COMPOSITION COMPRISING KAKKALIDE

Abstract

The present invention relates to a composition comprising kakkalide. In addition, the composition of the present invention comprising kakkalide can be variously used for brightening the skin, improving wrinkles in the skin, moisturizing the skin, inhibiting bacteria in the skin, preventing hair loss, or promoting hair growth.

Claims

1. A method of brightening the skin, comprising treating a subject with kakkalide represented by the following Chemical Formula 1: ##STR00002##

2. The method of claim 1, wherein the kakkalide is comprised in a composition.

3. The method of claim 1, wherein the treating with the kakkalide comprises orally administering the kakkalide or applying the kakkalide to the skin.

4. A method of preventing or treating a disease related to excessive pigmentation, comprising treating a subject with kakkalide represented by the following Chemical Formula 1: ##STR00003##

5. The method of claim 4, wherein the disease related to excessive pigmentation is melasma, freckles, or hyperpigmentation.

6. The method of claim 4, wherein the treating with the kakkalide comprises orally administering the kakkalide or applying the kakkalide to the skin.

7. A method of improving wrinkles in the skin, comprising treating a subject with kakkalide represented by the following Chemical Formula 1: ##STR00004##

8. The method of claim 7, wherein the treating with the kakkalide comprises orally administering the kakkalide or applying the kakkalide to the skin.

9. The method of claim 7, wherein the treating with the kakkalide promotes type I collagen synthesis and thus improves the wrinkles in the skin.

10. A method of preventing, improving, or treating symptoms of dry skin, comprising treating a subject with kakkalide represented by the following Chemical Formula 1: ##STR00005##

11. The method of claim 10, wherein the treating with the kakkalide comprises orally administering the kakkalide or applying the kakkalide to the skin.

12. The method of claim 10, wherein the symptoms of the dry skin comprise lack of moisture in the skin, atopic dermatitis, psoriasis, xeroderma, eczema, or xeroderma pigmentosum.

13. A method of inhibiting bacteria in the skin, comprising treating a subject with kakkalide represented by the following Chemical Formula 1: ##STR00006##

14. The method of claim 13, wherein the treating with the kakkalide comprises orally administering the kakkalide or applying the kakkalide to the skin.

15. A method of preventing or treating dandruff, comprising treating a subject with kakkalide represented by the following Chemical Formula 1: ##STR00007##

16. The method of claim 15, wherein the kakkalide inhibits growth of Pityrosporum ovale and thus prevents or treats dandruff

17. A method of preventing hair loss or promoting hair growth, comprising treating a subject with kakkalide represented by the following Chemical Formula 1: ##STR00008##

18. The method of claim 17, wherein the treating with the kakkalide comprises orally administering the kakkalide or applying the kakkalide to the skin.

19. The method of claim 17, wherein the kakkalide activates Wnt/β-catenin signaling pathway and/or promotes proliferation of dermal papilla cells, and thus prevents hair loss or promotes hair growth.

Description

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0087] Hereinafter, the present invention will be described in detail by way of exemplary embodiments. However, the following exemplary embodiments are only illustrative of the present invention, and the contents of the present invention are not limited to the following exemplary embodiments.

Experimental Example 1

Brightening Effect According to Reduction in Total Amount of melanin at Cellular Level

[0088] In order to evaluate a brightening effect according to inhibition of melanogenesis, an extract was added to a mouse melanoma cell (B-16 mouse melanoma cell) culture, and the total amount of melanin was measured according to a method described in Lotan R. et al. (Cancer Res. 40:3345-3350, 1980). For this experiment, toxicity with respect to the mouse melanoma cells was first evaluated, and the brightening effect was evaluated at a concentration not inducing toxicity. Dimethyl sulfoxide (DMSO) was used as a negative control, and arbutin was used as a positive control.

[0089] Specifically, A compound of Formula 1 was added to a medium of each sample so that the final concentration of the compound of Formula 1 is 1 ppm, 10 ppm, 100 ppm, or 500 ppm, and Daidzein was also added to the medium so that the final concentration of Daidzein is 1 ppm, 10 ppm, 100 ppm, or 500 ppm. Arbutin was added to the medium so that the final concentration was 100 ppm. In each of the mediums, melanoma cells were cultured for three days. Subsequently, the cells were treated with trypsin, detached from the culture plate, and centrifuged, and then melanin was extracted. The detached cells were boiled for 10 minutes by adding 1 ml of a sodium hydroxide solution (1 N concentration) so that melanin was dissolved, and by measuring an absorbance at 400 nm using a spectrophotometer, the amount of melanin production was measured.

[0090] The amount of melanin was measured by a method of indicating absorbance per unit cell count (1×10.sup.6 cells), The amount of melanin production in each experimental group with respect to the negative control was calculated as a relative value, and the value obtained by subtracting the amount of melanin production in each experimental group from 100% of the melanin production amount in the negative control group was expressed as the inhibition rate (%). The results are shown in Table 1. The experiment was performed three times for each sample, and averages of measurements were obtained.

TABLE-US-00001 TABLE 1 Sample Inhibition rate (%) Negative control 100 Positive control (Arbutin, 100 ppm) 56.05 Chemical Formula 1 (1 ppm) 35.29 Chemical Formula 1 (10 ppm) 48.05 Chemical Formula 1 (100 ppm) 57.62 Chemical Formula 1 (500 ppm) 68.42 Daidzein (1 ppm) 18.73 Daidzein (10 ppm) 16.45 Daidzein (100 ppm) 20.76 Daidzein (500 ppm) 30.22

[0091] Referring to Table 1, it can be seen that kakkalide represented by Chemical Formula 1 has an excellent melanin reduction effect even at a low concentration as compared to daidzein, which is also a component of Pueraria lobata flowers, and is thus useful for brightening the skin.

Experimental Example 2

Effect of Promoting Type I Collagen Synthesis in Human-Derived Fibroblasts

[0092] A sample was added to a human-derived fibroblast culture medium to evaluate the effect of promoting type I collagen synthesis at the cellular level. The synthesized collagen was quantified using a Procollagen Type I C-Peptide Enzyme Immunoassay kit (PICP EIA kit). In order to measure the amount of collagen synthesis, a compound of Formula 1 was added to the fibroblast culture medium (Dulbecco's Modified Eagle Medium (DMEM)) so that the final concentration of compound of Formula 1 was 0.1 ppm, 1 ppm, or 10 ppm in each medium, and Genistein was also added to the fibroblast culture medium (Dulbecco's Modified Eagle Medium (DMEM)) so that final concentration of Genistein was 0.1 ppm, 1 ppm, or 10 ppm. After culturing for 48 hours, and then the culture medium was taken, and, by using the PICP EIA kit, the degree of type I collagen synthesis at each concentration was measured at 450 nm using a spectrophotometer.

[0093] In order to compare the effect, the culture medium of fibroblasts without sample treatment was used as a negative control, and 10 ng/ml TGF-β was used as a positive control, and the degrees of collagen synthesis therein were measured in the same manner as for testing an added sample. A rate of increase in collagen synthesis was calculated as a ratio of the amount of collagen production relative to that of the negative control, and the results are shown in Table 2. The experiment was performed three times for each sample, and averages of measurements were obtained.

TABLE-US-00002 TABLE 2 Amount of type I collagen Increase Sample production (ng/ml) rate (%) Negative control 151.4 — Positive control (TGF-β 10 ng/ml) 172.35 13.84 Chemical Formula 1 (0.1 ppm) 157.74 4.19 Chemical Formula 1 (1 ppm) 170.41 12.56 Chemical Formula 1 (10 ppm) 175.72 16.07 Genistein (0.1 ppm) 155.24 2.53 Genistein (1 ppm) 158.75 4.85 Genistein (10 ppm) 165.93 8.59

[0094] Referring to Table 2, it can be seen that kakkalide represented by Chemical Formula 1 is more effective in synthesizing collagen than genistein, which is also a component of Pueraria lobata flowers, and thus is useful for improving wrinkles.

Experimental Example 3

Testing of Increase in HAS2, HAS3, and AQP3 Expression in Human Keratinocytes

[0095] Human keratinocytes (HaCaT) were dispensed into a 6-well cell culture plate at 2×10.sup.5 cells/well and cultured for 24 hours in a DMEM medium containing 10% fetal bovine serum (FBS). The medium was replaced with a DMEM medium without 10% FBS and calcium (Ca.sup.2+), and the cultured cells were treated with the compound of Chemical Formula 1, which had been diluted with a medium, for 24 hours. After the 24 hours had passed, the cells were recovered and then washed with a cold phosphate buffer solution (PBS), and total RNA was extracted using an RNeasy mini kit (Qiagen, Germany). A reverse transcription reaction was performed using RNA and a cDNA Synthesis kit (PhileKorea, Korea). The synthesized cDNA was diluted with water after being quantified, and the same amount thereof was used for all reactions. HAS2, HAS3, and AQP3 primers from a TaqMan® Universal Master Mix II and TaqMan® Gene Expression Assays (Thermo Fisher, USA) were used. Real-time quantitative polymerase chain reaction (PCR) was carried out using a StepOnePlus® Real-time PCR System (Applied Biosystems, USA). The experimental results were obtained through calculation performed by a ΔΔCt method based on a housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The levels of HAS2, HAS3, and AQP3 gene expression in each sample were quantified based on the mRNA expression level (normalized to 1.0) of the negative control to which the sample was not added, and the results are shown in Table 3. As a positive control, 10 ng/ml EGF recombinant human protein (Life Technologies, USA) was used.

TABLE-US-00003 TABLE 3 Increased Increased Increased HAS2 mRNA HAS3 mRNA AQP3 mRNA Sample amount amount amount Negative control 1 1 1 EGF 10 ng/ml 16.42 5.24 1.09 Chemical Formula 1 2.28 3.97 1.75 10 ppm Chemical Formula 1 6.64 5.23 4.17 100 ppm Genistein (10 ppm) 1.06 2.86 1.09 Genistein (100 ppm) 2.74 3.34 2.47

[0096] Referring to Table 3, it can be seen that the compound of Chemical Formula 1 is more effective in expressing hyaluronic acid synthesis than the negative control, considering the higher levels of HAS2, HAS3, and AQP3 expression.

[0097] Therefore, it can be seen that the composition containing kakkalide of the present invention represented by Chemical Formula 1 is useful for moisturizing the skin.

Experimental Example 4

Effect of Inhibiting Microbes (Effect of Inhibiting Dandruff-Causing Fungi)

[0098] This experiment was carried out by a serial dilution method and a method of measuring a minimum inhibitory concentration (MIC), using an MHB2 medium (for bacteria) or a PDB medium (for fungi). The bacteria or fungi were inoculated at about 10.sup.6/well. Three types of general bacteria, two types of fungi, and Pityrosporum ovale were inoculated and cultured, and the degree of bacterial/fungal growth at each sample concentration was observed to determine the minimum concentration for inhibiting bacterial/fungal growth.

TABLE-US-00004 TABLE 4 Minimum inhibitory concentration (%) Pityrosporum Sample E. coli S. aureus P. aeruginosa A. niger C. albicans ovale Zinc pyrithione 0.002 0.001 0.002 0.02 0.02 0.0002 Chemical 0.05 0.01 0.05 0.1 0.05 0.0003 Formula 1 Genistein 0.1 0.03 0.07 0.2 0.08 0.0005

[0099] Referring to Table 4, it can be seen that like zinc pyrithione (positive control) having an excellent antimicrobial effect against Pityrosporum ovale, which is a dandruff-causing fungus, the compound of Chemical Formula 1 has an excellent antimicrobial effect. Therefore, it can be seen that a composition comprising kakkalide of the present invention represented by Chemical Formula 1 is useful for inhibiting bacteria.

Experimental Example 5

Investigation of Wnt/β-Catenin Signaling Pathway Activation Effect of Extract

[0100] The effect of the compound of Chemical Formula 1 on the activation of the Wnt/β-catenin signaling pathway was investigated using TOPflash (T-cell factor reporter plasmid) Kit (Millipore, USA). Human dermal papilla cells were inoculated into a 24-well plate at 2×10.sup.5/well, cultured for 24 hours, treated with the compound of Chemical Formula 1 so that the concentration was 10 μg/ml, and then additionally cultured for 15 hours, and firefly luciferase activity was subsequently measured using a dual luciferase assay kit (Promega, USA).

[0101] In order to accurately measure the luciferase activity, the experiment was performed three times for each sample, an increase rate (%) was calculated as a ratio of activity relative to that of a control was calculated, and the results are shown in Table 5. The negative control was an untreated group. As a positive control, 6-bromoindirubin-3′-oxime (BIO) was used, and BIO is a material known to inhibit GSK-3 in the Wnt signaling system.

TABLE-US-00005 TABLE 5 Sample Increase rate (%) Control (not treated) 100 BIO 1 μM 153 Chemical Formula 1 (10 μg/ml) 160 Genistein (10 μg/ml) 140

[0102] Referring to Table 5, it can be seen that kakkalide represented by Chemical Formula 1 is more effective in activating the Wnt/β-catenin signaling pathway than the control. Therefore, it can be seen that a composition comprising kakkalide of the present invention represented by Chemical Formula 1 is useful for preventing hair loss or promoting hair growth.

Experimental Example 6

Effect of Proliferating Dermal Papilla Cells

[0103] This experiment was performed using the following media 1) to 3):

[0104] 1) DMEM (10% FBS, 1% P/S)

[0105] 2) Depletion medium: 5% charcoal-stripped, FBS-containing, phenol red-free DMEM

[0106] 3) Serum-free (SF) medium: DMEM (1% P/S).

[0107] On days 0 to 5, the following various experiments were performed to confirm a dermal papilla cell proliferation effect.

[0108] Day 0: At 10 a.m., cells were seeded (5,000 cells/well)

[0109] Day 1: At 10 a.m., after changing the depletion medium, cells were cultured for 24 hours.

[0110] Day 2: At 10 a.m., CCK-8 was measured (average initial value: 0.586) before the cells were treated with a sample, and subsequently, cells were treated with a sample.

[0111] Day 5: At 10 a.m., CCK-8 was measured (it is determined that cells do not die, and the initial number is maintained at the of the average control value of 0.556).

TABLE-US-00006 TABLE 6 Sample Average (ratio) stdev Control (DMEM) 1.00 0.03 Serum-free (SF) 0.68 0.04 Chemical Formula 1 100 ppm 1.34 0.07 Genistein 100 ppm 1.10 0.05 stdev: standard deviation

[0112] Referring to Table 6, it can be seen that in a group treated with kakkalide of the present invention represented by Chemical Formula 1, the dermal papilla cells proliferated more than in the control.

[0113] Therefore, it can be seen that a composition comprising the compound of the present invention represented by Chemical Formula 1 is useful for preventing hair loss.

[0114] A composition comprising kakkalide according to the present invention can be variously used for brightening the skin, improving wrinkles in the skin, moisturizing the skin, inhibiting bacteria in the skin, preventing hair loss, or promoting hair growth.