Method for evaluating the effect of light irradiation on melanin production by melanocytes

Abstract

The present invention aims to provide a method for evaluating the effect of light irradiation on melanin production by melanocytes. The method of the present invention includes: irradiating melanocytes with light, culturing the irradiated melanocytes in a culture medium, and quantifying the amount of melanin produced by the melanocytes.

Description

Technical Field

[0001] This disclosure relates to: methods for evaluating the effect of light irradiation on melanin production by melanocytes, methods for determining light irradiation conditions for inhibiting melanin production by melanocytes, methods for determining light irradiation conditions for promoting melanin production by melanocytes, skin treatment methods for inhibiting melanin production in the skin, skin treatment methods for promoting melanin production in the skin, and beauty devices. Background Technology

[0002] Melanin, produced by melanocytes present in the epidermis, is the cause of skin pigmentation. A cosmetic device has been developed to reduce melanin production by melanocytes through light irradiation (Patent Document 1).

[0003] Existing technical documents Patent Document 1: International Publication No. 2024 / 101301 Summary of the Invention

[0004] The problem the invention aims to solve However, a suitable method for evaluating the effect of light irradiation on melanin production in melanocytes is not yet known.

[0005] The subject of this disclosure is to provide a method for evaluating the effect of light irradiation on melanin production by melanocytes.

[0006] Technical means to solve the problem This disclosure includes the following implementation methods.

[0007] [1] A method for evaluating the effect of light irradiation on melanin production by melanocytes, comprising: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. The amount of melanin produced by melanocytes was quantified.

[0008] [2] A method for determining light irradiation conditions for inhibiting melanin production by melanocytes, comprising: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. Quantifying the amount of melanin produced by melanocytes When light exposure reduces the amount of melanin produced by melanocytes, the light exposure condition is selected as the light exposure condition for inhibiting melanin production by melanocytes.

[0009] [3] A method for determining light irradiation conditions for promoting melanin production by melanocytes, comprising: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. Quantifying the amount of melanin produced by melanocytes When light exposure leads to an increase in the amount of melanin produced by melanocytes, the light exposure condition is selected as the light exposure condition for promoting melanin production by melanocytes.

[0010] The effects of the invention According to this disclosure, a method for evaluating the effect of light irradiation on melanin production by melanocytes can be provided. Attached Figure Description

[0011] Figure 1 This indicates that the cell seeding density is 0.8 × 10⁻⁶. 4 cells / cm 2 (7×10) 4 Cells / well, 6-well plate), irradiated with light 3 times (505nm, illuminance: 12mW / cm²). 2 The appropriate FBS concentration (0%–10% (v / v)) in DMEM used to induce melanin production under the condition of 10 minutes per irradiation session, and the appropriate FBS concentration (0%–10% (v / v)) in DMEM used to induce melanin production under the condition of 3 irradiations (505 nm, illuminance: 12 mW / cm²). 2 1. Irradiation time per session: 10 minutes; 2. Suitable melanocyte seeding density in DMEM for inducing melanin production at an appropriate FBS concentration of 2% (v / v) (n=2 or 3).

[0012] Figure 2 This indicates that the cell seeding density is 0.8 × 10⁻⁶. 4 cells / cm 2 A graph showing the percentage of melanin production in melanocytes under various light irradiation conditions with an FBS of 2% (v / v) and a control group of 100%. A represents the evaluation system schedule, and B represents the production rate at an illuminance of 12 mW / cm². 2 The percentage of melanin production (n=4) under light irradiation (505nm) at 7.5 minutes or 10 minutes intervals, with the control group as 100%, is calculated. C represents the melanin production at an illuminance of 12mW / cm². 2 The percentage of melanin production (n=4) was calculated when the control group was 100% under light irradiation (505nm) for 3 minutes 3 times, 5 minutes 3 times, 7.5 minutes 3 times, or 10 minutes 3 times. All figures are expressed as mean + / - standard deviation.

[0013] Figure 3 This indicates that the cell seeding density is 0.8 × 10⁻⁶. 4 cells / cm 2 (7×10) 4 Graphs (n=4) showing intracellular melanin levels, melanin production in melanocytes, and melanin / protein ratio (%) under various light irradiation conditions (505 nm) with a 2% (v / v) FBS concentration in DMEM used to induce melanin production, with the control group as 100%. All graphs are presented as mean + / - standard deviation.

[0014] Figure 4 This indicates that the cell seeding density is 1.5 × 10⁻⁶. 4 cells / cm 2 (14×10) 4 Graphs (n=3) showing intracellular melanin levels, melanin production in melanocytes, and melanin / protein ratio (%) under various light irradiation conditions (505 nm) with a 4% (v / v) FBS concentration in DMEM used to induce melanin production (cells / wells, 6-well plates) and a control group as 100%. All graphs are presented as mean + / - standard deviation.

[0015] Figure 5 This is a schedule (A) showing the evaluation system under extended culture time before melanocyte recovery, and a graph showing the melanin production (%) of melanocytes with the control group at 100%. All graphs are presented as mean + / - standard deviation (n=2). Detailed Implementation

[0016] The following describes one embodiment of the present disclosure in detail, but the scope of the present disclosure is not limited to the one embodiment described herein, and various modifications can be made without departing from the scope of the present disclosure.

[0017] The various methods disclosed in this specification can be combined with any other features disclosed in this specification. The various configurations and combinations thereof in each embodiment are merely examples, and appropriate additions, omissions, substitutions, and other modifications can be made without departing from the spirit of this disclosure. For a specific parameter, where multiple upper and lower limits are provided, any combination of these upper and lower limits can be used to form a preferred numerical range. Furthermore, the lower and / or upper limits of the numerical range described in this disclosure are values ​​within that range, and can also be replaced with the values ​​shown in the embodiments. The expression "X~Y" representing a numerical range means "above X and below Y". In other embodiments, there may be instances where a specific description described for one embodiment is also applicable to other embodiments.

[0018] The various numerical indicators can be interpreted, at least in a manner that does not limit the application of the equivalence theory to the patent claims, taking into account at least the number of significant figures reported and applying the usual rounding. Any range of numerical values ​​may inherently include a range of errors arising from the standard deviations found in these respective experimental determinations.

[0019] Unless otherwise specified, the implementation methods and examples use the methods described in standard protocol collections such as J. Sambrook, EFFritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd., or modified and improved methods thereof. Furthermore, when using commercially available reagent kits and assay devices, unless otherwise specified, the accompanying protocols shall be used.

[0020] [First Implementation Method (Evaluation Method)] <Methods for evaluating the effects of light irradiation on melanin production by melanocytes> The evaluation method related to this embodiment is a method for evaluating the effect of light irradiation on melanin production by melanocytes, which includes: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. The amount of melanin produced by melanocytes was quantified.

[0021] In one embodiment, the effect of light irradiation on melanin production by melanocytes is that light irradiation leads to an increase or decrease in the amount of melanin produced by melanocytes.

[0022] (a) Irradiation of melanocytes with light (Melanocytes) Melanocytes are cells that produce melanin, also known as melanocytes or melanin-producing cells, and are mainly found in the basal layer of the epidermis of humans and other animals.

[0023] For melanocytes, the source animal is not limited within the range of animals containing melanocytes. Animals can be mammals, and other animals such as birds, reptiles, amphibians, and fish are also acceptable, with mammals being preferred. Mammals can be humans or non-human animals, but humans are preferred. Non-human animal species can include monkeys, dogs, cats, horses, cattle, pigs, sheep, goats, rabbits, groundhogs, hamsters, mice, and / or rats, and are not limited to domestic animals, pets, or laboratory animals.

[0024] In one implementation, the melanocytes are melanocytes derived from humans or mice.

[0025] Melanocytes are not limited by the stage of development or age of the animal from which they originate; for example, they can be fetuses or any age from newborns to adults.

[0026] Within the range of tissues containing melanocytes, melanocytes are not limited to the type of tissue from the animal of origin, such as any of the following: skin (including face, neck, trunk, lower limbs, vulva), eyes (including retina, choroid), or mucous membranes (including oral cavity, esophagus, intestines).

[0027] Melanocytes can be cells existing in the tissues of an animal individual, cells existing in the tissues isolated from an animal individual, or cells isolated from an animal individual. Alternatively, they can be any of the following: primary cultured cells, passaged cultured cells, or cultured cells of established lines isolated from an animal individual. In addition, they can be cells differentiated from stem cells or the like under conditions known to those skilled in the art.

[0028] When melanocytes are cultured cells, primary cultured, passaged, or established human or mouse melanocytes that can be purchased from reagent companies, cell banks, etc., can also be used.

[0029] Melanocytes can be normal cells or cancerous cells (e.g., malignant melanoma cells). Both normal and cancerous cells may contain gene mutations related to or unrelated to melanin production.

[0030] When melanocytes are melanoma cells, examples include mouse-derived melanoma cell lines (B16) (including B16 4A5) and human-derived melanoma cell lines (HMV-II, MNT-1, etc.). Established melanocyte and melanoma cell lines can be obtained from reagent companies and research institutions (such as RIKEN RBC).

[0031] When melanocytes are cells present in the tissues of an animal or in tissues separated from an animal, light can be applied to these cells. In this case, light can be applied from any side of the animal tissue, within the range where it can reach the melanocytes. However, for example, in the case of skin, light can also be applied to the skin from the outside of the skin in the animal, thus reaching the melanocytes located in the epidermis of the skin.

[0032] Melanocytes can be cells isolated from individual animals, or primary cultured cells, passaged cultured cells, or established cultured cells. They can be exposed to light under culture conditions known to those skilled in the art, or they can be cultured in liquid culture medium, with the liquid culture medium temporarily removed during light exposure to expose melanocytes adhering to the culture medium plate (culture plate, culture dish, test tube, etc.).

[0033] In cases of in vitro light irradiation, the culture medium used for culturing melanocytes before and / or during light irradiation, as well as its acidity, culture temperature, CO2 environment, etc., may be as described in “(b)” below.

[0034] In one embodiment, the culture of melanocytes before and / or during light irradiation is preferably performed using a culture medium containing a higher concentration of serum than that used in “(b)”. For example, the serum concentration is 2% to 15% (v / v), more preferably 5% to 12% (v / v), further preferably 8% to 10% (v / v), and even more preferably 10% (v / v). Examples and preferred embodiments of the serum type are described in “(b)” below.

[0035] In one embodiment, the culture of melanocytes before and / or during light irradiation can be carried out using liquid medium containing 8-10% (v / v) fetal bovine serum in DMEM (Dulbecco's Modified Eagle Medium) at 36-37°C and 3-10% CO2.

[0036] (light exposure) Regarding the wavelength of light irradiation, those skilled in the art can make an appropriate selection based on the wavelength of light irradiation they wish to evaluate the effect of light irradiation on melanocytes.

[0037] The light is preferably visible light. Visible light can be green or yellow wavelengths. Specifically, the green wavelength can be 490nm–570nm, or in one embodiment, 490nm–525nm, 495nm–525nm, or 505nm. Furthermore, the yellow wavelength can be 570nm–590nm.

[0038] The light can be light with a peak wavelength region of 490nm to 570nm. In one embodiment, the peak wavelength region can be 490nm to 525nm, or 495nm to 525nm, or 505nm. That is, the peak wavelength region of the light is 490nm to 570nm, and it can also be multiple types of light with different wavelengths within and / or outside this peak wavelength region.

[0039] Since the light is visible light, the effect of light irradiation on melanin production by melanocytes can be easily evaluated with higher sensitivity using the evaluation methods related to this embodiment.

[0040] In one implementation, multiple different wavelengths of light can be irradiated simultaneously and / or sequentially, and the effect of such light irradiation combinations on the light irradiation of melanocytes to produce melanin can be evaluated.

[0041] Light is not limited by its source; it can be natural light, light emitted by lighting fixtures, or light source provided on a light irradiation device. Examples of light irradiation devices include fluorescent lamps, LEDs (light-emitting diodes), lasers, and IPL (intense pulsed light).

[0042] In one implementation, the cumulative irradiation dose of light (mJ / cm²) 2 The range can be 1000-200000, 3000-200000, or above 3000 but below 180000, or 3000-150000, or 4000-100000, or 5000-90000, or 54000-90000. It can also be any range with one of these data as the lower limit and another as the upper limit, but it is not limited to these.

[0043] By cumulative radiation dose (mJ / cm) 2 The range is 3,000 to 200,000, which makes it easier to evaluate the effect of light irradiation on melanin production by melanocytes with higher sensitivity.

[0044] Furthermore, through cumulative radiation dose (mJ / cm) 2The light intensity is below 180,000, more preferably below 90,000, and even more preferably below 90,000, thus making it difficult to produce toxicity to melanocytes. Therefore, it is easy to evaluate the effect of light irradiation on melanin production by melanocytes within a range that is not affected by toxicity.

[0045] In one implementation, the cumulative irradiation dose (mJ / cm) is used. 2 The value is 1000 or more and less than 18000, more preferably 3000 or more and less than 180000, further preferably 3000 or more and less than 90000, even more preferably 3000 or more and less than 90000, even more preferably 4000 or more and less than 90000, even more preferably 5000 or more and less than 90000, even more preferably 54000 or more and less than 90000. Therefore, it is easy to evaluate the effect of light irradiation on melanin production by melanocytes with higher sensitivity, and it is easy to evaluate the effect of light irradiation on melanin production by melanocytes within the range that is not affected by toxicity.

[0046] Among them, the illuminance (mW / cm²) can be determined according to the following formula. 2 ), irradiation time (seconds), number of irradiations, calculation of cumulative irradiation dose (mJ / cm²) 2 ).

[0047] [Formula] Cumulative exposure (mJ / cm²) 2 = Illuminance × Irradiation time × Number of irradiations In cumulative radiation dose (mJ / cm) 2 When the irradiance is between 3,000 and 200,000, those skilled in the art can make appropriate adjustments to the irradiance, irradiation time, and number of irradiations to bring the cumulative irradiation to the above range.

[0048] Regarding the duration, frequency, and intensity of light irradiation, those skilled in the art can make appropriate selections based on the desired evaluation of the effect of light irradiation on melanocytes.

[0049] For example, the number of irradiations can be from 1 to 100 times, or it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times. It can also be any range with one of these data as the lower limit and another data as the upper limit, but it is not limited to these.

[0050] When performing multiple light irradiations, the interval between each irradiation can be, for example, 30 minutes to 3 days, or 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 50 hours, 70 hours or 72 hours (3 days), or any range where one of these data is the lower limit and the other is the upper limit, but it is not limited to these.

[0051] For example, the irradiation time for each irradiation can be from 30 seconds to 60 minutes (3600 seconds), or it can be 30 seconds, 1 minute, 3 minutes, 5 minutes, 7.5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes, or any range where one of these data is the lower limit and the other is the upper limit, but it is not limited to these.

[0052] For example, illuminance (mW / cm²) 2 The range can be 1 to 500, 1 to 400, 1 to 300, 1 to 200, or 3, 3.5, 3.7, 5, 6, 6.5, 10, 12, 15, 18, 20, 21, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200. It can also be any range with one of these values ​​as the lower limit and another as the upper limit, but it is not limited to these values.

[0053] In the case of multiple light irradiations, the irradiation time and / or illuminance of each irradiation may be different.

[0054] In one embodiment, at a wavelength of 505 nm, the light irradiation can be any combination of the following: illuminance, irradiation time, and number of irradiations.

[0055] Illuminance: 12mW / cm² 2 Irradiation time: 7.5 minutes; Number of irradiations: 1 time Illuminance: 12mW / cm² 2 Irradiation time: 10 minutes; Number of irradiations: 1 time Illuminance: 100mW / cm² 2 Irradiation time: 3 minutes; Number of irradiations: 1 time Illuminance: 12mW / cm² 2 Irradiation time: 5 minutes; Number of irradiations: 1 time Illuminance: 6.5 mW / cm² 2 Irradiation time: 10 minutes; Number of irradiations: 3 times Illuminance: 12mW / cm² 2、 Irradiation time: 5 minutes, Number of irradiations: 3 times Illuminance: 12mW / cm² 2 Irradiation time: 7.5 minutes; Number of irradiations: 3 times Illuminance: 12mW / cm² 2 Irradiation time: 10 minutes; Number of irradiations: 3 times Illuminance: 18mW / cm² 2 Irradiation time: 5 minutes; Number of irradiations: 3 times Illuminance: 18mW / cm² 2 Irradiation time: 10 minutes; Number of irradiations: 3 times Illuminance: 100mW / cm² 2 Irradiation time: 3 minutes; Number of irradiations: 3 times Illuminance: 100mW / cm² 2 Irradiation time: 5 minutes; Number of irradiations: 3 times Illuminance: 12mW / cm² 2 Irradiation time: 20 minutes; Number of irradiations: 3 times In one embodiment, if the melanocytes are cells present in the tissues of an animal individual or cells present in the tissues separated from the animal individual, the embodiment may further include a step of separating the melanocytes from the tissue after irradiating them with light.

[0056] For the method of isolating melanocytes from tissue, it can be carried out according to methods known to those skilled in the art.

[0057] For example, the tissue can be finely fragmented using a scalpel or similar tool, and then processed multiple times using proteolytic enzyme solutions to separate and disperse the epidermis. Cells can then be seeded, and during the initial passage, proteolytic enzyme solutions can be used to obtain a cell dispersion. Further, proteolytic enzyme solutions can be used to separate only the melanocytes.

[0058] (b) Culture of light-exposed melanocytes in a culture medium. (culture medium) As long as it is within the range where melanocytes can be cultured, there are no particular limitations on the culture medium, and an appropriate selection can be made from the culture media known to those skilled in the art for cell culture.

[0059] In one embodiment, even if the conditions for the culture medium described below are continuously maintained under the same conditions from the start to the end of the culture, the conditions can be adjusted as needed during a certain period of the culture.

[0060] Examples of non-limiting culture media include: DMEM (Dulbecco modified Eagle medium), RPMI 1640 medium, DMEM / F12 medium, MEM medium, Ham's F-10 medium, Ham's F-12 medium, 199 medium (M199), etc.

[0061] Regarding the acidity of the culture medium, it can be appropriately set by considering the source animal of the melanocytes, such as pH 6.0 to 8.0 or pH 6.8 to 7.5. In the case of mammals, pH 7.0 to 7.4 is preferred.

[0062] The culture medium is not limited by its form; for example, it can be a liquid culture medium or a solid culture medium.

[0063] In the case of a solid culture medium, it can be a culture medium that has been prepared into a solid form using agar, gelatin, etc.

[0064] Liquid culture medium is preferred when considering cell culture and post-culture treatment.

[0065] In one embodiment, the culture medium preferably contains serum.

[0066] When the culture medium contains serum, its concentration is preferably greater than 0.2% and less than 5% (v / v), more preferably 0.5% to 4.5% (v / v), and even more preferably 1% to 4% (v / v). When it is 1% to 4% (v / v), it can be 1%, 2%, 3% or 4%, or any range with one of these as the lower limit and another as the upper limit, but is not limited to these.

[0067] By containing serum in the culture medium at a concentration greater than 0.2% but less than 5% (v / v), the effect of light irradiation on melanin production by melanocytes can be evaluated with greater sensitivity.

[0068] For the serum, appropriate selections can be made from serums known to those skilled in the art that can be used in culture media for cell culture, without limitation on the animal source of the serum.

[0069] Non-limiting examples of animals that can be used as sources of serum include: cattle, horses, sheep, goats, pigs, llamas (Lamaglama), dogs, donkeys, cats, rabbits, marmots, hamsters, rats, mice, humans, and other mammals, as well as birds such as chickens.

[0070] The source animal for serum is not limited by its stage of development or age. For example, it can be any animal, such as a fetus, newborn, juvenile, or adult.

[0071] In one embodiment, the serum is fetal bovine serum (FBS, FCS).

[0072] In one embodiment, the culture medium contains FBS at a concentration of more than 0.2% and less than 5% (v / v), preferably at a concentration of 0.5 to 4.5% (v / v), more preferably at a concentration of 1 to 4% (v / v).

[0073] In one embodiment, the culture medium is a liquid culture medium containing FBS at a concentration of more than 0.2% and less than 5% (v / v), preferably at a concentration of 0.5 to 4.5% (v / v), more preferably at a concentration of 1 to 4% (v / v).

[0074] In one embodiment, the culture medium may contain one or more components capable of promoting and / or inducing melanocytes to produce melanin.

[0075] Examples of such components include: melanocyte-producing hormones, pituitary hormones, female hormones, and xanthine derivatives.

[0076] Examples of melanocyte-producing hormones (MSH, melanocyte-stimulating hormone) include α-MSH, β-MSH, or γ-MSH. Examples of pituitary hormones include ACTH (adrenocorticotropic hormone), LH (luteinizing hormone), or FSH (follicle-stimulating hormone). Examples of female hormones include estradiol, estriol, or progesterone. Examples of xanthine derivatives include theophylline or caffeine.

[0077] In one embodiment, the culture medium may also contain α-MSH and / or theophylline.

[0078] Those skilled in the art can appropriately set the concentration of the above-mentioned components in the culture medium. For example, when used as melanocyte-producing hormones, pituitary secretory hormones, or female hormones, the concentration can be 1 nM to 1000 nM (1 μM), 1 nM to 500 nM, 10 nM to 500 nM, or 50 nM to 200 nM, but preferably 80 nM to 120 nM, more preferably 90 nM to 110 nM, and even more preferably 100 nM.

[0079] For example, when used as xanthine derivatives, each can be 1 μM to 1000 μM (1 mM), 1 μM to 500 μM, 10 μM to 500 μM, or 50 μM to 200 μM, but preferably 80 μM to 120 μM, more preferably 90 μM to 110 μM, and even more preferably 100 μM.

[0080] Because the culture medium contains the above-mentioned components, it is easy to increase the amount of melanin produced by melanocytes, thus making it easier to evaluate the effect of light irradiation on melanin production by melanocytes with higher sensitivity.

[0081] In one embodiment, the culture medium preferably contains 80 nM to 120 nM, more preferably 90 nM to 110 nM, and even more preferably 100 nM of α-MSH, and / or in one embodiment, the culture medium preferably contains 80 μM to 120 μM, more preferably 90 μM to 110 μM, and even more preferably 100 μM (0.1 mM) of theophylline.

[0082] The culture medium may also contain additives that are typically added to the culture medium in cell cultures, not limited to melanocytes. Non-limiting examples of additives include: amino acids (containing L-glutamine), sodium bicarbonate, antibiotics, etc.

[0083] In one embodiment, the culture medium contains 1 to 10 mM of L-glutamine, preferably 1 to 5 mM, and more preferably 2 mM.

[0084] (nourish) Regarding the culture conditions for melanocytes, those skilled in the art can make appropriate settings, taking into account the types of melanocytes and the types of culture media, without being limited to the scope of being able to culture melanocytes.

[0085] In one embodiment, the culture temperature can be appropriately set by those skilled in the art, taking into account factors such as the type of animal from which the melanocytes originate. When the melanocytes are derived from mammals, the culture temperature is preferably 20°C to 50°C, more preferably 25°C to 45°C, even more preferably 30°C to 40°C, and even more preferably 36°C to 37°C. In one embodiment, the culture temperature is 37°C.

[0086] In one embodiment, for the gaseous environment of the culture, those skilled in the art can make appropriate settings considering the culture medium conditions and / or the species of animal from which the melanocytes are derived. The culture can be carried out in an environment with adjusted CO2 concentration, for example, in a 1-15% CO2 environment, preferably in a 3-10% CO2 environment, more preferably in a 4-7% CO2 environment, and even more preferably in a 5% CO2 environment.

[0087] For the culture, sufficient time is required for the melanocytes to produce melanin. In one embodiment, the culture time is 1 to 10 days from the first light exposure, preferably 2 to 5 days, more preferably 3 to 4 days, and even more preferably 3 days, or 24 to 240 hours from the first light exposure, preferably 48 to 120 hours, more preferably 72 to 96 hours, and even more preferably 72 hours.

[0088] In one embodiment, the process of alternating light exposure of melanocytes and culturing the light-exposed melanocytes in a culture medium can be repeated multiple times (refer to "light exposure" in "(a)").

[0089] For example, the seeding density of melanocytes can be 0.05 × 10⁻⁶. 4 cells / cm 2 ~6.0×10 4 cells / cm 2 Preferably exceeding 0.1×10 4 cells / cm 2 And below 2.2×10 4 cells / cm 2 More preferably 0.3×10 4 cells / cm 2 ~2.0×10 4 cells / cm 2 Further preferred is 0.4×10 4 cells / cm 2 ~1.5×10 4 cells / cm 2 .

[0090] At 0.4×10 4 cells / cm 2 ~1.5×10 4 cells / cm 2 In this case, it can be 0.4×10 4 cells / cm 2 0.5×10 4 cells / cm 2 0.6×10 4 cells / cm 2 0.7×10 4 cells / cm 2 0.8×10 4 cells / cm 2 0.9×10 4 cells / cm 2 1.0×10 4 cells / cm 2 Or 1.5×104 cells / cm 2 It can also be any range with one of these data as the lower limit and another data as the upper limit, but it is not limited to these.

[0091] When the seeding density of melanocytes exceeds 0.1 × 10⁻⁶ 4 cells / cm 2 And below 2.2×10 4 cells / cm 2 In this case, it is easier to evaluate the effect of light irradiation on melanin production by melanocytes with higher sensitivity.

[0092] The aforementioned components used to culture light-exposed melanocytes can also be combined with each other in any numerical range.

[0093] In one embodiment, light-irradiated melanocytes can reach a concentration exceeding 0.1 × 10⁻⁶. 4 cells / cm 2 And below 2.2×10 4 cells / cm 2 The inoculation density was determined by culturing melanocytes at a density exceeding 0.1 × 10⁻⁶ for 3 days from the start of light exposure in a liquid culture medium containing 1–4% (v / v) FBS, 80–120 nM α-MSH, and 80–120 μM theophylline. In one embodiment, the light-exposed melanocytes could be inoculated at a density exceeding 0.1 × 10⁻⁶. 4 cells / cm 2 And below 2.2×10 4 cells / cm 2 The inoculation density was determined by inoculating the medium with 1–4% (v / v) FBS, 80 nM–120 nM α-MSH, and 80 μM–120 μM theophylline. The second and third light irradiations were performed every 24 hours starting from the first light irradiation, and the medium was cultured for 3 days starting from the first light irradiation.

[0094] (c) Quantifying the amount of melanin produced by melanocytes. The amount of melanin produced by melanocytes can be quantified by methods known to those skilled in the art.

[0095] For example, after recovering cultured melanocytes, the melanin production can be determined by dissolving them in a 1 mol / L sodium hydroxide aqueous solution containing 10 wt% dimethyl sulfoxide (DMSO) and measuring the absorbance relative to 405 nm.

[0096] In addition, the amount of melanin produced by melanocytes can be determined by using protein quantification methods known to those skilled in the art, and the amount of melanin produced per unit of melanocyte-derived protein can be calculated (melanin production / amount of cell-derived protein) as the amount of melanin produced by melanocytes.

[0097] Proteins can also be quantified using commercially available kits, such as the Pierce (registered trademark) BCA Protein Assay Kit (catalog number: 23225, manufactured by ThermoScientific) and the RC DCTM Protein Assay Kit (manufactured by BioRad).

[0098] The amount of cell-derived protein reflects the number of viable cells (the number of viable melanoma cells). For example, in the light irradiation group, if the amount of cell-derived protein is lower than that in the control group, the light irradiation can be considered toxic to the cells. Therefore, in one embodiment, when the amount of melanin per unit of melanocyte-derived protein (melanin production / cell-derived protein amount) is used as the amount of melanin produced by melanocytes, it is difficult to determine whether the light irradiation is toxic.

[0099] The effect of light irradiation on melanin production by melanocytes can be evaluated based on a quantified amount of melanin.

[0100] More specifically, for example, light exposure can be used to evaluate whether melanin production in melanocytes increases or decreases. Evaluation can be made, for example, by comparing with a control group that has not undergone light exposure or with a pre-defined baseline or range. Comparisons with control groups, baselines, or ranges can be performed, for example, through any statistical processing.

[0101] The "influence of light irradiation" that is the evaluation object of the evaluation method related to this embodiment can be the influence of a specific light wavelength, cumulative irradiation, illuminance, irradiation time and / or number of irradiations. It can also be used based on this evaluation to determine the desired light irradiation conditions (e.g., light wavelength, cumulative irradiation, illuminance, irradiation time and / or number of irradiations).

[0102] The following method for evaluating the effect of light irradiation on melanin production by melanocytes, according to one embodiment, is described in the order of one embodiment.

[0103] (1) In a 6-well plate, with a strength of 0.4 × 10⁻⁶ 4 cells / cm 2 ~1.6×10 4 cells / cm 2B16 mouse melanoma cells (B16 4A5) were seeded at a density of 10% (v / v) FBS and cultured in DMEM at 37°C and 5% CO2.

[0104] (2) 24 hours after the start of culture, replace with DMEM containing 1-4% (v / v) FBS, 100 nM α-MSH, and 100 μM theophylline (DMEM used to induce melanin production).

[0105] (3) After 1 to 6 hours of cultivation, the first light irradiation is performed.

[0106] (4) Culture under the same conditions for 3 days after the first light exposure. If more than two light exposures are performed, the exposure should be performed every 24 hours after the first light exposure.

[0107] (5) Wash melanoma cells with PBS (-) and recover them, then dissolve them in a 1 mol / L sodium hydroxide aqueous solution containing 10 wt% DMSO. Determine the amount of melanin produced by melanoma cells based on absorbance at 405 nm.

[0108] (6) Proteins derived from melanoma cells were measured using a protein assay kit.

[0109] (7) Calculate the amount of melanin produced by each melanoma cell from the protein source, and use it as the amount of melanin produced by the melanocyte (melanoma cell).

[0110] (8) Evaluate the effect of light irradiation on melanin production by melanocytes based on the comparison with the control group (no light irradiation).

[0111] <Methods for determining light irradiation conditions to inhibit melanin production by melanocytes> In one embodiment, the above-described "method for evaluating the effect of light irradiation on melanin production by melanocytes" can be used in methods for determining light irradiation conditions for inhibiting melanin production by melanocytes, including this method.

[0112] That is, "a method for determining light irradiation conditions for inhibiting melanin production by melanocytes" includes: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. Quantifying the amount of melanin produced by melanocytes When light exposure reduces the amount of melanin produced by melanocytes, the light exposure condition is selected as the light exposure condition for inhibiting melanin production by melanocytes.

[0113] Examples and preferred practices for irradiating melanocytes with light, culturing irradiated melanocytes in a culture medium, and quantifying the amount of melanin produced by melanocytes are described, for example, in (a) to (c) above.

[0114] [(d-1) When light exposure reduces the amount of melanin produced by melanocytes, the light exposure condition is selected as the light exposure condition to inhibit melanin production by melanocytes.] Whether light exposure leads to a decrease in melanin levels can be determined, for example, by comparing with a control group that has not undergone light exposure or with a pre-defined baseline value or range. Comparisons with control groups, baseline values, or baseline ranges can be performed, for example, through any statistical processing.

[0115] When the amount of melanin produced by melanocytes is reduced, this light irradiation condition is selected as the light irradiation condition for inhibiting the production of melanocytes.

[0116] Such light irradiation conditions can be applied, for example, to the skin treatment method described in the second embodiment or the beauty device described in the third embodiment, and can be used to prevent or reduce melanin deposition in the skin, etc.

[0117] <Methods for determining light irradiation conditions to promote melanin production by melanocytes> In one embodiment, the above-described "method for evaluating the effect of light irradiation on melanin production by melanocytes" can be used in methods for determining light irradiation conditions that promote melanin production by melanocytes, including this method.

[0118] That is, "a method for determining light irradiation conditions for promoting melanin production by melanocytes" includes: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. Quantifying the amount of melanin produced by melanocytes When light exposure leads to an increase in the amount of melanin produced by melanocytes, the light exposure condition is selected as the light exposure condition for promoting melanin production by melanocytes.

[0119] Examples and preferred practices for irradiating melanocytes with light, culturing irradiated melanocytes in a culture medium, and quantifying the amount of melanin produced by melanocytes are described in "(a)" to "(c)" above.

[0120] [(d-2) When light irradiation leads to an increase in the amount of melanin produced by melanocytes, select that light irradiation condition as the light irradiation condition for promoting melanin production by melanocytes.] Whether light exposure promotes melanin production can be determined, for example, by comparing the individual with a control group that has not undergone light exposure or with a pre-defined baseline value or range. Comparisons with control groups, baseline values, or baseline ranges can be performed, for example, through any statistical processing.

[0121] When the amount of melanin produced by melanocytes increases, this light irradiation condition is selected as the light irradiation condition to promote the production of melanocytes.

[0122] Such selected light irradiation conditions can be applied, for example, to the skin treatment method described in the second embodiment or the beauty device described in the third embodiment, and can be used to promote melanin production and / or melanin deposition in hair, primarily human hair (prevention of gray hair or regeneration of gray hair).

[0123] [Second Embodiment (Skin Treatment Method)] The skin treatment method described in this embodiment is for inhibiting melanin production in the skin, and includes irradiating the skin with light according to the light irradiation conditions determined in the "Method for Determining Light Irradiation Conditions for Inhibiting Melanin Production by Melanocytes" described in the first embodiment. In one embodiment, this skin treatment method can be used to prevent or reduce melanin deposition in the skin or the like.

[0124] Other skin treatment methods related to this embodiment are skin treatment methods for promoting melanin production in the skin, which include irradiating the skin with light according to the light irradiation conditions determined in the "Method for Determining Light Irradiation Conditions for Promoting Melanin Production by Melanocytes" described in the first embodiment. In one embodiment, this skin treatment method can be used to promote melanin production and / or melanin deposition in hair, primarily human hair (prevention of gray hair or hair darkening).

[0125] In any of the above skin treatment methods, therapeutic actions can also be excluded.

[0126] Skin treatment is preferably performed on the skin of an organism. There are no limitations on the target animal, as long as it contains melanocytes. The animal can be any other species, such as mammals, birds, reptiles, amphibians, or fish, but mammals are preferred. Mammals can be humans or non-human animals, but humans are preferred. Non-human animal species can include, for example, monkeys, dogs, cats, horses, cattle, pigs, sheep, goats, rabbits, groundhogs, hamsters, mice, and / or rats, and are not limited to the purpose of being used as livestock, pets, or laboratory animals.

[0127] In one implementation, the skin is human skin.

[0128] The method of light irradiation of the skin is not limited; for example, it can be performed by a device having a light irradiation unit capable of performing light irradiation under defined conditions. As a non-limiting example of the device, the medical device or beauty device described in the third embodiment can be cited.

[0129] [Third Embodiment (Medical Device or Beauty Device)] The medical or cosmetic device related to this embodiment is a device that includes a light irradiation unit for performing light irradiation according to the light irradiation conditions determined in the "Method for Determining Light Irradiation Conditions for Inhibiting Melanin Production by Melanocytes" described in the first embodiment. In one embodiment, the device is a device that includes a light irradiation unit for performing light irradiation under any one or more of the various conditions described in "Light Irradiation" of the first embodiment. In one embodiment, the device is a medical or cosmetic device for inhibiting melanin production by melanocytes, and may be used, for example, to prevent or reduce melanin deposition in the skin.

[0130] Other medical or cosmetic devices related to this embodiment are devices that include a light irradiation unit for performing light irradiation according to the light irradiation conditions determined in the "Method for Determining Light Irradiation Conditions for Promoting Melanin Production by Melanocytes" described in the first embodiment. In one embodiment, the device is a medical or cosmetic device for promoting melanin production by melanocytes, for example, it can be used to promote melanin production and / or melanin deposition in hair, primarily human hair (prevention of gray hair or hair darkening).

[0131] The device may also be, for example, equipped with a light irradiation unit (light source) capable of irradiating light under defined conditions, and having a structure that can irradiate the skin with light emitted by the light source.

[0132] In one embodiment, the light irradiation unit may also be a structure controlled by a control unit (computer, etc.) equipped on the device.

[0133] As light irradiation under defined conditions, it includes conditions such as illuminance, the duration of each irradiation session, the total irradiation time over a certain period (hereinafter, the sum of the irradiation time and the duration of each irradiation session will also be referred to as "irradiation time"), the number of irradiations, and the cumulative irradiation amount. However, the device may also be equipped with a unit that, by quantifying or counting the illuminance, irradiation time, and / or the number of irradiations used by the user, senses that the light irradiation has reached an appropriate range (e.g., at least one of the defined conditions), automatically stops the light irradiation, and / or notifies the user that the appropriate range has been reached. Furthermore, the device may also be equipped with a unit that automatically calculates the cumulative irradiation amount based on the illuminance, irradiation time, and / or the number of irradiations used by the user, automatically stops the light irradiation when the light irradiation reaches an appropriate range, and / or notifies the user that the appropriate range has been reached. Such a notification unit is not limited to the scope within which the customer can be notified; it can be any of the senses acting on vision, hearing, touch, taste, or smell, such as a display on the device surface, a notification sound, or device vibration. By having such a unit, appropriate light irradiation can be provided to the user.

[0134] In one embodiment, when the appropriate range of other light irradiation conditions differs due to different light sources and / or wavelengths, the device may also be equipped with a control unit capable of providing appropriate light irradiation according to the light source and / or wavelength.

[0135] In one embodiment, the device may also have a structure capable of selecting the user's desired illuminance from an illuminance of 2 or higher. In this case, the device may also be equipped with a unit that counts the selected illuminance, irradiation time, and number of irradiations, and, as described above, senses that the light irradiation has reached an appropriate range, stops the light irradiation, and / or notifies the user.

[0136] In one embodiment, the device may also be equipped with a unit capable of registering one or more users. In this case, the device may further be equipped with a unit that can monitor the device usage status of each registered user over a certain period. This period may be, for example, one hour, one day, one week, one month, or one year. By monitoring the device usage status over this period, the device may also be equipped with a unit that, as described above, senses that the light exposure for each registered user during the period has reached an appropriate range (a defined condition), stops the light exposure, and / or notifies the user. With such a unit, even if the device is used intermittently during a certain period, light exposure within an appropriate range can be maintained. Furthermore, since multiple users can be registered, even if multiple users use the device intermittently, light exposure within an appropriate range can still be provided to each user.

[0137] In one embodiment, the device can be a handheld type that can be held by a user's hand as a whole, or a placement type consisting of a fixed device, a movable part, and an arm that connects the fixed device and the movable part.

[0138] In one embodiment, the device may also be equipped with various types of buttons for users to control light illumination, such as on / off buttons, mode switching buttons, and intensity adjustment buttons.

[0139] For equipment configurations other than those described above, those skilled in the art can make appropriate settings.

[0140] A non-limiting list of embodiments and combinations thereof illustrated in this disclosure is provided below.

[0141] [1] A method for evaluating the effect of light irradiation on melanin production by melanocytes, comprising: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. The amount of melanin produced by melanocytes was quantified.

[0142] [2] According to the method of [1], wherein the culture medium contains more than 0.2% and less than 5% (v / v) of serum.

[0143] [3] According to the method described in [1] or [2], light-irradiated melanocytes reach a concentration exceeding 0.1 × 10⁻⁶. 4 cells / cm 2 And below 2.2×10 4 cells / cm 2 The density was inoculated into the culture medium.

[0144] [4] The method described in [1] to [3] is arbitrary, wherein the light is visible light.

[0145] [5] The method described in [2] to [4] is optional, wherein the serum is fetal bovine serum.

[0146] [6] The method described in [2] to [5] in any way, wherein the serum is at a concentration of 1 to 4% (v / v).

[0147] [7] A method for determining light irradiation conditions for inhibiting melanin production by melanocytes, comprising: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. Quantifying the amount of melanin produced by melanocytes When light exposure reduces the amount of melanin produced by melanocytes, the light exposure condition is selected as the light exposure condition for inhibiting melanin production by melanocytes.

[0148] [8] A method for determining light irradiation conditions for promoting melanin production by melanocytes, comprising: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. Quantifying the amount of melanin produced by melanocytes When light exposure leads to an increase in the amount of melanin produced by melanocytes, the light exposure condition is selected as the light exposure condition for promoting melanin production by melanocytes.

[0149] [9] The method according to [7] or [8], wherein the culture medium contains more than 0.2% and less than 5% (v / v) of serum.

[0150]

[10] According to the method described in [7] to [9] (optional), the light-irradiated melanocytes reached a concentration exceeding 0.1 × 10⁻⁶. 4 cells / cm 2 And below 2.3×10 4 cells / cm 2 The density was inoculated into the culture medium.

[0151]

[11] The method described in [7] to

[10] in any form, wherein the light is visible light.

[0152]

[12] The method described in [9] to

[11] in any way, wherein the serum is fetal bovine serum.

[0153]

[13] The method described in [9] to

[12] in any form, wherein the serum is at a concentration of 1 to 4% (v / v).

[0154]

[14] A skin treatment method for inhibiting melanin production in the skin, comprising irradiating the skin with light according to light irradiation conditions determined by any one of [7] and [9] to

[13] (other than therapeutic actions).

[0155]

[15] A skin treatment method for promoting melanin production in the skin, comprising irradiating the skin with light according to light irradiation conditions determined by any one of [8] and [9] to

[13] (other than therapeutic actions).

[0156]

[16] A beauty device comprising a light irradiation unit for irradiating light according to light irradiation conditions determined by any one of [7] to

[13] .

[0157] [Example] The following illustrative embodiments further illustrate this disclosure in detail, but the interpretation of this disclosure is not limited to these embodiments.

[0158] Example 1: Evaluation system for the effects of light irradiation on melanin production by melanocytes [1. Evaluation Methods] In a 6-well plate (35mm in diameter, 9.6cm in diameter) 2 (Catalogue No.: 2-8588-01, Manufacturer: VIOLAMO or Catalogue No.: 3000-035, Manufacturer: IWAKI) B16 mouse melanoma cells (B164A5, obtained from Riken RBC) were seeded at the following density and cultured in DMEM (Catalogue No.: 040-30095, Fujifilm and Hikari Pure Chemicals) containing 10% (v / v) FBS (Catalogue No.: 26140-079, Gibco, USA) and 2 mM L-glutamine (Catalogue No.: 073-05391, Fujifilm and Hikari Pure Chemicals) at 37°C and 5% CO2.

[0159] (Melanoma cell seeding density) (1) 0.1×10 4 cells / cm 2 (1×10) 4 (cells / well, 6-well plate) (2) 0.4×10 4 cells / cm 2 (3.5×10) 4 (cells / well, 6-well plate) (3) 0.7×10 4 cells / cm 2 (5×10) 4 (cells / well, 6-well plate) (4) 0.8×10 4 cells / cm 2 (7×10) 4 (cells / well, 6-well plate) (5) 1.5×10 4 cells / cm 2 (14×10) 4 (cells / well, 6-well plate) (6) 2.2×10 4 cells / cm 2 (21×10) 4 (cells / well, 6-well plate) Twenty-four hours after the start of culture, the mixture was replaced with DMEM (for inducing melanin production) containing FBS, 100 nM α-MSH (catalog number: M4135, Sigma-Aldrich, USA), 100 μM theophylline (catalog number: T1633, Sigma-Aldrich, USA), and 2 mM L-glutamine (catalog number: 073-05391, Fujifilm and Koden Pharmaceuticals).

[0160] After 2 hours of cultivation, the first 505nm wavelength light irradiation was performed under the following conditions. For the light source LED, an SMT505 manufactured by USHIO Motors was used.

[0161] The FBS in the culture medium was set to 0, 0.2, 1, 2, 3, 4, 5 or 10% (v / v).

[0162] The illuminance (output) of the light source is set to 12 mW / cm². 2 .

[0163] The light exposure can be set to 1 or 3 times.

[0164] The duration of each light irradiation session is set to 3, 5, 7.5, or 10 minutes.

[0165] Following the first light exposure, melanoma cells were further cultured for 3 days (72 hours) at 37°C and 5% CO2 using the same culture medium. During this period, the group receiving three light exposures was exposed to the same illuminance (output) and exposure time as the first exposure, 24 hours and 48 hours after the first light exposure.

[0166] After 3 days of cultivation, a further 1-day cultivation was carried out under the same conditions.

[0167] In this evaluation method, the control group was the group that never received light irradiation but whose sequence of light irradiation was the same as that of the light irradiation group.

[0168] (Quantification of melanin production) Cells were washed and recovered using PBS (-) (calcium and magnesium phosphate-buffered saline) (catalog number: 2810305, manufactured by MPBiomedicals), and dissolved in a 1 mol / L sodium hydroxide aqueous solution containing 10 wt% dimethyl sulfoxide (DMSO). Melanin production in melanoma cells was determined based on absorbance at 405 nm.

[0169] For samples used to determine melanin production, cell-derived proteins were further measured using the Pierce (registered trademark) BCA protein assay kit (catalog number: 23225, manufactured by ThermoScientific). Based on the measurement results, the melanin content (melanin production / cell-derived protein content) of each melanoma cell-derived protein was calculated and set as the melanin production. Unless otherwise specified, the following evaluation results are expressed as % with the control group as 100%.

[0170] Among them, the amount of cell-derived protein reflects the number of living cells (the number of living melanoma cells). For example, in the light irradiation group, when the amount of cell-derived protein decreases compared to the control group, it can be said that the light irradiation is toxic to the cells.

[0171] [2. Evaluation Results] (Suitable fetal bovine serum concentration) Regarding the FBS concentration (0%–10% (v / v)) in the DMEM used to induce melanin production, the cell seeding density was 0.8 × 10⁻⁶. 4 cells / cm 2 (7×10) 4 Evaluation results for cells / well, 6-well plate) are as follows: Figure 1 As shown. With an illuminance of 12 mW / cm². 2 The evaluation was conducted by subjecting the subjects to three light irradiations, each lasting 10 minutes (n=2 or 3).

[0172] A significant decrease in melanin production in melanocytes (melanoma cells) could be detected when the FBS concentration was above 0.2% but below 5% (v / v).

[0173] (Suitable melanocyte seeding density) The evaluation results regarding the seeding density of melanocytes (melanoma cells) at a FBS concentration of 2% (v / v) in the DMEM used to induce melanin production are as follows: Figure 1 As shown. With an illuminance of 12 mW / cm². 2 The evaluation was conducted after three light exposures, each lasting 10 minutes. n=2 or 3.

[0174] When the inoculation density exceeds 0.1×10 4 cells / cm 2 And below 2.2×10 4 cells / cm 2 In this case, a significant reduction in melanin production in melanocytes (melanoma cells) can be detected.

[0175] (At an inoculation density of 0.8 × 10⁻⁶) 4 cells / cm 2 Evaluation results when FBS is 2% (v / v) At a cell seeding density of 0.8 × 10⁻⁶ 4 cells / cm 2 (7×10) 4 (cells / well, 6-well plate), with an FBS concentration of 2% (v / v) in DMEM used to induce melanin production (refer to) Figure 2 A) Data on melanin production obtained under different light conditions, such as Figure 2 As shown in B and C (n=4).

[0176] Figure 2 B indicates an illuminance of 12 mW / cm². 2 The amount of melanin produced in the groups that received light irradiation for 7.5 minutes or 10 minutes at a time. Figure 2 C indicates an illuminance of 12 mW / cm². 2 The amount of melanin produced in groups that received light irradiation for 3 minutes 3 times, 5 minutes 3 times, 7.5 minutes 3 times, or 10 minutes 3 times.

[0177] like Figure 2 As shown in B, at an illuminance of 12 mW / cm² 2 In the case of light exposure at intervals of 7.5 minutes or 10 minutes, a significant decrease in melanin production was detected compared to the control group (*p<0.05).

[0178] like Figure 2 As shown in C, at an illuminance of 12 mW / cm² 2 In the case of light exposure, after 3 exposures of 5 minutes, 3 exposures of 7.5 minutes, or 3 exposures of 10 minutes, a significant decrease in melanin production was detected compared to the control group (*p<0.05).

[0179] (Suitable light conditions) At an inoculation density of 0.8 × 10 4 cells / cm 2 (7×10) 4 Cells / well, 6-well plate) or 1.5 × 10⁻⁶ cells / well 4 cells / cm 2 (14×10) 4The study investigated the intracellular melanin content, protein content, and melanin production (melanin content / protein content) (%) of melanocytes (melanoma cells) under various light irradiation conditions (illuminance, irradiation time per session, number of irradiations (1 or 3 times)) with the control group as 100%. The FBS concentration in the DMEM used to induce melanin production was 2% (v / v) or 4% (v / v).

[0180] At least under the following light irradiation conditions, a significant decrease in melanin production in melanocytes relative to the control group could be detected (*p<0.05).

[0181] (1) At an inoculation density of 0.8 × 10 4 cells / cm 2 When FBS is 2% (v / v) Illuminance: 12mW / cm² 2 Irradiation time: 7.5 minutes; Number of irradiations: 1 time Illuminance: 12mW / cm² 2 Irradiation time: 10 minutes; Number of irradiations: 1 time Illuminance: 100mW / cm² 2 Irradiation time: 3 minutes; Number of irradiations: 1 time Illuminance: 12mW / cm² 2 Irradiation time: 5 minutes; Number of irradiations: 1 time Illuminance: 6.5 mW / cm² 2 Irradiation time: 10 minutes; Number of irradiations: 3 times Illuminance: 12mW / cm² 2、 Irradiation time: 5 minutes, Number of irradiations: 3 times Illuminance: 12mW / cm² 2 Irradiation time: 7.5 minutes; Number of irradiations: 3 times Illuminance: 12mW / cm² 2 Irradiation time: 10 minutes; Number of irradiations: 3 times Illuminance: 18mW / cm² 2 Irradiation time: 5 minutes; Number of irradiations: 3 times Illuminance: 18mW / cm² 2 Irradiation time: 10 minutes; Number of irradiations: 3 times Illuminance: 100mW / cm² 2 Irradiation time: 3 minutes; Number of irradiations: 3 times Illuminance: 100mW / cm² 2 Irradiation time: 5 minutes; Number of irradiations: 3 times (2) At an inoculation density of 1.5 × 10 4 cells / cm 2 When FBS is 4% (v / v) Illuminance: 12mW / cm² 2 Irradiation time: 10 minutes; Number of irradiations: 3 times Illuminance: 12mW / cm² 2 Irradiation time: 20 minutes; Number of irradiations: 3 times Illuminance: 100mW / cm² 2 Irradiation time: 5 minutes; Number of irradiations: 3 times A portion of the data obtained based on the aforementioned light irradiation conditions is as follows: Figure 3 and Figure 4 As shown.

[0182] As mentioned above, and Figure 3 As shown, at an inoculation density of 0.8 × 10⁻⁶ 4 cells / cm 2 At an FBS concentration of 2% (v / v), the cumulative radiation dose (mJ / cm²) 2 When the illuminance × time (seconds) × number of irradiations is 3000 to 200000 (e.g., 5400 to 180000), a significant decrease in melanin production in melanocytes relative to the control group can be detected (*p<0.05).

[0183] As mentioned above, and Figure 4 As shown, at an inoculation density of 1.5 × 10⁻⁶ 4 cells / cm 2 At an FBS concentration of 4% (v / v), the cumulative radiation dose (mJ / cm²) 2 When the illuminance × time (seconds) × number of irradiations is 3000 to 200000 (e.g., 43200 to 90000), a significant decrease in melanin production in melanocytes relative to the control group can be detected (*p<0.05).

[0184] Furthermore, such as Figure 3 and Figure 4 As shown, in the cumulative radiation dose (mJ / cm) 2 At concentrations below 180,000, it is difficult to achieve a significant decrease in the amount of protein in melanocytes (*p<0.05), and only an inhibitory effect on melanocyte production is easily obtained. Therefore, when the cumulative irradiation dose (mJ / cm²) is below 180,000, it is difficult to achieve a significant decrease in the amount of protein in melanocytes (*p<0.05), and only an inhibitory effect on the amount of melanocytes produced is easily obtained. 2When the value is above 3000 and below 180000 (e.g., 5400–90000), a significant decrease in melanin production in melanocytes (melanoma cells) relative to the control group can be detected under conditions where they are less susceptible to the toxic effects of light-induced melanocyte irradiation (*p<0.05).

[0185] In this embodiment, light irradiation is used as an example, with a wavelength of only 505nm. However, even if it is visible light, preferably green or yellow wavelength, more preferably wavelength of 490-570nm (e.g., 490nm-525nm) or light in the peak wavelength region of these wavelengths, the amount of melanin production can also be evaluated.

[0186] Example 2. Study on culture time The seeding density of melanocytes was set at 0.8 × 10⁸. 4 cells / cm 2 (7×10) 4 Cells / well, 6-well plate), with FBS concentration in DMEM used to induce melanin production set to 2% (v / v), and irradiated twice every 10 minutes (at the time of the first irradiation in Example 1 and 24 hours later) at 505 nm (illuminance: 12 mW / cm²). 2 After adding 24 hours of light irradiation to the culture time starting from Example 1, the culture was then recovered.

[0187] Except as described above, the evaluation was conducted in the same manner as in Example 1 (n=2).

[0188] like Figure 5 As shown, no significant change in melanin production was detected in melanocytes (melanoma cells) in the light-irradiated group during extended culture periods.

[0189] [Industry availability] Since the evaluation method of this embodiment can evaluate the effect of light irradiation on melanin production by melanocytes, it can be applied to determine light irradiation conditions that inhibit or promote melanin production by melanocytes, and thus has industrial applicability.

Claims

1. A method for evaluating the effect of light irradiation on melanin production by melanocytes, comprising: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. The amount of melanin produced by melanocytes was quantified.

2. The method according to claim 1, wherein, The culture medium contains more than 0.2% but less than 5% v / v serum.

3. The method according to claim 1 or 2, wherein the light-irradiated melanocytes reach a concentration exceeding 0.1 × 10⁻⁶. 4 cells / cm 2 And below 2.2×10 4 cells / cm 2 The density was inoculated into the culture medium.

4. The method according to any one of claims 1 to 3, wherein, The light is visible light.

5. The method according to any one of claims 2 to 4, wherein, The serum was fetal bovine serum.

6. The method according to any one of claims 2 to 5, wherein, The serum concentration is 1-4% v / v.

7. A method for determining light irradiation conditions for inhibiting melanin production by melanocytes, comprising: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. Quantifying the amount of melanin produced by melanocytes When light exposure reduces the amount of melanin produced by melanocytes, the light exposure condition is selected as the light exposure condition for inhibiting melanin production by melanocytes.

8. A method for determining light irradiation conditions for promoting melanin production by melanocytes, comprising: Irradiating melanocytes with light Melanocytes exposed to light were cultured using a culture medium. Quantifying the amount of melanin produced by melanocytes When light exposure leads to an increase in the amount of melanin produced by melanocytes, the light exposure condition is selected as the light exposure condition for promoting melanin production by melanocytes.

9. The method according to claim 7 or 8, wherein, The culture medium contains more than 0.2% but less than 5% v / v serum.

10. The method according to any one of claims 7 to 9, wherein the light-irradiated melanocytes reach a concentration exceeding 0.1 × 10⁻⁶. 4 cells / cm 2 And below 2.2×10 4 cells / cm 2 The density was inoculated into the culture medium.

11. The method according to any one of claims 7 to 10, wherein, The light is visible light.

12. The method according to any one of claims 9 to 11, wherein, The serum was fetal bovine serum.

13. The method according to any one of claims 9 to 12, wherein, The serum concentration is 1-4% v / v.

14. A non-therapeutic skin treatment method for inhibiting melanin production in the skin, comprising irradiating the skin with light according to the light irradiation conditions determined by the method of claim 7.

15. A non-therapeutic skin treatment method for promoting melanin production in the skin, comprising irradiating the skin with light according to the light irradiation conditions determined by the method of claim 8.

16. A beauty device comprising a light irradiation unit for performing light irradiation according to light irradiation conditions determined by the method of any one of claims 7 to 13.

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