WO2023112724A1

WO2023112724A1 - Method of suppressing pigment production by high frequency electric stimulation

Info

Publication number: WO2023112724A1
Application number: PCT/JP2022/044547
Authority: WO
Inventors: 咲綾小池
Original assignee: 株式会社資生堂
Priority date: 2021-12-17
Filing date: 2022-12-02
Publication date: 2023-06-22

Abstract

The present disclosure relates to a cosmetic method based on a whitening effect or a wrinkle suppressing or reducing effect from the application of high frequency waves to the skin. Specifically, pigment formation is suppressed by applying high frequency waves to the skin. More specifically, it is possible to suppress pigment formation by decreasing the number of senescent fibroblasts and/or increasing the number of normal fibroblasts.

Description

Pigment production suppression method by high-frequency electrical stimulation

The present invention relates to a cosmetic method using high frequencies, and more particularly to a method for suppressing or reducing spots or whitening.

A high-frequency beauty method is known that can improve wrinkles and sagging of the skin by applying a high-frequency (RF) current to the skin of the face, hands and feet. When a high-frequency current is applied to the skin, the temperature of the dermis layer and subcutaneous tissue rises. The heating effect is expected to have effects such as promoting blood flow and thermally damaging collagen in the dermis layer to promote regeneration of collagen in the dermis layer. In recent years, various cosmetic treatment apparatuses using such high frequencies have been proposed (eg, Patent Document 1).

Patent Document 2 discloses a system and method for skin electrosurgical treatment. In this document, in particular, high-frequency voltage is applied to the electrode terminals to raise the temperature of the collagen fibers in the tissue of the target site from body temperature (about 37°C) to usually about 60-70°C, thereby Collagen fibers are disclosed to contract substantially irreversibly. The methods disclosed in this document involve cutting, ablating, peeling or removing a volume of skin tissue or hair (i.e., peeling or dissociating molecules of the tissue structure) to separate the tissue layers from the underlying tissue layers. It is used to remove layers of tissue, contract or contact collagenous connective tissue, and/or coagulate blood vessels beneath the surface of the skin, and is highly invasive to the subject of application.

Patent Document 3 discloses a liquid agent permeation device and method for cosmetic use. It is described that by applying a pulse voltage to an electrode coated with a liquid agent and attached to the surface of the skin, it is possible to improve cosmetic effects such as a pigmentation suppressing effect and a whitening effect.

Patent Literature 4 discloses a high-frequency cosmetic treatment apparatus capable of preventing the skin from being locally heated.
Patent Document 5 discloses an RF generation device that generates RF output, an applicator body, an applicator body that is arranged in front of the applicator body, is inserted to the dermis layer of the skin of a treatment subject, and is electrically connected to the RF generation device. An RF-based skin treatment device is described that includes a bipolar applicator including a needle connected to and transmitting said RF power.
US Pat. No. 6,200,000 discloses a mask-shaped skin treatment device and system that includes a coupling agent detector configured to detect the presence of a coupling agent and a heating element configured to cause localized heating of a portion of skin. is disclosed. Locally raising the temperature of the skin, either mild thermal effects (40°C to 45°C) or large thermal effects (50°C to 90°C) on the dermis, can reduce collagen production from fibroblast activation. It is capable of increasing, denaturing and contracting collagen.

Patent Document 7 discloses a high-frequency therapy device consisting of a main body, a plate, a probe, a footswitch, and a band. This high-frequency therapy device increases skin elasticity, raises the temperature of the dermis to 50-60°C, helps contraction and production of collagen, increases binding force with elastin, and improves skin problems. It is stated that it is

Patent Document 8 discloses a new pulse high frequency beauty machine control system consisting of a sine wave high frequency power supply, a square wave drive power supply, a voltage pre-combustion module, a pulse generator, a high frequency generator, a liquid crystal display, a central processing unit and a cooling water machine. disclosed.

Patent Literature 9 discloses a massaging device which has a simple structure, is easy to operate, and not only makes the face thinner, but also lifts and tightens the skin.
However, until now, by applying high frequency, the reduction of the number of senescent fibroblasts and / or the induction of the increase of the number of normal fibroblasts and the suppression of pigmentation have been investigated. do not have.

Japanese Patent Application Laid-Open No. 2018-489 Japanese Patent Publication No. 2001-523513 Japanese Patent Application Laid-Open No. 2007-319474 WO2014/196195 Japanese Patent Publication No. 2021-533845 Japanese Patent Publication No. 2021-506517 Korean Patent No. 10-2018-0047181 Chinese Patent Application Publication No. 105920732 Chinese Patent Application Publication No. 112426341

One of the causes of age spots is thought to be the chronic abnormal aging of dermal fibroblasts. It has been difficult for conventional cosmetics and laser treatments to act directly on the dermis. In addition, for example, the conventional high-frequency cosmetic method as described in Patent Document 2 cuts, excises, exfoliates or removes a certain volume of skin tissue or hair (that is, exfoliates or dissociates molecules of the tissue structure). are used to separate and remove tissue layers from underlying tissue layers, to contract or contact collagenous connective tissue, and/or to coagulate blood vessels beneath the surface of the skin. , so to speak, was highly invasive to the target.

One of the objects of the present invention is to provide a cosmetic method based on the suppression or reduction of blemishes obtained by applying physical stimulation to the skin by applying high frequency waves, or on the basis of the whitening effect.

According to one aspect of the present invention, there is provided a cosmetic method including applying high frequency waves to the skin to suppress or reduce blemishes or to whiten the skin.

In addition, according to one aspect of the present invention, there is provided a cosmetic method including applying high frequency waves to the skin to suppress pigmentation.

In addition, according to one aspect of the present invention, there is provided a method for suppressing pigmentation, including the step of applying high frequency waves to the skin.

Furthermore, according to one aspect of the present invention, there is provided a method of reducing the number of aged fibroblasts and/or increasing the number of normal fibroblasts, comprising applying radiofrequency waves to the skin.

According to another aspect of the present invention, there is provided a method for operating a high-frequency cosmetic treatment apparatus having a pair of electrodes and a control circuit for applying a high-frequency voltage to the pair of electrodes, wherein the control circuit controls the A method of operating a high-frequency cosmetic treatment device, comprising: applying a high-frequency voltage that suppresses pigmentation in the skin to a pair of electrodes, wherein the high-frequency voltage is applied while the pair of electrodes are in contact with the skin. is provided.

Furthermore, according to one aspect of the present invention, a device comprising a pair of electrodes and a control circuit for applying a high frequency voltage to the pair of electrodes is caused to execute the operation method of the high frequency cosmetic treatment device according to the present disclosure. A computer program is provided.

Also according to one aspect of the present invention, there is provided a non-transitory computer-readable medium having instructions stored thereon which, when executed by a processor, cause a high frequency cosmetic treatment apparatus according to the present disclosure. A computer readable medium is provided capable of executing a computer program for performing the method of operation of.

Further in accordance with one aspect of the present invention is a radio frequency cosmetic treatment apparatus for use in a cosmetic method for inhibiting pigmentation in the skin comprising applying radio frequency to the skin, comprising: a pair of electrodes; and a control circuit for applying a high frequency voltage to the pair of electrodes, the high frequency cosmetic treatment device being configured to apply a high frequency voltage to the skin to suppress skin pigmentation.

(definition)
As used herein, the terms "applying high frequency" and "applying high frequency" refer to applying a high frequency voltage between a pair of electrodes spaced apart from each other and in contact with the object, thereby It means to pass high frequency current.

In addition, the word "about" used when indicating a numerical value indicates that it includes 5% of the numerical value before and after the numerical value modified by this word.

According to the present invention, cosmetic effects can be obtained by improving the stability of dermal stem cells.

1 is a block diagram showing one form of a high-frequency device that can be used in the present invention; FIG. FIG. 2 is a photograph showing cell proliferation after RF application to PUVA-treated fibroblasts (Fb). RF-: No RF applied, RF+: Frequency: 1 MHz/Voltage level: 70 Vpp/Output pattern: 4 seconds ON/4 seconds OFF RF applied, RF++: Frequency: 950 kHz/Voltage level: 168 Vpp/Output pattern: 1 second ON・ Apply RF with 2 seconds off. Graph quantifying cell proliferation after application of RF to PUVA-treated fibroblasts (Fb). Relative values relative to RF- are shown. FIG. 2 is a photograph showing cell proliferation after RF application to non-PUVA-treated fibroblasts (Fb). RF-: No RF applied, RF+: Frequency: 1 MHz/Voltage level: 70 Vpp/Output pattern: 4 seconds ON/4 seconds OFF RF applied, RF++: Frequency: 950 kHz/Voltage level: 168 Vpp/Output pattern: 1 second ON・ Apply RF with 2 seconds off. Graph quantifying cell proliferation after application of RF to non-PUVA-treated fibroblasts (Fb). Relative values relative to RF- are shown. Fig. 3 is a photograph showing the number of p21-positive fibroblasts (Fb) after application of RF. Graph quantifying the number of p21-positive Fb cells after application of RF. Relative values relative to RF- are shown. Fig. 3 is a graph quantifying the amount of melanin in an epidermal model co-cultured with RF-stimulated, PUVA-treated fibroblasts. Relative values relative to RF- are shown. FIG. 10 is a graph showing the results of RT-PCR analysis of gene expression of TYR, TYRP1, DCT, and MITF in an epidermal model co-cultured with RF-stimulated, PUVA-treated fibroblasts. FIG. Relative values relative to RF- are shown. Fig. 3 is a graph quantifying the amount of melanin in an epidermal model co-cultured with RF-stimulated, PUVA-treated fibroblasts. Relative values relative to RF- are shown. Fig. 3 is a graph quantifying the amount of melanin in an epidermal model co-cultured with RF-stimulated, non-PUVA-treated fibroblasts. Relative values relative to RF- are shown. Figure 3 shows the results of temperature measurements on PUVA-treated fresh skin (NSA11) with RF+ stimulation. Figure 3 shows the results of temperature measurements on PUVA-treated fresh skin (NSA11) with RF++ stimulation. Fig. 3 is a photograph showing the color tone after application of RF to PUVA-treated fresh skin (NSA11). Figure 2 shows images of Fontana-Masson staining of untreated or PUVA-treated fresh skin (NSA11) with senescence induced. Images of Fontana Masson staining of PUVA-treated fresh skin (NSA11) after RF- (no RF applied), RF+, RF++ applications are shown. Fig. 10 is a photograph showing changes in the number of p21-positive senescent fibroblasts (Fb) in PUVA-treated fresh skin (NSA11) after application of RF+ and RF++.

The present invention is based on the present inventors' new finding that application of radio frequency (RF) to the skin greatly contributes to suppressing pigmentation by skin cells, and is one aspect of the present invention. , a cosmetic method comprising applying radio frequency to the skin to suppress or reduce blemishes or whitening, and a cosmetic method comprising applying radio frequency to the skin to inhibit pigmentation. Suppression of pigmentation can be due to a decrease in the number of senescent fibroblasts and/or an increase in the number of normal fibroblasts. Senescent fibroblasts can also be p21 positive cells. A reduction in the number of senescent fibroblasts (which can be identified as p21 positive cells) and/or an increase in the number of normal fibroblasts can improve skin blemishes.

The frequency of radio frequency applied to the skin is in the range of 0.5 MHz to 4 MHz, such as any frequency of 0.5 MHZ, 0.95 MHz, 1 MHz, 2 MHZ, 3 MHz or 4 MHz, or the range defined by any of the above frequencies. Any frequency included (eg, in the range of 1-4 MHz) may be used. In some forms of the invention, the frequency is about 1 MHz.

The application of high frequency to the skin may be combined with the application of drugs, various extracts, etc. to the skin. For example, applicable drugs and various extracts include inositol, retinol derivatives, xylitol, hyaluronic acid, glycerin, yeast extract, cherry leaf extract, cinnamon extract, pine extract, Bulgarian rose water, okra extract, musk extract, burnet extract, glycyrrhizin. Dipotassium acid, ginkgo biloba extract, tencha extract, coix seed extract, licorice extract, panax ginseng extract, clara extract, star fruit leaf extract, carrot extract, juyaku extract, neem leaf extract, saffron extract, cinchona extract, comfrey extract, thyme extract, winter strawberry , Yomena, Mube, Ryukyu Strawberry, Indian Yomena, Matebashii, Scutellaria root extract, Rosemary extract, Apricot kernel, Gentian extract, Mint powder, Taisou extract, Hop extract, Kiwi extract, Roman chamomile extract, Apple extract, Hawthorn extract, Examples include turmeric extract, bayberry, myrtaceae, and konara. These can be used singly or in combination.

A high-frequency device can be used to apply high-frequency waves to the skin. One form of a high-frequency device will be described with reference to FIG.

A high-frequency device 1 has an electrode section 10 and a control circuit 20 . The electrode section 10 has a first electrode 10a and a second electrode 10b connected to the control circuit 20. As shown in FIG. A contact surface that contacts the user's skin is provided on the electrode unit 10, and the first electrode 10a and the second electrode 10b are arranged on this contact surface with a space therebetween, thereby forming the first electrode 10a and the second electrode 10b. They can be easily spaced apart from each other and contacted to the skin. The first electrode 10a and the second electrode 10b may be connected to the control circuit 20 via a wiring cable instead of the contact surface, so that the first electrode 10a and the second electrode 10b can be arranged freely. can do.

The control circuit 20 can have a power supply section, a voltage control section and a resistance measurement section. The power supply section applies a high frequency voltage (voltage indicating high frequency) between the first electrode 10a and the second electrode 10b. The power supply unit has an oscillation circuit that generates a high-frequency voltage, a booster circuit that boosts the oscillated voltage, and the like. is configured to be applied between By applying a high-frequency voltage between the first electrode 10a and the second electrode 10b while the first electrode 10a and the second electrode 10b are in contact with the user's skin, a high-frequency current can flow through the skin. can. The application of radio frequency can be non-invasive, for example by applying electrodes against the skin surface of the skin. For example, the application of radio frequency can be performed while the electrode is in vertical contact with the skin and moved so that the tip of the electrode touches the skin. The speed at which the electrodes are moved can be, for example, about 2 cm per second.

The voltage control unit controls the high-frequency voltage applied by the power supply unit by controlling the power supply unit. In addition, the voltage control section uses the measurement result of the resistance measurement section to control the voltage. The resistance measurement unit measures the resistance value (electrical resistance value) between the first electrode 10a and the second electrode 10b while the first electrode 10a and the second electrode 10b are in contact with the user's skin.

This resistance value changes depending on the user's skin condition and the type of external skin preparation such as lotion applied to the skin. For example, it is known that when a high-frequency current is applied to the skin of a human body, the skin generates heat, and the impedance inside the skin decreases as the temperature of the skin rises. Therefore, as the skin temperature rises, the current flowing between the first electrode 10a and the second electrode 10b increases, and the measured resistance value decreases. The resistance measurement unit measures the resistance value at predetermined time intervals (for example, every 0.5 seconds), and supplies the measured resistance value to the voltage control unit each time. In the method according to the present disclosure, the temperature rise of the skin can be set to remain below 45°C, or below 40°C, for example.

The voltage control unit controls the high-frequency voltage according to the resistance value measured by the resistance measurement unit, for example, to increase the high-frequency voltage (increase the amplitude of the high-frequency voltage) as the measured resistance value increases.

The lower limit of the high-frequency voltage applied between the first electrode 10a and the second electrode 10b is preferably 50 Vpp or more, more preferably 70 Vpp or more in terms of peak-to-peak voltage. The upper limit of the high-frequency voltage applied between the first electrode 10a and the second electrode 10b is preferably 200 Vpp or less, more preferably 170 Vpp or less in terms of peak-to-peak voltage. The high frequency voltage applied can be, for example, 70 Vpp.

The application of the high-frequency voltage may be continuous application or pulse application. In the case of pulse application, application (ON) and non-application (OFF) of the high frequency voltage are periodically repeated. The repetition period of pulse application may be 0.1 sec to 10 sec, for example 0.1 sec, 0.2 sec, 0.3 sec, 0.5 sec, 0.8 sec, 1 sec, 1 sec .2 seconds, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, or 10 seconds. The duty ratio, which is the ON time ratio of the high-frequency voltage in pulse application, is preferably in the range of 10% to 99%, more preferably in the range of 20% to 80%. A duty ratio can be, for example, 40%.

A user may be able to set high frequency application conditions such as high frequency voltage and duty ratio. In order to allow the user to set the application conditions of the radio frequency, the radio frequency apparatus can have an input device operated by the user. Any device such as a switch can be used as the input device. When the application condition of high frequency can be set by the user, the control circuit 20 accepts an input operation through the input device, and according to the accepted input operation, the high frequency voltage, continuous application or pulse application, and pulse application. Control parameters such as period and duty ratio. When setting the high frequency application conditions, each parameter may be set independently, and a plurality of combinations of the high frequency voltage, period and duty ratio in pulse application are preset in the control circuit 20. A user may select a preset combination.

As described above, it has a pair of electrodes and a control circuit that applies a high frequency voltage to the pair of electrodes, and is configured to apply to the skin a high frequency voltage that suppresses pigmentation of the skin. High frequency waves can be easily applied to the user's skin by using the cosmetic treatment device. A high frequency device can be operated as follows. First, the first electrode 10a and the second electrode 10b are placed in contact with the user's skin with a space between them. Next, the control circuit 20 applies a high-frequency voltage between the first electrode 10a and the second electrode 10b placed in contact with the user's skin so as to enhance the stability of the dermal stem cells. In this way, by applying high frequency waves to the skin, pigmentation in the skin can be suppressed, and a suppression or reduction of age spots or a whitening effect can be achieved.

The above operating method can be controlled by executing a computer program. A computer program according to the present disclosure may be stored in a non-transitory computer-readable medium. Instructions defined by a computer program are executed by a processor, such as a CPU. Based on control programs such as the OS (Operating System) and execution programs stored in the memory device, the CPU controls the processing of the entire computer, such as various calculations and data input/output with each hardware component. Each process can be realized by The memory device can store an execution program read from the auxiliary storage device by the CPU. Note that the memory device can be composed of ROM (Read Only Memory), RAM (Random Access Memory), or the like. The auxiliary storage device is storage means such as a hard disk, and can store the execution program according to the present disclosure, control programs provided in the computer, and the like, and can input/output them as necessary. Various information required during program execution can be obtained from an auxiliary storage device, and execution results can be stored. The execution program may be downloaded in whole or in part from an external device via any network when necessary. Furthermore, the external device may be provided with an auxiliary storage device in which the execution program is installed and/or a memory device for storing the installed execution program. The external device may be a server. The server may be a specific server, or may be an indefinite server such as a cloud infrastructure. Furthermore, the execution program may be provided by a subscription type service. A subscription-type service is one of the usage forms of computer software, and is a service in which fees are paid according to the period of use of the software. In this way, the execution program may be used in any form and is not limited.

[Confirmation of improved stability of dermal stem cells by application of high frequency]
An experiment conducted by the inventors to confirm suppression, reduction, and improvement of spots by applying high frequency will be described below.

[1-1 Culture of epidermal cells]
Normal human epidermal melanocytes (Kurabo) were cultured in medium Medium 254 (Thermo Fisher Scientific) supplemented with human melanocyte growth supplement (HMGS-2). Normal human epidermal keratinocytes (Kurabo) were cultured in Epilife® medium (Thermo Fisher Scientific) containing 60 μM calcium supplemented with keratinocyte growth supplement (EDGS).

[1-2 Culture of fibroblasts (Fb) and PUVA treatment]
Normal human fibroblasts (Cell Research Corp) were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum (hereinafter referred to as 10% DMEM), and seeded in T25 flasks at 3×10 ⁵ cells. Before the cell density reached 100% confluency, the cells were replaced with 10% DMEM containing a photosensitizer, psoralen (final concentration 25 ng/mL), and cultured. After 24 hours, the tissue was swollen with 4 mL of PBS, removed, replaced with 1 mL of PBS containing psoralen (final concentration: 25 ng/mL), and irradiated with 6 J/cm ² UVA (hereinafter referred to as PUVA treatment). Then, after swelling with 4 mL of PBS, the PBS was removed and replaced with 10% DMEM, followed by culturing for 3 days, and the culture supernatant and cells were collected. PUVA-untreated fibroblasts from the same donor served as a control for the PUVA-treated fibroblasts.

[1-3 RF irradiation]
1×10 ⁵ cells of the fibroblasts cultured by the above method after 3 days of PUVA treatment or untreated were seeded on culture jigs equipped with electrodes. Before the cell density reaches 80% confluency, two types of high-frequency equipment ([RF+] frequency: 1 MHz / voltage level: 70 Vpp / output pattern: apply RF for 4 seconds ON / 4 seconds OFF, [RF++] frequency: 950 kHz / Voltage level: 168 Vpp/output pattern: 1 second ON/2 seconds OFF RF applied) were connected to the electrodes of the jig, and RF stimulation was applied for 3 minutes each. After culturing for 3 days, the culture supernatant and cells were collected.

[1-4 Cell Viability Assay]
2 ml of DMEM supplemented with 10% Alamar Blue reagent (Thermo Fisher Scientific Inc.) was added directly to each jig in which fibroblasts were cultured, and the cells were further cultured at 37° C. for 4 hours in the dark. After culturing, the medium was recovered and measured at OD570 nm and OD600 nm to measure the intensity of fluorescence emitted by the cells.

[1-5 Creation of three-dimensional culture models of epidermal cells and fibroblasts]
Three days after PUVA treatment, or untreated fibroblasts stimulated with RF were seeded in 24-well plates at 3×10 ⁴ cells and cultured for about 24 hours to adhere the cells. A three-dimensional epidermal model was created by seeding and culturing epidermal cells in a 24-well culture insert. Keratinocytes and melanocytes were seeded on the culture inserts at a ratio of 10:1 to form an epidermal layer. Keratinocytes and melanocytes were seeded at densities of 1×10 ⁵ cells/cm ² and 1×10 ⁶ cells/cm ² , respectively. The cells were cultured for 3 days, and when the cells became confluent, they were cultured by exposing the epidermal layer to the gas phase and bringing the culture medium into contact with the outside of the insert membrane. At the start of the liquid phase culture of the epidermis model, the culture inserts were transferred onto fibroblasts pre-seeded in 24-well plates and co-cultured for 14 days (see also the description of WO2020/111265, which is incorporated herein by reference). . After culturing, the color tone of the model was evaluated visually and by melanin quantification. A 1:1:1 mixture of 10% DMEM fibroblast culture medium, M254 melanocyte culture medium, and EpiLife keratinocyte culture medium was used to culture the three-dimensional skin model.

[1-6 Immunoantibody staining]
The medium was removed from the cell-seeded chamber slide, washed with PBS, added with 4% paraformaldehyde (Wako), and allowed to stand at room temperature for about 1 hour to fix the cells. Then, after washing three times with PBS, 1 mL of a blocking agent for immunological experiments, Immunoblock (registered trademark) (DS Pharma Biomedical Co., Ltd.) was added per well, and the plate was allowed to stand at room temperature for 1 hour. Then, after washing with PBS three times, the primary antibody p21 (Abcam) was diluted 1:100 with a diluent (1% BSA-PBS), added to the fixed cells, and allowed to stand overnight at 4°C. After washing with PBS three times, the secondary antibody Alexa Fluor (registered trademark) 488 (Abcam) was diluted at a ratio of 1:1000, added to the fixed cells, and allowed to stand at room temperature for 1 hour. After washing again with PBS three times, add 500 μl of 0.1% Hoechst (registered trademark) 33342 (Thermo Fisher Scientific Inc.)-PBS solution and incubate at 37°C, 5% CO ₂ for 15 minutes to stain cell nuclei. bottom. After that, the cells were washed with PBS three times, and fluorescence observation and imaging were performed using a microscope. Immunofluorescent staining results were observed with an LSM700 laser scanning confocal microscope and images were acquired with Zen2011 software (Carl Zeiss, Thornwood, NY, USA). Image analysis software ImageJ (NIH) was used to quantify the ratio of p21-positive cells from the photographs taken. Representative pictures are shown as results after four independent experiments.

[1-7 RT-PCR]
Total RNA was extracted using the RNeasy Mini Kit (Qiagen). 200 ng of total RNA was reverse transcribed using PrimeScript RT Reagent Kit (Takara). PCR was performed using the PrimeScript RT-PCR Kit (Takara). Gene expression of MITF, TYR, TYRP1 and DCT in keratinocytes after fibroblast treatment was analyzed by RT-PCR assay. PCR results were obtained from three independent experiments. The sequences of the PCR primers are shown below.

[1-8 Measurement of intracellular melanin content]
Melanocytes or three-dimensional skin models were collected and centrifuged (4°C, 2,300g, 5min). 120 μl of 0.2N NaOH was added to the resulting cell mass and boiled in hot water at 80° C. for 30 minutes to extract intracellular melanin. The obtained supernatant was measured at OD405 nm, and this was defined as the amount of melanin contained in cells. The amount of melanin was quantified with a calibration curve obtained with synthetic melanin (Sigma Aldrich, St. Louis, MO, USA) and then standardized by the amount of protein. Results were obtained from three independent experiments.

[1-9 Ex vivo fresh skin PUVA treatment and temperature distribution measurement]
Fresh skin (NSA11) was purchased from Genoskin. 1 mL of PBS containing a photosensitizer/psoralen (final concentration: 0.5 ug/mL) was added to the top of the tube and allowed to stand in an incubator for 30 minutes, after which the solution was removed with a cotton swab. After that, 6 J/cm ² UVA irradiation (hereinafter referred to as PUVA treatment) was performed, followed by further incubation for 24 hours. For RF irradiation (RF+: frequency 1MHz, voltage 70Vpp, RF++: frequency 950kHz, voltage 168Vpp), two types of high-frequency devices are connected and electrodes with a width of 3mm are used to stimulate from the top of the skin for 3 minutes. was added. During the irradiation, a thermography camera (Optris) was used to take pictures, and a temperature distribution image was acquired using the attached software PIX Connect.

[1-10 Preparation and staining of tissue sections]
Immediately after RF irradiation, 70% 1,3-BG was added to the top and further incubated for 24 hours. After repeating this RF irradiation and addition of 70% 1,3-BG for 3 days, skin samples were collected.
Skin samples were fixed in 4% PFA at 4°C overnight and embedded in paraffin. Paraffin sections (5 μm) were prepared, deparaffinized, and Fontana-Masson stained. After being immersed in Fontana ammonia silver solution (Muto Kagaku) for 1 hour, it was immersed in 0.2% gold chloride (Muto Kagaku) for 5 seconds to dye melanin. After encapsulation, photography and virtualization were performed to confirm changes in melanin localization and amount depending on the presence or absence of stimulation.
Changes in the number of senescent fibroblasts induced by RF irradiation in PUVA-treated fresh skin (NSA11) were evaluated by immunofluorescence staining. Deparaffinized slides were subjected to antigen retrieval and permeabilization. After washing with TBS three times, 10% goat serum (Nichirei Biosciences) was added and blocking was performed at room temperature for 1 hour. After three more washes with TBS, the primary antibodies p21 (Abcam) and Vimentin (Abcam) were diluted 1:100 in diluent (1% BSA-TBS), added onto the slides and allowed to stand at room temperature for 1 hour. placed. After three TBS washes, secondary antibodies Alexa Fluor® 488 and Alexa Fluor® 594 (Abcam) were diluted 1:1000 and added onto slides for 1 hour at room temperature. left undisturbed. After washing again with TBS three times, the cells were mounted with a DAPI-containing mounting medium, Fluoro-KEEPER Antifade Reagent (Nacalai Tesque, Inc.), and fluorescence observation and imaging were performed with a microscope.
Immunofluorescent staining results were observed with an LSM700 laser scanning confocal microscope and images were acquired with Zen2011 software (Carl Zeiss, Thornwood, NY, USA). Representative pictures are shown as results after four independent experiments.

[1-11 Statistical processing]
Statistical processing was performed by one-dimensional ANOVA using Graph Pad Prism 5 (Graph Pad Software, La Jolla, Calif.). When p<0.05, it was determined that there was a statistically significant difference, and the degree was indicated by the following asterisk notation. (*p<0.05, **p<0.01, ***p<0.001) All experiments were repeated three times or more to confirm reproducibility.

[2-1 RF irradiation reduces aging Fb proliferation in the dermis, which is one of the root causes of age spots]
Three days after the PUVA treatment, 1×10 ⁵ cells of fibroblasts were seeded on a culture jig equipped with electrodes. Before the cell density reaches 80% confluency, two types of high-frequency devices (RF+: frequency 1MHz, voltage 70Vpp, RF++: frequency 950kHz, voltage 168Vpp) are connected to the electrodes of the jig, and RF stimulation is performed for 3 minutes each. added. Figures 2-1 and 2-2 show the results of examining cells after 3 days of culture. As a result, it was clarified that RF irradiation reduces the proliferation of senescent fibroblasts in the dermis, which is one of the root causes of age spots.

[2-2 RF promotes an increase in normal Fb]
PUVA-untreated fibroblasts were seeded at 1×10 ⁵ cells on a culture jig equipped with electrodes. Before the cell density reaches 80% confluency, two types of high-frequency devices (RF+: frequency 1MHz, voltage 70Vpp, RF++: frequency 950kHz, voltage 168Vpp) are connected to the electrodes of the jig, and RF stimulation is performed for 3 minutes each. added. Figures 3-1 and 3-2 show the results of examining cells after 3 days of culture. As a result, it was revealed that RF promotes the increase of normal fibroblasts (RF selectively removes only senescent Fb).

[2-3 RF irradiation is mainly involved in the removal of stagnant Fb due to accelerated aging]
Three days after the PUVA treatment, 1×10 ⁵ cells of fibroblasts were seeded on a culture jig equipped with electrodes. Before the cell density reaches 80% confluency, two types of high-frequency devices (RF+: frequency 1MHz, voltage 70Vpp, RF++: frequency 950kHz, voltage 168Vpp) are connected to the electrodes of the jig, and RF stimulation is performed for 3 minutes each. added. Figures 4-1 and 4-2 show the results of examining cells after 3 days of culture. Image analysis software ImageJ (NIH) was used to quantify the ratio of p21-positive cells from the photographs of the cells. As a result, it was clarified that RF irradiation is mainly involved in the removal of stagnant fibroblasts (p21-positive cells) due to accelerated senescence.

[2-4 RF irradiation inhibits pigment formation through the phenomenon of aging Fb]
RF-stimulated fibroblasts 3 days after PUVA treatment were co-cultured with epidermal models for 14 days. After culturing, the color tone of the model was evaluated visually and by melanin quantification. As a result, it became clear that the amount of melanin decreased due to the RF treatment (Fig. 5-1). In addition, RT-PCR analysis of gene expression of TYR, TYRP1, DCT, and MITF revealed that gene expression of TYR, TYRP1, and DCT was significantly reduced by RF treatment (Fig. 5-2).

[2-5 RF irradiation inhibits pigmentation by approaching senescent Fb, whereas RF irradiation induces proliferation of normal Fb, but does not contribute to pigmentation alone]
RF-stimulated fibroblasts 3 days after PUVA treatment or untreated were co-cultured with epidermal models for 14 days. After culturing, the color tone of the model was evaluated by visual observation and melanin quantification. As a result, the amount of melanin in PUVA-treated senescent Fb cells decreased due to RF treatment, but in normal Fb cells not treated with PUVA, the amount of melanin due to RF treatment decreased. It was found that no effect occurred (Figures 6-1 and 6-2).

[2-6 Temperature distribution of ex vivo fresh skin by RF irradiation]
Fresh skin (NSA11) was purchased from Genoskin. 1 mL of PBS containing a photosensitizer/psoralen (final concentration: 0.5 ug/mL) was added to the top of the tube and allowed to stand in an incubator for 30 minutes, after which the solution was removed with a cotton swab. After that, 6 J/cm ² UVA irradiation (hereinafter referred to as PUVA treatment) was performed, followed by further incubation for 24 hours. For RF irradiation (RF+: frequency 1MHz, voltage 70Vpp, RF++: frequency 950kHz, voltage 168Vpp), two types of high-frequency devices are connected and electrodes with a width of 3mm are used to stimulate from the top of the skin for 3 minutes. was added. During the irradiation, a thermography camera (Optris) was used to take pictures, and temperature distribution images were acquired using the attached software PIX Connect (Figs. 7-1 and 7-2). It was found that both RF+ and RF++ remained below 40°C without excessive temperature rise even after 3 minutes of continuous stimulation.

[2-10 RF irradiation suppresses blackening effect]
Immediately after RF irradiation, 70% 1,3-BG was added to the top and further incubated for 24 hours. After repeating this RF irradiation and addition of 70% 1,3-BG for 3 days, skin samples were collected. The color tone of the skin samples was visually observed (Fig. 8). Blackening suppression and whitening effects were observed with RF irradiation.

[2-10 RF irradiation decreases the abundance ratio of aging Fb (p21 ⁺ Fb)]
Skin samples were fixed in 4% PFA at 4°C overnight and embedded in paraffin. Paraffin sections (5 μm) were prepared, deparaffinized, and Fontana-Masson stained. After being immersed in Fontana ammonia silver solution (Muto Kagaku) for 1 hour, it was immersed in 0.2% gold chloride (Muto Kagaku) for 5 seconds to dye melanin. After encapsulation, photographing and virtualization were performed to confirm changes in melanin localization and amount depending on the presence or absence of stimulation (Figs. 9-1 and 9-2). PUVA treatment induced fibroblast senescence (Fig. 9-1). Subsequent RF irradiation reduced the number of senescent fibroblasts (Fig. 9-2).

Immunofluorescence staining was performed to demonstrate the number of senescent fibroblasts by PUVA treatment. Deparaffinized slides were subjected to antigen retrieval and permeabilization. After washing with TBS three times, 10% goat serum (Nichirei Biosciences) was added and blocking was performed at room temperature for 1 hour. After three more washes with TBS, the primary antibodies p21 (Abcam) and Vimentin (Abcam) were diluted 1:100 in diluent (1% BSA-TBS), added onto the slides and allowed to stand at room temperature for 1 hour. placed. After three TBS washes, secondary antibodies Alexa Fluor® 488 and Alexa Fluor® 594 (Abcam) were diluted 1:1000 and added onto slides for 1 hour at room temperature. left undisturbed. After washing again with TBS three times, the cells were mounted with a DAPI-containing mounting medium, Fluoro-KEEPER Antifade Reagent (Nacalai Tesque, Inc.), and fluorescence observation and imaging were performed with a microscope.
Immunofluorescence staining results were observed with an LSM700 laser scanning confocal microscope and images were acquired with Zen2011 software (Carl Zeiss, Thornwood, NY, USA) (Fig. 10). A decrease in senescent fibroblasts was observed with RF irradiation.

[Summary of experimental results]
From the results of the above experiments, it was clarified that the application of high frequency waves is effective in suppressing and improving blemishes, that is, in whitening the skin, and is particularly effective against aged cells.

1 high-frequency device 10 electrode section 10a first electrode 10b second electrode 20 control circuit

Claims

A cosmetic method that includes applying high frequency waves to the skin to suppress or reduce blemishes or to whiten the skin.
A cosmetic method that involves applying high frequency waves to the skin to suppress pigmentation.
The cosmetic method according to claim 1 or 2, wherein the frequency of the high frequency is approximately 1 MHz.
The cosmetic method according to claim 1 or 2, wherein the high-frequency peak-to-peak voltage is 50 Vpp or more.
The cosmetic method according to claim 4, wherein the high-frequency peak-to-peak voltage is 50 to 200 Vpp.
The cosmetic method according to claim 1 or 2, wherein the application of high frequency is pulse application with a duty ratio of 10% to 99%.
The cosmetic method according to claim 6, wherein the pulse application period is 0.1 seconds to 10 seconds.
The cosmetic method according to claim 1 or 2, wherein the suppression or reduction of age spots, whitening, or suppression of pigmentation is due to a decrease in the number of aged fibroblasts and/or an increase in the number of normal fibroblasts.
The cosmetic method according to claim 8, wherein the aged fibroblasts are p21-positive cells.
The cosmetic method according to claim 1 or 2, which improves skin spots.
The cosmetic method according to claim 1 or 2, wherein the high frequency is noninvasively applied to the skin surface.
The cosmetic method according to claim 1 or 2, wherein the skin temperature rise is less than 40°C.
A method of inhibiting pigmentation, including the step of applying radio frequency to the skin.
A method of reducing the number of aged fibroblasts and/or increasing the number of normal fibroblasts comprising applying radiofrequency to the skin.
A method of operating a high-frequency cosmetic treatment apparatus having a pair of electrodes and a control circuit for applying a high-frequency voltage to the pair of electrodes, comprising:
applying, by the control circuit, to the pair of electrodes a high-frequency voltage that suppresses pigmentation in the skin;
The high-frequency voltage is applied with the pair of electrodes in contact with the skin,
A method of operating a high frequency cosmetic treatment device.
The operating method according to claim 15, wherein the high-frequency peak-to-peak voltage is 50 Vpp or more.
The operating method according to claim 16, wherein the high-frequency peak-to-peak voltage is 50-200 Vpp.
The operating method according to claim 15, wherein the application of the high-frequency voltage is pulse application with a period of 0.1 to 10 seconds and a duty ratio of 10 to 99%.
A computer program that causes a device comprising a pair of electrodes and a control circuit that applies a high-frequency voltage to the pair of electrodes to execute the operating method according to claim 15.
A non-transitory computer-readable medium having instructions stored thereon, the computer-readable medium being capable of executing a computer program according to claim 19 when the instructions are executed by a processor.
A radio frequency cosmetic treatment device for use in a cosmetic method for inhibiting pigmentation in skin comprising applying radio frequency to the skin, comprising:
Having a pair of electrodes and a control circuit that applies a high frequency voltage to the pair of electrodes,
A radiofrequency cosmetic treatment device configured to apply a radiofrequency voltage to the skin to inhibit skin pigmentation.