JP6320833B2 - Screening method for whitening ingredients - Google Patents

Description

  The present invention relates to a screening method for whitening components, characterized by using fibroblasts.

  In general, skin pigmentation in spots, freckles, sunburn, etc. is caused by excessive melanin pigment produced by melanin pigment cells (melanocytes) present in the skin due to hormonal abnormalities or stimulation of ultraviolet rays. It is thought to be caused by deposition. As one method for preventing such pigmentation, a method for suppressing excessive production of melanin is known. Conventionally, arbutin, hydroquinone, kojic acid, tranexamic acid, and derivatives thereof have been used for the purpose of suppressing excessive production of melanin.

  neurogulin 1 (NRG1) is a neurotrophic factor that regulates proliferation and differentiation of nerve cells and glial cells in the central nervous system, but its expression is recently seen in dermal fibroblasts, It has been found that it acts on melanocytes to cause melanin production (Non-patent Documents 1 and 2).

  As a screening method for whitening components, a screening method based on a melanin production inhibition test using B16 mouse melanoma (Patent Document 1), a screening method using melanosome structural protein gp100 in melanocytes as an index (Patent Document 2), adrenomedullin A screening method (Patent Document 3) and the like using an inactivation effect on a signal transduction system via a binding receptor as an index is already known. However, all of these indicators were keratinocytes and melanocytes present in the epidermis.

  Recent studies have revealed that the darker the skin color, the higher the expression of NRG1 in the dermis and that factors other than the epidermis are involved in melanin production (Non-patent Documents 1 and 2). However, until now, no screening method for a whitening component has been developed that uses a factor produced from fibroblasts present in the dermis as an index.

JP2008-280249 JP2008-232693 JP2012-255710A

J. et al. Cell Sci. , 123 (Pt18), 3102-3111 (2010) Pigment Cell. Melanoma Res. , 25 (4), 477-481 (2012)

  Then, this invention makes it a subject to establish the novel technique which leads to discovery of the outstanding whitening component. In particular, it is considered difficult to find an excellent whitening component only by using a whitening action based on a known mechanism of action using keratinocytes and melanocytes. That is, an object of the present invention is to establish a method capable of evaluating a whitening action based on a novel action mechanism.

  As a result of intensive studies aimed at solving the above problems, the present inventors have made a novel whitening using the neurotrophic factor NRG1 as an index and focusing on fibroblasts present in the dermis rather than keratinocytes and melanocytes present in the epidermis. It came to complete the screening method of an ingredient. NRG1 is considered to be a dermal-derived melanin production-promoting factor, and control of this dermis-derived melanin production-promoting factor is also important for suppressing excessive production of melanin in melanocytes. Furthermore, it evaluated by this method and it confirmed that the composition containing the selected component exhibited the prevention or improvement effect of pigmentation.

  That is, the present invention comprises the following (1) to (5).

(1) A screening method for whitening components, comprising using fibroblasts.
(2) The method according to (1), wherein the production amount of neurotrophic factor in fibroblasts is used as an index.
(3) The method according to (2), wherein the neurotrophic factor is neuroglin1 (NRG1).
(4) The method according to (2) or (3), wherein the production inhibitory effect of neurotrophic factor is used as an index.
(5) The method according to any one of (1) to (4), wherein fibroblasts previously loaded with oxidative stress are used.

  According to the present invention, by using the neurotrophic factor NRG1 in fibroblasts as an index, it is possible to evaluate or screen for whitening components used in cosmetics, skin external preparations and the like.

It is a figure of the immuno-staining of NRG1 protein in a human skin fibroblast. One spindle shape is one cell.

  The whitening component screening method in the present invention is a method for selecting a component having a whitening effect. The whitening component screened by the method of the present invention can be blended and prepared in cosmetics or skin external preparations.

  The neurotrophic factor in the present invention refers to a factor that delivers nutrients to nerve cells and is a factor for maintaining the function and growth of nerves. Typical examples include neurogulin, neurotrophin, brain-derived neurotrophic factor, nave growth factor, and linear neurotrophic factor. Preferably, it is neurogulin.

  In the present invention, neuroglin (NRG) is an axon growth factor for nerve cells, and plays an important role in determining proliferation, metastasis and fate of nerves and glia. So far, NRG 1, 2, 3, and 4 are known as subtypes of NRG. From recent studies, NRG1 is expressed not only in the nervous system but also in the fibroblasts of the skin. The darker the skin color, the higher the expression, and the effect of NRG1 protein on melanocytes can affect melanin production. It has been reported.

  The production inhibitory effect of the neurotrophic factor in the present invention is an effect of inhibiting the production by some component using the neurotrophic factor NRG1 produced in fibroblasts existing in the dermis as an index. For this purpose, NRG1 needs to be measured. The method used for this measurement is not particularly limited, and any method can be used. Representative examples include a method for measuring NRG1 mRNA expression level by real-time RT-PCR, a method for measuring NRG1 protein level by Western blotting, a method for measuring NRG1 protein level by ELISA, and the like. . Preferably, the NRG1 mRNA expression level is measured by a real-time RT-PCR method.

The oxidative stress in the present invention is an action harmful to a living body caused by an oxidative reaction. In a test using cells, a technique of applying oxidative stress by exposure to ultraviolet rays, active oxygen, lipid peroxide, or the like is used. A method using hydrogen peroxide (H ₂ O ₂ ) is preferable.

  Next, in order to describe the present invention in detail, specific examples will be given and described. These examples specifically illustrate the effect and do not limit the scope of the invention. The compounding quantity in an Example is weight%.

  Tsukishita Bijin Extract was used as a screening target drug. The beauty of the moon is a genus of cactiaceae peafowl cactus (Epiphyllum oxypetalum Haw.) And related species thereof, and the whole cultivar and dried flowers can be used.

  The following extraction can be performed as an example of the production of a moonlight beauty.

Production Example 1 Tsukishita Bijin Extract 3 g of purified water was added to 100 g of the dried ginger flower, and extracted at 95-100 ° C. for 2 hours. After filtration, the filtrate was concentrated under reduced pressure and further freeze-dried to obtain 48 g of a moon moon beauty extract.

  As a prescription example, the beauty of the moon moon extract used as a screening target drug can be formulated as follows.

Formulation Example 1 Cream Formulation Amount (%)
1. Moonlight Beauty Extract (Production Example 1) 0.1
2. Squalane 5.5
3. Olive oil 3.0
4). Stearic acid 2.0
5. Beeswax 2.0
6). Octyldodecyl myristate 3.5
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Behenyl alcohol 1.5
9. Glycerol monostearate2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.25
12.1,3-Butylene glycol 8.5
13. [Manufacturing method] Components 2 to 9 are heated and dissolved and mixed with purified water to a total amount of 100, and kept at 70 ° C to obtain an oil phase. Ingredients 1 and 11 to 13 are heated and dissolved and mixed, and kept at 75 ° C. to obtain an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. The component 10 is added at 45 ° C., and further cooled to 30 ° C. to obtain a product.

Comparative Example 1 Conventional cream 1
In Formulation Example 1, a conventional cream 1 was obtained by replacing the moon beauty extract (Production Example 1) with a birch extract (Ichimaru Falcos).

Comparative Example 2 Conventional Cream 2
In Formulation Example 1, a conventional cream 2 was prepared by replacing the beautiful moon extract (Production Example 1) with purified water.

  Next, in order to describe the present invention in detail, a specific experimental example will be described. The present invention is not limited to these experimental examples.

Experimental Example 1 Detection of NRG1 protein in fibroblasts Human skin fibroblasts (NB1RGB) were fixed with 10% formalin and then immunostained for NRG1 protein. The primary antibody used was NRG1 (R & D SYSTEMS), and the secondary antibody was Alexa Fluor 594 Donkey Anti-Goat IgG (H + L) (Molecular Probes). After staining, it was observed with a fluorescence microscope.

  The experimental results are shown in FIG. As a result, NRG1 protein was detected in fibroblasts. Therefore, it was shown that NRG1 is certainly expressed in fibroblasts.

Experimental Example 2 Effect of NRG1 Protein on Promoting Melanin Production In melanin production, tyrosinase (TYR), tyrosinase-related protein 1 (TRP1) and tyrosinase-related protein 2 (TRP2) are involved as major enzymes, and the expression control of these enzymes is involved. Microphthalmia-related transcription factor (MITF) plays an important role. NRG1 (PROSPEC) prepared to a final concentration of 100 and 200 ng / mL is added to human normal melanocytes (NHEM) in a confluent state, cultured for 24 hours, and then total RNA is extracted using RNAiso plus (TAKARA). Extraction was performed. Based on the total RNA, TYR, TRP1, TRP2, and MITF mRNA expression levels were measured by real-time RT-PCR. SYBR Select Master Mix (Life Technologies) was used for the real-time RT-PCR method, and the following primers were used for TYR, TRP1, TRP2, and MITF. Β-actin was used as an internal standard. The operation of real-time RT-PCR was determined according to a predetermined method, and the expression levels of TYR, TRP1, TRP2, and MITF mRNA were determined as a ratio with respect to the expression level of β-actin mRNA as an internal standard. The expression rate of TYR, TRP1, TRP2, and MITF was calculated as the ratio of the mRNA expression level of the NRG1 added group to the unadded mRNA expression level.

Primer set TGCGGTGGGAACAAGAAATC for TYR (SEQ ID NO: 1)
GAAGAATGATGCTGGGCTGAGT (SEQ ID NO: 2)
Primer set CGAAACACAGTGGAAGGTTACAGT for TRP1 (SEQ ID NO: 3)
CTCCTCAGCCCTTCATCAAAAGACT (SEQ ID NO: 4)
Primer set GGAATGCTTTGGAAGGGTTTG for TRP2 (SEQ ID NO: 5)
AAAGCGTTTGTCCCGTTCAG (SEQ ID NO: 6)
Primer set GAGGCAGTGGTTTGGGCTT for MITF (SEQ ID NO: 7)
AATTCTGCACCCGGGAATC (SEQ ID NO: 8)
Primer set CACTCTCCCAGCCTCTCTCCC for β-actin (SEQ ID NO: 9)
GTGTTGCGCGTACAGGTCTTTG (SEQ ID NO: 10)

  The experimental results are shown in Tables 1-4. As a result, NRG1 protein promoted the expression of TYR, TRP1, TRP2, and MITF in melanocytes. Therefore, it was shown that NRG1 acts on melanocytes and has a melanin production promoting effect.

Experimental Example 3 Effect of Promoting NRG1 Production by Oxidative Stress Hydrogen peroxide (H ₂ O ₂ ) prepared to final concentrations of 250 and 500 μM was added to human skin fibroblasts (NB1RGB) in a confluent state by adding 24 After time culture, total RNA was extracted using RNAiso plus (TAKARA). Based on the total RNA, the NRG1 mRNA expression level was measured by the real-time RT-PCR method. For the real-time RT-PCR method, SYBR Select Master Mix (Life Technologies) was used, and the following primers were used for NRG1. Β-actin was used as an internal standard. The operation of real-time RT-PCR was determined according to a predetermined method, and the expression level of NRG1 mRNA was determined as a ratio with respect to the expression level of β-actin mRNA as an internal standard. The expression rate of NRG1 was calculated as the ratio of the expression level of mRNA in the H ₂ O ₂ added group to the expression level of unadded mRNA.

Primer set GCCCATCACTCCACTACTGTCA for NRG1 (SEQ ID NO: 11)
GCTTTCAGTGTGTCCGTTGCT (SEQ ID NO: 12)

The experimental results are shown in Table 5. As a result, the oxidative stress due to the addition of H ₂ O ₂ promoted the expression of NRG1. Therefore, it was shown that when oxidative stress is applied to fibroblasts, the production of NRG1 is enhanced.

Experimental Example 4 Screening method for whitening ingredients using NRG1 production as an index Extraction of beauty of the moon under beautiful human skin fibroblasts (NB1RGB) prepared to final concentrations of 50, 100 and 200 μg / mL Product (Production Example 1) and birch extract (Comparative Example 3, Ichimaru Falcos), which is known to have a melanin production inhibitory effect, were cultured for 24 hours, and then total RNA was extracted using RNAiso plus (TAKARA). Extraction was performed. Based on the total RNA, the NRG1 mRNA expression level was measured by the real-time RT-PCR method. For the real-time RT-PCR method, SYBR Select Master Mix (Life Technologies) was used, and the primer for NRG1 was the same as in Experimental Example 3. Β-actin was used as an internal standard. The operation of real-time RT-PCR was determined according to a predetermined method, and the expression level of NRG1 mRNA was determined as a ratio with respect to the expression level of β-actin mRNA as an internal standard. The expression rate of NRG1 was calculated as the ratio of the expression level of mRNA in the sample addition group to the expression level of unadded mRNA.

  The experimental results are shown in Table 6. As a result, the birch extract had no effect on the expression of NRG1, but the Tsukishita beauties extract significantly suppressed the expression of NRG1. Therefore, Tsukishita Beauty extract showed an excellent inhibitory effect on NRG1 production.

Experimental Example 5 Screening Method for Whitening Component Characterized by Loading Oxidative Stress Hydrogen peroxide (H ₂ O ₂₎ prepared to a final concentration of 500 μM in confluent human skin fibroblasts (NB1RGB) At the same time, Tsukishita Bijin extract (Production Example 1) prepared so as to have final concentrations of 100 and 200 μg / mL was added. After incubation for 24 hours, total RNA was extracted using RNAiso plus (TAKARA). Based on the total RNA, the NRG1 mRNA expression level was measured by the real-time RT-PCR method. For the real-time RT-PCR method, SYBR Select Master Mix (Life Technologies) was used, and the primer for NRG1 was the same as in Experimental Example 3. Β-actin was used as an internal standard. The operation of real-time RT-PCR was determined according to a predetermined method, and the expression level of NRG1 mRNA was determined as a ratio with respect to the expression level of β-actin mRNA as an internal standard. The expression rate of NRG1 was calculated as the ratio of the expression level of mRNA in the sample addition group to the expression level of unadded mRNA.

  The experimental results are shown in Table 7. As a result, the beautiful moon extract suppressed the increase in NRG1 expression due to oxidative stress. Therefore, Tsukishita Beauty Extract showed an excellent inhibitory effect against NRG1 that increases when oxidative stress is applied.

Experimental Example 6 Inhibition test of melanin production using B16 mouse melanoma 3 × 10 ⁴ B16 cells, which are a mouse melanoma-derived cell line, were seeded in a 6 cm dish, and 5% CO ₂ , 37 in MEM with NEAA containing 10% FBS. The cells were cultured for 5 days under the condition of 0C. At that time, the beautiful moon extract (Production Example 1) prepared to have final concentrations of 50, 100 and 200 μg / mL and the birch extract known to have a melanin production inhibitory effect (Comparative Example 3, Ichimaru) Falcos) was added. Next, after the cells were washed twice with PBS (−), 1 mL of PBS (−) was added and collected with a rubber policeman. PBS (-) 0.5mL was added to the pellet obtained by centrifugation, and the pellet was dissolved by ultrasonic crushing operation. The amount of protein was determined by the Lowry method {J. Biol. Chem. , 193, 265-275 (1951)}. When measuring the amount of melanin per mg of protein, 0.5 mL of 4N sodium hydroxide was added to the remaining cell disruption solution taken for protein determination and reacted at 60 ° C. for 2 hours. D. 475 was measured, and melanin was determined from the calibration curve. The data calculated the amount of melanin per 1 mg protein, the amount of melanin not added to the sample was taken as a control, and the inhibition rate of melanin generation was calculated from the value of the amount of melanin generated when the sample was added to the control.

  These test results are shown in Table 8. As a result, Tsukishita beautiful woman extract did not affect melanin production in melanoma, but birch extract suppressed melanin production. Therefore, Tsukishita Beauty extract did not show an inhibitory effect on melanin production in melanoma.

Experimental Example 7 Use Test Using the cream of Formulation Example 1, the conventional cream 1 of Comparative Example 1 and the conventional cream 2 of Comparative Example 2, each for 30 women (20-45 years) for 3 months A test was conducted. After use, a questionnaire survey on the improvement of spots and freckles was conducted to determine the whitening effect. The evaluation criteria of the questionnaire were evaluated as “excellent” for valid, “good” for slightly effective, “good” for slightly effective, and “impossible” for invalid.

  These results are shown in Table 9. The cream of the present invention containing the extract of beautiful moon moon of Formulation Example 1 showed an excellent whitening effect. Moreover, although the conventional cream 1 containing the birch extract of the comparative example 1 showed the whitening effect, the effect was lower than the cream of the present invention containing the moon beautiful woman extract of the prescription example 1. During the test period, there was no skin problem and there was no problem with safety.

  From FIG. 1 and Tables 1 to 4, it was confirmed that NRG1 was produced from fibroblasts and involved in melanin production. From Table 5, it was found that the expression of NRG1 was enhanced by exposure to the oxidizing agent. Further, from Table 9, it was confirmed that the moonlight beauty extract is a drug having a whitening effect. However, from Table 8, it was found that the mechanism is not solely due to melanocytes. On the other hand, from Tables 6 and 7 above, it was confirmed that the whitening effect of the moon moon bean extract was correlated with the expression level of NRG1 in dermal fibroblasts.

  From the above, it became possible to screen for whitening components by using the expression level of neurotrophic factor NRG1 in fibroblasts as an index. Similarly, the whitening component can be screened by using the production inhibitory effect of the neurotrophic factor NRG1 as an index.

  The screening method for whitening ingredients characterized by using the fibroblasts of the present invention is used for cosmetics and skin external preparations that could not be evaluated by conventional screening methods for whitening ingredients using keratinocytes and melanocytes. It is used for the development of whitening ingredients.

Claims (1)

Whitening, characterized in that hydrogen peroxide and a sample are added to fibroblasts, and the inhibitory effect of the sample on the increase in production of the neurotrophic factor neuroglin1 (NRG1) by hydrogen peroxide is evaluated using the NRG1 production inhibitory effect as an index. Ingredient screening method .