Topical skin treatment composition comprising dendranthema indicum extract

Abstract

The present invention relates to a topical skin treatment composition comprising Dendranthema indicum extract and, more particularly, to a topical skin treatment composition containing Dendranthema indicum extract which is excellent for the anti-oxidation, anti-aging, whitening, and moisturizing of skin, and also does not cause skin irritation.

Claims

1. A method for improving skin condition of a subject, comprising applying a composition comprising an extract of a plant as an active ingredient and a cosmetically acceptable additive to skin of the subject, wherein the plant consists of Chrysanthemum indicum var. albescens; wherein the improvement of skin condition is selected from the group consisting of improved moisturization and improved brightness; and wherein the composition is selected from the group consisting of a solution, a gel, a solid, a paste anhydride, an oil-in-water emulsion, a suspension, a microemulsion, microcapsules, microgranules, liposome, non-ionic vesicles, cream, skin toner, lotion, powder, ointment, spray, conceal stick, foamed composition, and an aerosol composition, wherein the extract of plant is obtained by extracting the plant with 30 to 70% ethanol.

2. The method of claim 1, wherein the extract of plant is contained in an amount of 0.001-10 wt % based on the total weight of the composition.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a graphic diagram showing the results of analyzing the components of an extract of Chrysanthemum indicum var. albescens (white Chrysanthemum) by HPLC.

(2) FIG. 2 is a graphic diagram showing the results of analyzing the components of an extract of Chrysanthemum indicum (yellow Chrysanthemum) by HPLC.

BEST MODE

(3) The composition for external skin application according to the present invention contains an extract of Chrysanthemum indicum var. albescens or Chrysanthemum indicum var. albescens Makino as an active ingredient.

(4) The extract of Chrysanthemum indicum var. albescens according to the present invention can be obtained by a method known in the art. For example, the extract of the present invention can be prepared by washing Chrysanthemum indicum var. albescens with purified water, drying the washed plant with sunlight or hot air, finely powdering the dried plant, and extracting the powder with an extraction solvent. The extraction solvent that is used in the present invention may be selected from among organic solvents, including ethanol, methanol, butanol, ether, ethyl acetate and chloroform, and mixed solvents of these organic solvents and water. In view of the safety of the raw material, water or 30-70% ethanol is preferably used in the present invention. The extract obtained using the solvent as described above may be extracted under reflux, filtered, and concentrated under reduced pressure at a temperature of 4045 C., thereby obtaining a dry extract of Chrysanthemum indicum var. albescens.

(5) The composition of the present invention preferably contains the extract of Chrysanthemum indicum var. albescens in an amount of 0.001-10 wt % based on the total weight of the composition. If the content of the extract of Chrysanthemum indicum var. albescens in the composition is less than 0.001 wt %, the efficacy and effect of the extract will be insignificant, and if the content of the extract is more than 10 wt %, it will cause problems in terms of skin safety and formulation.

(6) The composition of the present invention may be used as an external skin application composition for anti-oxidation, which shows an excellent antioxidant effect by scavenging or inhibiting free radicals.

(7) The composition of the present invention may be used as a composition may be used an external skin application composition for anti-aging, which has excellent effects of improving skin elasticity and reducing wrinkles.

(8) The composition of the present invention may be used as a composition may be used an external skin application composition for moisturization, which can enhance skin barrier function and induce the differentiation of skin keratinocytes.

(9) The composition of the present invention may be used as a composition may be used an external skin application composition for skin whitening, which exhibits excellent whitening effects of inhibiting tyrosinase activity and melanin production.

(10) The composition according to the present invention contains a cosmetically and skin-scientifically acceptable medium or base. The composition may be formulated as a preparation for local application. Examples of formulations for local application include a solution, a gel, a solid, a paste anhydride, an oil-in-water emulsion, a suspension, a microemulsion, microcapsules, microgranules, ionic (liposome) and non-ionic vesicles, cream, skin toner, lotion, powder, ointment, spray, or conceal stick. Also, the composition according to the present invention can be formulated as a foamed composition or an aerosol composition further containing a compressed propellant. In addition, the composition of the present invention can be formulated according to a conventional method known in the art.

(11) Further, the composition according to the present invention may contain additives which are conventionally used in the cosmetic field or the skin science field, for example, a fatty substance, an organic solvent, a solubilizing agent, a thickener, a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, a foaming agent, an aromatic, a surfactant, water, an ionic or non-ionic emulsifying agent, a filler, a sequestering agent, a chelating agent, a preservative, vitamins, a blocker, a moisturizing agent, essential oil, a dye, a pigment, a hydrophilic or hydrophobic activator, a lipid vesicle, or other components which are generally used in cosmetics. These additives are contained in amounts which are generally used in the cosmetic field or the skin science field.

(12) Further, the composition of the present invention may contain a skin absorption-promoting material in order to increase the effects of improving skin conditions.

(13) In addition, the composition of the present invention may contain, in addition to the extract of Chrysanthemum indicum var. albescens, other components capable of increasing the skin protective effect, within a range that does not impair the antioxidant, anti-aging, moisturizing and whitening effects of the present invention.

MODE FOR INVENTION

(14) Hereinafter, the construction and effect of the present invention will be described in further detail with reference to test examples and formulation examples. It is to be understood, however, that these test examples and formulation examples are for illustrative purposes and are not intended to limit the scope of the present invention.

Preparation Example 1: Preparation of Extract of Chrysanthemum indicum Var. Albescens

(15) Chrysanthemum indicum var. albescens harvested in a fresh state was washed with purified water, dried by sunlight, and then finely powdered. To 100 g of the powder was added 1000 ml of 70% ethanol, extracted under reflux, filtered, and concentrated under reduced pressure at a temperature of 4045 C., thereby obtaining 17.1 g of an extract of Chrysanthemum indicum var. albescens.

Comparative Preparation Example 1: Preparation of Extract of Chrysanthemum indicum (Yellow Chrysanthemum)

(16) An extract was prepared in the same manner as described in Preparation Example 1, except that Chrysanthemum indicum (yellow chrysanthemum) was used in place of Chrysanthemum indicum var. albescens.

Test Example 1: Analysis of Components of Chrysanthemum Indicum Extract and Chrysanthemum indicum Var. Albescens Extract

(17) The components of 10 mg/me of each of the Chrysanthemum indicum var. albescens extract prepared in Preparation Example 1 and the Chrysanthemum indicum extract prepared in Comparative Preparation Example 1 were analyzed by HPLC, and the results of the analysis are shown in Table 1 below and FIGS. 1 and 2.

(18) TABLE-US-00001 TABLE 1 Linarin (acacetin-7-O- Unit (mg/g) rutinoside) Luteolin Apigenin Comparative 2.5 5.7 Preparation Example 1 Preparation 14.9 6.4 1.6 Example 1

(19) As can be seen in Table 1 above, the Chrysanthemum indicum extract does not contain linarin known as an antioxidant active substance, whereas the Chrysanthemum indicum var. albescens extract contains a large amount of linarin.

Test Example 2: Test for Antioxidant Effect (DPPH Test)

(20) In order to examine the antioxidant effect of the Chrysanthemum indicum var. albescens extract prepared in Preparation Example 1 and the Chrysanthemum indicum extract prepared in Comparative Preparation Example 1, the antioxidant effect was measured based on the change in absorbance resulting from reduction of the organic radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) (the antioxidant is oxidized). Specifically, the decrease in absorbance resulting from the inhibition of DPPH oxidation caused by each of the Chrysanthemum indicum var. albescens extract prepared in Preparation Example 1 and the Chrysanthemum indicum extract prepared in Comparative Preparation Example 1 was measured, and the concentration at which the absorbance decreased to 50% of control was determined as the effective antioxidant concentration.

(21) 10 l of each of the Chrysanthemum indicum var. albescens extract prepared in Preparation Example 1, the Chrysanthemum indicum extract prepared in Comparative Preparation Example 1, and a positive control, was added to 190 l of 100 M DPPH solution (in ethanol) to make reaction solutions. Each of the reaction solutions was allowed to react at 37 C. for 30 minutes, and the absorbance at 540 nm was measured. The positive control used was the synthetic antioxidant Trolox which is widely used. The results of DPPH analysis of each test sample are shown in Table 2 below, and IC.sub.50 in Table 2 means the sample concentration at which the absorbance was decreased by 50% due to the sample added.

(22) TABLE-US-00002 TABLE 2 Test sample IC.sub.50 (ppm) Trolox 43 Chrysanthemum indicum var. albescens 48 extract (Preparation Example 1) Chrysanthemum indicum extract 107 (Comparative Preparation Example 1)

(23) As can be seen in Table 2 above, the antioxidant effect of the Chrysanthemum indicum var. albescens extract of Preparation Example 1 was similar to that of the synthetic antioxidant Trolox used as the positive control and was at least two times higher than that of the Chrysanthemum indicum extract of Comparative Preparation Example 1. This suggests that the Chrysanthemum indicum var. albescens extract according to the present invention has an excellent antioxidant effect.

Test Example 3: Anti-Aging EffectEffect on Inhibition of Collagenase Expression

(24) The collagenase expression inhibitory activities of the Chrysanthemum indicum var. albescens extract prepared in Preparation Example 1 and the Chrysanthemum indicum extract prepared in Comparative Preparation Example 1 were measured comparatively with those of tocopherol and EGCG. Tocopherol and EGCG are antioxidant substances known to function to regenerate the epidermal cells of the skin to prevent the aging of the skin.

(25) In the test, human fibroblasts were added to a 96-well microtiter plate containing 2.5% FBS-containing DMEM (Dulbecco's Modified Eagle's Media) medium at a density of 5,000 cells/well and were cultured to a confluence of about 90%. Next, the cells were cultured in serum-free medium for 24 hours, and then treated with 50 ppm of each of the Chrysanthemum indicum var. albescens extract of Preparation Example 1 and the Chrysanthemum indicum extract of Comparative Preparation Example 1 in serum-free DMEM medium or 10.sup.4 M of each of tocopherol and EGCG for 24 hours, after which the cell cultures were harvested.

(26) The production of collagenase in the harvested cell cultures was measured using a commercially available collagenase measuring device (Amersham Pharmacia, USA). Specifically, the harvested cells were added to a 96-well plate having primary collagenase antibody applied uniformly thereto and were subjected to an antigen-antibody reaction in a constant-temperature bath for 3 hours

(27) After 3 hours, chromophore-conjugated secondary collagen antibody was added to the 96-well plate and allowed to react for 15 minutes. After 15 minutes, a substance inducing color development was added to the 96-well plate, and color development was induced at room temperature for 15 minutes. When 1M sulfuric acid was added to the 96-well plate to stop the reaction (color development), the reaction solution had a yellow color, and the intensity of the yellow color varied depending on the degree of progression of the reaction.

(28) The absorbance of the 96-well plate having a yellow color was measured at 405 nm using a spectrophotometer, and the degree of synthesis of collagenase was calculated using the following Equation 1. Herein, the absorbance of the cell culture broth harvested from the group not treated with the test material was used as a control. That is, the expression level of collagenase in the untreated group was set as 100, and the expression level of collagenase in the group treated with the test material was calculated relative to the untreated group. The calculation results are shown in Table 3 below.
Collagenase expression level(%)=absorbance of group treated with test material/absorbance of control group100Equation 1

(29) TABLE-US-00003 TABLE 3 Collagenase expression Test material level (%) Untreated group 100 Tocopherol 76 EGCG 69 Chrysanthemum indicum var. albescens 64 extract (Preparation Example 1) Chrysanthemum indicum extract 81 (Comparative Preparation Example 1)

(30) As the expression level of collagenase becomes lower, the ability of the test material to inhibit collagenase expression is higher, the degradation of collagen in the skin is reduced so that a decrease in skin elasticity is inhibited and the formation of wrinkles is reduced. As can be seen in Table 3 above, the Chrysanthemum indicum var. albescens extract of the present invention effectively inhibited the expression of collagenase in vitro and had excellent inhibitory effects on collagenase expression compared to tocopherol and EGCG.

(31) Particularly, the Chrysanthemum indicum var. albescens extract more effectively inhibited the expression of collagenase compared to the Chrysanthemum indicum extract, suggesting that it exhibits excellent anti-aging effects by reducing the degradation of collagen in the skin to improve skin elasticity and reduce skin wrinkles.

Test Example 4: Whitening EffectTest for Melanin Production Inhibitory Effect Using Mouse Melanocytes

(32) The melanin production inhibitory activities of the Chrysanthemum indicum var. albescens extract prepared in Preparation Example 1 and the Chrysanthemum indicum extract prepared in Comparative Preparation Example 1 were measured comparatively with those of the known whitening substance hydroquinone.

(33) C57BL/6 mouse melanocytes (Mel-Ab cells) (Dooley, T. P. et al, Skin pharmacol, 7, pp 188-200) were cultured in DMEM, containing 10% fetal bovine serum, 100 nM 12-O-tetradecanoylphorbol-13-acetate and 1 nM cholera toxin, under the conditions of 37 C. and 5% CO.sub.2. The cultured Mel-Ab cells were detached with 0.25% trypsin-EDTA and cultured in a 24-well plate at a concentration of 10 cells/well. Then, during three consecutive days from 2 days of culture, each of the test materials was added thereto and cultured. As the test materials, 25 ppm of each of the Chrysanthemum indicum var. albescens extract of Preparation Example 1, the Chrysanthemum indicum extract of Comparative Preparation Example 1, and hydroquinone were used. Herein, the hydroquinone was used as a positive control. Then, the media were removed, and the cells were washed with PBS and lysed with 1N sodium hydroxide. The lysed cells were measured for absorbance at 400 nm, and based on the measured absorbance, the percent inhibition of melanin production was calculated according to the following equation 2. The results of the calculation are shown in Table 4 (Dooley's method).
Percent inhibition of melanin production=100(absorbance of test material/absorbance of control100)Equation 2

(34) TABLE-US-00004 TABLE 4 Inhibition (%) of Test material melanin production Untreated group 100 Hydroquinone (positive control) 48.2 Chrysanthemum indicum var. albescens 54.9 extract (Preparation Example 1) Chrysanthemum indicum extract 29.4 (Comparative Preparation Example 1)

(35) As can be seen in Table 4 above, the Chrysanthemum indicum var. albescens extract of the present invention showed melanin production inhibitory activity similar to that of the known whitening substance hydroquinone and more effectively inhibited melanin production compared to the Chrysanthemum indicum extract, suggesting that it has an excellent whitening effect.

Test Example 5: Irritation Test

(36) In order to compare the sensory feeling of the Chrysanthemum indicum var. albescens extract of the present invention with that of the known whitening substance kojic acid, 15 panels sensitive to irritation, such as stinging, burning, etc., were subjected to a test for determining irritation, such as stinging, burning, etc.

(37) Each test panel was allowed to apply 0.5 mL of each of kojic acid (available from YM Chemical Co.) and the Chrysanthemum indicum var. albescens extract to his/her skin randomly at the left side or right side, and then evaluate the test sample by grading from 0 to 3.0 at an interval of 0.1. The results are shown in Table 5 below.

(38) Criteria for Evaluation

(39) 0-0.4: no irritation;

(40) 0.5-1.0: slight irritation;

(41) 1.1-2.0: mild irritation;

(42) 2.1-3.0: severe irritation.

(43) TABLE-US-00005 TABLE 5 Kojic Chrysanthemum indicum var. albescens acid extract (Preparation Example 1) Stinging 0.91 0.23 Burning 0.43 0.14 Average 0.67 0.19

(44) As can be seen in Table 5 above, kojic acid caused slight stinging and burning and a perceptible degree of irritation. On the contrary, the Chrysanthemum indicum var. albescens extract of the present invention caused little or no irritation with respect to stinging and burning. This suggests that the Chrysanthemum indicum var. albescens extract of the present invention causes no irritation, and thus can give a good sensory feeling, compared to kojic acid.

Test Example 6: Skin Moisturization TestTest for Induction of Differentiation of Human Keratinocytes

(45) In order to examine the skin barrier function and skin moisturizing activity of each of the Chrysanthemum indicum var. albescens extract prepared in Preparation Example 1 and the Chrysanthemum indicum extract prepared in Comparative Preparation Example 1, the following test was performed using absorbance.

(46) Human neonatal epidermal keratinocytes (HEK; Lonza, NHEK-Neo-Neonatal Normal Human Epidermal Keratinocytes, Pooled) were dispensed in medium (KBM-gold, Lonza) in a 6-well plate at a density of 510.sup.4 cells per well and cultured for 24 hours under the conditions of 37 C. and 5% CO.sub.2. Next, each of the Chrysanthemum indicum var. albescens extract and the Chrysanthemum indicum extract was added to the cell culture at concentrations of 50 ppm and 100 ppm, and the cells were cultured to a confluence of 80-90% for 5 days. The cells were harvested and washed with PBS (phosphate buffered saline), and then 1 ml of 10 mM Tris-HCl buffer (pH 7.4) containing 2% SDS (sodium dodecyl sulfate) and 20 mM DTT (dithiothreitol) was added to the cells, followed by sonication for 3 minutes and boiling for 10 minutes. The resulting solution was centrifuged at 1200 rpm for 30 minutes, and the precipitate was suspended in 1 ml of PBS. The absorbance of the suspension at 340 nm was measured.

(47) Meanwhile, a portion of the solution after the sonication was taken and the protein content thereof was measured and used as a standard for evaluating the differentiation of the cells. A low-calcium (0.03 mM) group and a high-calcium (1.2 mM) group were used as negative/positive control groups, respectively. Each of the test materials was added at low calcium concentration, and the amount of CE (cornified envelope) produced during keratinocyte differentiation was measured to determine the cell differentiation-promoting effect of the test material. The results of the measurement are shown in Table 6 below.

(48) TABLE-US-00006 TABLE 6 Keratinocyte Test material Concentration differentiation (%) Control Low-calcium 100 group (0.03 mM) High-calcium 208 group (1.2 mM) Chrysanthemum indicum 50 ppm 128 var. albescens extract 100 ppm 153 (Preparation Example 1) Chrysanthemum indicum 50 ppm 109 extract (Comparative 100 ppm 124 Preparation Example 1)

(49) As can be seen in Table 6 above, the Chrysanthemum indicum var. albescens extract of the present invention promoted the differentiation of keratinocytes. Particularly, the Chrysanthemum indicum var. albescens extract more effectively promoted the differentiation of keratinocytes, suggesting that it enhances skin barrier function and skin moisturization.

INDUSTRIAL APPLICABILITY

(50) As described above, the present invention provides the composition that has excellent antioxidant, anti-aging, whitening and moisturizing effects on the skin without causing skin irritation.