KR20120049634A - Method for evaluating the effectiveness of uv screening agents by measuring the rate of cell apoptosis and necrosis - Google Patents

Abstract

PURPOSE: A method for evaluating efficiency of UV protector is provided to measure cell apoptosis rate and to accurately protect UV ray. CONSTITUTION: A method for evaluating efficiency of UV protector comprises: a step of applying an UV protector onto UV permeable material; a step of irradiating UV onto the upper portion of the UV permeable material; and a step of measuring cell death of skin cells. The UV permeable material is a quartz plate or band pass filter. The skin cells include keratinocytes, fibroblasts, or melanocytes. The cell death includes apoptosis and necrosis.

Description

Method for evaluating the effectiveness of UV screening agents by measuring the rate of cell apoptosis and necrosis}

The present invention relates to a method for evaluating the efficacy of a sunscreen, and more particularly, applying a sunscreen to a UV-transmitting material; Placing skin cells under the UV permeable material and then irradiating UV over the UV permeable material; And it relates to a method for evaluating the efficacy of the sunscreen comprising the step of obtaining the skin cells to measure the cell death rate.

Efficacy evaluation of all sunscreens at present is called erythema induced by UVB region (290-320 nm) called "sun protection factor" and "Protection of UVA" (PA). Persistent Pigment Darkening induced by the UVA region (320-400 nm) is evaluated through the naked eye evaluation of the product to evaluate the UV blocking ability of the product.

However, the light source transmitted through the sunscreen does not cause phenomena such as erythema and blackening, but the degree of affect on the skin is different and there is no method for evaluating a new evaluation method.

Accordingly, the present inventors confirmed that the degree of inducing apoptosis or necrosis of the cells is different even when the values indicating the blocking ability of SPF or PA are the same, and the rate of cell death due to cell death or necrosis is different. The present invention was completed by discovering that the effect of the sunscreen can be measured accurately and in detail.

Accordingly, it is an object of the present invention to provide a method for evaluating the efficacy of a sunscreen that can accurately identify the difference in efficacy even with a sunscreen having the same value indicating the blocking ability of SPF or PA.

In order to achieve the above object, the present invention comprises the steps of applying a sunscreen to the UV-transmitting material; Placing skin cells under the UV permeable material and then irradiating UV over the UV permeable material; And it provides a method for evaluating the efficacy of the sunscreen comprising the step of obtaining the skin cells to measure the cell death rate.

In the present invention, by measuring the cell death rate in the skin cells it was possible to further subdivide the effect of the sunscreen on the skin to evaluate the accurate and reliable sunscreen effect.

FIG. 1 is a graph showing the difference in the Sunscreen Transmittance of similar or identical SPF clinical index.
Figure 2 is a schematic of the degree of cell self-killing and necrosis by the amount of detection (%) of Annexin V and PI staining costimulatory molecules.
Figures 3a to 3g is a result of measuring the cell death rate and necrosis rate of the similar or identical clinical index sunscreen of Comparative Examples 1-2 and Examples 1-5.

In the present invention, it is insufficient to accurately represent the effect on the skin of the method of measuring SPF or PA through the visual evaluation of erythema or blackening simply by seeing the amount of sunlight penetration or prevention of penetration in artificial skin and laboratory animals including the existing human body. In order to supplement the above, it was proved that the degree of inducing cell death or necrosis is different even for the same values indicating the blocking ability of SPF or PA, thereby providing a new evaluation method to evaluate the efficacy of sunscreen. Could.

The present invention provides a method for evaluating the efficacy of a sunscreen comprising the following steps:

Applying a sunscreen to the UV transmissive material;

Placing skin cells under the UV permeable material and then irradiating UV over the UV permeable material; And

Obtaining the skin cells to measure cell death rate.

In the above, the UV-transmitting material can be used as long as the UV-transmitting material has a property that can be applied to the sunscreen while transmitting UV, for example, a quartz plate or a band for transmitting only a target ray region. A band pass filter may be used, but the present invention is not limited thereto.

The skin cells may be used both human or animal-derived skin cells, for example, keratinocytes, fibroblasts or melanocytes, etc., but are not limited thereto. It is not.

The apoptosis rate can be calculated by measuring the rate of self-killing or necrosis of cells by UV irradiation. At this time, the self-killing or necrosis of the cells changes the shape of the cells through visual evaluation, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide, MTT) assay, WST-1 There may be various methods, such as assays, Propidium iodide (PI) assays, and methods of viewing related proteins and genes, but it is more preferable to evaluate them by flow cytometry through antibodies and staining. The WST-1 assay is a Premix WST-1 cell growth assay that can determine cell proliferation ability or cell viability using the principle of changing from tetrazolium salt (WST-1) to formazan pigment. The MTT assay is a method for analyzing the color of a pigment formed from a yellow tetrazolium salt which is reduced to a formazan crystal insoluble in living cells, and the iodide propidium assay is a damaged iodide propidium. It is a method of measuring the stained cells using the principle of penetrating the cell membrane and binding to the intracellular DNA to give a bright red color.

The substance to be evaluated in the present invention may be any substance to be applied as a constituent to products or formulations applied to human skin, such as cosmetics, in addition to sunscreen.

Hereinafter, the present invention will be described more specifically with reference to examples and test examples. These examples are provided only for understanding the contents of the present invention, but the scope of the present invention is not limited to these examples and test examples, and modifications, substitutions, and insertions commonly known in the art may be performed. It may be included in the scope of the present invention.

Example 1 to 5 Preparation of sunscreen

A sunscreen having the components shown in Table 1 below was prepared in a conventional manner (unit: wt%).

ingredient Example 1 Example 2 Example 3 Example 4 Example 5 TiO ₂ 1.1 2 8.8 3.4 1.4 ZnO 2.8 12 8 - - Octyl methoxy cinnamate (OMC) 7 7.5 - 3 5 Isoamyl p-methoxycinnamate
(Isoamyl p-Methoxycinnamate) 2 - - - 2 Ethylhexyl salicylate
(Ethylhexyl Salicylate) 4.5 - - - - Tinosorb S 3 3 - 6 3 Octocrylene - 2 - - - Polysilicone-15 - One - - -

[Test Example 1]

SPF and PA clinical index, and sunscreen transmittance of the sunscreen prepared in Examples 1 to 5 were investigated, respectively, and the results are shown in Table 2 and FIG. 1, respectively. At this time, the transmittance of the sunscreen was applied to the bandpass filter in an amount of 2 mg / ㎠, and then measured with a spectrophotometer SPF-290S sunscreen analyzer system (Sunscreen Analyzer System).

Similar or identical SPF and PA clinical index Test substance SPF PA Example 1 54 +++ Example 2 52 +++ Example 3 57 +++ Example 4 54 +++ Example 5 35 ++

In the results of Table 2 and FIG. 1, the sunscreen agents of Examples 1 to 5 all exhibited SPF and PA clinical indexes and transmittances of similar or identical categories.

Test Example 2 Measurement of Apoptosis

Human normal keratin skin cells used in the present invention was commercially secured and recommended, and purchased from Lonza, Inc., Walkersville, Maryland.

After subculture the cells were cultured in a cell under a CO ₂ constant temperature incubator (CO ₂ incubator) at 37 ℃, 5% CO ₂ condition. Cell cultures were cultured according to Lonza's instructions. In 500 ml of KBM-2 (Clonetics CC-3103) medium, KGM-2 Bullet Kit (Bullet kit: 2 ml of Bovine pituitary extract (BPE), 0.5 ml of human epidermal growth factor (hEGF), 0.5 ml of insulin (Insulin), hydro 0.5 ml of Cortison (Hydrocortisone), 0.5 ml of Transferrin, 0.5 ml of Epinephrine and 0.5 ml of Gentamycin Sulfate + Amphofericin-B (Gentamycin Suflate + Amphofericin-B: GA-1000) were used.

After applying the sunscreen of Examples 1-5 on the bandpass filter, human epidermal neonatal keratinocyte cells were placed on the bottom, and then the light source (UVB and UVA) was applied above the sunscreen. ) Was irradiated (50 MED, 1MED = 21 mJ / ㎠), and then the cells placed below were obtained to measure the rate of cell death and necrosis. Comparative Example 1 is a cell not treated with UV, and Comparative Example 2 is a cell irradiated with UV without treating the sunscreen.

The cells obtained above were washed with phosphate buffer (PBS) and stained for 30 minutes at 4 ° C. using an anti-Annexin V-FITC antibody (Sigma) fluorescently labeled with FITC, followed by 1 μg iodide propidium with phosphate buffer. After staining at 4 ° C. for 30 minutes with (PI; Sigma), cells were washed in the same manner as above. The fluorescence intensity of the washed cells was measured by flow cytometry to measure the detection amount (%) of Annexin V and PI staining costimulatory molecules expressed in the cells, thereby apoptosis and necrosis. ), And the results are shown in FIGS. 3A to 3G. In this case, the meanings of the terms indicated in FIGS. 3A to 3G are as follows:

Forward light scatter (FSC-H): A measure related to the size of a cell or particle.

Side light scatter (SSCH): A measure of the internal structure and granularity of a cell or particle.

-FL1-H (fluorescence 1): Among the fluorescence of various wavelengths that can be detected by flow cytometer, fluorescence corresponding to green, fluorescent dyes such as FITC and CFSE emit light of this wavelength .

-FL3-H (fluorescence 3): Among the fluorescence of various wavelengths that can be detected by flow cytometer, fluorescence corresponding to red, propidium iodide and texas red emit light of this wavelength. (H is an insignificant mark).

Example of result analysis: For cells in the apoptotic phase, the FSC value becomes smaller and the SSC value increases because the granule inside and the cell itself become smaller.

In the results of FIGS. 3A to 3G, it was confirmed that SPF and PA clinical indexes of similar or identical categories, and apoptosis rates of the sunscreen treatment groups of Examples 1 to 5, which showed transmittance, were all measured differently.

Claims (4)

Applying a sunscreen to the UV transmissive material;
Placing skin cells under the UV permeable material and then irradiating UV over the UV permeable material; And
Efficacy evaluation method of the sunscreen comprising the step of obtaining the skin cells to measure the cell death rate. The method of claim 1, wherein the UV-transmitting material is a quartz plate or a band pass filter. According to claim 1, wherein the skin cells are keratinocyte (Keratinocytes), fibroblasts (fibroblast) or melanocytes (Melanocytes) method for evaluating the efficacy of the sunscreen. The method of claim 1, wherein the cell death rate is due to cell death or necrosis.

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