Composition for skin soothing containing liquid-phase plasma

Abstract

The present invention is to provide a composition for skin soothing, which is safe from a risk of side effects, and relates to a composition for skin soothing containing a liquid-phase plasma. The liquid-phase plasma of the present invention is obtained by applying plasma as pure energy to a liquid-phase material, and can be used in the skin sensitive to a chemical material without a risk of side effects. Furthermore, the liquid-phase plasma has remarkable effects on the prevention or treatment of dermatitis or photo-aging due to ultraviolet light, and thus is expected to be greatly used in beauty care and mechanical fields.

Claims

1. A cosmetic composition for soothing skin, comprising a plasma-conditioned liquid material prepared by a method comprising: (a) filling a plasma generator with a carrier gas; (b) generating a plasma by providing a voltage of 1 kV to 30 kV and a frequency of 10 to 30 kHz to the plasma generator; and (c) irradiating a liquid-phase material with the generated plasma, as an active ingredient.

2. A cosmetic composition for preventing or improving photoaging, comprising a plasma-conditioned liquid material prepared by a method comprising: (a) filling a plasma generator with a carrier gas; (b) generating a plasma by providing a voltage of 1 kV to 30 kV and a frequency of 10 to 30 kHz to the plasma generator; and (c) irradiating a liquid-phase material with the generated plasma, as an active ingredient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the result of confirming the activity of ERK and p38 in epidermal cells treated with a liquid-type plasma (LTP) before and after UV stimulation according to an example of the present invention.

(2) FIG. 2 shows the result of confirming the activity of NF-kB in epidermal cells treated with LTP before and after UV stimulation according to an example of the present invention.

(3) FIG. 3 shows the result of confirming the activity of EGFR in epidermal cells treated with LTP before and after UV stimulation according to an example of the present invention.

(4) FIG. 4 shows the result of confirming the expression levels of INF-α and IL-6 in epidermal cells treated with LTP before and after UV stimulation according to an example of the present invention.

(5) FIG. 5 shows the result of confirming the expression levels of IL-1β, IL-6 and IL-8 in epidermal cells treated with LTP (argon plasma) before and after UV stimulation according to an example of the present invention.

(6) FIG. 6 shows the result of confirming the activity of p-NFkB, p-ERK and IL-1β in epidermal cells treated with LTP (argon plasma) before and after UV stimulation according to an example of the present invention.

MODES OF THE INVENTION

(7) Hereinafter, the present invention will be described in further detail with reference to examples. These examples are merely provided to illustrate the present invention, and it should be obvious to those of ordinary skill in the art that the scope of the present invention is not limited by the following examples according to the gist of the present invention.

Example 1

Preparation of Liquid Plasma

(8) A liquid-type plasma (LTP) was prepared using an atmospheric plasma generator equipped with a quartz or ceramic tube as a dielectric and including a multi-nozzle non-thermal plasma source. The generator was designed to have a gas supply nozzle diameter of less than 3 mm, and to generate a uniform plasma having a 1-inch size for medical research. The LTP was prepared by a method of supplying nitrogen (N.sub.2) as a carrier gas to the generator at a flow rate of 10 L/min, and applying the plasma for 60 seconds per mL at a distance of 4 cm from the bottom surface of a culture dish (12-well plate, TPP, Renner, Dannstadt, Germany) into which Dulbecco's Modified Eagle Medium (DMEM) was dispensed. Here, the power supply specifications of the plasma generator preferably are a voltage of 1 to 30 kV and an average frequency of 10 to 30 kHz, and most preferably, a voltage of 15 kV and an operation frequency of 15 kHz, but the present invention was not limited thereto.

Example 2

UV Treatment for Immortalized Human Keratinocyte Cell Line

(9) An immortalized human keratinocyte (HaCaT) cell line was provided from the Korean Cell Line Bank, and cultured in DMEM containing 10 vol % FBS and 1 vol % antibiotic/antibacterial agent.

(10) For UV treatment, 1×10.sup.6 of the HaCaT cells were seeded in a 100-mm plate, after 24 hours, the cells were washed with phosphate-buffered saline (PBS), and the medium was replaced with a 1% FBS-containing medium. After 24 hours, the cells were irradiated with 20 mJ/cm.sup.2 or 100 mJ/cm.sup.2 UVB (280-320 nm) in a minimal amount of PBS (˜2 ml), immediately after the irradiation, incubated in DMEM for 15 minutes, and collected for Western blotting. For an LTP experimental group, 1 hour prior to the UVB irradiation, cells were pretreated with LTP for 1 hour, and immediately after the UVB irradiation, incubated with LTP, instead of DMEM, for 15 minutes.

Example 3

Confirmation of LTP (Nitrogen Plasma) Effect on UV-Damaged Epidermal Cells

(11) Cells further cultured for 15 minutes after treatment with 20 mJ/cm.sup.2 UV rays were lysed with a RIPA buffer (150 mM NaCl, 1.0% Nonidet-P 40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Iris, pH 8.0, and a protease inhibitor cocktail, and PhoSTOP (Roche Molecular Biochemicals, Basel, Switzerland)), 10 μg of a protein lysate was loaded in a 10% SDS/PAGE gel to isolate proteins, and then proteins were transferred to a PVDF membrane (Amersham, Arlington Heights, Ill., USA). The protein-transferred PVDF membrane was reacted with a pERK, ERK, p-p38, p38, p-NFκB, NFκB, p-EGFR, EGFR or GAPDH (Cell Signaling Technology) antibody, the extent of the expression of each signal was measured with ECL Western blotting detection reagents (Amersham). The expression level of each signal was recalculated based on a GAPDH signal. The results are shown in FIGS. 1 to 3.

(12) As the experimental result, it was seen that, compared with a control only treated with UV irradiation, in the group treated with LTP before and after the UV treatment, the expression of pERK, p-p38, p-NFκB and p-EGFR decreases. NF-kB is known as a transcription factor causing the expression of a tumor necrosis factor (TNF) or an interleukin-6 (IL-6) molecule, which is involved in an immune response or inflammatory response by activating a gene expression system causing inflammation. Therefore, compared to a control not treated with LTP, in cells treated with LTP before and after UV damage, it can be understood that the reduced phosphorylation of ERK, p38 and EGFR as well as NF-kB is an effect of reducing the inflammatory response caused by skin damage.

(13) In addition, the expression levels of the TNF-α and IL-6 genes of the cells further cultured for 4 hours after the treatment with 20 mJ/cm.sup.2 UV rays were confirmed by qPCR. TNF-α and IL-6 are known to be associated with the etiological cause of inflammation. Primer sets of TNF-α, IL-6 and GAPDH used herein were commercially available (Qiagen, Hilden, Germany), and expression levels were recalculated based on GAPDH signals of TNF-α and IL-6. The results are shown in FIG. 4.

(14) As the experimental result, compared to the control only treated with UV irradiation, in the group treated with LTP before and after UV treatment, it was confirmed that the TNF-α and IL-6 expression was significantly reduced.

Example 4

Confirmation of LTP (Argon Plasma) Effect on UV-Damaged Epidermal Cells

(15) LTP was prepared by applying the same method as described in Example 1, except argon was supplied as a carrier gas, and an effect on UV-damaged epidermal cells was analyzed. FIG. 5 shows the result of confirming IL-1β, IL-6 and IL-8 expression levels in epidermal cells (HaCaT cells) treated with LTP (argon plasma) before and after UV irradiation through qPCR. To investigate an expression level of a transcriptome, the expression levels of the IL-1β, IL-6 and IL-8 genes of the epidermal cells treated with 20 mJ/cm.sup.2 UV rays and then further cultured for 4 hours were confirmed by qPCR. Primer sets of IL-1β, IL-6, IL-8 and GAPDH were commercially available (Qiagen, Hilden, Germany), and the expression levels of IL-1β, IL-8 and IL-6 were recalculated based on a 18 s RNA level. As the experimental result, compared with a group only treated with UV treatment, in the group treated with argon plasma before and after UV irradiation (UVB, 20 mJ/cm.sup.2), it was confirmed that the IL-1β, IL-6 and IL-8 expression corresponding to a proinflammatory cytokine was significantly reduced.

(16) FIG. 6 shows the result of confirming the p-NFkB, p-ERK and IL-1β activity in epidermal cells treated with LTP (argon plasma) before and after UV irradiation (HaCaT cells). In this experiment, cells further incubated for 15 minutes or 4 hours (IL-1β) after treatment with 20 mJ/cm.sup.2 UN rays were lysed with an RIPA buffer (150 mM NaCl, 1.0% Nonidet-P 40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0, and a protease inhibitor cocktail, and PhoSTOP (Roche Molecular Biochemicals, Basel, Switzerland)), 10 μg of a protein lysate was loaded in a 10% SDS/PAGE gel to isolate proteins, and the proteins were transferred to a PVDF membrane (Amersham, Arlington Heights, Ill., USA). A pERK, ERK, p-p38, p38, p-NFκB, NFκB, p-EGFR, EGFR, or GAPDH (Cell Signaling Technology) antibody was reacted with the protein-transferred. PVDF membrane, and the extent of the expression of each signal was measured with ECL Western blotting detection reagents (Amersham). The expression level of each signal was recalculated based on a GAPDH signal. As the experimental result, it was confirmed that, compared with the group only treated with UV rays, in the group treated with an argon plasma before and after UV irradiation (UVB, 20 mJ/cm.sup.2), in epidermal cells treated with LTP (argon plasma) before and after the UV irradiation (UVB, 20 mJ/cm.sup.2), the activity of p-NF-kB and p-ERK corresponding to skin inflammatory molecules is reduced, and the production of IL-1β corresponding to a proinflammatory cytokine is reduced.

(17) From the results of FIGS. 1 to 6, it was seen that the LTP treatment of the skin before and after UV damage has a significant effect on the prevention or treatment of dermatitis or photoaging caused by UV rays.