JP6978015B1 - Composition for skin application to increase the expression of CCN1 and CCN2 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel composition for skin application for increasing the expression of CCN1 and CCN2. It increases the expression of CCN1 and CCN2, which comprises water extract of Clitoria ternatea or less than 10% alcohol extract and / or both of water extract of Pandan or less than 10% alcohol extract. Composition for skin application. [Selection diagram] None

Description

The present disclosure relates to a composition for skin application to increase the expression of CCN1 and CCN2.

The skin is composed of the epidermis, the dermis and the hypodermis from the outermost layer. The epidermis plays a role in protecting the body from the external environment and ensuring its healthy condition. The epidermis also plays a role in preventing the skin from drying out by suppressing the invasion of microorganisms and chemical substances and preventing the evaporation of water.

The dermis is composed of fibroblasts, tissue spheres / macrophages, mast cells, and plasma cells. Fibroblasts are cells that produce collagen, elastin, hyaluronic acid, etc., and play a role in forming the dermis structure with collagen as a fiber bundle. Fibroblasts also play a role in migrating to the injured area when the skin tissue is damaged and initiating the production of extracellular matrix such as collagen. Due to these roles, fibroblasts play an important role in the process of wound healing.

Fibroblasts are further reduced in proliferative capacity by the effects of UV light, desiccation, stress, and aging. Therefore, if the proliferative ability of fibroblasts is affected, it may cause skin aging such as a decrease in skin elasticity, or the rate of wound healing may be slowed down.

Studies for activating the proliferation of fibroblasts have been reported (Patent Document 1).

Japanese Unexamined Patent Publication No. 2013-194008

The present disclosure is intended to provide novel compositions for novel skin applications.

The present disclosure provides the following inventions:
[Item 1]
Skin for increasing expression of CCN1 and CCN2, comprising water extract of Clitoria ternatea or less than 10% alcohol extract and / or both of water extract of Pandan or less than 10% alcohol extract. Composition for application.
[Item 2]
Item 2. The composition according to Item 1, which is used for preventing skin aging, healing wounds, or promoting hair growth.
[Item 3]
For promoting skin differentiation from epidermal stem cells to epidermal keratinocytes, or for preventing skin aging by promoting proliferation of epidermal stem cells and / or epidermal keratinocytes; for wound healing by promoting proliferation of fibroblasts; or from hair follicle stem cells to hair papillae Item 2. The composition according to Item 2, which is used for promoting hair growth by promoting differentiation into cells or promoting proliferation of hair follicle stem cells and / or hair papilla cells.
[Item 4]
Item 6. The composition according to any one of Items 1 to 3, which comprises a water extract of Clitoria ternatea.
[Item 5]
Item 6. The composition according to any one of Items 1 to 3, which comprises a water extract of Pandanaceae.
[Item 6]
Less than 10% alcohol extract of the butterfly bean is less than 10% ethanol extract of Pandan, and less than 10% alcohol extract of Pandan is less than 10% ethanol extract of Pandan. Item 2. The composition according to any one of Items 1 to 3.

A bar graph showing the expression ratio of CCN1 by the water extract. A bar graph showing the expression ratio of CCN1 with 70% ethanol extract. A bar graph showing the expression ratio of CCN2 by the water extract. A bar graph showing the expression ratio of CCN2 with 70% ethanol extract.

"Clitoria ternatea L." is a leguminous plant belonging to the genus C. ternatea of the genus Clitoria. Clitoria ternatea can be cultivated and harvested, for example, by known methods, or is commercially available. In one embodiment, the Clitoria ternatea may be at least one site selected from the group consisting of flowers, leaves, stems, and roots, preferably one or both of the flowers and leaves, more preferably. Is a flower. The weight of Clitoria ternatea is the weight of the dried form.

The water extract of Clitoria ternatea can be obtained, for example, by mixing pulverized C. elegans and water, and separating and removing the solid matter from the obtained mixture by a separation method such as centrifugation or filtration. In one embodiment, the water extract of Clitoria ternatea (preferably dried flower) is obtained by mixing the pulverized C. elegans and 10 times the weight of water of the pulverized product, and the resulting mixed solution is 20 at room temperature. It can be obtained by separating and removing the solid matter from the mixed solution by a separation method such as centrifugation or filtration after leaving it for a minute. The water extract of Clitoria ternatea may be liquid or solid. The water extract of Clitoria ternatea (solid) can be prepared, for example, by freeze-drying the water extract of Clitoria ternatea (liquid).

For the ethanol extract of less than 10% of Clitoria ternatea, for example, a pulverized C. elegans and an aqueous ethanol solution of less than 10% w / w are mixed, and a solid substance is separated from the obtained mixed solution by a separation method such as centrifugation or filtration. It can be obtained by separation and removal. In one embodiment, the ethanol extract of less than 10% of Clitoria ternatea is a pulverized product of Clitoria ternatea (preferably dried flower) and an aqueous ethanol solution of less than 10% w / w, which is 10 times the weight of the pulverized product. It can be obtained by mixing and allowing the obtained mixed solution to stand at room temperature for 20 minutes, and then separating and removing solid matter from the mixed solution by a separation method such as centrifugation or filtration. The ethanol extract of less than 10% w / w of Clitoria ternatea may be liquid or solid. The ethanol extract (solid) of less than 10% w / w of Clitoria ternatea can be prepared, for example, by freeze-drying the ethanol extract (liquid) of less than 10% w / w of Clitoria ternatea. The ethanol extract (liquid) of less than 10% w / w of Clitoria ternatea is, for example, a liquid obtained by solidifying the ethanol extract (liquid) of less than 10% w / w by freeze-drying and dissolving the solid in water. May be.

Ethanol extract of Clitoria ternatea is, for example, ethanol of less than 8% w / w, ethanol of less than 5% w / w, ethanol of less than 3% w / w, or ethanol of less than 1% w / w, and crushed C. elegans. It may be prepared by mixing with a substance and separating and removing the solid substance from the obtained mixed solution.

"Pandanus" is a monocotyledonous plant belonging to the genus Pandanaceae of the Pandanaceae family, and is also called panda leaf. Pandanaceae can be cultivated and harvested, for example, by known methods, or are commercially available. In one embodiment, Pandanaceae may be at least one site selected from the group consisting of leaves, stems, and roots, preferably one or both of the leaves and stems, more preferably. It is a leaf. The weight of Pandanaceae is the weight of the dried form.

The water extract of Pandanaceae can be obtained, for example, by mixing pulverized Pandanaceae with water and separating and removing the solid matter from the obtained mixed solution by a separation method such as centrifugation or filtration. .. In one embodiment, the water extract of Pandan (preferably dried leaves) is prepared by mixing a pulverized product of Pandan and 10 times the weight of water of the crushed product, and the resulting mixed solution is brought to room temperature. It can be obtained by separating and removing the solid matter from the mixed solution by a separation method such as centrifugation or filtration after leaving it for 20 minutes. The water extract of Pandanaceae may be liquid or solid. The water extract (solid) of Pandanaceae can be prepared, for example, by freeze-drying the water extract (liquid) of Pandanaceae.

The ethanol extract of less than 10% of Pandanaceae is obtained by mixing, for example, a pulverized product of Pandanaceae and an aqueous ethanol solution of less than 10% w / w, and solidifying the obtained mixed solution by a separation method such as centrifugation or filtration. It can be obtained by separating and removing objects. In one embodiment, the ethanol extract of less than 10% of Pandan is a pulverized product of Pandan (preferably dried leaves) and an aqueous ethanol solution of less than 10% w / w, which is 10 times the weight of the pulverized product. It can be obtained by mixing and leaving the obtained mixed solution at room temperature for 20 minutes, and then separating and removing the solid matter from the mixed solution by a separation method such as centrifugation or filtration. The ethanol extract of Pandanaceae less than 10% w / w may be liquid or solid. An ethanol extract (solid) of less than 10% w / w of Pandanaceae can be prepared, for example, an ethanol extract (liquid) of less than 10% w / w of Pandanaceae by freeze-drying. For the ethanol extract (liquid) of less than 10% w / w of Pandan, for example, the ethanol extract (liquid) of less than 10% w / w was solidified by freeze-drying or the like, and the solid was dissolved in water. It may be a liquid.

Ethanol extracts of Pandanaceae include, for example, ethanol of less than 8% w / w, ethanol of less than 5% w / w, ethanol of less than 3% w / w, or ethanol of less than 1% w / w, and Pandanaceae. It may be prepared by mixing with the pulverized product of the above and separating the solid substance from the obtained mixed solution.

The "composition for skin application" may be in a form applicable to the skin, and may be a solution system, an emulsifying system, a powder system, a gel system, an ointment system, a cream, a mist, an aerosol, a water-oil two-layer system, or water. -Oil-powder may be a three-layer system. The composition for skin application is, for example, a water extract of butterfly bean or an alcohol extract of less than 10%, and either or both of a water extract of Pandan or an alcohol extract of less than 10%. 95% w / w or less, 90% w / w or less, 80% w / w or less, 70% w / w or less, 60% w / w or less, 50% w / w or less, 40% w / w or less per weight , 30% w / w or less, 20% w / w or less, 10% w / w or less, or 5% w / w or less. The composition for skin application may further contain a carrier or excipient applicable to the skin and, if necessary, a suitable additive as appropriate. The composition for skin application can be prepared by a known method. In one example, the composition for skin application is applied to the skin with either or both of a water extract of Clitoria ternatea or an alcohol extract of less than 10% and a water extract of Pandan or an alcohol extract of less than 10%. It can be prepared by mixing with a possible carrier or excipient.

"CCN1" is a protein belonging to the CCN family, also known as Cysteine rich 61 (Cyr61). CCN1 is involved in cell proliferation, cell motility, cell differentiation, and cell aging through interaction with cell surface integrin receptors and heparan sulfate proteoglycans. The amino acid sequence and nucleic acid sequence of CCN1 are known.

"CCN2" is a protein belonging to the CCN family, also known as connective tissue growth factor (CTGF). CCN2 is involved in promoting fibroblast proliferation, promoting extracellular air mass production, promoting connective tissue proliferation, chondrocyte proliferation, promoting cell differentiation, wound healing, and fibrotic diseases. The amino acid and nucleic acid sequences of CCN2 are known.

The "increased expression" of CCN1 and CCN2 means that the expression level of CCN1 and the expression level of CCN2 when the composition according to the present disclosure is added to cultured human skin fibroblasts is the addition of the composition according to the present disclosure. It may be increased by at least 20%, 30%, 40%, or 50% as compared with the expression level of CCN1 and the expression level of CCN2 when not used. The expression level of CCN1 and CCN2 means the amount of RNA in cultured human fibroblasts corresponding to CCN1 or CCN2. The amount of RNA can be measured by RT-PCR as described in the examples herein.

"Skin anti-aging" means that the elasticity of the skin is maintained or improved. To prevent skin aging, the expression of CCN1 and CCN2 is increased, the differentiation of epidermal stem cells into epidermal keratinocytes is promoted, the proliferation of epidermal stem cells and / or epidermal keratinocytes is promoted, and the proliferation of dermal stem cells and / Alternatively, it can be achieved by promoting the differentiation into fibroblasts, or by promoting the production of collagen, elastin, and hyaluronic acid by the differentiation of fibroblasts. For skin aging prevention, for example, when the composition according to the present disclosure is applied to human skin once to three times a day for 3 weeks, as compared with the case where the composition according to the present disclosure is not applied. The elasticity of the skin may be maintained or improved by 5% or more, 10% or more or 15% or more.

"Wound healing" means that wound healing is promoted. Wound healing can be achieved by increased expression of CCN1 and CCN2, accelerated proliferation of fibroblasts, or migration and proliferation of epidermal cells. Wound healing is enhanced, for example, when the composition according to the present disclosure is applied to a wound on the skin, as compared to when the composition according to the present disclosure is not applied to a wound on the same degree of skin. It may be that.

"Promoting hair growth" means promoting hair growth. Hair promotion can be achieved by increasing the expression of CCN1 and CCN2, promoting the differentiation of hair follicle stem cells into hair papilla cells, or promoting the proliferation of hair follicle stem cells and / or hair papilla cells. The promotion of hair growth may be, for example, when the composition according to the present disclosure is applied to the scalp, the hair on the scalp is increased as compared with the case before the composition according to the present disclosure is applied.

In one embodiment, the skin-applied composition according to the present disclosure is for skin aging prevention, wound healing, or hair growth promotion, preferably for skin aging prevention or wound healing, and more preferably. It is for preventing skin aging.

Specific examples will be described below, but they show preferred embodiments of the present invention and do not limit the inventions described in the appended claims in any way.

[Preparation example]
No. in the table below. 1-No. 2 g of the dried plant sample corresponding to No. 8 was crushed by using a mortar and a pestle or by packing in a bag and tapping. Each crushed plant sample was mixed with 20 ml of water. The obtained mixed solution was left at room temperature for 20 minutes. The mixed solution was centrifuged (3000 rpm, 10 minutes), and the supernatant was used as an aqueous extract of each plant sample (Comparative Examples 1 to 6, Examples 1 and 2).

No. in the table below. 1-No. 2 g of the dried plant sample corresponding to No. 8 was crushed by using a mortar and a pestle or by packing in a bag and tapping. Each crushed plant sample was mixed with 20 ml of 70% ethanol. In the same manner as above, ethanol extracts (Comparative Examples 7-14) of each plant sample were obtained from the obtained mixed solution.

[Cell culture]
Human skin fibroblasts were cultured in a petri dish containing 3 ml of culture medium. 0.25 ml of the water extract of Comparative Example 1 was added to the culture broth. 0.25 ml of each water extract of Comparative Examples 2 to 6, Examples 1 and 2 was added to the culture broth. As a control, 0.25 ml of water was used (C1). Similarly, 0.25 ml of each ethanol extract of Comparative Examples 7 to 14 was added to the culture broth. As a control, 0.25 ml of 70% ethanol was used (C2). The skin fibroblasts were further cultured for 24 hours at _{37 ° C. under 5% CO 2} in the culture medium to which each extract was added.

After further culture, human skin fibroblasts were harvested from each petri dish. The expression levels of CCN1 and CCN2 in the recovered human skin fibroblasts were quantified by RT-PCT using Applied Biosystems 7000 Sequence Detection System. RT-PCR was performed using the One Step TB Green® PrimeScript ™ RT-PCR Kit according to the instructions attached to the kit. For RT-PCR of CCN1, primers having each nucleic acid sequence shown in SEQ ID NOs: 1 and 2 were used. For RT-PCR of CCN2, primers having each nucleic acid sequence shown in SEQ ID NOs: 3 and 4 were used.

[result]
From the CCN1 expression level when the water extract of each plant sample (No. 1 to No. 8) was used, a relative value with respect to the CCN1 expression level when the control C1 was used was calculated. The calculated result is shown in FIG. FIG. 1 shows the plant sample No. 1-No. 3 and No. 5 shows that the expression level of CCN1 was increased as compared with the control C1.

From the CCN1 expression level when the 70% ethanol extract of each plant sample (No. 1 to No. 8) was used, a relative value with respect to the CCN1 expression level when the control C2 was used was calculated. The calculated result is shown in FIG. FIG. 2 shows the plant sample No. 1 and No. 3 to No. 8 shows that the expression level of CCN1 was increased as compared with the control C2.

1 and 2 show the plant sample No. 1, No. 3 and No. 5 showed that both the water extract and the 70% ethanol extract increased the expression level of CCN1 as compared to controls C1 and C2, respectively. 1 and 2 also show the plant sample No. 2 shows that in the case of a water extract, the expression of CCN1 is increased, but in the case of a 70% ethanol extract, the expression of CCN1 is decreased.

From the CCN2 expression level when the water extract of each plant sample (No. 1 to No. 8) was used, a relative value with respect to the CCN2 expression level when the control C1 was used was calculated. The calculated result is shown in FIG. FIG. 3 shows the plant sample No. 2. No. 3 and No. 8 shows that the expression level of CCN2 was increased as compared with the control C1.

From the CCN2 expression level when the 70% ethanol extract of each plant sample (No. 1 to No. 8) was used, a relative value with respect to the CCN2 expression level when the control C2 was used was calculated. The calculated result is shown in FIG. FIG. 4 shows the plant sample No. 1 indicates that the expression level of CCN2 was increased as compared with the control C2.

3 and 4 show the plant sample No. 2. No. 3 and No. 8 shows that in the case of a water extract, the expression of CCN2 is increased, but in the case of a 70% ethanol extract, the expression of CCN2 is decreased.

1 to 4 show No. 2 and No. It is shown that the water extract of No. 3 increased the expression level of CCN1 and CCN2, respectively, as compared with the controls C1 and C2.

Claims (2)

A composition for skin application, comprising a water extract of Clitoria ternatea, for increasing expression of CCN1 and CCN2, wherein the composition for skin application is for wound healing. The composition according to claim 1, wherein the composition is for promoting the proliferation of fibroblasts or for wound healing by migration and proliferation of epidermal cells.

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