JP7084595B2 - RhoGDIβ inhibitor - Google Patents

Description

The present invention relates to RhoGDIβ inhibitors and cosmetics.

In various tissues of our body, homeostasis is maintained by constantly replacing cells in the tissues with new cells.

For example, in skin tissue, an undifferentiated group of cells called the basal layer (basal cells), which is in contact with the basement membrane inside the skin epidermis, repeatedly divides and proliferates. The basal layer (basal cells) gradually differentiates from the basement membrane side to the stratum spinosum side outside the skin epidermis, and peels off from the outermost surface of the skin tissue to repeat the metabolism that supplies new cells.

That is, in skin tissue, homeostasis is maintained by repeating division, proliferation, and differentiation of the basal layer (basal cells), and the barrier function and transparency are maintained.

Cell division in the basal layer (basal cells) is directional and usually divides in the parallel or perpendicular direction to the basement membrane. The former is symmetric division, and since both daughter cells are in contact with the basement membrane, they maintain proliferative capacity and contribute to the expansion of the epidermal sheet.

On the other hand, the latter is asymmetric division, and daughter cells dissociated from the basement membrane contribute to the multi-layering of skin tissue by stopping proliferation and gradually differentiating. This direction of division is important for the formation of skin tissue and maintenance of homeostasis.

Then, for example, the direction of cell division of the skin tissue is disturbed by aging or exposure to ultraviolet rays, and the structure of the formed epidermis is disturbed, which is one of the unfavorable skin symptoms such as dryness and dullness of the skin. Can be a cause.

In recent years, it has become known that the Rho guanine nucleotide dissociation inhibitor β (Rho guanine nucleice dissociation inhibitor β; RhoGDIβ), which is a regulator of the Rho family low molecular weight G protein, is involved in the disorder of the direction of cell division. rice field.

RhoGDI regulates RhoGTPases, a group of important intracellular signaling factors that control cell morphology and cell motility, and suppresses dissociation of GDP from Rho and hydrolysis of GTP. Most Rho proteins are thought to bind to RhoGDI and be present in the cytoplasm. RhoGDI is thought to function as a switch for cellular signal transduction.

In mammals, three types of RhoGDI (RhoGDIα, RhoGDIβ, RhoGDIγ) are known.

As a document relating to RhoGDIβ that disturbs the direction of cell division, for example, Japanese Patent Application Laid-Open No. 2014-141430 (Patent Document 1) is known.

Patent Document 1 discloses an agent for suppressing the production of RhoGDIβ protein, which can suppress the production of RhoGDIβ protein.

The agent for suppressing the production of RhoGDIβ protein described in Patent Document 1 is characterized by containing an extract of Scutellaria baicalensis. The RhoGDIβ protein production inhibitor containing the Ogon extract is also used as a skin epidermal cell division direction control agent, an external skin preparation agent, and a skin epidermal cell division direction control method.

Further, in recent years, it has become known that RhoGDIβ, which has an action of disturbing the direction of cell division, is often found in cancer cells.

The RhoGDIβ protein is degraded into ΔN-RhoGDIβ protein under the influence of ultraviolet irradiation and the like. The ΔN-RhoGDIβ protein, which is a fragmented product of the RhoGDIβ protein, has an action of binding to Cdc42, which is one of the Rho family G proteins. Cdc42 is a protein that plays an important role in various cell morphogenesis and other processes, and is also involved in cell morphogenesis in the division of skin epidermal cells.

More specifically, Cdc42 is usually placed in two places along a straight line through the nucleus of the cell as the skin epidermal cells divide into two. The cell then divides so that the two Cdc42s are separated from each other along the straight line. That is, the extending direction of the straight line is the splitting direction.

Therefore, if the function of Cdc42 is inhibited by binding to the ΔN-RhoGDIβ protein or the like, the cell division direction may be more random.

Furthermore, the association between RhoGDIβ and cancer has also been reported. As literature on abnormal cell division direction and canceration, RhoGDIβ, for example, J. Cell. Physiology. 231: 2493-2505, 2016. (Non-Patent Document 1) is known. Non-Patent Document 1 discloses the following contents.

RhoGDIβ is known as a direct cleavage substrate for caspase 3, and N-terminally truncated RhoGDIβ (ΔN-RhoGDIβ protein) accumulates in caspase-3 activated cells.

RhoGDIβ is overexpressed in many epithelial cancers. In addition, it is already known that caspase 3 is activated by radiation therapy or cytotoxic anticancer treatment.

As a result of experiments using human-derived cervical cancer cells Hela cells, it was revealed that stable expression of ΔN-RhoGDIβ protein constitutively inhibits the activity of Cdc42 and enhances radiation-induced compensatory proliferation. did.

That is, it is considered that the RhoGDIβ protein (ΔN-RhoGDIβ protein) is involved in the regrowth of cancer cells by promoting compensatory growth during cytotoxic anticancer treatment.

Japanese Unexamined Patent Publication No. 2014-141430

J. Cell. Physiology. 231: 2493-2505, 2016.

In Patent Document 1, the inventors have found an Ogon extract as a material for suppressing the generation of RhoGDIβ, which disturbs the direction of cell division of epidermal cells. Further elucidation of materials that suppress the generation of RhoGDIβ is desired.

In addition, the above-mentioned Non-Patent Document 1 suggests that the RhoGDIβ protein overexpressed in many epithelial cancers may be a novel mechanism of cancer progression induced by activation of caspase 3.

However, little is known about materials that suppress the generation of RhoGDIβ. Therefore, further elucidation of the material that suppresses the generation of RhoGDIβ is desired.

Therefore, in view of such circumstances, the inventors have screened a material that suppresses the generation of RhoGDIβ and found a material having an excellent effect of suppressing the generation of RhoGDIβ.

That is, the inventors further screened for materials that suppress the generation of RhoGDIβ, and obtained new findings that certain vitamin C derivatives (ascorbic acid derivatives) are materials that suppress the generation of RhoGDIβ. rice field.

In view of such circumstances, the present invention provides RhoGDIβ inhibitors and cosmetics capable of adjusting the direction of skin cell division, enhancing the skin barrier function and moisturizing power by suppressing the generation of RhoGDIβ. The purpose is to provide.

The RhoGDIβ inhibitor of the present invention is characterized by containing a vitamin C derivative having an alkyl group having 16 carbon atoms.

Vitamin C is a water-soluble vitamin that is essential for living organisms, has excellent antioxidant power, and has excellent cosmetic effects such as whitening (suppressing the production of melanin that causes age spots), collagen synthesis, and suppressing sebum. It is known. However, vitamin C has a characteristic that it is difficult to be absorbed transdermally because it lacks permeability and is unstable.

Therefore, vitamin C derivatives have been used as vitamin Cs having a modifying group to improve the stability and permeability of vitamin C. Examples of the type of modifying group include glycerin, phosphoric acid, and an alkyl group (carbon (organic substance) linked in a chain).

According to the RhoGDIβ inhibitor having the above configuration, since it contains a vitamin C derivative having an alkyl group having 16 carbon atoms, it suppresses the generation of RhoGDIβ in addition to its function as a vitamin C derivative, thereby causing the division of skin cells. It can adjust the directionality and enhance the barrier function and moisturizing power of the skin.

As one aspect of the present invention, it is preferable that the vitamin C derivative having an alkyl group having 16 carbon atoms has a linear alkyl group having 16 carbon atoms.

According to the RhoGDIβ inhibitor having the above configuration, the vitamin C derivative having an alkyl group having 16 carbon atoms has a linear alkyl group having 16 carbon atoms, so that the vitamin C derivative having a linear alkyl group having 16 carbon atoms can be used. By suppressing the generation of RhoGDIβ, the direction of division of skin cells can be adjusted, and the barrier function and moisturizing power of the skin can be enhanced. It may be important that the vitamin C derivative having an alkyl group having 16 carbon atoms is modified with a linear alkyl group and has no modifying group at the 6-position. In addition, the modification mode (ether bond) and the number of modifications may also be important factors.

As one aspect of the present invention, it is preferable that the vitamin C derivative having an alkyl group having 16 carbon atoms has an alkyl group having 16 carbon atoms only at the modified position at the 2-position or the 3-position.

According to the RhoGDIβ inhibitor having the above configuration, the vitamin C derivative having an alkyl group having 16 carbon atoms has an alkyl group having 16 carbon atoms only at the modified position at the 2-position or 3-position, and therefore has an alkyl group having 16 carbon atoms at the 2-position and 3-position. On at least one of them, a vitamin C derivative having an alkyl group having 16 carbon atoms suppresses the generation of RhoGDIβ, thereby adjusting the direction of division of skin cells, and enhancing the barrier function and moisturizing power of the skin. ..

As one aspect of the present invention, a vitamin C derivative having an alkyl group having 16 carbon atoms at at least one of the 2-position and 3-position includes 3-O-cetyl ascorbic acid and 2-O-cetyl ascorbic acid. Is preferable.

According to the RhoGDIβ inhibitor having the above constitution, a vitamin C derivative having an alkyl group having 16 carbon atoms at at least one of the 2-position and 3-position contains 3-O-cetyl ascorbic acid and 2-O-cetyl ascorbic acid. Therefore, 3-O-cetyl ascorbic acid or 2-O-cetyl ascorbic acid suppresses the generation of RhoGDIβ, thereby adjusting the direction of skin cell division and enhancing the barrier function and moisturizing power of the skin. Can be done.

As yet another aspect of the present invention, it is preferable that the cosmetic contains the above-mentioned RhoGDIβ inhibitor.

According to the cosmetics having the above constitution, since the cosmetics contain the above-mentioned RhoGDIβ inhibitor, they can exhibit excellent cosmetic effects of vitamin C derivatives. Further, by suppressing the generation of RhoGDIβ, the direction of skin cell division can be adjusted, and the barrier function and moisturizing power of the skin can be enhanced.

As described above, according to the present invention, by suppressing the generation of RhoGDIβ, it is possible to adjust the direction of division of skin cells, and to enhance the barrier function and moisturizing power of the skin. Can play.

It is a graph which shows the inhibitory action on RhoGDIβ of the vitamin C derivative which has various modifying groups about the RhoGDIβ inhibitor which concerns on one Embodiment of this invention. 6 is a graph showing the relative expression level of RhoGDIβ in the difference in the number of carbon atoms of the alkyl group as a modifying group of a vitamin C derivative for the RhoGDIβ inhibitor according to the same embodiment. 3 is a three-dimensional bar graph showing the number of dividing cells in the direction perpendicular to the cell division direction (80 to 90 °) and the horizontal direction (0 to 10 °) for the RhoGDIβ inhibitor according to the same embodiment.

Hereinafter, the RhoGDIβ inhibitor according to the embodiment of the present invention will be described in detail with reference to the accompanying drawings.

As described above, the inventors have obtained new findings regarding a material that suppresses the generation of RhoGDIβ. That is, the inventors further screened the materials that suppress the generation of RhoGDIβ, and further found those having the effect of suppressing the generation of RhoGDIβ.

As a result of further screening of materials that suppress the generation of RhoGDIβ, the inventors have found that certain vitamin C derivatives suppress the generation of RhoGDIβ.

Vitamin C is a water-soluble vitamin that is essential for living organisms, has excellent antioxidant power, and has excellent cosmetic effects such as whitening (suppressing the production of melanin that causes age spots), collagen synthesis, and suppressing sebum. It is known. However, vitamin C has a characteristic that it is difficult to be absorbed transdermally because it lacks permeability and is unstable.

Therefore, vitamin C derivatives have been used as vitamin Cs having a modifying group to improve the stability and permeability of vitamin C. Vitamin C derivatives enhance the osmotic power of vitamin C and excellent effects such as excellent antioxidant power, whitening, collagen synthesis, and sebum suppression. Examples of the type of modifying group include glycerin, phosphoric acid, and an alkyl group (carbon (organic substance) linked in a chain).

Furthermore, the inventors have begun searching for vitamin C derivatives that suppress the development of RhoGDIβ in epidermal cells.

The RhoGDIβ inhibitor according to this embodiment contains a vitamin C derivative having an alkyl group having 16 carbon atoms.

In this embodiment, the vitamin C derivative having an alkyl group having 16 carbon atoms has a linear alkyl group having 16 carbon atoms. In the present embodiment, the vitamin C derivative having an alkyl group having 16 carbon atoms has a linear alkyl group having 16 carbon atoms, but is not limited thereto. A vitamin C derivative having an alkyl group having 16 carbon atoms may have an alkyl group having a branched chain having 16 carbon atoms.

In this embodiment, a vitamin C derivative having an alkyl group having 16 carbon atoms has an alkyl group having 16 carbon atoms only at the modified position at the 2-position or the 3-position. Examples of vitamin C derivatives having an alkyl group having 16 carbon atoms only at the modified positions at the 2- and 3-positions include 3-O-cetyl ascorbic acid (3-O-hexadecyl ascorbic acid) and 2-O-cetyl. Ascorbic acid (2-O-hexadecyl ascorbic acid) and the like can be mentioned, but the present invention is not limited thereto.

3-O-cetyl ascorbic acid (3-O-hexadecyl ascorbic acid) is a highly stable vitamin C derivative of normal type I collagen fiber bundles through the promotion of NO in dermal fibroblasts. It is known to promote formation and prevent wrinkles and sagging.

As a method for producing 3-O-cetyl ascorbic acid (3-O-hexadecyl ascorbic acid), a conventional general production method can be used.

In the present embodiment, the content of 3-O-cetyl ascorbic acid or 2-O-cetyl ascorbic acid is 0.0004% by weight or more. When the content of 3-O-cetyl ascorbic acid or 2-O-cetyl ascorbic acid is 0.0004% by weight or more, the direction of skin cell division is adjusted by suppressing the generation of RhoGDIβ. , The barrier function of the skin and the moisturizing power can be enhanced. Preferably, the content of 3-O-cetyl ascorbic acid or 2-O-cetyl ascorbic acid is 0.0004 to 10% by weight. More preferably, the content of 3-O-cetyl ascorbic acid or 2-O-cetyl ascorbic acid is 0.1 to 10% by weight.

The basis for the concentration range of the content of 3-O-cetyl ascorbic acid or 2-O-cetyl ascorbic acid is 0 when converted to 10 μM having an effect on RhoGDIβ expression in the cell experiments shown in the following examples. It is 0004% by weight, the maximum compounding record of the inventors in cosmetics is 10% among other similar vitamin C derivatives, and 0.1% of 3-O-cetyl ascorbic acid. Then, when the penetration into the skin was confirmed, the penetration into the stratum corneum could be confirmed.

In the present embodiment, the cosmetic contains the above-mentioned RhoGDIβ inhibitor.

As long as the effects of the present invention are not impaired, various arbitrary ingredients generally used in cosmetics or used in quasi-drugs and the like can be appropriately blended as needed. Examples of such optional ingredients include purified water, ethanol, oily ingredients, moisturizers, thickeners, preservatives, emulsifiers, medicinal ingredients, powders, ultraviolet absorbers, pigments, fragrances, emulsion stabilizers and the like. Can be done.

Examples of prescriptions for cosmetics containing, for example, 3-O-cetyl ascorbic acid as a RhoGDIβ inhibitor are shown below, but the present invention is not limited thereto.

(Prescription example (cream))
Ingredients Ingredients Amount (% by weight)
Squalene 5.0
Octyldodecanol 4.5
Octyldodecyl myristate 3.5
Dimethicone 2.0
Cetanol 4.0
Beeswax 1.5
Glyceryl stearate 2.5
3-O-cetyl ascorbic acid 1.0
(Na acrylate / acryloyldimethyltaurine) copolymer 0.7
1,3-butylene glycol 5.0
Concentrated glycerin 4.0
Polysorbate 60 2.0
Isohexadecane 0.4
Palm fatty acid Na glutamic acid 0.5
Methyl paraoxybenzoate 0.2
Polysorbate 80 0.1
Sorbitan oleate 0.05
Disodium edetate 0.1
Purified water balance

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

<Action of modifying groups of vitamin C derivatives on the development of RhoGDIβ>
Regarding the inhibitory effect on RhoGDIβ due to the difference in modifying groups (glycerin, phosphate, alkyl group) of vitamin C derivatives, the inhibitory effect on RhoGDIβ was compared and examined using various vitamin C derivatives shown in Table 1 below as test substances. .. Table 1 shows the compound names of vitamin C derivatives having various modifying groups, the modifying groups of each vitamin C derivative, and the modification positions.

(Test substance: various vitamin C derivatives)

(experimental method)
Normal human epidermal keratinized cells (NHEK cells) (manufactured by Keratinocyte) were seeded on a 24-well culture plate at 1 × 10 ⁵ cells / well and cultured at 37 ° C. under 5% CO ₂ for 24 hours. As a medium, HuMedia-KG2 (manufactured by Kurabo Industries Ltd.) was used.

After 1 day of culturing, the compounds of various vitamin C derivatives shown in Table 1 were replaced with HuMedia-KG2 (manufactured by Kurabo Industries, Ltd.) in a medium for drug treatment prepared to 5 to 25 μM, and at 37 ° C. under 5% CO ₂ . It was cultured for 24 hours.

Then, the next day, RNA was extracted / purified in the same manner as above using RNAiso Plus kit (manufactured by Takara Bio Inc.).

Furthermore, the mRNA expression levels of RhoGDIβ and GAPDH (control) were measured by the real-time RT-PCR method using the TaKaRa One Step SYBR Prime Script PLUS RT-PCR kit (manufactured by Takara Bio Inc.) as follows. did. The results are shown in FIGS. 1 (a) to 1 (c). 1 (a) to 1 (c) show relative values when the control value is 1. As a PCR device, a 7300 Real Time PCR System (manufactured by Applied Biosystems) was used.

(Results: Relative expression level of RhoGDIβ by various vitamin C derivatives)
As shown in FIGS. 1 (a) to 1 (c), glyceryl ascorbic acid, 3-glyceryl ascorbic acid, bisglyceryl ascorbic acid, hexyl 3-glyceryl ascorbic acid and L-ascorbic phosphate Mg are compared with the control. However, the expression of RhoGDIβ was equal or increased at all concentrations. In addition, ascorbyl tetra-2-hexyldecanoate and ascorbyl dipalmitate also showed the same or increased expression of RhoGDIβ at all concentrations as compared with the control.

On the other hand, for 3-O-cetyl ascorbic acid, the expression of RhoGDIβ was significantly (about 0.5 times) decreased at a concentration of 10 μM as compared with the control.

Based on the above results, among the various vitamin C derivatives shown in Table 1, various types of modifying groups (glycerin / branched alkyl groups), modification modes (ether bond / ester bond), modification positions and modification numbers of vitamin C derivatives are selected. As evaluated, only 3-O-cetyl ascorbic acid (3-O-hexadecyl ascorbic acid) was found to have an inhibitory effect on RhoGDIβ.

3-O-cetyl ascorbic acid (3-O-hexadecyl ascorbic acid) is a vitamin C derivative on which a linear alkyl group is ether-bonded.

From the above, in order for a vitamin C derivative to exhibit a RhoGDIβ inhibitory effect, it may be important that the vitamin C derivative is modified with a linear alkyl group and has no modifying group at the 6-position. Was inferred. In addition, the modification mode (ether bond) and the number of modifications may also be important factors.

In the development of raw materials for vitamin C derivatives, it is most likely that 3-O-cetyl ascorbic acid (3-O-hexadecyl ascorbic acid) will be used as a commercially available cosmetic raw material. In addition to commercially available cosmetic raw materials, 3-O-cetyl ascorbic acid (3-O-hexadecyl ascorbic acid) is an arbitrarily produced or similarly produced 3-O-cetyl ascorbic acid (3-O-cetyl ascorbic acid). 3-O-hexadecylascorbic acid) can also be expected to have the same effect as described above.

<Comparison of Alkyl Group Lengths of Vitamin C Derivatives>
As described above, since only vitamin C derivatives having a linear alkyl group suppressed the generation of RhoGDIβ in epidermal cells, we next focused on the number of carbon atoms and the modification position in the alkyl group. That is, among the vitamin C derivatives, vitamin C derivatives having an alkyl group having a different carbon number only at the modified position at the 2- or 3-position as a modifying group and not having glycerin as a modifying group are examined. did.

More specifically, as shown in Table 2 below, vitamin C derivatives having an alkyl group having each carbon number (C2, C12, C18) at the modified position at the 2-position were synthesized, and their inhibitory effects on RhoGDIβ were compared. investigated. Further, as shown in Table 2 below, vitamin C derivatives having an alkyl group having each carbon number (C2, C12, C16, C18) at the modified position at the 3-position were synthesized, and their inhibitory effects on RhoGDIβ were compared and examined. ..

(experimental method)
Normal human epidermal keratinized cells (NHEK cells) (manufactured by Keratinocyte) were seeded on a 12-well culture plate at 1 × 10 ⁵ cells / well and cultured at 37 ° C. under 5% CO ₂ for 24 hours. As a medium, HuMedia-KG2 (manufactured by Kurabo Industries Ltd.) was used.

A medium for drug treatment prepared from a compound having an alkyl group having each carbon number (C2, C12, C16, C18) only at the modified position at the 2-position or 3-position with HuMedia-KG2 (manufactured by Kurabo Industries Ltd.) to 5 to 25 μM. And cultured at 37 ° C. under 5% CO ₂ for 24 hours.

Then, RNA was extracted / purified in the same manner as above using RNAiso Plus kit (manufactured by Takara Bio Inc.).

Furthermore, the mRNA expression levels of RhoGDIβ and GAPDH (control) were measured by the real-time RT-PCR method using the TaKaRa One Step SYBR Prime Script PLUS RT-PCR kit (manufactured by Takara Bio Inc.) as follows. did. The results are shown in FIG. FIG. 2 shows the relative value of RhoGDIβ when the control value is 1. As a PCR device, a 7300 Real Time PCR System (manufactured by Applied Biosystems) was used.

The sequence of the primers used in this experiment is shown below.

<Arrangement of primers used>
RhoGDIβ fw: GAG ACA GAG GCA CCC CGG ACA
RhoGDIβ rv: CAG GGA CTT CTG TGG TGG AGG C
GAPDH ww: GCT CTC TGC TCC TCC TGT TC
GAPDH rv: ACG ACC AAA TCC GTT GAC TC

(Result: Relative expression level of RhoGDIβ due to difference in carbon number of vitamin C + alkyl group)
As shown in FIG. 2, for vitamin C derivatives having an alkyl group at the modified position at the 2-position, the relative expression level of RhoGDIβ is a vitamin C derivative having an alkyl group having C12 or more carbon atoms (C12, compared to the control). It can be seen that C18) suppressed the generation of RhoGDIβ (about 0.55 times and about 0.75 times, respectively) at a concentration of 25 μM or more, respectively. A vitamin C derivative having an alkyl group having C16 carbon atoms at the modified position at the 2-position has not been evaluated because it is not commercially available and cannot be easily obtained. However, it is considered that a vitamin C derivative having an alkyl group having a carbon number of C16 at the modified position at the 2-position also has a similar RhoGDIβ inhibitory effect, inferring from the results of other vitamin C derivatives.

Further, as shown in FIG. 2, regarding the vitamin C derivative having an alkyl group at the modified position at the 3-position, the vitamin C derivative having an alkyl group having C12 or more carbon atoms (compared to the control) in the relative expression level of RhoGDIβ ( It can be seen that C12, C16, and C18) suppressed the generation of RhoGDIβ (about 0.8 times, about 0.55 times, and about 0.75 times, respectively) at concentrations of 5 μM, 10 μM, and 25 μM or more, respectively. .. Further, it can be seen that the vitamin C derivative having an alkyl group having C16 carbon atoms suppressed the generation of RhoGDIβ most (about 0.55 times).

<Verification of action on cell division direction>
Next, using a 3D skin model, the action of a vitamin C derivative (3-O-cetyl ascorbic acid) (alkyl group having C16 carbon atoms) exhibiting an RhoGDIβ inhibitory action on the cell division direction was evaluated.

That is, the cell division of the basal layer (basal cell) of the skin is directional, and it always divides in the parallel direction or the vertical direction with respect to the basement membrane. Therefore, using a 3D skin model, vertical (80 to 90 °) and horizontal (0 to 10 °) division of the basal layer (basal cells) of the skin by a vitamin C derivative (3-O-cetyl ascorbic acid). The number of cells was measured as follows, and the effect of 3-O-cetyl ascorbic acid on the cell division direction was examined.

(Action of 3-O-cetyl ascorbic acid on cell division direction)

(experimental method)
A KGF-containing EPI-100 medium (manufactured by Kurabo) was added to a 6-well culture plate (manufactured by FALCON, model number 353046) as a medium for short-term culture, and a three-dimensional skin model (EPI-200) (EPI-200) was added to the medium. (Made by Kurabo) was added and pre-cultured for 1 hour.

Then, the skin three-dimensional model was transferred to a 6-well culture plate supplemented with 1 ml of PBS (-) (manufactured by DS Pharma Biomedical).

Next, the lid of the above plate was removed, and the skin 3D model was irradiated with UVB100 mJ / cm ² and UVB200 mJ / cm ² using DARMARAY 200 type A / B (manufactured by Toko Denki Co., Ltd.) (UVB100 mJ / cm ² ). , 500 μW / cm ² for 3 minutes and 20 seconds, UVB 200 mJ / cm ² at 500 μW / cm ² for 6 minutes and 40 seconds).

Then, replace PBS (-) with a medium in which a drug was added to EPI-100-NMM (manufactured by Kurabo Industries), put two washers on top of each other, put the skin three-dimensional model back on it, and place it at 37 ° C., 5 ° C. Cultured at% CO ₂ .

The medium was changed on the 3rd, 5th, 8th and 10th days of the culture, and the number of cells was measured on the 11th day of the culture. More specifically, EPI-100 and CellTiter 96 AQueuous One Solution CellProliferation Assay (manufactured by Promega) were mixed at a ratio of 9: 1 to prepare a reaction solution, and the reaction solution was placed on a 24-well culture plate (manufactured by Thermo Scientific). 500 μl / well was added. A 3D skin model was transferred to a 24-well culture plate and incubated for 90 minutes.

Then, the reaction solution after incubation was collected on a 96-well culture plate (manufactured by Thermo Scientific), and the absorbance at 450 nm was measured with POWERSCAN HT (manufactured by Dainippon Pharmaceutical Co., Ltd.) to calculate the cell viability.

The three-dimensional skin model after incubation was cut out together with the membrane using a biopsy trepan 8.0 mm (manufactured by Kai Industries, Ltd.) and washed with PBS (−).

A three-dimensional skin model cut out together with the membrane was fixed with a 4% paraformaldehyde phosphate buffer solution (manufactured by Nakaraitesk Co., Ltd.), embedded in paraffin, and then sections were prepared and HE-stained.

Take a picture of the basal cells that are likely to be dividing or just after dividing with a microscope (model number: IX71 manufactured by OLYMPUS), and use the image processing software Image-J to connect the cells that are dividing into two. The angle between the membrane and the membrane (0 ° to 180 °) was measured (N = 80).

Next, regarding the results, the number of dividing cells in the vertical direction (80 to 90 °) and the horizontal direction (0 to 10 °) using a three-dimensional bar graph is shown in FIG. The culture conditions in FIG. 3 are briefly summarized below.

(Culture conditions: All samples contain 0.01% DMSO)
(A) (Control (EPI-100-NMM medium only)
(B) UVB 100 mJ / cm ² (500 μW / cm ² for 3 minutes and 20 seconds, the same applies hereinafter)
(C) UVB 200 mJ / cm ² (500 μW / cm ² for 6 minutes and 40 seconds, the same applies hereinafter)
(D) Add 5 μM 3-O-cetyl ascorbic acid (e) UVB 100 mJ / cm ² + 3-O-cetyl ascorbic acid 5 μM
(F) UVB 200 mJ / cm ² + 3-O-cetyl ascorbic acid 5 μM
(G) Add 10 μM 3-O-cetyl ascorbic acid (h) UVB 100 mJ / cm ² + 3-O-cetyl ascorbic acid 10 μM
(I) UVB 200 mJ / cm ² + 3-O-cetyl ascorbic acid 10 μM

(Result: Number of dividing cells in the vertical and horizontal directions using a 3D bar graph)
As shown in FIGS. 3 (a) to 3 (c), when 3-O-cetyl ascorbic acid was not added, the number of dividing cells in the vertical direction (80 to 90 °) decreased in proportion to the UVB irradiation intensity. rice field.

As shown in FIGS. 3 (d) to 3 (f), when 5 μM of 3-O-cetyl ascorbic acid is added, the horizontal direction (0 to 10 °) is proportional to the concentration of 3-O-cetyl ascorbic acid. The number of normal dividing cells was increasing. From this result, by adding 5 μM of 3-O-cetyl ascorbic acid, the number of normal dividing cells in the horizontal direction (0 to 10 °) increased, and the cells recovered normally in the horizontal direction (0 to 10 °). It can be seen that the number of cells is increasing.

As shown in FIGS. 3 (g) to 3 (i)), when 10 μM of 3-O-cetyl ascorbic acid is added, the vertical direction (80 to 90 °) depends on the concentration of 3-O-cetyl ascorbic acid. The number of normal dividing cells was increasing. From this result, by adding 10 μM of acid to 3-O-cetyl ascorbine, the number of normal dividing cells in the vertical direction (80 to 90 °) increased, and the cells recovered normally in the vertical direction (80 to 90 °). It can be seen that the number of cells is increasing.

As described above, according to the RhoGDIβ inhibitor according to the present embodiment, a vitamin C derivative having a linear alkyl group having C16 carbon atoms suppresses the generation of RhoGDIβ only at the modified position at the 2-position or 3-position. Therefore, it is possible to adjust the direction of skin cell division and enhance the barrier function and moisturizing power of the skin.

In addition, 3-O-cetyl ascorbic acid (3-O-hexadecylascorbic acid) as a vitamin C derivative having a linear alkyl group having C16 carbon atoms generates RhoGDIβ only at the modified position at the 2-position or 3-position. By suppressing the above, it is highly possible that the direction of skin cell division can be adjusted and the barrier function and moisturizing power of the skin can be enhanced.

If the content of 3-O-cetyl ascorbic acid (3-O-hexadecyl ascorbic acid) is 0.0004% by weight or more, the generation of RhoGDIβ is suppressed, and the direction of skin cell division. It is highly possible that it can adjust the sex and enhance the barrier function and moisturizing power of the skin.

In this example, 3-O-cetyl ascorbic acid (3-O-hexadecyl ascorbic acid) was used to study the inhibitory effect on RhoGDIβ, but the present invention is not limited to this, and for example, In addition to 2-O-cetyl ascorbic acid (2-O-hexadecyl ascorbic acid) having a linear alkyl group having C16 carbon atoms at the modified position at the 2-position, the number of carbon atoms at at least one of the modified positions at the 2-position and 3-position is carbon. Similar results can be expected even when a vitamin C derivative having a linear alkyl group of C16 and having no modifying group at the modified position at the 6-position is used.

The form of the RhoGDIβ inhibitor according to the present embodiment is not particularly limited, and various forms adopted in general RhoGDIβ inhibitors and cosmetics shall be adopted within a range that does not impair the effects of the present invention. Can be done.

For example, the form of the RhoGDIβ inhibitor can be liquid, lotion, milky, creamy, ointment, semi-solid, oil gel, sheet or the like. Further, for example, oils, dyes, preservatives, surfactants, fragrances, pigments and the like can be appropriately blended according to the dosage form of the RhoGDIβ inhibitor according to the present embodiment.

The RhoGDIβ inhibitor of the present embodiment can be applied not only to the cosmetics of the present invention but also to existing (conventional) cosmetics, and the versatility of cosmetics containing vitamin C derivatives is enhanced. Furthermore, it can be applied not only to cosmetics but also to external agents and drugs.

It should be noted that the RhoGDIβ inhibitor of the present invention is not limited to the above-mentioned embodiment, and of course, it can be appropriately changed without departing from the gist of the present invention.

INDUSTRIAL APPLICABILITY The present invention is effectively used in cosmetics containing a RhoGDIβ inhibitor and a RhoGDIβ inhibitor that can enhance the barrier function and moisturizing power of the skin by suppressing the generation of RhoGDIβ.

Claims (2)

Of the hydroxy groups at the 2- or 3-positions of vitamin C, only the hydrogen group of one of the hydroxy groups has 12, 16 or 18 carbon atoms to orient the cell division in the basal layer of the skin. A RhoGDIβ inhibitor comprising a vitamin C derivative substituted with a linear alkyl group of the same and the hydrogen group of the other hydroxy group not substituted. The RhoGDIβ inhibitor according to claim 1, wherein the vitamin C derivative is either 3-O-cetyl ascorbic acid or 2-O-cetyl ascorbic acid.

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