% at a sample concentration of 4 μM, 21% at 6 μM and 63% at 8 μM respectively, and an IC50 value was 7 μM. On the other hand, in case of arbutin, a control substance, its inhibitory effect on melanin production, which was induced by α-MSH stimulation, was indicated as inhibitory rate of 13% at a sample concentration of 100 μM, 44% at 200 μM and 73% at 400 μM respectively, and an IC50 value was 242 μM. Thus, it was confirmed that a whitening effect of α-viniferin on melanin production, which was induced by α-MSH, was about 35 times higher than that of arbutin.

Formulation Example 1: Preparing of a Solubilized Formulation Comprising the Fraction of Caragana Sinica Root Extract

(34) In order to confirm a stability of a cosmetic composition comprising a fraction of Caragana Sinica root extract as an active ingredient, the ethyl acetate fraction of Caragana Sinica root extract of the above Example 4 was dissolved in the mixed solvent of buthylene glycol and purified water at a ratio of 50:50, and then a solubilized formulation was prepared such as that shown in the following Table 4.

(35) TABLE-US-00004 TABLE 4 Phase Ingredient Content (%) Water Purified water To 100 phase Glycerin 10-25 Dipropylene Glycol Betaine D-panthenol Sodium Hyaluronate Thickener Suitable amount Metal ion sequestering agent Suitable amount Solubilized PEG-60 hydrogenated castor oil 0-2 phase PEG-40 hydrogenated castor oil 0-2 Polyhydric alcohol  0-10 Fragrance Suitable amount Ethanol  0-20 Addition I Ethyl acetate fraction 3.2 of Caragana Sinica root extract of Example 4 Addition II Preservative Suitable amount Other additive Suitable amount
Preparation Method

(36) (1) Water phase and solubilized phase were evenly mixed and dissolved, respectively.

(37) (2) The solubilized phase was inserted into the water phase to be mixed and solubilized together.

(38) (3) An addition I, comprising the ethyl acetate fraction of Caragana Sinica root extract of the Example 4, was solubilized and inserted into the resulting solution to be mixed together, after which an addition II was mixed into the resulting mixture to complete a solubilized formulation.

Formulation Example 2: Preparing of an Essence Formulation Comprising the Fraction of Caragana Sinica Root Extract

(39) In order to confirm a stability of the cosmetic composition comprising a fraction of Caragana Sinica root extract as an active ingredient, the ethyl acetate fraction of Caragana Sinica root extract of the above Example 4 was dissolved in the mixed solvent of buthylene glycol and purified water at a ratio of 50:50, and then an essence formulation was prepared such as that shown in the following Table 5.

(40) TABLE-US-00005 TABLE 5 Phase Ingredient Content (%) Water phase Purified water To 100 Ceteareth-6 olivate 0.1-3 Glycerin .sup. 10-25 1,2-propanediol Sodium Hyaluronate Thickener Suitable amount Metal ion sequestering agent Suitable amount Oil phase PEG-100 Stearate 0.1-1 Glyceryl Stearate 0.1-1 Polysorbate 60 0.1-1 Cetyl alcohol 0.1-1 Behenyl alcohol 0.1-1 Squalane   5-20 Tocopheryl Acetate .sup. 0.1-0.5 Ethyl acetate fraction 3.2 of Caragana Sinica root extract of Example 4 Additional phase I Fragrance Suitable amount Additional phase II Preservative Suitable amount Other additive Suitable amount
Preparation Method

(41) (1) Water phase and oil phase were heated to be evenly mixed and dissolved, respectively.

(42) (2) The oil phase was inserted into the water phase at 75° C. to be mixed and emulsified together.

(43) (3) An additional phase I, comprising the ethyl acetate fraction of Caragana Sinica root extract of the Example 4 as an active ingredient, was inserted into the resulting solution at 50° C. to be mixed together, after which an additional phase II was mixed into the resulting mixture.

Formulation Example 3: Preparing of a Cream Formulation Comprising the Fraction of Caragana Sinica Root Extract

(44) In order to confirm a stability of the cosmetic composition comprising a fraction of Caragana Sinica root extract as an active ingredient, the ethyl acetate fraction of Caragana Sinica root extract of the above Example 4 was dissolved in the mixed solvent of buthylene glycol and purified water at a ratio of 50:50, and then a cream formulation was prepared such as that shown in the following Table 6.

(45) TABLE-US-00006 TABLE 6 Phase Ingredient Content (%) Water phase Purified water To 100 Glycerin .sup. 10-25 Betaine Sodium Hyaluronate Thickener Suitable amount Metal ion sequestering agent Suitable amount Oil phase PEG-100 Stearate 0.1-2 Glyceryl Stearate 0.1-2 Polysorbate 60 0.1-2 Stearic Acid 0.1-2 Cetearyl Alcohol 0.1-2 Capric/Caprylic Triglyceride .sup. 10-30 Tocopheryl Acetate .sup. 0.1-0.5 Addition I Ethyl acetate fraction 3.2 of Caragana Sinica root extract of Example 4 Addition II Fragrance Suitable amount Preservative Suitable amount Other additive Suitable amount
Preparation Method

(46) (1) Water phase and oil phase were heated to evenly mixed and dissolved, respectively.

(47) (2) The oil phase was inserted into the water phase at 75° C. to be mixed and emulsified together.

(48) (3) An additional phase I, comprising the ethyl acetate fraction of Caragana Sinica root extract of the Example 4 as an active ingredient, was inserted into the resulting solution at 50° C. to be mixed together, after which an additional phase II was mixed into the resulting mixture.

Experimental Example 3: Confirming a Stability of the Cosmetic Composition Comprising a Fraction of Caragana Sinica Root Extract

Experimental Example 3-1: Confirming a Raw Material Stability of the Cosmetic Composition Comprising the Fraction of Caragana Sinica Root Extract

(49) In order to confirm a raw material stability, the solubilized formulation, essence formulation and cream formulation of the above Formulation Examples 1 to 3 were stored under a condition of 4° C., 30° C., 45° C. and sunlight for 12 weeks, and then changes of α-viniferin ingredient comprised in the ethyl acetate fraction of Caragana Sinica root extract with the passage of time were observed through an HPLC analysis by the same method of the above Experimental Example 1, wherein results thereof were shown in the following Table 7.

(50) TABLE-US-00007 TABLE 7 α-viniferin (compared to the initial content %) Solubilized formulation Essence formulation Cream formulation Elapsed Sun- Sun- Sun- time 4° C. 30° C. 45° C. light 4° C. 30° C. 45° C. light 4° C. 30° C. 45° C. light Initial 99.9 100 99 99.2 99 99.8 100 99.3 100 99.5 99.3 100 1 week 96.2 96.7 97.2 97 97.3 96.6 96 97.7 97 96.2 95.7 94.4 3 week 97 97.6 97.3 94.3 97 98.1 98.5 97.1 96.7 97.7 98.3 92.4 5 week 97.6 98.2 97.7 90.9 96.1 96.1 98.8 96.3 95.8 96.8 99.4 92 8 week 96.6 99 94.6 86.4 94.3 96 96.6 96.9 96.4 95.2 100.2 90.9 12 week  94.9 94.2 98.7 92.3 97 95.7 98.1 94.2 95.9 93.9 101.7 88.9 Result Stable Stable Stable Stable Stable Stable Stable Stable Stable Stable Stable Stable

(51) As a result, as shown in the Table 7, it was confirmed that the solubilized formulation, essence formulation and cream formulation all showed an excellent stability of α-viniferin, an active ingredient inside the ethyl acetate fraction of Caragana Sinica root extract, under a high temperature condition and a sunlight condition.

Experimental Example 3-2: Confirming a Formulation Stability of the Cosmetic Composition Comprising a Fraction of Caragana Sinica Root Extract

(52) In order to confirm a formulation stability, the solubilized formulation, essence formulation and cream formulation of the above Formulation Examples 1 to 3 were stored under a condition of 4° C., 30° C., 45° C. and sunlight for 12 weeks, after which changes in color, odor and formulation thereof were observed through a visual evaluation and a sensory evaluation, wherein results thereof were shown in the following Tables 8 to 10. For this, a degree of color, odor and formulation changes was classified and evaluated according to following evaluation criteria.

(53) <Evaluation Criteria>

(54) No change: ∘

(55) Slight change: Δ

(56) Significant change: x

(57) TABLE-US-00008 TABLE 8 Solubilized formulation Change in odor Change in color Change in formulation Elapsed Sun- Sun- Sun- time 4° C. 30° C. 45° C. light 4° C. 30° C. 45° C. light 4° C. 30° C. 45° C. light 1 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 2 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 4 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 8 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 12 week  ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘

(58) TABLE-US-00009 TABLE 9 Essence formulation Change in odor Change in color Change in formulation Elapsed Sun- Sun- Sun- time 4° C. 30° C. 45° C. light 4° C. 30° C. 45° C. light 4° C. 30° C. 45° C. light 1 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 2 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 4 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 8 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 12 week  ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘

(59) TABLE-US-00010 TABLE 10 Cream formulation Change in odor Change in color Change in formulation Elapsed Sun- Sun- Sun- time 4° C. 30° C. 45° C. light 4° C. 30° C. 45° C. light 4° C. 30° C. 45°c light 1 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 2 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 4 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 8 week ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ 12 week  ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘

(60) As a result, as shown in the Tables 8 to 10, it was confirmed that the solubilized formulation, essence formulation and cream formulation all showed an excellent stability under a high temperature condition and a sunlight condition.

Experimental Example 3-3: Confirming a Stability of α-Viniferin Isolated from the Fraction of Caragana Sinica Root Extract

(61) In order to confirm a stability of α-viniferin, the α-viniferin isolated from the ethyl acetate fraction of the ethanol extract of Caragana Sinica root obtained from the above Experimental Example 1 was stored under a condition of 50° C. for 2 weeks, after which content changes thereof were observed through an HPLC analysis by the same method of the above Experimental Example 1. A control group was kobophenol A isolated from the Caragana Sinica root extract, which was stored under the same condition with that of the α-viniferin, after which content changes thereof were observed by the same method. Results thereof were shown in the following Table 11.

(62) TABLE-US-00011 TABLE 11 Test Condition and Period Kobophenol A α-viniferin Initial 121.4 ppm  252.8 ppm 1 week 115.6 ppm 249.60 ppm 95.2%  98.7% 2 weeks 103.6 ppm 253.45 ppm 85.3% 100.3%

(63) As a result, as shown in the Table 11, a content of the α-viniferin was not decreased at a high temperature of 50° C. for 2 weeks, and thus it was confirmed that the α-viniferin had an excellent stability compared to kobophenol A, i.e. another whitening substance of Caragana Sinica root extract.

Experimental Example 4: Confirming Skin Irritation of the Cosmetic Composition Comprising the Fraction of Caragana Sinica Root Extract

(64) In order to confirm a skin irritation of the cosmetic composition comprising the fraction of Caragana Sinica root extract, evaluation on the skin irritation of a cream formulation of the above Formulation Example 3 was conducted.

(65) Specifically, a 5×20 cm patch with a certain amount (20 μl) of the cream formulations, comprising the ethyl acetate fraction of the ethanol extract of Caragana Sinica root prepared from the above Formulation Example 3 in a concentration-dependent manner, were attached onto the back of 30 adults for 24 hours, after which the patch was removed therefrom and changes in skin conditions were read with naked eyes at 1 and 24 hours later, wherein the results were shown in the following Table 12. For this, a degree of changes in skin conditions was classified and evaluated according to following evaluation criteria for skin conditions.

(66) <Evaluation Criteria for Skin Conditions>

(67) 0: No change

(68) 1: Extremely slight change

(69) 2: Slight change

(70) 3: Slightly severe change

(71) 4: Severe change

(72) 5: Extremely severe change

(73) TABLE-US-00012 TABLE 12 Skin Condition according Subject (gender to Fraction Content and skin type) Elapsed Time 3.2% 6.4% 16%  1 (M, Neutral) 1 hour 0 0 0 24 hours 0 0 0  2 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0  3 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0  4 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0  5 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0  6 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0  7 (M, Neutral) 1 hour 0 0 0 24 hours 0 0 0  8 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0  9 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 16 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 11 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 12 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 13 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 14 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 15 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 1 16 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 17 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 18 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 19 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 20 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 21 (M, Neutral) 1 hour 0 0 0 24 hours 0 0 0 22 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 23 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 1 24 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 25 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 26 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 27 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 28 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 29 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 0 30 (F, Neutral) 1 hour 0 0 0 24 hours 0 0 1

(74) As a result, as shown in the Table 12, it was identified that the cream formulation comprising high content of α-viniferin had a little skin irritation, too.

Experimental Example 5: Clinical Evaluation of Effectiveness of the Cosmetic Composition Comprising the Fraction of Caragana Sinica Root Extract

(75) In order to evaluate an effectiveness of the cosmetic composition comprising the fraction of Caragana Sinica root extract, a clinical test was conducted with 23 women with an average age of 44. A test product was a cosmetic composition comprising the fraction of Caragana Sinica root extract by 3.2%, and a control product was a placebo cream comprising purified water instead of the fraction of Caragana Sinica root extract of the test product. The test product and the control product were respectively applied onto left and right sides of a subject face at random, and were applied thereto again at an interval of twice a day, wherein the changes were observed for 8 weeks.

Experimental Example 5-1: Clinical Evaluation of Skin Brightness Changes According to Application of the Cosmetic Composition Comprising the Fraction of Caragana Sinica Root Extract

(76) In order to confirm a change in skin brightness according to a use of the test and control products, the skin brightness was measured before use and in 4, 6 and 8 weeks after use employing Chroma meter CM700d for a skin color measuring apparatus. In every measurement, skin brightness of the same region was measured three times, wherein an average value thereof was used as an evaluation material.

(77) TABLE-US-00013 TABLE 13 Significance Test Product Control Product Probability of Brightness Significance Brightness Significance Test-Control Classification (L*) Probability (L*) Probability Comparison Test of within- — 0.000* —  0.000* — subject effect Before use 62.567 ± 3.117 — 63.114 ± 2.802 — — 4 weeks after use 65.235 ± 2.761 0.121  63.544 ± 2.265 0.696 0.554  6 weeks after use 67.346 ± 2.912 0.045* 62.239 ± 2.450 0.062 0.000** 8 weeks after use 68.565 ± 2.559 0.000* 62.507 ± 2.215 0.306 0.000** *p < 0.05 Post-hoc test results of a non-parametric test, which was repeatedly measured by a parametric method ANOVA according to a test of normality **p < 0.05 Independent sample t test result

(78) In the Table 13, data showed that the results of measuring a skin brightness of 23 subjects were indicated as an average±standard error (SE), wherein a degree of their changes was illustrated in graph in FIG. 5.

(79) As seen in Table 13 and FIG. 5, measured value of skin brightness after use of the test product were significantly increased (significance probability <0.05) in 6 and 8 weeks after use in comparison with before use, and also showed a significant difference (significance probability <0.05) in 6 and 8 weeks after use in comparison with the control product. Accordingly, it was confirmed that the cosmetic composition comprising the fraction of Caragana Sinica root extract gave a benefit to the skin brightness from 6 weeks after use.

Experimental Example 5-2: Clinical Evaluation of Skin Melanin Content Changes According to Application of the Cosmetic Composition Comprising the Fraction of Caragana Sinica Root Extract

(80) In order to confirm a change in skin brightness according to the use of the test and control products, an amount of skin melanin was measured before use and in 4, 6 and 8 weeks after use by using a reflected light from a probe at 568 nm (green), 660 nm (red), 880 nm (infrared) employing a narrow-band reflectance spectrophotometer called Mexameter. In every measurement, an amount of melanin of the same region was measured three times, wherein an average value thereof was used as an evaluation material.

(81) TABLE-US-00014 TABLE 14 Significance Test Product Control Product Probability of Significance Significance Test-Control Classification Brightness (L*) Probability Brightness (L*) Probability Comparison Test of within- — .sup. 0.000* — 0.198 — subject effect Before use 113.652 ± 30.693 — 117.783 ± 32.342 — — 4 weeks after use 108.913 ± 26.258 0.068.sup.  118.478 ± 27.860 — 0.008.sup.## 6 weeks after use 105.188 ± 26.619 0.000.sup.# 117.232 ± 32.130 — 0.000.sup.#  8 weeks after use 101.873 ± 26.170 0.001.sup.# 117.043 ± 32.472 — 0.003.sup.## .sup.#p < 0.017 (=5%/3) Results of conducting a Friedman test as a non-parametric method and then a post-hoc test with a non-parametric test method according to a test of normality .sup.##p < 0.05 Mann-Whitney U test results

(82) In the Table 14, data showed that the results of 23 subjects were indicated as an average±standard error (SE), wherein a degree of their changes was illustrated in graph in FIG. 6.

(83) As seen in Table 14 and FIG. 6, measured value of skin melanin after use of the test product were significantly decreased (significance probability <0.017) in 6 and 8 weeks after use in comparison with before use, and also showed a significant difference (significance probability <0.05) in 4, 6 and 8 weeks after use in comparison with the control product. Accordingly, it was confirmed that the cosmetic composition comprising the fraction of Caragana Sinica root extract was beneficial to decrease a content of skin melanin from 6 weeks after use.

Experimental Example 5-3: Visual Evaluation of Skin Color Changes According to Application of the Cosmetic Composition Comprising the Fraction of Caragana Sinica Root Extract

(84) In order to confirm a change in skin brightness according to the use of the test and control products, a visual evaluation on skin was made before use and in 4, 6 and 8 weeks after use.

(85) The visual evaluation on a skin color of a test region was made by two experts on the basis of an intensity score table, which is a skin color evaluation reference table, wherein a brightness of skin color was evaluated by ranging from scores 1 (bright and transparent) to 10 (dark and dull), depending on the skin color. If there is any difference of evaluation between two investigators, a lower score was selected as an evaluation material. Table 15

(86) TABLE-US-00015 TABLE 15 Significance Test Product Control Product Probability of Significance Significance Test-Control Classification Score Probability Score Probability Comparison Test of within- — .sup. 0.000* — 0.697 — subject effect Before use 3.174 ± 0.576 — 3.087 ± 0.793 — — 4 weeks after use 3.000 ± 0.739 0.046.sup.  3.043 ± 0.878 — 0.252 6 weeks after use 2.826 ± 0.778 0.005.sup.# 3.000 ± 0.953 — 0.108 8 weeks after use 2.564 ± 0.752 0.002.sup.# 3.130 ± 0.920 — .sup. 0.006.sup.## .sup.#p < 0.017 (=5%/3) Results of conducting a Friedman test as a non-parametric method and then a post-hoc test with a non-parametric test method according to a test of normality .sup.##p < 0.05 Mann-Whitney U test results

(87) In the Table 15, data showed that the results of 23 subjects were indicated as an average±standard error (SE), wherein a degree of their changes was illustrated in graph in FIG. 7.

(88) As seen in Table 15 and FIG. 7, scores of visual evaluations on whitening of the test product group made by experts were significantly decreased (significance probability <0.017) in 6 and 8 weeks after use in comparison with before use, and also showed a significant difference (significance probability <0.05) in 8 weeks after use in comparison with the control product. Accordingly, it was confirmed that the cosmetic composition comprising the fraction of Caragana Sinica root extract gave a benefit to a skin whitening from 6 weeks after use.