WO2021215411A1

WO2021215411A1 - Skin anti-aging agent, beautification method for skin anti-aging, and beautification instrument for use in method

Info

Publication number: WO2021215411A1
Application number: PCT/JP2021/015926
Authority: WO
Inventors: 聡堀場; 大悟井上; 曽我部　敦; 智哉内山; 基治青木; 大谷　毅
Original assignee: 株式会社資生堂
Priority date: 2020-04-20
Filing date: 2021-04-19
Publication date: 2021-10-28
Also published as: CN115427009A; JPWO2021215411A1

Abstract

The present invention provides a skin anti-aging agent that includes arabinoxylan as an active ingredient, the skin anti-aging agent being characterized by increasing the number of cells having nuclear localized YAP that decreases upon receipt of physical stimulation on skin, and/or the number of cells having actin stress fibers. The present invention also provides: a beautification method that includes a step for applying, on the skin of a subject, the skin anti-aging agent containing arabinoxylan as an active ingredient; and a beautification instrument employed in the method.

Description

Skin anti-aging agents, cosmetological methods for skin anti-aging and cosmetological devices for use in those methods

The present invention relates to a skin anti-aging agent, a cosmetological method for skin anti-aging, and a cosmetological device for applying to the method.

Skin aging is a change in the skin mediated by various factors including internal factors such as aging, hormones and metabolism, and external factors such as ultraviolet rays and dryness. Skin aging may be judged from the appearance such as spots, wrinkles, sagging, and deflation, but invisible aging such as changes in the structure and constituents of the internal tissues of the skin may be progressing. It is difficult to take preventive measures because such aging often progresses without being noticed. It is important to take appropriate measures to prevent and improve skin aging.

Here, there are currently various beauty methods. For example, Patent Document 1 discloses an electroactive material actuator that causes displacement of a keratinous substance. Patent Document 2 discloses a light irradiation beauty device that irradiates light with the skin stretched. Patent Document 3 discloses a slimming method for reducing subcutaneous fat by combining a stretch that stretches subcutaneous adipocytes and an external skin preparation containing an adipocyte accumulation inhibitor and / or an adipocyte differentiation inhibitor. Patent Document 4 discloses an extension-stimulating-mediated collagen-degrading enzyme production inhibitor containing an extract of a plant of the genus Oat or an extract of a plant of the genus Psoralea. Patent Document 5 discloses a wrinkle improving agent or an NF-κB activity inhibitor caused by extension stress. Non-Patent Document 1 recommends mechanical stimulation at 65 to 85 Hz. Further, Patent Document 6 related to the apparatus described in Non-Patent Document 1 discloses a terminal effector that stimulates the skin at a frequency of about 65 to about 120 Hz.

However, when stimulating from the skin surface, the stimulus is first applied to the epidermis, which is the surface layer, so there is a problem that the epidermis is damaged. In addition, there is also a structurally very characteristic basement membrane just below the epidermis (undulation structure), and it is highly likely that it will receive mechanical stimuli. It has also been reported that the surface layer is significantly changed by actually giving a physical stimulus according to the prior literature (Non-Patent Document 2). Therefore, there is a need for skin anti-aging agents, methods and devices that have a favorable anti-aging effect on the skin including the epidermis and basement membrane.

Japanese Patent Application Laid-Open No. 2011-505897 Japanese Unexamined Patent Publication No. 2009-28271 Japanese Unexamined Patent Publication No. 2015-127315 Japanese Unexamined Patent Publication No. 2011-162507 Japanese Unexamined Patent Publication No. 2014-108924 Special Table 2018-501000

An object of the present invention is to provide a skin anti-aging agent, a cosmetological method for skin anti-aging, and a cosmetological device for applying to the method.

As a result of diligent research, the present inventors have conducted a physical stimulus on the skin when a composition containing arabinoxylan is applied to the skin or when a physical stimulus such as extension is applied to the skin at a specific extension rate and frequency. It was discovered that the number of cells having a decreasing nuclear localization YAP and / or a cell having an actin stress fiber is increased to exert a skin anti-aging effect, and the present invention was conceived.
That is, the present application provides the following inventions:
[1] A skin anti-aging agent containing arabinoxylan as an active ingredient.
A skin anti-aging agent characterized by increasing cells with nuclear localized YAP and / or actin stress fibers that are reduced by physical stimulation in the skin.
[2] The skin anti-aging agent according to item 1, wherein the arabinoxylan is derived from rye.
[3] The skin anti-aging agent according to

item

1 or 2, wherein the arabinoxylan is derived from Kohiris ™ GR.
[4] A cosmetic method for skin anti-aging that increases cells having nuclear-localized YAP and / or actin stress fibers that decrease due to physical stimulation in the skin.
(A) Applying the skin anti-aging agent according to any one of items 1 to 3 to the target skin,
Cosmetology methods, including.
[5] The cosmetological method further includes a step of applying a weak physical stimulus to the skin, wherein the step of applying the physical stimulus is, for example, for example.
(B-1) The target skin is stretched to a stretch rate of 0.1% or more and 50.0% or less, where the stretch rate is defined as.

(In the equation, the fixed points A and B are arbitrary positions on the epidermis or the matrix to which the epidermis is adhered, and the straight line passing through the fixed points A and B is parallel to the extension direction).
And (c-1) recovering the subject's skin from the stretched state;
And / or
(B-2) Pressing the subject's skin by 1 μm to 1000 μm; and (c-2) Recovering the subject's skin from the pressed state;
Including
Here, the cycles (b-1) and (c-1) and / or the cycles (b-2) and (c-2) are carried out at a frequency of 60 Hz or less.
4. The cosmetology method according to item 4.
[6] The beauty method according to item 5, wherein one or a plurality of sets are performed by performing the cycle for 2 to 500 cycles.
[7] When the skin anti-aging is measured using the ratio of the number of cells having nuclear localization YAP to the total number of keratinized cells as an index, the nucleus measured in the extended keratinized cells after applying the cosmetic method to the skin. The proportion of cells having localized YAP is increased as compared with before the application of the cosmetic method, and here, the proportion of cells having localized YAP is determined.

The beauty method according to any one of items 4 to 6, which is calculated in 1.
[8] When the skin anti-aging is measured using the ratio of the number of cells having actin stress fibers to the total number of keratinized cells as an index, actin stress measured in extended keratinized cells after applying the cosmetic method to the skin. The proportion of cells with fibers increases, where the proportion of cells with actin stress fibers is

The beauty method according to any one of items 4 to 6, which is calculated in 1.
[9] A cosmetological device for applying to the cosmetological method according to item 5 or 6.
The device is
The stimulus generator that causes physical stimulus and
It is equipped with a stimulus-giving part that gives the physical stimulus generated in the stimulus-generating part to the skin.
Here, the device is a device for performing a step of applying a weak physical stimulus to the skin, and the step is described here, for example.
(I-1) Stretching the skin to a stretch rate of 0.1% or more and 50.0% or less; and (ii-1) Restoring the skin from the stretched state;
And / or
A cycle including (i-2) pressing the skin by 1 μm to 1000 μm; and (ii-2) recovering the skin from the pressed state; is performed at a frequency of 60 Hz or less.
Here, the extension rate is calculated by the above formula 1, the beauty device.
[10] A cosmetological method for skin anti-aging, which comprises applying the cosmetological device according to item 9 to the target skin.
[11]
The cosmetological method according to any one of items 4 to 8 and 10, for increasing hyaluronic acid in the skin.
[12] Presenting to the subject the skin anti-aging agent according to any one of items 1 to 3, the cosmetological method according to any one of items 4 to 8 and 10, or the cosmetological device according to item 9. Cosmetology counseling methods that support the subject's cosmetology, including.

According to the present invention, a skin anti-aging agent that increases cells having nuclear-localized YAP and / or cells having actin stress fibers, which are reduced by physical stimulation in the skin, and exerts a preferable skin anti-aging effect on a shallow layer such as the epidermis. Beauty methods and beauty devices for use in those methods are provided. As a result, prevention and improvement of skin aging such as age spots, wrinkles, and sagging are expected.

FIG. 1 shows the setting of the extension stimulus performed in

Experiments

1 and 3. FIG. 2a shows the localization of YAP (red) and actin (green) in a monolayer culture of keratinocytes obtained from a 44-year-old donor in Experiment 1 without stretching stimulus and immediately after giving. FIG. 2b shows YAP (red) and actin in a state in which the monolayer culture of keratinocytes obtained from donors (0 years old, 18 years old, 64 years old) of each age in Experiment 1 was not stimulated for extension and immediately after the application. (Green) indicates localization. FIG. 3 shows the number of cells having nuclear localization YAP immediately after the keratinocyte culture obtained from donors (0 years old, 18 years old, 62 years old) of each age in Experiment 1 without extension stimulation. Shows the percentage. In the figure, * indicates that there is a significant difference by the two-sided t-test (when the variances of the two populations are equal) (p <0.05%). FIG. 4 shows the ratio of the number of cells having actin stress fibers immediately after the keratinized cell culture obtained from donors (0 years old, 18 years old, 62 years old) of each age in Experiment 1 without the extension stimulation. Is shown. In the figure, * indicates that there is a significant difference by the two-sided t-test (when the variances of the two populations are equal) (p <0.05%). FIG. 5 shows YAP (red), actin (green), and actin (green) in the state in which the culture of fibroblasts obtained from donors (0 years old, 68 years old) of each age in Experiment 1 was not stimulated for extension and immediately after the culture was given. Shows the localization of the nucleus (blue). FIG. 6 shows the apparatus used in Experiment 2 and an ex vivo skin sample. FIG. 7 shows the setting of the extension stimulus performed in

Experiments

2, 4 and 5. FIG. 8 shows the localization of YAP (green) and nucleus (blue) immediately after the skin sample obtained from a 44-year-old donor in Experiment 2 without pressing stimulation. FIG. 9 shows a condition in which a monolayer culture of keratinocytes obtained from a 44-year-old donor in Experiment 3 was not given a stretch stimulus, a condition in which one stretch stimulus was given, and a condition in which two stretch stimuli were given. After that, the localization of YAP and BrdU after normal culture for 1 week is shown. FIG. 10 shows the device and skin sample used in Experiment 4. In FIG. 11, two skin samples (samples # 1 and # 2) obtained from a 44-year-old donor in Experiment 4 were subjected to normal culture for one week after a single extension stimulus was not applied and under the conditions. Later, the localization of YAP (green), Ki67 (red) and nucleus (blue) is shown. In FIG. 12, two skin samples (samples # 1 and # 2) obtained from a 44-year-old donor in Experiment 4 were subjected to normal culture for one week after being given two stretch stimuli without and under the conditions. Later, the localization of YAP (green), Ki67 (red) and nucleus (blue) is shown. In FIG. 13, two skin samples (samples # 1 and # 2) obtained from a 44-year-old donor in Experiment 5 were subjected to normal culture for one week after being given two stretch stimuli without and under the conditions. Later, hyaluronic acid (green), collagen (red), and nucleus (blue) are shown. FIG. 14 shows an example of the apparatus of the present invention. FIG. 15 shows immediately after adding 0.3% by weight and 1.0% by weight of Kohiris ™ GR to the monolayer culture obtained from a 44-year-old donor in Experiment 6 and giving a single extension stimulus. The localization of YAP (red) and actin (green) is shown (top). The lower left of FIG. 15 shows the ratio of the number of cells having nuclear localization YAP. The lower right of FIG. 15 shows the ratio of the number of cells having actin stress fibers. In the figure, *** indicates that there is a significant difference due to the two-sided t-test (when the variances of the two populations are equal) (p <0.05%). FIG. 16a shows the measurement results by the vaporometer and the corneometer in Experiment 7. FIG. 16b shows the R0 value by the cute meter in Experiment 7. In FIG. 16ab, * indicates that there is a significant difference from the value on the 0th day in Dunnett's test (***: p <0.005, **: p <0.01, *: p <0. 05). FIG. 16c shows the results of subjective evaluation in Experiment 7. In FIG. 16c, * indicates that there is a significant difference from the value on day 0 in the Wilcoxon signed rank test (***: p <0.005, **: p <0.01, *: p < 0.05). The upper left of FIG. 17 shows the setting of Experiment 8. The upper right of FIG. 17 shows an example (rectangular wave) of the waveform of the pressing stimulus of Experiment 8. The lower part of FIG. 17 shows the conditions of the pressing stimulus of Experiment 8 (waveform: Shape of wave, frequency: Frequency / Hz, depth: Displacement / μm, period: Day). FIG. 18 shows a condition in which no pressing stimulus was applied to two skin samples (samples # 1 and # 2) obtained from a 44-year-old donor in Experiment 8 (Cont) and a condition in which a square wave pressing stimulus was applied (0.5 Hz / The localization of Ki67 (green) and nucleus (blue) on the 1st day (d1) and the 5th day (d5) at 100 μm) is shown. FIG. 19 shows one day under the conditions (Cont) in which a single pressing stimulus was not applied to the skin sample obtained from a 44-year-old donor in Experiment 8 and the conditions (square wave: 0.5 Hz, 5 Hz, 50 Hz / 100 μm). The ratio of the number of cells showing Ki67 positive to the total number of cells of the eyes (left) and the 5th day (right) is shown. The upper part of FIG. 20 shows the setting of Experiment 9, and the lower part shows the waveform (rectangular wave) of the adopted pressing stimulus. FIG. 21 shows the results of Experiment 9. In each figure, the left is the result when the pressing stimulus is applied (apply), and the right is the result when the pressing stimulus is not applied (control) on the 0th and 7th days. Is shown. FIG. 21a shows the measurement result by the corneometer. FIG. 21b shows the measurement result by the vapor meter. FIG. 21c shows the R0 value by the cute meter. In FIG. 21c, * indicates that there is a significant difference from the value on the 0th day in Dunnett's test (*: p <0.05).

Currently, there are many beauty methods that target layers deeper than the dermis, such as those that aim to strengthen facial muscles, promote fat decomposition, and strengthen dermis fibers. Further, for example, although there is a beauty method focusing on keratin substances as in Patent Document 1, there are many methods by electrical stimulation and strong stimulation that gives a high frequency. However, as described above, there is a problem that the epidermis is damaged when the skin is stimulated from the surface of the skin. Furthermore, as described in

Patent Documents

4 and 5, there is a finding that the extension stimulus has an adverse effect on the skin. In fact, in Non-Patent Document 2, the surface layer is significantly changed by the extension stimulus. Has been reported.

Here, the present inventors focused on the surface layer of the skin and conducted diligent research. As a result, it was discovered that the behavior of YAP in keratinocytes and the structure of actin fibers are related to aging. For example, when a physical stimulus such as extension was applied to the skin, the proportion of cells with nuclear-localized YAP and cells with actin stress fibers decreased significantly in aging keratinocytes. Furthermore, the present inventors searched for a new skin anti-aging agent and a method for skin anti-aging by using the behavior of YAP and the structure of actin fiber as an index of skin aging, and found a composition containing arabinoxylan. It was also found that when applied to the target skin or when a specific physical stimulus was applied to the target skin, a favorable effect was exerted on the skin including the epidermis. More specifically, even aged epidermis has nuclear-localized YAP and / or actin stress fibers characteristic of aging keratinocytes by applying a skin anti-aging agent containing arabinoxylan as an active ingredient. As a result, the effect of suppressing the decrease in the proportion of cell numbers was suggested. Even in the case of aged epidermis, when a weak physical stimulus is given, for example, a physical stimulus performed at a specific extension rate and a frequency of 60 Hz or less such as 1 Hz or less and 10 Hz or less, it is characteristic of aging keratinocytes. It was suggested that the effect of suppressing the decrease in the proportion of cells having nuclear-localized YAP and / or actin stress fibers was suggested. Furthermore, it was found that such physical stimulation increases the amount of hyaluronic acid not only in the epidermis but also in the dermis, demonstrating that it is effective in preventing and improving skin aging.

Skin anti-aging may be determined by measuring the ratio of the number of keratinized cells having nuclear localized YAP to the total number of keratinized cells. The presence or absence of nuclear localized YAP is determined by staining intracellular YAP, observing and imaging the endogenous YAP in the nucleus or cytoplasm with a microscope, and using image processing analysis software. It can be determined by quantifying the ratio of YAP to intracellular YAP by the degree of fluorescence intensity. Alternatively, it can be measured by the method described in Non-Patent Document 6 and Non-Patent Document 7. The ratio of the number of keratinized cells having nuclear localization YAP to the total number of keratinized cells is calculated by the above formula 2. For example, the number of cells determined to have nuclear localization YAP by the above method is counted, and the cells whose nuclei are stained by nuclear staining such as DAPI or Hoechst33342 are counted as the total number of cells, and the formula 2 It may be calculated by.

In one embodiment, the nuclear localization of YAP is that the endogenous YAP in keratinocytes is localized in the nucleus as a transcriptional conjugating factor, with the increase or decrease in the total expression level remaining within a range of 2 times or less before and after the implementation. Point to. From Non-Patent Documents 3 to 5, even if the inhibitor of YAP is deleted and the nuclear localization of YAP is promoted, the expression level is not significantly increased at the protein level (for example, in Non-Patent Document 5, YAP This is because it has been confirmed that the amount of protein in the above is less than twice on average).

YAP is a Yes-associated protein, which is a protein of about 65 kDa, which is sometimes called YAP1, YAP65, or the like. It has been reported that YAP functions as a transcriptional regulator, is involved in cell proliferation and apoptosis, is associated with the maintenance and establishment of stem cells (nitch), and is associated with the development of tumors and cancers. YAP is degraded when it is present in the cytoplasm, but when it translocates into the nucleus, it is activated and regulates transcription. It has also been reported that such localization of YAP is associated with extracellular matrix hardness, cell density, cell proliferation and self-renewal (Non-Patent Documents 11 to 13).

It is also known that stress fiber formation in the cytoplasm of actin, which is an upstream factor of YAP, is correlated and actively involved in YAP nuclear localization. Therefore, the skin anti-aging of the present invention may be determined using the presence or absence of actin stress fibers in keratinized cells as an index. For example, skin anti-aging may be determined by measuring the ratio of actin stress fiber-bearing keratinized cells to total keratinized cells. The presence or absence of actin stress fibers is determined by staining actin fibers with phalloidin cross-linked with a fluorescent compound and containing at least one actin stress fibers present in the cytoplasm or around the nucleus, as described in Examples. Can be determined to have. Alternatively, it can be measured by the method described in Non-Patent Document 9 or Non-Patent Document 10. The ratio of the number of keratinized cells having actin stress fibers to the total number of keratinized cells is calculated by the above formula 3. For example, the number of cells determined to have actin stress fibers by the method as described above may be counted, and the total number of cells may be counted by DAPI staining as described above and calculated by the formula 3.

In one embodiment, the keratinized cell having actin stress fiber refers to the keratinized cell in which actin stress fiber is formed without increasing the expression level of actin protein. This is because, as described in Non-Patent Document 8, the amount of actin protein does not change even if the presence or absence of YAP and the position of YAP (nucleus, cytoplasm, etc.) are different.

Therefore, the skin anti-aging of the present invention refers to, for example, a decrease in the ratio of the number of cells having nuclear-localized YAP or actin stress fibers to the total number of keratinized cells in the keratinized cells after applying a stretch stimulus to a skin sample. It may also mean suppression by the method / apparatus of the invention. Suppression is, for example, suppression of the decrease in the above percentage having a statistically significant difference (eg Student's t-test) with a significance level of 5%, and / or, for example, 10% or more, 20% or more, 30% or more, The suppression may be 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

Nuclear localization YAP and actin stress fibers are known to act indirectly or directly on cell proliferation and self-renewal (Non-Patent Document 11). On the other hand, it has become clear that the non-formation of YAP localized in the nucleus or cytoplasm, YAP degraded in the cytoplasm, or actin stress fiber is involved in the mechanism of exclusion from the cell population by cell death or differentiation. (Non-Patent Document 12). Based on the findings of research on keratinocytes in the skin and various cell types, keratinocytes with phenomena such as YAP not being localized in the nucleus and actin stress fibers not being formed have low self-renewal ability and self-renewal ability. , Aging or differentiation-like cells have been suggested (Non-Patent Document 13). In addition, in the examples of the present application, the number of aging keratinocytes having a weak nuclear localization YAP retention ability or a weak actin stress fiber forming ability was significantly reduced by the method / apparatus of the present invention. Therefore, although not bound by theory, the skin anti-aging agents and methods / devices of the present invention selectively eliminate the aging keratinocytes as described above, and have weak nuclear localized YAP retention ability or actin. It was suggested that the selection of "young" keratinocytes with retained stress fiber-forming ability and subsequent reactivation and cell proliferation were promoted. That is, keratinization in which YAP is not localized in the nucleus or actin stress fibers are not formed in the elongated keratinocytes by giving the composition containing the arabinoxylan of the present invention to the skin or further giving a physical stimulus. As a result of selectively eliminating cells and selectively leaving keratinocytes localized in the nucleus and keratinocytes having actin stress fibers, it is possible that keratinocytes are proliferating or self-renewing. .. That is, the skin anti-aging agent and method / device of the present invention selectively eliminate aging keratinocytes and select keratinocytes that proliferate and self-replicate, and as a result, proliferate and recover keratinocytes. It is possible that they are urging.

Here, when measuring the number of cells having nuclear-localized YAP or actin stress fibers, it is preferable to give a physical stimulus such as a stretch stimulus to the skin sample. The physical stimulus for measuring the degree of skin aging and the skin anti-aging effect may be the same as or different from the physical stimulus used in the anti-aging method / apparatus of the present invention, and the degree of skin aging is measured. Sufficient stimulation should be achieved. For example, contact with skin samples such as living skin, post-collection skin, or cultured keratinocytes, such as tension, pressing, tapping, kneading, suction, and / or, for example, ultrasound, air pressure, or water pressure. The extension of the skin sample may be achieved by giving a physical stimulus by non-contact such as displacement by applying a shock wave to the skin, and the direction in which the physical stimulus is given is not limited.

In one embodiment, the present invention is a skin anti-aging agent comprising arabinoxylan as an active ingredient.
Provided are skin anti-aging agents characterized by increasing cells with nuclear localized YAP and / or actin stress fibers that are reduced by physical stimulation in the skin.

The arabinoxylan that can be used in the present invention is a kind of hemicellulose, which improves the elasticity of the skin, gives the skin suppleness, restores the ability of the skin to respond to physical stress, improves sagging and wrinkles, and the like. Is a known component (Non-Patent Document 14). However, it is not known that arabinoxylan increases cells having nuclear-localized YAP and / or actin stress fibers, which are reduced by physical stimulation in the skin, and the present inventors have found for the first time. Is. The arabinoxylan that can be used in the present invention may be derived from rye (preferably rye seeds), and for example, an extract of rye seeds commercially available from Silab (trade name: Kohiris ™ GR, or coheriss). (Registered trademark) (collectively referred to as "Kohiris" in the present specification)) may be derived.

The agent of the present invention may be a composition in combination with other components of the above-mentioned active ingredients, such as excipients, carriers and / or diluents. The composition and form of the composition are arbitrary, and may be appropriately selected according to the conditions such as the active ingredient and the intended use. The composition can be produced by a conventional method with a formulation appropriately combined with an excipient, a carrier and / or a diluent and the like and other components according to the dosage form.

The agent of the present invention can be blended in cosmetics or the like and used for humans and animals, or can be administered to humans and animals as a pharmaceutical preparation. In addition, it may be mixed with various foods and drinks and feeds and ingested by humans and animals.

When the present invention is applied to external preparations for skin such as cosmetics, pharmaceuticals, and quasi-drugs, the blending amount thereof can be appropriately determined according to their types, purposes, forms, usage methods, and the like. For example, when Kohiris (trademark) GR is used, the amount of Kohiris (trademark) GR is 0.00001% to 50% (v / v) (for example, 0.1 to 10%, preferably 1 to 1) in the total amount of cosmetics. 4% (v / v)) may be blended.

In addition to the above ingredients, if necessary, ingredients usually used for skin external preparations such as cosmetics, pharmaceuticals, quasi-drugs, etc., such as antioxidants, oils, UV protective agents, within the range that does not impair the effects of the present invention. , Surfactants, thickeners, alcohols, powder components, coloring materials, aqueous components, water, various skin nutrients, etc. can be appropriately blended as needed.

The external preparation for skin of the present invention can be applied as a cosmetic, a non-medicinal product, etc., which is applied to the outer skin, particularly preferably as a cosmetic, and the dosage form is not limited as long as it can be applied to the skin. Any formulation such as solution system, solubilization system, emulsification system, powder dispersion system, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, aerosol, etc. is applied.

When the agent of the present invention is used as cosmetics, it includes lotions, milky lotions, foundations, lipsticks, lip balms, cleansing creams, massage creams, packs, hand creams, hand powders, body shampoos, body lotions, body creams, bath cosmetics, etc. It may be used as a form.

However, the possible forms of the agent and composition of the present invention are not limited to the above-mentioned dosage forms and forms.

In another aspect, the invention is a cosmetic method for skin anti-aging that increases cells with nuclear localized YAP and / or actin stress fibers that are reduced by physical stimulation in the skin.
(A) Applying the above-mentioned skin anti-aging agent containing arabinoxylan as an active ingredient to the target skin,
Provide beauty methods, including.

The beauty method of the present invention in one embodiment provides a method including, in addition to the above step (a), a step of applying a weak physical stimulus to the skin. .. Further, the present invention is an apparatus for skin anti-aging, which comprises a stimulus generating portion that generates a physical stimulus and a stimulating portion that imparts a physical stimulus generated in the stimulus generating portion to the skin, which can be applied to the above-mentioned beauty method. I will provide a.

For example, the step of applying a weak physical stimulus to the skin is to (b-1) stretch the skin to a stretch rate of 0.1% or more and 50.0% or less; and (c-1) from the stretched state. The cycle including recovery; may be performed at a frequency of 60 Hz or less.

The extension rate is calculated by the above formula 1. Physical stimulation is 0.001% or more and 80.0% or less, 0.01% or more and 60.0% or less, 0.1% or more and 50.0% or less, preferably 0.1% or more and 50.0% or less. It may be done at the extension rate. For example, 0.1% or more and 1.0% or less, 0.1% or more and 5.0% or less, 0.1% or more and 10.0% or less, 0.1% or more and 20.0% or less, 0.1% 30.0% or less, 1.0% or more and 5.0% or less, 1.0% or more and 10.0% or less, 1.0% or more and 20.0% or less, 1.0% or more and 30.0% or less , 1.0% or more and 50.0% or less, 10.0% or more and 20.0% or less, 10.0% or more and 30.0% or less, and any range of extension rate can be adopted.

Extension speed refers to the speed (% / s) until the maximum extension rate (%) is reached in one cycle. The recovery rate refers to the rate (% / s) of returning from the maximum extension rate to the non-extension state. Extension speed / recovery speed is 0.010% / s to 40% / s, 0.05% / s to 30% / s, 0.10% / s to 20% / s, 0.2% / s to It may be carried out at any speed such as 15% / s and 0.3% / s to 10% / s. The extension rate and recovery rate may be the same or different.

For example, the step of applying a weak physical stimulus to the skin is (b-2) pressing the target skin by 1 μm to 1000 μm; and (c-2) recovering the target skin from the pressed state. The cycle including; may be performed at a frequency of 60 Hz or less.

"Pressing the skin from 1 μm to 1000 μm" means pressing the skin to a depth of 1 μm to 1000 μm from the outermost surface of the skin. The depth of pressing can be arbitrarily set from 1 μm to 1000 μm, 10 μm to 1000 μm, 10 μm to 300 μm, 10 μm to 100 μm, etc. from the outermost surface of the skin.

The frequency refers to the number of cycles per second when one cycle is the cycle of recovery from the start of extension or pressing to the non-extension or non-pressing state. One cycle may include maintaining a stretched or pressed state for a period of time and / or resting in a non-extended or non-pressed state. For example, after (b-1) and before (c-1), or after (b-2) and before (c-2) in one cycle, 0 seconds to 30 minutes, 1 second to Hold the stretched or pressed state for 20 minutes, 5 seconds to 10 minutes, or 10 seconds to 5 minutes; and / or after (c-1) and before (b-1) of the next cycle, or ( After b-2) and before (c-2), 0 seconds to 30 seconds, 0 seconds to 20 seconds, 0 seconds to 10 seconds, 1 second to 10 seconds, 1 second to 20 seconds, 1 second to 10 It may further include resting in a non-extended or non-pressed state for a second. The frequency may be, for example, 0.0000001 Hz or more and 10 kHz or less, 0.000001 Hz or more and 1 kHz or less, 0.00001 Hz or more and 100 Hz or less, preferably 0.0001 Hz or more and 60 Hz or less, 0.0001 Hz or more and 10 Hz or less. For example, 0.001 Hz or more and 60 Hz or less, 0.01 Hz or more and 60 Hz or less, 0.001 Hz or more and 10 Hz or less, 0.01 Hz or more and 10 Hz or less, 0.1 Hz or more and 60 Hz or less, 0.1 Hz or more and 10 Hz or less, 0.5 Hz or more and 60 Hz or less. , 0.5Hz or more and 50Hz or less, 0.5Hz or more and 10Hz or less, 0.5Hz or more and 5Hz or less, 0.5Hz or more and 1Hz or less, 0.001Hz or more and 0.01Hz or less, 0.001Hz or more and 0.1Hz or less, 0.001Hz Frequencies in any range such as 1 Hz or less, 0.01 Hz or more and 1 Hz or less, 0.1 Hz or more and 1 Hz or less, 1 Hz or more and 60 Hz or less, 1 Hz or more and 10 Hz or less, 1 Hz or more and 5 Hz or less can be adopted.

Commercially available facial equipment, massage equipment, etc. include those using electromagnetic waves having a frequency of about 0.3 to 300 MHz such as RF waves, and those using ultrasonic waves having a frequency of about 1 MHz to 7 MHz. Compared with these frequencies / frequencies, the frequencies adopted by the method / apparatus of the present invention are extremely low. If a strong frequency is applied to the skin like a conventional facial device, there is a risk of adverse effects such as redness, pressure marks, scratches, pain, and inflammation on the skin, but if a frequency like the present invention is adopted, the above risks Is low and allows non-invasive physical stimulation. This is because the present inventors have discovered that if the extension rate, the degree of pressing, the frequency, etc. are too high, the stimulation is too strong, and it is preferable to gently stimulate the skin by adjusting these values to appropriate values. ..

In addition, it has been a common general knowledge of those skilled in the art that in beauty equipment that uses a mechanism such as a motor that is generally used in the field, only a frequency exceeding 60 Hz can be selected from the mechanical mechanism of the motor. .. In order to adopt a frequency of "60 Hz" or less, which is the limit of beauty equipment in the prior art, for example, a frequency of 60 Hz or less, 10 Hz or less, 1 Hz or less as in the present invention, it is necessary to create a special machine. Was required. Furthermore, there is a fixed concept that such a low frequency is "too weak" to achieve the effect of the present invention, and so far, little study has been made. However, when the present inventors actually applied a physical stimulus to the skin using a very low frequency considering the conventional common sense of technology, surprisingly, even with such a gentle stimulus with a low frequency, the present inventors tried to give a physical stimulus to the skin. A good effect was achieved.

Although low and medium frequency devices are commercially available as EMS devices, these are designed to act in deep layers such as muscle and subcutaneous fat, and the effect on the skin surface layer as in the present invention is unknown. .. Further, such a device may be accompanied by a tingling stimulus when an electric current is passed even if the frequency is low, which is different from the present invention that gives a gentle stimulus to the skin. On the other hand, the present invention enables a beauty method by a simple method of directly applying an extension or pressing stimulus to the skin without applying energy such as ultrasonic waves, electric current, or magnetic field. Further, the extension or pressing stimulus having such a frequency is a gentle stimulus, but has a good effect on both epidermal cells and dermis cells as described in Examples. Therefore, the use of the method / apparatus of the present invention is expected to have a cosmetic effect on the epidermis of the skin in the short term and on the dermis in the long term without adversely affecting the skin.

The physical stimulus may be by an instrument such as a facial device, an experimental device, a massage using human hands or instruments, a facial exercise, or may be achieved by contact or non-contact. .. In one aspect, a mechanically generated physical stimulus is applied to the skin by using a device having a stimulus generating part that generates a physical stimulus and a stimulating part that applies the physical stimulus by contact or non-contact. Can be done. Physical irritation may be achieved, for example, by contact such as pulling, pressing, tapping, kneading, sucking the skin, and / or displacement by applying a shock wave to the skin, for example, by ultrasonic waves or pneumatic pressure. It may be achieved by non-contact such as letting. Facial exercises include swelling the cheeks and opening the eyes. Examples of the massage include a massage using an instrument such as a hand or a roller by the subject to be treated or a practitioner such as a cosmetologist. However, it is not limited as long as it is within the range of the physical stimulus of the present invention.

Examples of the device of the present invention include a beauty device provided with a skin contact portion that comes into contact with the user's skin and imparts the physical stimulus of the present invention. For example, it may be composed of a grip portion and a skin extension portion or a skin pressing portion. For example, the device shown on the left of FIG. 14 is designed to stretch the skin at a specific frequency and extension rate when the skin contact portion touches the skin.

Further, for example, the device of the present invention includes a power supply, a stimulus generating part, and a skin stimulating part, the power source generates an electric signal, and the stimulus generating part converts the electric signal from the power source into a physical stimulus. The skin stimulating part may receive the physical stimulus generated by the stimulating part and give the physical stimulus to the user's skin.

For example, the device shown on the left of FIG. 14 includes a grip portion, a power source, a control unit that controls physical stimulation, a stimulation generation unit, and a skin contact portion including a skin stimulation portion and a skin fixing portion. The user holds the grip part and puts the skin contact part on the skin, fixes the skin with the skin fixing part, and operates the control part so that the electrical signal from the power supply is physically stimulated by the stimulus generating part. It is designed so that the physical stimulus is transmitted to the skin stimulating part, and the skin is stretched at a specific frequency and extension rate by the skin stimulating part while being fixed to the skin fixing part. For example, the stimulus generator may be driven by a motor or the like to convert an electrical signal into a physical stimulus. The skin stimulating portion shown on the left side of FIG. 14 gives a stretching stimulus to the skin, but may, for example, give a pressing stimulus to the skin.

Alternatively, the device of the present invention may be a cosmetological device including a power supply, a control unit for controlling physical stimulation, a stimulation generating unit, and a skin contact portion including a skin contact surface made of a sheet-like material. good. As an example, the skin contact portion of such a beauty device is shown on the right side of FIG. The sheet-like material can carry an electric current and may convert an electrical signal from a power source into a physical stimulus. Examples of such sheet-like materials include dielectric elastomer actuators (DEA), conductive polymers, IPMC, PVC gel, McCinnen type and the like.

The power source of the device of the present invention may be an internal power source or an external power source, or may be a rechargeable type. Further, the device of the present invention may, for example, use data stored in a mobile phone, cloud, or the like, or may be remotely operated wirelessly.

The physical stimulus may be one that achieves the stretching or pressing of the skin by giving the physical stimulus by contact or non-contact as described above. The physical stimulus may be applied in the direction parallel to the skin surface, that is, in the lateral direction, in the direction perpendicular to the skin surface, that is, in the vertical direction, or in any direction such as an oblique direction or a twisting direction. can.

The number of cycles to perform physical stimulation is not limited. For example, any number of cycles such as 2 to 500 cycles, 10 to 500 cycles, 20 to 400 cycles, 30 to 300 cycles, 40 to 200 cycles, and 50 to 100 cycles may be performed. For example, as described in Examples, 27 cycles may be sufficient.

Further, any number of these cycles may be regarded as one set, and this set may be performed with an arbitrary number of sets, for example, 1 to 100 sets, 2 to 50 sets, or 3 to 10 sets.

The time to perform physical stimulation is not limited. For example, the cycle may be repeated with or without a pause time for a certain period of time such as 5 minutes to 3 hours, 10 minutes to 2 hours, and 30 minutes to 1 hour.

The time interval between cycles or sets is not limited. For example, the extension or pressing stimulus may be one set or multiple sets alone, with one or more sets daily, 2, 3, 4, 5, 6, 6, and 7. It may be performed once, once every 1, 2, 3, or 4 weeks, etc., continuously or intermittently, regularly or irregularly.

However, if sufficient stimulation is achieved to exert the skin anti-aging effect, the frequency, extension rate, number of cycles, and frequency are not limited to those described above. The waveform for physical stimulation can be set arbitrarily, such as a square wave, sine wave, triangle wave, and sawtooth wave.

The object to which the method of the present invention is applied may be an object in which skin aging is objectively or subjectively recognized, or an object in which skin aging is desired to be prevented. For example, the subject may be judged to be deficient in skin constituents such as hyaluronic acid. In one embodiment, the subject may be a subject judged to have a high degree of skin aging by using the behavior of YAP in the stratum corneum cells and / or the structure of actin fibers as an index. For example, when measured with stretched keratinized cells, it was determined that the ratio of the number of cells having nuclear localization YAP to the total number of keratinized cells and / or the ratio of the number of cells having actin stress fiber to the total number of keratinized cells was low. It may be a target. Alternatively, the subject may be concerned about skin aging, such as spots, wrinkles, sagging, dullness, conspicuous pores, or smoothness, elasticity, firmness, gloss, and decreased elasticity. Spots, wrinkles, sagging, dullness, conspicuous pores, smoothness, elasticity, firmness, luster, elasticity, etc. can be determined by using known indicators such as visual judgment, subjective or objective evaluation, and cute meter values. Can be decided.

Alternatively and / or additionally, the skin anti-aging effect may mean an increase in the amount of hyaluronic acid in the dermis according to the present invention. The increase is, for example, an increase in hyaluronic acid having a statistically significant difference (eg, Student's t-test) with a significance level of 5%, and / or, for example, 10% or more, 20% or more, 30% or more, 40%. The increase may be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more.

Alternatively and / or additionally, the skin anti-aging effect may mean an increase in indicators such as skin viscoelasticity, elasticity, firmness, luster, smoothness, less noticeable pores, and less dullness according to the present invention. be. Skin viscoelasticity, elasticity, firmness, luster, smoothness, etc. can be measured by a cute meter or a subjective or objective index. The increase is, for example, an increase with a statistically significant difference (eg Dunnett's test or Wilcoxon signed rank test) with a significance level of 5%, and / or, for example, 10% or more, 20% or more, 30%. The increase may be 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more.

The present invention can prevent and improve skin aging while exerting a favorable effect on a shallow layer such as the epidermis. For example, to improve the behavior of YAP and the structure of actin fibers in keratinized cells, especially for keratinized cells when a skin antiaging agent containing arabinoxylan as an active ingredient is applied to the skin or when physical stimulation is applied. It is possible to suppress the decrease in the proportion of cells having nuclear-localized YAP or actin stress fibers, or to recover the subsequent increase. In addition, it can lead to an increase in hyaluronic acid in the dermis, an increase in skin viscoelasticity and the above subjective or objective indicators.

Further, the present invention may be used in combination with other treatments. Other treatments include, but are not limited to, application of stimuli such as light stimulation, electrical stimulation, mechanical stimulation, human hand stimulation, administration of anti-aging agents, application of cosmetics, and the like. The anti-aging agent may be a naturally or chemically synthesized compound, an extract of animals and plants, a simple substance or a mixture, a state contained in a solvent such as an aqueous solution, cosmetics, etc. It may be in the form of. In addition, the administration route of the anti-aging agent can be arbitrarily selected, and examples thereof include oral administration, transdermal administration, subcutaneous administration, transmucosal administration, and intramuscular administration.

The present invention may be aimed at beauty, medical treatment, stress relief, relaxation, and the like. In certain embodiments, the present invention is intended for cosmetology and may not be used by physicians or healthcare professionals. The present invention also provides a cosmetology counseling method that supports a cosmetological act of the subject, including presenting the present invention to the subject.

All documents cited herein are hereby incorporated by reference in their entirety.

Next, the present invention will be described in more detail by way of examples. The examples of the present invention are for illustration purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the claims. The present invention may be modified, for example, the constituent elements of the present invention may be added, deleted or replaced, provided that the gist of the present invention is not deviated.

Experiment 1: Changes in in vitro of keratinized cells due to aging Sample: Monolayer cultured keratinized cells from donors (0, 18, 44, 64 years old) of different ages purchased from KAC Co., Ltd. And fibroblast cultures were used.

Stretching conditions: Using ShellPa manufactured by Menicon Life Science, stretch stimulation was given to the above-mentioned keratinocytes and fibroblasts. The extension stimulation was performed at an extension rate of 10% as shown in the center of the lower figure of FIG. In the cycle, as shown on the left side of the upper figure of FIG. 1, the culture chamber was pulled in one direction at an extension rate of 0.33% / s, and the cells were extended to an extension rate of 10%. Then, the extended state was maintained for 5 minutes, and the cells were returned to the original non-extended state at a recovery rate of 0.33% / s. This was regarded as one cycle, and a total of 27 cycles were performed over 3 hours. The rest time between cycles was 0 to 10 seconds.

Observation method: The intracellular YAP after non-extension stimulation and extension stimulation was observed by the fluorescent antibody staining method as follows. YAP in cells uses anti-YAP antibody (Santa Cruz Biotechnology, YAP antibody (63.7): sc-10199), and actin fiber uses phalloidin (ThermoFisher Scientific, Alexa Fluor ^TM 488 P) cross-linked with a fluorescent compound. Detection, observation and image acquisition with a confocal laser scanning microscope (ZEISS, LSM700). In addition, the image processing analysis software ImageJ was used to quantify the nucleus-cytoplasm ratio of YAP by the degree of fluorescence intensity. The fluorescence intensity ratio between the nucleus and the cytoplasm (fluorescence intensity of nuclear YAP / fluorescence intensity of intracellular YAP) was calculated, and the number of cells whose value was 1 ≧ was counted as a cell having nuclear YAP. The total number of cells is stained using DAPI (Vector Laboratories, VECTASHIELD Mounting Medium with DAPI: H-1200), the number of cells stained with nuclei is counted, and the ratio of the number of cells having nuclear localized YAP is calculated. Calculated according to 2. In addition, cells containing at least one actin stress fiber in the cytoplasm or around the nucleus are counted as cells having actin stress fiber, the total number of cells is counted in the same manner, and the ratio of the number of cells having actin stress fiber is calculated by Equation 3. Calculated according to.

result:
Micrographs of keratinocytes are shown in FIGS. 2a and 2b. As can be seen from these figures, in keratinocytes obtained from young donors (0 and 18 years old), YAP is localized in the nucleus in almost all cells with and without extension stimulation. However, in the keratinocytes obtained from middle-aged and elderly donors (44 years old and 64 years old), the number of cells having nuclear-localized YAP decreased sharply when extension stimulation was given. FIG. 3 shows the percentage of cells having nuclear localization YAP in keratinocytes. Although there was no change in the number of cells with nuclear localization YAP in the younger generation, it was significantly decreased in the keratinized cells in the elderly. However, although the number decreased sharply, the number of cells having nuclear localization YAP did not become zero and existed at a constant rate. In FIG. 4, which shows the ratio of the number of cells having actin stress fibers in the keratinocytes, there is no change in the number of cells having actin stress fibers in the younger generation, and it is significantly decreased in the keratinocytes in the elderly. I can't do it anymore. On the other hand, in fibroblasts, as shown in FIG. 5, no change was observed in YAP and actin stress fibers under the condition with and without extension stimulation, and almost no age difference was observed.

Experiment 2: Changes in ex vivo of keratinized cells due to aging Sample: Ex vivo human abdominal skin tissue pieces (NativeSkin, 12well size, about 1 cm in diameter) obtained from Genoskin were used. The piece of tissue is 38 years old.
Extension conditions: A plastic pressing portion as shown in FIG. 6 was created. The bottom surface of the pressing portion was pressed vertically from the top of the sample so as to be pressed against the top surface of the sample, and the skin was adjusted to be stretched at an extension rate of 0.1% or less. Such a pressing motion is performed at a speed of 10% / s as shown in FIG. 7, then the pressing portion is held and the extended state is maintained for 10 seconds, and then lifted at a speed of 10% / s to the original non-extended state. It was returned to, and rested for 10 seconds until the next pressing exercise. This was regarded as one cycle, and 90 cycles were carried out over 30 minutes. These 90 cycles were regarded as one set, and a total of 3 sets of pressing exercises (total of 270 cycles) were performed with a rest period of 30 minutes to 1 hour between sets.

Observation method: YAP and DAPI were stained, observed, and imaged in the same manner as in Experiment 1 under the conditions with and without extension stimulation.

result:
The change in YAP behavior due to aging observed in Experiment 1 was also observed in the ex vivo sample. As can be seen in FIG. 8, even in the ex vivo skin model obtained from a middle-aged donor (38 years old), in the keratinocytes in the epidermis, the nuclear localization YAP decreased and many parts were not stained when the extension stimulation was given. It was seen. Such changes were particularly remarkable near the basal layer.

Experiment 3: Effect of in vitro on keratinocytes by extension stimulation Sample: A monolayer culture of keratinocytes of the same middle-aged donor (44 years old) as in Experiment 1 was used.
Extension conditions: The same extension stimulus as in Experiment 1 was applied. That is, a set of 27 cycles of extension stimulation performed in Experiment 1 was given. However, 12 to 24 hours after giving one set of extension stimuli consisting of 27 cycles for 3 hours, the same extension stimulus was further given to the sample, and a total of two sets were performed.

Observation method: YAP after non-extension stimulation and extension stimulation was stained in the same manner as in Experiment 1. In addition, BrdU was stained with an anti-BrdU antibody (Abcam, Anti-BrdU antibody [BU1 / 75 (ICR1)]: ab6326) as a growth marker by the fluorescent antibody method. The treatment of BrdU was carried out for 6 hours, and 7 days later, it was fixed at 4 ° C. for 24 hours with 4% paraformaldehyde. The fixed sample was observed and imaged using a confocal laser scanning microscope (ZEISS, LSM700).

The results are shown in Fig. 9. In the first extension stimulus, as in Experiment 1, there was a large change in the number of cells having nuclear localization YAP before and after the extension stimulus. BrdU also decreased sharply due to extension stimulation, similar to nuclear localization YAP. On the other hand, when the second extension stimulus was given 24 hours after the first extension stimulus, the nuclear localization YAP and BrdU showed no significant change between the non-extension stimulus condition and the second extension stimulus condition. It was increasing. It has been suggested that there is some relationship between nuclear localized YAP and proliferation cells, and the number of cells capable of self-renewal increases in cells after two extension stimuli are given rather than one extension stimulus. It is suggested that the cell proliferation ability is restored or maintained similar to that of non-extended or young keratinocytes. Although not bound by theory, the first extension stimulation promotes cell death of aging keratinocytes with low growth maintenance and self-renewal ability, or causes aging keratinocytes to peel off by some mechanism. It is considered that the proliferation and recovery of keratinocytes are promoted by selectively eliminating cells showing nuclear-localized YAP having high growth maintenance and self-renewal ability.

Experiment 4: Effect of ex vivo on epidermis by extension stimulation Sample: Ex vivo human abdominal skin tissue piece (NativeSkin, 6well size, about 2 to 2.5 cm in diameter) of a middle-aged donor (42 years old) purchased from Genoskin. used.
Stretching conditions: A stretching device having grips for gripping both ends of the skin in the well as shown in FIG. 10 and stretching the skin by pulling the grips was created. The well containing the above tissue piece was placed horizontally, and the grip portion was operated to stretch the skin from both ends, and as shown in FIG. 7, stretching with a stretching rate of 10% was performed at a rate of 10% / s. The sample was returned to its original non-extended state at a recovery rate of% / s. This was regarded as one cycle, and a total of 90 cycles were performed over 30 minutes. These 90 cycles were regarded as one set, and a total of 3 sets (270 cycles in total) were performed with a rest time of 30 minutes to 1 hour between the sets. After 12 to 24 hours after giving these 3 sets of extension stimuli for 3 hours, the same 3 sets of extension stimuli were further applied to the sample twice for 2 consecutive days.

Observation method: On the 0th day of the non-extension stimulation, immediately after the extension stimulation (the 0th day of the same), after culturing the skin tissue treated with the non-extension stimulation and the extension stimulation for 1 week, the same YAP and DAPI treatment as in Experiment 1 was performed, and further. Ki67 was stained with an anti-Ki67 antibody (Abcam, Anti-Ki67 antibody [SP6]: ab16667) as a growth marker, and observation and image acquisition were performed in the same procedure as in Experiment 1.

FIG. 11 shows the state of the non-extension stimulation on the 0th day and immediately after the extension stimulation (the 0th day). The number of epidermal cells in the basal layer having YAP decreased, and the proliferation marker Ki67 increased immediately after tension. Although not bound by theory, it is possible that stretching stimulation promoted the process of cell proliferation and subsequent cell differentiation. FIG. 12 shows the state after culturing for 1 week after giving the extension stimulus twice for 2 consecutive days. Before extension, YAP was observed not only in the basal layer but also in the entire epidermal cells. YAP is usually abundant in the basal layer, but was observed in all layers of skin cultured for 1 week. Although not bound by theory, in non-extended skin tissue, cell aging may cause, for example, some abnormalities in cell proliferation and cell differentiation in each layer of the epidermis, resulting in altered YAP distribution. Is also possible. However, the expression distribution of YAP was maintained in the basal lamina after two stretching stimuli. Although not bound by theory, physical stimulation activates and maintains homeostasis of cell proliferation and cell differentiation in the epidermis, resulting in a normal distribution of YAP in early fresh skin tissue even after 1 week of culture. It is also possible that the condition is maintained.

Experiment 5: Effect of ex vivo on dermis by extension stimulation Sample and extension conditions: Ex vivo human abdominal skin tissue piece (NativeSkin, 6well size, about diameter) of a middle-aged donor (44 years old) purchased from the same Genoskin company as in Experiment 4. 2 to 2.5 cm) was used. Three sets of extension stimulation, which is the same extension as in Experiment 4, was performed twice for two consecutive days.

Observation method: DAPI, hyaluronic acid (HOKUDO, Biotin-HABP: BC41), collagen (Merck Millipore, Anti-human COL1N-terminal antibody: MAB1912) were observed before the extension stimulation and after culturing for 1 week after the extension stimulation. As for DAPI, hyaluronic acid and collagen were stained by the fluorescent antibody method as in Experiment 1, and observed and imaged with a confocal laser scanning microscope (ZEISS, LSM700).

The results are shown in FIG. Hyaluronic acid in the dermis was increased when the extension stimulus was given. It is shown that the extension stimulation not only promotes the proliferation of epidermal cells but also has a favorable effect on the dermis.

Experiment 6: Effect of in vitro on keratinized cells by administration of Kohiris ™ GR Sample: Using a monolayer culture of keratinized cells of the same middle-aged donor (44 years old) as in

Experiment

1, 1 of the initiation of extension stimulation A day ago, Kohiris ™ GR (water (84.50%), petylene glycol (10.00%), limegi seed extract (5.50%)) was added in 0.3% by volume and 1.0% by volume. Normal human keratinized cells were cultured for 1 day in a culture medium dissolved in a medium for exclusive use of keratinized cells (Epilife, Thermo Fisher Scientific, HuMedia-KG growth additive set, Kurabo). For control, a culture medium in which (water (90.00%), petylene glycol (10.00%)) was dissolved in a medium for keratinized cells at concentrations of 0.3% by volume and 1.0% by volume was used. , Normal human keratinized cells were cultured in the same manner.
Extension conditions: The same extension stimulus as in Experiment 1 was applied. That is, a set of 27 cycles of extension stimulation performed in Experiment 1 was given.

Observation method: The intracellular YAP after non-extension stimulation and extension stimulation was stained and observed in the same manner as in Experiment 1, and the ratio of the number of cells having nuclear localized YAP and the ratio of the number of cells having actin stress fiber were calculated. ..

result:
The results are shown in FIG. As shown in Experiment 1, in the keratinized cells obtained from a middle-aged donor (44 years old), the ratio of the number of cells having nuclear localized YAP and the ratio of the number of cells having actin stress fiber suddenly increased when extension stimulation was given. Although it decreased, by adding Kohiris ™ GR, all the ratios were about the same as before the extension stimulation, and when 1% was administered, the ratio of the number of cells having actin stress fibers was higher than before the extension stimulation. rice field.

Experiment 7: Effect of in vivo by extension stimulation Subjects: 17 healthy women in their 30s and 50s were selected.
Extension conditions: The extension device shown on the left side of FIG. 14 was used. In this device, the skin contact part indicated by the arrow on the left of FIG. 14 touches the skin, and the skin fixing part fixes the skin while appropriately setting the amount of vertical pushing of the skin extension part with respect to the skin within a range of a maximum of about 5 mm. It is designed to stretch the skin at a frequency of 1.2 Hz with a horizontal stretch rate of 0.1 to 20%. The subject applied this device to the half face to give a stretching stimulus for 30 minutes every day for 10 days.

Measurement method: On the 3rd and 10th days after the start of the experiment, the skin condition of each half face with and without stretching stimulation (treated) was measured. The skin condition is measured by measuring the percutaneous water evaporation (TEWL) with a vapor meter of Delfin Technologies, measuring the skin water with a Corneometer (CM825) of Course + Khazaka, and measuring the skin viscoelasticity with a cute meter (Courage + Khazaka). This was done by measuring with SEM575). The obtained data was statistically processed by comparing each with the value on the 0th day in Dunnett's test (***: p <0.005, **: p <0.01, *: p. <0.05).

Furthermore, the subjects made a subjective evaluation by entering the impressions of the cheeks before and 10 days after the extension stimulus on a 5-point scale for each of the following evaluation items.

The results of the subjective evaluation were statistically processed by Wilcoxon signed rank test (***: p <0.005, **: p <0.01, *: p <0.05).

Result: The results are shown in FIGS. 16a-c. As shown in FIG. 16a, neither TEWL nor the skin water content was changed by the extension stimulus, but as shown in FIG. 16b, the R0 value was significantly increased and the skin viscoelasticity was improved as measured by the cute meter. I found out that there is. This tendency was also seen in the R2 and R7 values. In addition, as shown in FIG. 16c, it was found that the subjects felt that the skin condition was improved in both the appearance and the tactile sensation.

Experiment 8: Effect of ex vivo by pressing stimulus Sample: Ex vivo human abdominal skin tissue piece (NativeSkin, 6well size, about 2 to 2.5 cm in diameter) of a middle-aged donor (44 years old) purchased from the same Genoskin company as in Experiment 4. )It was used.

Pressing condition: Dielectric elastomer actuator (manufactured by Sumitomo Riko Co., Ltd .: SRHP074-001) was used. As shown in the upper left of FIG. 17, the dielectric elastomer actuator is applied to the skin tissue piece and repeatedly contracted and expanded to press various waveforms of 0 to 100 μm under the conditions described in the lower part of FIG. The stimulus was given at a frequency of 0.5 Hz, 5 Hz, or 50 Hz for 1 or 5 days.

Observation method: DAPI and Ki67 before and after the pressing stimulus were observed by the same method as in Experiment 4.

result:
The results are shown in FIGS. 18 and 19. When no pressing stimulus was given (Cont: cellent), the number of Ki67 on the 5th day decreased compared to the 1st day, but when the pressing stimulus was given (0.5Hz / 100μm: square), the number of Ki67 on the 5th day was reduced. It was shown that the decrease of Ki67 in epidermal cells was suppressed, and that the suppression of epidermal cell proliferation was suppressed by the pressing stimulus. In addition, the pressing stimulus showed a higher effect at a low frequency of about 0.5 Hz than at 5 Hz and 50 Hz. The results shown in FIGS. 18 and 19 show the case of a rectangular wave (square), but the same result was obtained even in the case of a sine wave (sin).

Experiment 9: Effect of in vivo by pressing stimulus Subjects: 17 healthy women in their 30s and 50s were selected.
Extension conditions: The pressing device shown in FIG. 20 and a dielectric elastomer actuator (manufactured by Sumitomo Riko Co., Ltd .: SRHP074-001) were used. In this device, the skin contact portion of the device shown in FIG. 20 pushes the skin with a force of 10 gf, so that the pressing stimulus of the rectangular wave shown in the lower part of FIG. 20 is given to the skin at a frequency of 5 Hz and a depth of 100 μm. Is designed for. As shown in FIG. 20, this device was applied to the medial part of the forearm of the subject, and the operation of applying a pressing stimulus for 30 minutes every day was performed for 5 days.

Measurement method: The skin condition of the medial part of the forearm with and without the pressing stimulus was measured before the start of the experiment and on the 7th day. The skin condition is measured by measuring the percutaneous water evaporation (TEWL) with a vapor meter of Delfin Technologies, measuring the skin water with a Corneometer (CM825) of Course + Khazaka, and measuring the skin viscoelasticity with a cute meter (Courage + Khazaka). This was done by measuring with SEM575).

Result: The results are shown in FIGS. 21a-c. Neither TEWL nor skin water content was changed by the pressure stimulus, but the results of the cute meter showed that the R0 value decreased significantly on the 7th day without the pressure stimulus, but it decreased by the pressure stimulus. Was suppressed, and improvement in viscoelasticity was observed.

It was suggested that in the skin to which the present invention was applied, senescent cells were selectively eliminated by extension stimulation and keratinocytes having nuclear localized YAP and actin stress fibers were selectively retained. We also obtained results suggesting cell proliferation of keratinized cells having these selected nuclear localization YAPs and actin stress fibers. Furthermore, the amount of hyaluronic acid in the dermis as well as the epidermis where keratinocytes are present was increased. In other words, it is suggested that the present invention exerts an anti-aging effect not only on the epidermis but also on the skin including the dermis. When the present invention was actually applied to humans in vivo, the viscoelasticity of the skin, the appearance and tactile sensation such as elasticity, luster, dullness, and smoothness were improved.

Claims

A skin anti-aging agent containing arabinoxylan as an active ingredient.
A skin anti-aging agent characterized by increasing cells with nuclear localized YAP and / or actin stress fibers that are reduced by physical stimulation in the skin.
The skin anti-aging agent according to claim 1, wherein the arabinoxylan is derived from rye.
The skin anti-aging agent according to claim 1 or 2, wherein the arabinoxylan is derived from Kohiris ™ GR.
A cosmetic method for skin anti-aging that increases cells with nuclear localized YAP and / or actin stress fibers that decrease with physical stimulation in the skin.
(A) Applying the skin anti-aging agent according to any one of claims 1 to 3 to the target skin,
Cosmetology methods, including.
The cosmetological method further includes a step of applying a weak physical stimulus to the skin, wherein the step of applying the physical stimulus is, for example, for example.
(B-1) The target skin is stretched to a stretch rate of 0.1% or more and 50.0% or less, where the stretch rate is defined as.

(In the equation, the fixed points A and B are arbitrary positions on the epidermis or the matrix to which the epidermis is adhered, and the straight line passing through the fixed points A and B is parallel to the extension direction).
And (c-1) recovering the subject's skin from the stretched state;
And / or
(B-2) Pressing the subject's skin by 1 μm to 1000 μm; and (c-2) Recovering the subject's skin from the pressed state;
Including
Here, the cycles (b-1) and (c-1) and / or the cycles (b-2) and (c-2) are carried out at a frequency of 60 Hz or less.
The beauty method according to claim 4, which comprises.
The beauty method according to claim 5, wherein one or a plurality of sets are performed by performing the cycle from 2 to 500 cycles.
When the skin anti-aging is measured using the ratio of the number of cells having nuclear-localized YAP to the total number of keratinized cells as an index, the nuclear-localized YAP measured in extended keratinized cells after applying the cosmetic method to the skin. The proportion of cells having nuclear localization YAP is increased as compared with that before applying the cosmetic method.

The beauty method according to any one of claims 4 to 6, which is calculated in 1.
The skin anti-aging has actin stress fibers measured in extended keratinized cells after applying the cosmetic method to the skin when measured using the ratio of the number of cells having actin stress fibers to the total number of keratinized cells as an index. The proportion of cells is increasing, where the proportion of cells with actin stress fibers is

The beauty method according to any one of claims 4 to 6, which is calculated in 1.
A cosmetological device for applying to the cosmetological method according to claim 5 or 6.
The device is
The stimulus generator that causes physical stimulus and
It is equipped with a stimulus-giving part that gives the physical stimulus generated in the stimulus-generating part to the skin.
Here, the device is a device for performing a step of applying a weak physical stimulus to the skin, and the step is described here, for example.
(I-1) Stretching the skin to a stretch rate of 0.1% or more and 50.0% or less; and (ii-1) Restoring the skin from the stretched state;
And / or
A cycle including (i-2) pressing the skin by 1 μm to 1000 μm; and (ii-2) recovering the skin from the pressed state; is performed at a frequency of 60 Hz or less.
Here, the extension rate is calculated by the above equation 1.
The beauty device.
A beauty method for skin anti-aging, which comprises applying the beauty device according to claim 9 to the target skin.
The cosmetological method according to any one of claims 4 to 8 and 10, for increasing hyaluronic acid in the skin.
The skin anti-aging agent according to any one of claims 1 to 3, the cosmetological method according to any one of claims 4 to 8 and 10, or the cosmetological device according to claim 9 is presented to the subject. Cosmetology counseling methods that support the subject's cosmetology, including.