COSMETIC COMPOSITION FOR IMPROVING SKIN CONTAINING EXTRACTS OF POLYGALA TENUIFOLIA, ANGELICA DAHURICA AND ELSHOLTZIA SPLENDENS

Abstract

A composition for improving skin comprising extracts of Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens is described. The composition is excellent in effects of improving skin inflammation, antioxidant effects, antibacterial effects on skin, and skin moisturizing effects, and thus can be used as a cosmetic for improving skin, and further, as a pharmaceutical composition for treating inflammatory skin diseases.

Claims

1-12. (canceled)

13. A method for improving skin inflammation, antioxidation, antibacterial effects on skin, or moisturizing skin, comprising step of: administering to a subject, a cosmetic composition comprising a Polygala tenuifolia extract, an Angelica dahurica extract and an Elsholtzia splendens extract as active ingredients.

14. The method of claim 13, wherein the Polygala tenuifolia extract, the Angelica dahurica extract and the Elsholtzia splendens extract are extracted with one or more solvents selected from water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

15. The method of claim 13, wherein the cosmetic composition inhibits DPPH (1-1-diphenyl-2-picryl-hydrazyl) and ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) activities.

16. The method of claim 13, wherein the cosmetic composition inhibits NO (nitric oxide) production.

17. The method of claim 13, wherein the Polygala tenuifolia extract, the Angelica dahurica extract and the Elsholtzia splendens extract are mixed at a weight ratio of 0.05-3:0.05-3:0.05-3 (w/w).

18. The method of claim 13, wherein the cosmetic composition has antibacterial activity against strains of the genus Staphylococcus.

19. The method of claim 13, wherein the cosmetic composition is one or more formulation selected from the group consisting of serums, tonics, essences, pastes, mask packs, patches, gels, creams, lotions, nourishing lotions, nourishing creams, moisturizing creams, massage creams, powders, soaps, cleansers, oils, foundations, makeup bases, waxes, and sprays.

20. The method of claim 13, wherein the skin inflammation is any one or more selected from the group consisting of dermatitis, allergic dermatitis, irritant dermatitis, seborrheic dermatitis, atopic dermatitis, sensitive skin diseases, pruritus, eczematous skin diseases, dry eczema, erythema, urticaria, psoriasis, drug rash, and acne.

21. A method for improving treating inflammatory skin diseases, comprising step of: administering to a subject, a composition comprising a Polygala tenuifolia extract, an Angelica dahurica extract and an Elsholtzia splendens extract as active ingredients.

22. The method of claim 21, wherein the inflammatory skin disease is any one or more selected from the group consisting of dermatitis, allergic dermatitis, irritant dermatitis, seborrheic dermatitis, atopic dermatitis, sensitive skin diseases, pruritus, eczematous skin diseases, dry eczema, erythema, urticaria, psoriasis, drug rash, and acne.

23. The method of claim 21, wherein the Polygala tenuifolia extract, the Angelica dahurica extract and the Elsholtzia splendens extract are mixed at a weight ratio of 0.05-3:0.05-3:0.05-3 (w/w).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0066] FIG. 1 is a graph showing the results of measuring the DPPH radical scavenging activity of the samples of mixed extracts of Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens according to an example of the present invention.

[0067] FIG. 2 is a graph showing the results of measuring the ABTS radical scavenging activity of the samples of mixed extracts of Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens according to an example of the present invention.

[0068] FIG. 3 is a graph showing the results of measuring the cell viability after treating Raw 264.7 cells with the samples of mixed extracts of Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens according to an example of the present invention.

[0069] FIG. 4 is a graph showing the results of measuring the NO production in Raw 264.7 cells after treating the Raw 264.7 cells with the samples of mixed extracts of Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens according to an example of the present invention.

[0070] FIG. 5 is a graph showing the results of measuring the cell viability of the cells of the human keratinocyte cell line (HaCaT) after treating the cells with the samples of mixed extracts of Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens according to an example of the present invention.

[0071] FIG. 6 is a graph showing the results of measuring the hyaluronic acid production in HaCaT cells after treating the cells with the samples of mixed extracts of Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens according to an example of the present invention.

DESCRIPTION OF EMBODIMENTS

Examples

[0072] Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Materials and Methods

1-1. Reagents and Equipment

[0073] The reagents used were ascorbic acid (AA, Sigma-Aldrich, USA), 1,1-diphenyl-2-picryl-hydrazyl (DPPH, Sigma, USA), 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma, USA), Dulbecco's Modified Eagles Medium (DMEM, Welgene, Korea), fetal bovine serum (FBS, Gibco, USA), FBS (Samchun, Korea), penicillin/streptomycin (Lonza, Switzerland), water-soluble tetrazolium salt (WST)-8 (Biomax, Korea), lipopolysaccharide (LPS, SigmaAldrich, USA), Griess reagent system (Promega, USA), hyaluronic acid (HA), enzyme linked immunosorbent assay (ELISA) kit (Minneapolis, USA).

[0074] The experimental equipment used was a grinder (Hanil, Korea), a rotary vacuum evaporator (BUCHI, Germany), an incubator (Thermo, USA), a microplate reader (BioTek, USA), and a pipette (Eppendorf, Germany).

1-2. Materials

[0075] The Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens used for extraction were purchased from Jecheon Oriental Herbal Medicine (Jecheon, Korea) after going through a washing and drying process, and were used after grinding.

1-3. Sample Extraction and Mixing

[0076] 400 mL of 70% ethanol was added to 100 g of each of ground Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens and extraction was performed at room temperature for 48 hours. The extract was filtered through filter paper and concentrated at 45 C. using a rotary vacuum evaporator, and the concentrated powder was stored at 50 C. for use in the experiment.

[0077] With regard to the mixing ratio of Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens, seven mixing ratios as shown in Table 1 were used for the Polygala tenuifolia extract, the Angelica dahurica extract and the Elsholtzia splendens extract. Mixtures were prepared according to the ratios.

TABLE-US-00001 TABLE 1 Polygala Angelicae Elsholtzia Sample teuifolia dahurica splendens M1 1 1 1 M2 0.5 1.5 1 M3 1.5 0.5 1 M4 0.1 1.95 0.95 M5 0.5 0.5 2 M6 0.95 1.95 0.1 M7 1.45 0.1 1.45

1-4. Evaluation of Antioxidant Efficacy

DPPH Radical Scavenging Assay

[0078] Antioxidant efficacy was measured by DPPH radical scavenging activity based on the Blois' method. This assay is one of the generation methods. DPPH is purple in the radical state, and when reduced by receiving electrons, it changes its color to yellow (DPPH-H). 100 L of 0.2 mM DPPH solution and 80 L of samples of each concentration were mixed in a 96-well plate, reacted for 30 minutes at room temperature in the dark, and then absorbance was measured at 517 nm to calculate radical scavenging activity according to Equation 1 below. Ascorbic acid, an antioxidant, was used as a positive control, and a DPPH solution which was not treated with a sample was used as a negative control.

[00001]$\begin{array}{cc}\mathrm{DPPH}\mathrm{radical}\mathrm{scavenging}\mathrm{assay}\left(\%\right)=\left(1-\mathrm{ansorbance}\mathrm{of}\mathrm{the}\mathrm{group}\mathrm{with}\mathrm{sample}\mathrm{added}/\mathrm{absorbance}\mathrm{of}\mathrm{the}\mathrm{group}\mathrm{without}\mathrm{sample}\right)100& \left[\mathrm{Equation}1\right]\end{array}$

ABTS Radical Scavenging Assay

[0079] Antioxidant activity was measured using 2,2-azino-biazoline-6-sulphonic acid (ABTS) radical with reference to the Van den Berg method. This method uses the principle that ABTS+ radical, which has a blue-green color, is made to be transparent by antioxidants. 7 mM ABTS and 2.4 mM potassium persulfate were mixed and left at room temperature in the dark for 24 hours to form ABTS. Absorbance was measured at 734 nm, and the material was used for the experiment after adjusting the absorbance to 0.70.1.

[0080] 100 L of ABTS working solution and 100 L of samples of each concentration were mixed and reacted in the dark for 10 minutes. Then, the absorbance was measured at 732 nm and the radical scavenging activity was calculated as shown in Equation 2 below. Ascorbic acid, an antioxidant, was used as a positive control, and an ABTS solution which was not treated with a sample was used as a negative control.

[00002]$\begin{array}{cc}\mathrm{ABTS}\mathrm{radical}\mathrm{scavenging}\mathrm{assay}\left(\%\right)=\left(1-\mathrm{absorbance}\mathrm{of}\mathrm{the}\mathrm{group}\mathrm{with}\mathrm{sample}\mathrm{added}/\mathrm{absorbance}\mathrm{of}\mathrm{the}\mathrm{group}\mathrm{without}\mathrm{sample}\right)100& \left[\mathrm{Equation}2\right]\end{array}$

1-5. Cell Culture

[0081] Raw 264.7 cells were provided by the Korea Cell Line Bank (KCLB; Seoul, Korea), and human keratinocyte cell line (HaCaT) cells were provided by Amore Pacific (Korea). For cell culture, DMEM containing 10% FBS and 1% penicillin/streptomycin was used, and the cells were cultured in an incubator at 5% carbon dioxide and 37 C.

1-6. Cell Viability Test

[0082] Cells were dispensed into a 96-well plate at 110.sup.4 cells/well, and then cultured in an incubator for 24 hours to allow complete attachment, and thereafter treated with samples for 48 hours. WST-8 was added at a 10% concentration and reacted for 2 hours, and then absorbance was measured at 450 nm with a microplate reader.

1-7. Measurement of Nitric Oxide (NO) Inhibitory Activity

[0083] Raw 264.7 cells were dispensed into a 96-well plate at 110.sup.4 cells/well, and then cultured in an incubator for 24 hours to allow complete attachment, and thereafter treated with samples for 48 hours. The stimulating source was LPS at a concentration of 10 ng/mL. The culture supernatant was taken and the amount of NO produced was quantified using the Griess reagent system according to the manufacturer's guidelines.

1-8. Measurement of Hyaluronic Acid (HA)

[0084] HaCaT cells were dispensed into a 96-well plate at 110.sup.4 cells/well, and then cultured in an incubator for 24 hours to allow complete attachment, and thereafter treated with samples for 48 hours. Retinoic acid was used as a positive control. The supernatant of the cultured cells was taken, and the amount of hyaluronic acid produced was measured by the ELISA assay according to the manufacturer's guidelines.

1-9. Antibacterial Activity

[0085] The antibacterial effect was measured against Staphylococcus aureus (S. aureus). The paper disc method was used to measure the antibacterial activity of the samples. Each strain cultured on a solid plate medium was taken by a platinum loop, then inoculated into a 4 mL liquid medium, and cultured for 20 hours to activate it. Then, 500 L of the strain was inoculated into 3 mL of liquid medium and cultured for 4 hours. The strain was inoculated onto a solid plate medium so that the number of bacteria was 110.sup.6-510.sup.6/mL, and then spread evenly using a spreader. Each sample was prepared according to the concentrations, and 50 L of the sample was absorbed into a paper disc (Toyo, Japan) with a diameter of 8 mm. The paper disc was placed on a solid plate medium inoculated with the strain and cultured at 37 C. for 20 hours. Then, the diameter of the clear zone around the disc was measured.

1-10. Statistical Processing

[0086] All experiments were repeated three times, and the results were expressed as meanstandard deviation. Results were considered statistically significant when the p value was less than 0.05 in independent sample t-test and one-way ANOVA using SPSS 18.0 program.

Example 2: DPPH and ABTS Radical Scavenging Activities

[0087] Radicals are unstable and thus easily react with cellular components, causing non-selective and irreversible damage. The DPPH radical, which displays a purple color, is a relatively stable compound and is used to test radical activity through reaction with antioxidants. The DPPH and ABTS radical scavenging activities of the extracts are shown in FIG. 1 and FIG. 2.

[0088] As for DPPH radical scavenging activity, M6, which has a high mixing ratio of Polygala tenuifolia and Elsholtzia splendens, showed the highest activity of 9.052.78-75.373.79% at a concentration of 50-1,000 g/mL. Ascorbic acid, which is used as a positive control (PC), showed antioxidant activity of 87.210.56% at a concentration of 50 g/mL. DPPH radical scavenging activity of other mixed extracts at a concentration of 50-1,000 g/mL was high in the order of M7 (2.104.91-67.833.44%), M1 (2.961.71-58.212.81%), M5 (1.344.48-57.741.87%), M3 (2.483.58-56.211.48%), M4 (0.474.12-52.953.29%) and M2 (1.731.33-51.350.36%). The activity increased in a concentration-dependent manner in all samples.

[0089] ABTS radical scavenging activity increased with increasing concentration in all mixed extracts. In particular, at a concentration of 50-1,000 g/mL, M6 showed the highest activity of 22.322.62-99.190.20%, showing a similar trend to its DPPH radical scavenging activity. ABTS radical scavenging activity increased in a concentration-dependent manner in all mixed extracts, and ascorbic acid, used as a positive control (PC), showed antioxidant activity of 99.680.21% at a concentration of 50 g/mL. At a concentration of 500 g/mL, M6 showed a significantly high activity of 94.610.13%, followed by M1 (92.301.04%), M3 (87.871.32%), M2 (80.420.11%), M4 (80.311.22%), M5 (76.271.44%), and M7 (70.592.06%).

Example 3: Raw 264.7 Cell Viability

[0090] WST-8 assay was performed to determine the effect of mixed extracts of Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens on the viability of Raw 264.7 cells, and the results are shown in FIG. 3. The concentrations of all mixed extracts were set to 50, 100, and 200 g/mL. Since they did not show toxicity at any concentrations, the maximum concentration was set to 200 g/mL, and NO production was measured.

Example 4: NO Production in Raw 264.7 Cells

[0091] To compare the effects of mixed extracts at various ratios on NO production, an inflammatory response was induced in Raw 264.7 cells by LPS, and then the cells were treated with the mixed extracts at concentrations of 50, 100, and 200 g/mL. Thereafter, the Griess assay was performed. As shown in FIGS. 4, M4 and M6 showed high NO production inhibitory activity in a concentration-dependent manner, and in particular, at a concentration of 200 g/mL, M6 showed about 50% production compared to the LPS-treated group and displayed statistically significant NO production inhibitory activity.

Example 5: HaCaT Cell Viability

[0092] WST-8 assay was performed to determine the effect of mixed extracts of Polygala tenuifolia, Angelica dahurica and Elsholtzia splendens on the viability of HaCaT cells. The concentrations were set to 50, 100, and 200 g/mL. As shown in FIG. 5, the mixed extracts did not show toxicity at a concentration range of 12.5-50.0 g/mL, and thus the maximum concentration was set to 50.0 g/mL, and hyaluronic acid production was measured.

Example 6: Hyaluronic Acid Production by HaCaT Cells

[0093] In order to determine the effect of the mixed extracts at various ratios on moisturizing effect, HaCaT cells were treated with 12.5, 25.0, and 50.0 g/mL of each mixed extract. The culture medium recovered after culturing the cells for 48 hours was analyzed for hyaluronic acid production using an ELISA kit. The results are shown in FIG. 6. The content of HA contained in skin tissues amounts to 50% of the total HA in the human body, and when HA in the skin decreases due to various factors, it is directly linked to skin aging, including loss of skin moisture. HA is widely used as a general indicator in the search for skin moisturizing materials in various in vitro models. After treating HaCaT cells with the mixed extracts at a concentration range that did not show cytotoxicity, HA production was evaluated. The results showed that all mixed extracts displayed concentration-dependent HA production enhancing activity in the concentration range of 12.5-50.0 g/mL, and at a concentration of 50 g/mL, M3 and M6 displayed statistically significantly increased (1.47-fold, 1.49-fold) HA production activity compared to the control.

Example 7: Antibacterial Test Results

[0094] Disc diffusion test is performed by placing a disc into which a sample was absorbed on an agar plate medium inoculated with bacteria and culturing them, and then measuring the diameter of the inhibition zone (clear zone) of the sample that diffused around the disc as much as it inhibited the growth of the bacteria.

[0095] Table 2 below shows the experimental results for Staphylococcus aureus strain. Methyl paraben, used as a positive control, showed 8.5 mm at 4 mg/mL, and M4 and M6 showed 8.750.25 and 9.750.25 mm at 8 mg/mL, showing the effect of inhibiting the growth of Staphylococcus aureus strain.

TABLE-US-00002 TABLE 2 Concen- Clear tration zone Sample (mg/mL) (mm) Methyl paraben 4 8.5 M1 8 M2 8 M3 8 M4 8 8.75 0.25 M5 8 M6 8 9.75 0.25 M7 8