KR102012166B1 - Method for screening the skin-whitening materials - Google Patents
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- KR102012166B1 KR102012166B1 KR1020120131433A KR20120131433A KR102012166B1 KR 102012166 B1 KR102012166 B1 KR 102012166B1 KR 1020120131433 A KR1020120131433 A KR 1020120131433A KR 20120131433 A KR20120131433 A KR 20120131433A KR 102012166 B1 KR102012166 B1 KR 102012166B1
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Abstract
The present invention relates to a method of screening for an agonist related to skin whitening, and more specifically, to determine whether the production of PGE 2 (prostaglandin E 2 ) associated with melanin production and migration is determined by measuring TRPM8 gene expression level, This relates to a method for screening an effective material for skin whitening.
Description
The present invention relates to a method of screening for an agonist related to skin whitening, and more specifically, to determine whether the production of PGE 2 (prostaglandin E 2 ) associated with melanin production and migration is determined by measuring TRPM8 gene expression level, This relates to a method for screening an effective material for skin whitening.
Human skin color is determined by red blood cells, carotene, and melanin in the blood, but differences in race color, hyperpigmentation such as blemishes and freckles are caused by melanin. The key enzyme in melanogenesis is tyrosinase, which leads to a reaction cascade that converts tyrosine into raw polymer melanin. The two tyrosinase-related proteins (TRP's) are known as TRP-1 and TRP-2, which share about 40% homology with tyrosinase and play a role in regulating melanogenesis as well as catalytic activity in melanogenesis. Have Melanin is produced in the organelle called melanosome (melanosome) in which the enzymes are distributed, and melanocytes containing melanin are later transferred to keratinocytes through the dendrite of melanocytes. This melanin delivered to keratinocytes is an important factor that actually determines the skin color.
As described above, the process of synthesizing melanin is very complicated, and in addition to the melanocytes from which melanin is synthesized, it is very much influenced by surrounding keratinocytes.
Melanin in the epidermal layer, the outer skin of the skin, protects the skin organs under the dermis by acting as a sunscreen, and catches free radicals generated in the skin's living body, thus protecting proteins and genes in the skin. . However, melanin, produced by internal and external stress stimuli, is a stable substance that does not disappear until it is released to the outside through skin keratinization even when stress disappears. In addition, when free radicals are generated in the skin, or when there is an inflammatory reaction or ultraviolet rays, in vivo, tyrosine or dopa is used as a substrate and enzymes such as tyrosinase are catalyzed. The melanin production is increased through the polymerization oxidation process. In particular, the production of melanin, which is partially increased due to ultraviolet rays, may develop into melanoma and the like, resulting in undesired aesthetic effects, or worse, skin cancer, which may be dangerous to life.
For these reasons, various screening methods have been used to develop substances that can control melanin synthesis. Representative examples thereof include tyrosinase activity and melanin synthesis regulator screening using melanocytes.
Recently, as the human genome research reaches the completion stage, an inhibitor that can control the synthesis of tyrosinase protein by controlling the promoter region of the tyrosinase gene, which plays the most important role in the skin whitening mechanism, Development of methods that utilize research results at the gene level, such as inhibitors that inactivate tyrosinase mRNA, is progressing.
Accordingly, the present inventors found that the TRPM8 gene, which is activated at a temperature of 22 ° C. or lower, among genes present in human skin is related to the generation of PGE 2 , an inflammatory pigmentation trigger, and completed the present invention.
Accordingly, an object of the present invention is to provide a method for screening a substance that ultimately helps skin whitening by activating the TRPM8 gene.
In order to achieve the above object, the present invention comprises the steps of 1) confirming the level of TRPM8 expressed in test skin cells; 2) treating the test skin cells with candidate substances; 3) confirming the level of TRPM8 in the cells of step 2); And 4) comparing the results of steps 1) and 3).
In the screening method according to the present invention, by measuring the level of TRPM8 in which the expression level varies depending on the skin temperature, an effective substance useful for skin whitening can be screened in vitro effectively, and thus, the substance found By not only lowering the concentration of melanin in the skin, but also lowering the skin temperature itself, it can help in the development of products that are effective in preventing heat aging due to skin sensation.
Figure 1 shows the temperature of activating TRPM8 and the expression amount of TRPM8 when treated with menthol.
Figure 2 shows the temperature of activating TRPM8 and TRPM8 activity when treated with menthol as the inflow of Ca.
Figure 3 shows the expression of PGE 2 after UV treatment to cells treated with 22 ℃ and menthol.
Figure 4 shows the expression level of TRPM8 when treated with UVB.
Figure 5 shows the expression level of TRPM8 after treatment of Geumchocho extract.
Figure 6 shows the TRPM8 activity after treatment of Geumchocho extract.
Figure 7 shows the expression of PGE 2 after UV treatment to cells treated with Geumchocho extract.
Figure 8 shows the melanin production after treatment of Geumchocho extract.
The present invention relates to a method for screening a substance that helps to whiten skin by measuring the expression level of TRPM8 gene present in the skin.
Existing skin whitening agents aim to protect the skin from the irritation caused by ultraviolet rays, and to reduce the amount of melanin produced in melanocytes by the stimulation of ultraviolet rays. Recently, however, there have been many studies showing whitening efficacy by inhibiting the migration of melanin produced from melanocytes to epidermal cells. At this time, the substance in the body that acts in connection with the transfer of melanin is PGE 2 (prostaglandin E 2 ).
When the skin is irritated by UV light, the production of PGE 2 in keratinocytes increases, which activates the G-protein-coupled receptors EP 1 and EP 3 , which stimulate melanocyte dendrite formation. Done. When the dendrite of melanocytes is expanded, the melanosome is transported to the end of the dendrite, and the melanin is excessively moved to the epidermis, and the skin color is darkened by the melanin accumulated in the epidermis.
On the other hand, the temperature of healthy skin is around 31 ℃, but in summer, the skin temperature rises to 43 ℃, especially in summer, as the number of skin melanin increases, the skin brightness and redness decrease, but the skin tone becomes dark and dull due to the rise of yellow phase. The tone changes to yellow.
Based on this fact, the present inventors tried to select genes that are activated at low temperature, especially below 22 ° C, among genes present in the skin, and selected TRPM8 (transient receptor potential melastatin 8) as an available gene. When activated, it was found that the level of PGE 2 increased in expression due to UV stimulation.
Thus, when the TRPM8 gene is activated, it is possible to reduce the production of PGE 2 , which is an inflammatory factor and promotes the transfer of melanin from melanocytes, thereby darkening the skin color by inhibiting the transfer of melanin produced from the dermis to the epidermis. You can prevent it. Therefore, by screening for a substance that activates the TRPM8 gene, it is easy to find a substance effective for skin whitening.
Specifically, the present invention provides a method for screening a whitening efficacy substance, and consists of the following steps.
1) identifying the level of TRPM8 expressed in test skin cells;
2) treating the test skin cells with candidate substances;
3) confirming the level of TRPM8 in the cells of step 2); And
4) comparing the results of steps 1) and 3).
Confirmation of the level of TRPM8 expressed in the cells can be made through conventional experimental methods known in the art such as PCR, Western blot, etc., and the level of TRPM8 measured in step 3) is different from that measured in step 1). The larger it can be expected to have a greater effect on skin whitening.
In addition, as described above, by measuring the expression level of TRPM8 after treating the candidate substance, it is possible to determine the effect on the concentration of melanin in the skin, in particular melanin produced by UV stimulation in accordance with the increase of TRPM8 expression It can be determined whether the amount of and the amount of melanin produced is reduced to the epidermis. In addition, it can be easily determined whether the skin temperature itself can be reduced to reduce yellowishness, redness, and the like of the skin.
In this way, it is possible to find a potent substance related to skin whitening and to develop a differentiated product that can prevent skin aging by simply lowering skin color as well as skin color.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and various modifications or changes may be made within the spirit and scope of the present invention.
Test Example 1 Expression of TRPM8 and its Activity by Cooling
In order to examine the expression and activity of TRPM8 by cooling, the following test was carried out. At this time, the cooling stimulus was used as a direct stimulus of 22 ℃ and menthol, an agonist of TRPM8.
First, in order to measure the amount of TRPM8 expression, human keratinocytes were grown using a medium containing HKGS added to Epilife® medium, and then 500,000 cells were seeded in a 35 mm dish. The day after seeding, the cells were incubated for 30 minutes in an incubator set at 22 ° C. for 24 hours at low temperature, and then incubated for 24 hours at 37 ° C. at normal temperature, and menthol stimulation was performed for 24 hours with
On the other hand, since TRPM8 activity is related to Ca channel activity, Ca inflow was measured through experiments to determine TRPM8 activity. Human keratinocytes were grown using HKGS added to Epilife® medium and seeded in 96 wells. The following day, it was measured using a Fluo-4 NW calcium assay kit (Molecular probe, F36205). The loading dye solution included in the kit was put into the cells and incubated for 30 minutes at 37 ℃, and further incubated for 30 minutes at room temperature. The stained cells and the material to be measured were prepared and placed in FlexStation3 (Molecular Device, USA), respectively, and the relative fluorescence value (relative fluorescence unit, RFU) was measured at 494 nm and emission 516 nm, and the results are shown in FIG. 2.
1 and 2, when the treatment using a material or temperature that activates TRPM8, it can be seen that while the expression level of TRPM8 increases, TRPM8 is activated at the same time.
Test Example 2 Effect of TRPM8 Activity on PGE 2 by UV Light
In order to confirm the effect of ultraviolet rays on the expression of TRPM8 and PGE 2 , in the procedure of Test Example 1, the cells were incubated for 30 minutes in an incubator previously set to 22 ° C or pretreated with
In addition, in this case, in order to confirm the expression change of TRPM8 by UVB, the same experimental set as the above-mentioned test set confirming the effect on the expression of PGE 2 was constructed, and after culturing at 37 ° C. for 24 hours after UV irradiation, cells were collected and subjected to qPCR. The measurement results are shown in FIG. 4.
3 and 4, it can be seen that when treated with a substance or temperature that activates TRPM8, PGE 2 increased by ultraviolet rays decreases to a level before irradiation with ultraviolet rays, whereby TRPM8 expression by UVB. You can see that there is no change in quantity.
[Test Example 3] Application to candidate material (using mosquitoes)
To measure the amount of TRPM8 expression, human keratinocytes were grown in Epilife® medium with HKGS added, and then 500,000 cells were seeded in 35 mm dishes. The day after seeding, the cells were pretreated with 100 ppm of Geumchocho extract for 24 hours, then irradiated with UVB 20mJ and incubated at 37 ° C. for 24 hours, and then the cells were harvested. Trizol (Trisol) was added to the cells by 500 μl, and the cells were homogenized at room temperature for 5 minutes. 100 μl of chloroform was added to each tube, shaken by hand for 15 seconds, left at room temperature for 3 minutes, and centrifuged at 4 ° C. and 15,000 × g for 15 minutes. After only the supernatant was transferred to a new tube, 250 μl of isopropyl alcohol was added thereto, mixed well, and left at room temperature for 10 minutes, and centrifuged at 4 ° C. and 15,000 × g for 10 minutes. The supernatant was discarded, leaving only the pellet and then vortexed after adding 500 μl of 75% alcohol. Thereafter, centrifugation was carried out at 4 ° C. and 7,500 × g for 5 minutes, and the supernatant was carefully discarded, leaving only the pellets. After drying, the mixture was dried well, dissolved in RNA in deionized water, and measured for absorbance at 260 nm. 2 μl of oligo dT was added to 4 μg of total RNA, reacted at 70 ° C. for 10 minutes, and then cooled quickly. DTT, dNTP, 10x RT buffer, MgCl 2 , RNAase Out was added, and after 2 minutes at 42 ° C, Superscript III Reverse Transcriptase (Invitrogen) was added and reacted at 50 ° C for 60 minutes. QPCR was performed using the synthesized cDNA, and Taqman's commercial primers (Hs00375481_m1; TaqMan Gene Expression Assays) were used. The qPCR results were obtained by calibrating each value using GAPDH, and the results are shown in FIG. 5.
In addition, Ca inflow was measured by the same method as described in Test Example 1 to determine the TRPM8 activity, the results are shown in FIG.
On the other hand, in order to measure the level of PGE 2 , pre-treatment with Geumchocho extract, irradiated with UVB 20mJ and incubated for 48 hours at 37 ℃ normal temperature, the medium was collected and used EL EL kit (# RPN222) of GE, as a result Is shown in FIG.
In addition, the melanin production inhibitory effect of Geumchocho itself was confirmed by the following method.
Melan a cells were seeded to 15,000 in 48 wells using RPMI medium. The next day, after treatment with 100ppm Geumchocho extract, or 50ppm of arbutin was incubated for 3 days, and then treated with Geumchocho extract or Arbutin once again, an additional 3 days were incubated. After discarding the medium and washing the cells with PBS, 100 μl of 1N NaOH was added to dissolve the melanin in the cells. Melanin dissolved in the cell was measured by absorbance at 475 nm to convert the melanin amount, and the total protein amount was also measured to calculate the amount of melanin per protein. The results are shown in FIG.
5 and 6, it was confirmed that the expression amount of TRPM8 is increased and activated by the Geumchocho extract.
In addition, it can be seen that the Geumchocho extract that activates TRPM8 inhibits PGE 2 increased by UVB.
In addition, it can be seen that Geumchocho itself has the effect of inhibiting melanin production, and melanin production inhibition effect is related to the degree of activation of TRPM8 gene.
Claims (4)
2) treating the test skin cells with candidate substances;
3) confirming the expression level of TRPM8 in the cells of step 2); And
4) comparing the expression levels of steps 1) and 3) to determine whether they are anti-aging and whitening efficacy substances;
Including;
In step 4), if the TRPM8 expression level of step 3) is higher than the TRPM8 expression level of step 1), the candidate substance is determined as an anti-aging and whitening efficacy substance,
The anti-aging and whitening effect substance is characterized in that it inhibits skin aging due to heat aging and photo-aging, and improves skin whitening, anti-aging and whitening effect substance.
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| [얼굴하얘지는법] 얼굴 화이트닝 비법 대공개! ! 하얀피부만들기 뷰티 tip★ (네이버 블로그, https://blog.naver.com/glin2000/50146232776) 1부.* |
| Huai-hu Chuang et al., 'The Super-Cooling Agent Icilin Reveals a Mechanism of Coincidence Detection by a Temperature-Sensitive TRP Channel', Neuron, 2004, Vol. 43, pp 859-869. 1부.* |
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