JP6969042B2 - Claudin-1 production promoter, occludin production promoter, epidermal tight junction constituent protein production promoter in human skin three-dimensional model, and skin barrier function decline inhibitor - Google Patents

Description

The present invention relates to a hyaluronidase activity inhibitor, a hydrogen peroxide scavenger, a whitening agent, an anti-aging agent, and a hair restorer.

Hyaluronate, which maintains its affinity for body tissues, is decomposed by ultraviolet rays, enzymes, etc. in the water-containing system, and the water retention effect decreases as the molecular weight decreases. Hyaluronic acid also exists as an intercellular tissue and is involved in vascular permeability. Furthermore, hyaluronidase is thought to be involved in degranulation from mast cells by activation in mast cells. Therefore, by inhibiting the activity of hyaluronidase, which is a hydrolase of hyaluronic acid, hyaluronic acid can be stabilized, various chemical mediators can be prevented from being released from mast cells, and anti-inflammatory can be expected. Examples of crude drugs having such a hyaluronidase activity inhibitory effect include extracts of plants of the genus Osvecchia (see Patent Document 1), wisteria tea extracts (see Patent Document 2), rosemary extracts, thyme extracts and Melissa extracts. Things (see Patent Document 3), etc. have been reported.

Until now, the development of whitening agents has been promoted by focusing on tyrosinase, which is a rate-determining enzyme for melanin production. Recently, after irradiation with ultraviolet UVB, production from epidermal keratinocytes increases and activates pigment cells (melanocytes). As cytokines, α-melanocyte stimulating hormone (α-MSH), endoserin-1 (ET-1), nitrogen monoxide (NO), basic fibroblast growth factor (bFGF), granulocyte / macrophage / colony stimulating factor (GM) -CSF), stem cell factor (SCF), etc. have been reported, and the development of substances that suppress melanin production and lead to a whitening effect by blocking the signal transduction system in which they are involved has been actively carried out. ing. For example, chamomile extract and altea extract have been reported as extracts of crude drugs that inhibit the action of endothelin-1 (ET-1) on pigment cells (melanocytes) (see Non-Patent Document 1). ..

In addition, conventionally, it was thought that the barrier function of the skin is carried only by the stratum granulosum, but the constituent proteins of tight junctions (hereinafter, may be abbreviated as TJ) existing in the stratum granulosum of the epidermis are at the gene level. In recent years, it is considered that TJs also play an important role in the barrier function of the skin (see Non-Patent Document 2), because the barrier function of the skin is disrupted when the skin is defective. The TJ is a binding device that not only brings adjacent cells into close contact with each other but also controls the permeation of a substance by sealing the gap between cells. The proteins constituting this TJ include claudin, occludin, ZO-1 and ZO-2, and these proteins are thought to constitute the skeleton of the TJ strand and control the barrier function of the TJ. (See Non-Patent Document 3). From the above, when the expression of claudin, occludin, ZO-1, and ZO-2 is reduced for some reason, structural destruction of TJ occurs and it does not function as a permeation barrier for substances, resulting in dry skin. It is expected to cause skin symptoms such as rough skin, atopic dermatitis and various infectious diseases.

Therefore, by promoting the production of claudin, occludin, ZO-1 and ZO-2 in the epidermis to promote the formation of TJ in epidermal keratinized cells, the barrier function and water retention function of the skin are enhanced, and the skin symptoms are described. Is considered to be able to prevent or improve. Based on this idea, Coptis chinensis extract derived from natural products (see Patent Document 4), Tohi extract (see Patent Document 5) and the like are disclosed as those for improving the skin barrier function through the TJ formation promoting action. ing.

The reaction of amino groups of amino acids, peptides, and proteins with reducing sugars such as ketones, aldehydes, and especially glucose to produce brown pigments is called the Maillard reaction. Substances produced as the final product of the Maillard reaction are referred to as advanced glycation end products (hereinafter, also referred to as "AGEs"). The Maillard reaction is an early reaction in which an amino group and glucose react non-enzymatically to form a Schiff base and then cause an Amadori rearrangement, and an active intermediate having a dicarbonyl group such as 3-deoxyglucoson (3-DG). It consists of a middle-term reaction that produces AGEs, and a late-stage reaction in which the active intermediate reacts non-enzymatically with the amino group and repeats dehydration and condensation reactions to form AGEs.
Examples of AGEs include imidazolone (see Non-Patent Document 4), N ^ε -carboxymethyl lysine (CML) (see Non-Patent Document 5), pentosidine, pyrarin, crosulin, N ^ε -carboxyethyl lysine, and methylglyoxal lysine dimer. Glyoxal lysine dimer and the like have been identified. It has been reported that imidazolone is produced by reacting 3-DG with arginine (see Non-Patent Document 4).

Aging symptoms are one of the pathological conditions involved in the onset and progression of AGEs. The progress of the Maillard reaction in living tissue causes aging (decreased elasticity) due to cross-linking of skin elastic fibers in the skin tissue, and the deposition of AGEs on the blood vessel wall tissue and neurofibrils leads to arteriosclerosis and Alzheimer's disease. It is also said to be.

As a natural product-derived substance having an AGEs production inhibitory action, for example, a pericarp extract of the leguminous dialium indam is disclosed (see Patent Document 6).
Further, as a compound having an AGEs production inhibitory action, for example, compounds such as aminoguanidine, OPB-9195, and pyridoxamine are known, but these compounds have problems such as side effects (Non-Patent Documents 4 to 6). reference).

Many steroid hormones are molecular types secreted from producing organs and bind to receptors to exert their actions, but in the case of male hormones collectively called androgens, for example, testosterone enters the cells of the target organ and testosterone. It is reduced to 5α-dihydrotestosterone (5α-DHT) by 5α-reductase, then binds to the receptor and exerts its action as an androgen.
The androgen is an important hormone, but when it acts excessively, it causes various diseases such as androgenetic alopecia, hyperplasia, seborrhea, acne (acne, etc.), benign prostatic hyperplasia, prostate tumor, and precocious puberty. Induces unwanted symptoms. Therefore, it is active by suppressing the action of excess androgen in order to improve these various symptoms, specifically, by inhibiting the action of testosterone 5α-reductase, which reduces testosterone to active 5α-DHT. A method for suppressing the generation of 5α-DHT and a method for inhibiting the binding of 5α-DHT generated from testosterone to a receptor to prevent the expression of androgen activity are disclosed. As a plant extract having an action of inhibiting the binding between 5α-DHT and its receptor, for example, at least one of Magito and Katya is disclosed (see Patent Document 7).

Hair repeats growth and shedding according to a periodic hair cycle (hair cycle) consisting of a growth phase, a catagen phase, and a resting phase. Of this hair cycle, the stage where new hair follicles are formed from the telogen to the growth phase is considered to be the most important for hair growth, and it is responsible for the proliferation or differentiation of hair follicle epithelial cells at this stage. It is believed that telogen cells play an important role. Hair follicle cells are cells located inside the hair follicle epithelial cells consisting of outer hair follicle sheath cells and matrix cells near the hair root, and are located at the root of the hair follicle surrounded by the basal membrane. It plays an important role in the proliferation and differentiation of hair follicle epithelial cells and the formation of hair, such as acting on epithelial cells to promote their proliferation. The hair papilla cells play the most important role in the proliferation and differentiation of hair follicle epithelial cells and the formation of hair, and the target substance is brought into contact with the cultured hair papilla cells to determine the presence or absence or strength of the proliferation activity of the cells. By specifying, a method for testing the hair growth effect of the target substance has been proposed. As a crude drug having such a dermal papilla cell proliferation promoting action, for example, an augin extract, a Coptis chinensis extract, a Wedelia chinensis extract and the like are disclosed (see Patent Documents 8 and 9).

However, to date, excellent substances that have at least one of the above-mentioned effects and are highly safe and therefore can be widely used as components of cosmetics, foods and drinks, research reagents, etc. are still available. It has not been obtained, and its prompt provision is strongly required.

Japanese Unexamined Patent Publication No. 2003-55242 Japanese Unexamined Patent Publication No. 2003-12532 Japanese Unexamined Patent Publication No. 8-333267 Japanese Unexamined Patent Publication No. 2007-176830 Japanese Unexamined Patent Publication No. 2007-176835 Japanese Unexamined Patent Publication No. 2010-1111615 Japanese Unexamined Patent Publication No. 2002-241297 Japanese Unexamined Patent Publication No. 9-208431 Japanese Unexamined Patent Publication No. 11-12134

"Fragrance Journal", Vol. 28, No. 9, p65-71, published in 2000 J. Cell Biol. , Vol. 156, pp. 1099-1111 (2002) Journal of the Japanese Society of Cosmetic Science, vol. 31, pp. 296-301 (2007) J. Clin. Invest. , Vol. 99, pp. 1272-1280 (1997) Kidney Int. , Vol. 50, pp. 1303-1309 (1996) J. Biol. Chem. , Vol. 275, pp. 21177-21184 (2000)

An object of the present invention is to solve the above-mentioned conventional problems and to achieve the following objects. That is, the present invention is an excellent hyaluronidase activity inhibitor having an excellent hyaluronidase activity inhibitory action and a highly safe hyaluronidase activity inhibitor, an excellent hyaluronidase scavenging agent having an excellent hydrogen peroxide scavenging action, and an excellent hydrogen peroxide scavenging agent. Provided are a whitening agent having a whitening effect and a high safety, an anti-aging agent having an excellent anti-aging effect and a high safety, and a hair growing agent having an excellent hair-growth effect and a high safety. The purpose is.

As a result of diligent studies to solve the above problems, the present inventors have found that the hibiscus extract has an excellent hyaluronidase activity inhibitory action, hydrogen peroxide scavenging action, whitening action, anti-aging action, and hair growth action. It was found to have.

The present invention is based on the above-mentioned findings of the present inventors, and the means for solving the above-mentioned problems are as follows. That is,
<1> A hyaluronidase activity inhibitor characterized by containing an extract of hibiscus.
<2> A hydrogen peroxide scavenger characterized by containing an extract of hibiscus.
<3> A whitening agent characterized by containing an extract of hibiscus.
<4> Suppressing melanin production on B16 melanoma cells, suppressing the expression of endoserin-1 mRNA, suppressing the expression of stem cell growth factor mRNA, suppressing the expression of basic fibroblast growth factor mRNA, and suppressing the expression of proopiomelanocortin mRNA. The whitening agent according to <3>, which has at least one of the actions.
<5> An anti-aging agent characterized by containing an extract of hibiscus.
<6> Epidermal hyaluronic acid production promoting action, glutathione production promoting action, seryllumitoyltransferase mRNA expression promoting action, Maillard reaction inhibitory action, advanced glycation end product formation inhibitory action, advanced glycation end product decomposition promoting action, claudin-1 production promoting action, The anti-aging agent according to <5>, which has at least one of an occludin production promoting action, an epidermal tight junction constituent protein production promoting action in a human skin three-dimensional model, and a skin barrier function deterioration suppressing action.
<7> A hair restorer characterized by containing an extract of Shoeblackplant.
<8> The hair restorer according to <7>, which has at least one of a testosterone 5α-reductase activity inhibitory action and a dermal papilla cell proliferation action.

According to the present invention, the above-mentioned problems in the prior art can be solved, the above-mentioned object can be achieved, an excellent hyaluronidase activity inhibitory action, a highly safe hyaluronidase activity inhibitor, and an excellent hydrogen peroxide scavenging action. A hydrogen scavenger with high safety, a whitening agent with excellent whitening effect, a whitening agent with high safety, an anti-aging agent with excellent anti-aging effect, and an excellent anti-aging agent. It is possible to provide a hair-growth agent having a hair-growth effect and having high safety.

FIG. 1A is a photograph showing the claudin-4 immunofluorescent staining image in Example 16 and showing the control result on the 4th day of culture. FIG. 1B shows a claudin-4 immunofluorescent stained image in Example 16 and is a photograph showing the results of a hibiscus extract having a sample concentration of 100 μg / mL on the 4th day of culture. FIG. 1C shows a claudin-4 immunofluorescent stained image in Example 16 and is a photograph showing the results of an extract of hibiscus having a sample concentration of 500 μg / mL on the 4th day of culture. FIG. 1D is a photograph showing the claudin-4 immunofluorescent staining image in Example 16 and showing the control results on the 7th day of culture. FIG. 1E shows a claudin-4 immunofluorescent stained image in Example 16 and is a photograph showing the results of a hibiscus extract having a sample concentration of 100 μg / mL on the 7th day of culture. FIG. 1F shows a claudin-4 immunofluorescent stained image in Example 16 and is a photograph showing the results of an extract of hibiscus having a sample concentration of 500 μg / mL on the 7th day of culture. FIG. 2A is a photograph showing the ZO-1 immunofluorescent staining image in Example 16 and showing the control result on the 4th day of culture. FIG. 2B shows the ZO-1 immunofluorescent staining image in Example 16 and is a photograph showing the result of the hibiscus extract having a sample concentration of 100 μg / mL on the 4th day of culture. FIG. 2C shows the ZO-1 immunofluorescent staining image in Example 16 and is a photograph showing the result of the hibiscus extract having a sample concentration of 500 μg / mL on the 4th day of culture. FIG. 2D is a photograph showing the ZO-1 immunofluorescent staining image in Example 16 and showing the control result on the 7th day of culture. FIG. 2E shows the ZO-1 immunofluorescent staining image in Example 16 and is a photograph showing the result of the hibiscus extract having a sample concentration of 100 μg / mL on the 7th day of culture. FIG. 2F shows the ZO-1 immunofluorescent staining image in Example 16 and is a photograph showing the result of the hibiscus extract having a sample concentration of 500 μg / mL on the 7th day of culture. FIG. 3A is a photograph showing the ZO-2 immunofluorescent staining image in Example 16 and showing the control result on the 4th day of culture. FIG. 3B shows the ZO-2 immunofluorescent staining image in Example 16 and is a photograph showing the result of the hibiscus extract having a sample concentration of 100 μg / mL on the 4th day of culture. FIG. 3C shows the ZO-2 immunofluorescent staining image in Example 16 and is a photograph showing the result of the hibiscus extract having a sample concentration of 500 μg / mL on the 4th day of culture. FIG. 3D shows the ZO-2 immunofluorescent staining image in Example 16 and is a photograph showing the result of the control on the 7th day of culture. FIG. 3E shows the ZO-2 immunofluorescent staining image in Example 16 and is a photograph showing the result of the hibiscus extract having a sample concentration of 100 μg / mL on the 7th day of culture. FIG. 3F shows the ZO-2 immunofluorescent staining image in Example 16 and is a photograph showing the result of the hibiscus extract having a sample concentration of 500 μg / mL on the 7th day of culture.

(Hyaluronidase activity inhibitor, hydrogen peroxide scavenger, whitening agent, anti-aging agent, and hair restorer)
The hyaluronidase activity inhibitor, hydrogen peroxide scavenger, whitening agent, anti-aging agent, and hair restorer of the present invention all contain an extract of hibiscus, and further contain other components as necessary.

The hyaluronidase activity inhibitor has a hyaluronidase activity inhibitory action.
The hydrogen peroxide scavenging agent has a hydrogen peroxide scavenging action.
The whitening agent has a melanin production inhibitory effect on B16 melanoma cells, an endoserin-1 mRNA expression increase inhibitory effect, a stem cell growth factor mRNA expression increase inhibitory effect, a basic fibroblast growth factor mRNA expression increase inhibitory effect, and proopiomelanocortin mRNA expression. It has a whitening effect based on at least one of the ascending inhibitory effects.
The anti-aging agent has an epidermal hyaluronic acid production promoting action, a glutathione production promoting action, a seryllumitoyltransferase mRNA expression promoting action, a Maillard reaction inhibitory action, an advanced glycation end product formation inhibitory action, an advanced glycation end product decomposition promoting action, and a claudin-1 production. It has an anti-aging action based on at least one of a promoting action, an occludin production promoting action, an epidermal tight junction constituent protein production promoting action in a human skin three-dimensional model, and a skin barrier function deterioration suppressing action.
The hair growth agent has a hair growth action based on at least one of a testosterone 5α-reductase activity inhibitory action and a dermal papilla cell proliferation action.

The details of the substance contained in the hibiscus extract that exerts at least one of hyaluronidase activity inhibitory action, hydrogen peroxide scavenging action, whitening action, anti-aging action, and hair growth action are unknown. It has not been known at all that the extract has such an excellent action and is useful as a hyaluronidase activity inhibitor, a hydrogen peroxide scavenger, a whitening agent, an anti-aging agent, and a hair restorer. This is a new finding by the present inventors.

The hibiscus is a evergreen shrub belonging to the Malvaceae hibiscus, Scientific name: the Africa called the Hibiscus subduction Reference (Hibiscus sabdarifa) as the origin, the Japanese name is called Roselle. The hibiscus has a refreshing acidity and is also used as a sauce for French and Italian cuisine, and is used as an herb.

The site of the hibiscus to be used as an extraction raw material is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include branches and leaves, trunks, bark, roots, rhizomes, root bark, and mixtures thereof, and among these, flower parts such as flowers, buds, and buds are preferable.

The hibiscus, which is a raw material for extraction, can be used for solvent extraction, for example, after being dried, as it is or in a state of being crushed using a coarse crusher or the like. Above all, as the extraction raw material, those which are dried and crushed immediately after collection are preferable. The drying may be performed in the sun, for example, or may be performed using a commonly used dryer. The hibiscus may be used as an extraction raw material after being subjected to pretreatment such as degreasing with a non-polar solvent such as hexane or benzene. By performing pretreatment such as degreasing, the extraction treatment of the hibiscus with a polar solvent can be efficiently performed.

The extract of Shoeblackplant can be easily obtained by using a method generally used for extracting plants. Further, as the extract of the hibiscus, a commercially available product may be used. The hibiscus extract includes the hibiscus extract, the diluted or concentrated solution of the extract, the dried product of the extract, or any of these crude or purified products.

The solvent used for the extraction is not particularly limited and may be appropriately selected depending on the intended purpose. For example, water, a hydrophilic organic solvent, or a mixed solvent thereof is used at room temperature or a temperature equal to or lower than the boiling point of the solvent. It is preferable to use it. The component containing at least one of the hyaluronidase activity inhibitory action, hydrogen peroxide scavenging action, whitening action, anti-aging action, and hair growth action contained in the hibiscus is easily extracted by an extraction treatment using a polar solvent as an extraction solvent. be able to.

The water that can be used as the extraction solvent is not particularly limited and may be appropriately selected depending on the intended purpose. For example, pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water and the like can be selected. In addition, those that have undergone various treatments are included. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering and the like. Therefore, the water that can be used as the extraction solvent includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.

The hydrophilic organic solvent that can be used as the extraction solvent is not particularly limited and may be appropriately selected depending on the intended purpose. For example, lower grades having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol and isopropyl alcohol. Alcohols; lower aliphatic ketones such as acetone and methyl ethyl ketone; polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol and glycerin, and the like, and a mixed solvent of the hydrophilic organic solvent and water. Etc. can also be used. When a mixed solvent of the water and the hydrophilic organic solvent is used, 1 part by mass to 90 parts by mass with respect to 10 parts by mass of water in the case of a lower alcohol, and 10 parts by mass of water in the case of a lower aliphatic ketone. It is preferable to use a mixture of 1 part by mass to 40 parts by mass with respect to parts by mass. In the case of polyhydric alcohol, it is preferable to use a mixture of 1 part by mass to 90 parts by mass with 10 parts by mass of water.

The method for extracting the hibiscus extract is not particularly limited as long as the fat-soluble component contained in the hibiscus extraction raw material can be eluted into the solvent, and the method can be carried out according to a conventional method. .. Further, in the extraction process, it is not necessary to adopt a special extraction method, and any device can be used at room temperature or under reflux heating.
Specifically, as a method for extracting the hibiscus extract, for example, an extraction raw material such as a hibiscus flower is put into a treatment tank filled with the solvent such as an aqueous ethanol solution, and the mixture is appropriately stirred as necessary. , A method of heat-extracting at 80 ° C. for 2 hours with a reflux extractor, filtering at hot time to elute the fat-soluble component, concentrating under reduced pressure using an evaporator, and further performing the same filtration treatment.
At this time, the extraction conditions can be appropriately adjusted according to the extraction raw material and the like, but the amount of the extraction solvent is preferably 5 to 20 times the amount (mass ratio) of the extraction raw material, and the extraction time is 1. The time is preferably 3 hours, and the extraction temperature is preferably 20 ° C to 95 ° C.

The obtained hibiscus extract is diluted, concentrated, dried, purified, etc. according to a conventional method in order to obtain a diluted product, a concentrate, a dried product, a crude product, a purified product, or the like. May be processed.
Further, the obtained extract of hibiscus can be used as it is as any one of the hyaluronidase activity inhibitor, the hydrogen peroxide scavenger, the whitening agent, the anti-aging agent, and the hair restorer. The concentrate and the dried product are preferable because they are easy to use. In obtaining the dried product, a carrier such as dextrin or cyclodextrin may be added in order to improve hygroscopicity.

The other components are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the intended purpose. For example, excipients, binders, disintegrants, and lubricants. Agents, stabilizers, flavoring agents, flavoring agents, etc. may be mentioned.

Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinylpyrrolidone, and the like. Can be mentioned. Examples of the disintegrant include dried starch, sodium alginate, canten powder, sodium hydrogencarbonate, calcium carbonate, sodium lauryl sulfate, stearate monoglyceride, lactose, and the like. Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol and the like. Examples of the stabilizer include sodium metabisulfite, EDTA, thioglycolic acid, thiolactic acid, and the like. Examples of the flavoring agent or odorizing agent include sucrose, orange peel, citric acid, tartaric acid, and the like.

The hibiscus extract obtained as described above has a hyaluronidase activity inhibitory effect, a hydrogen peroxide scavenging effect, a melanin production inhibitory effect on B16 melanoma cells, an endoserin-1 mRNA expression increase inhibitory effect, and a stem cell growth factor mRNA expression increase inhibitory effect. , Basic fibroblast growth factor mRNA expression increase inhibitory action, proopiomelanocortin mRNA expression increase inhibitory action, epidermal hyaluronic acid production promoting action, glutathione production promoting action, seryllumitoyltransferase mRNA expression promoting action, mailard reaction inhibitory action, final glycation Product formation inhibitory action, final saccharified product decomposition promoting action, claudin-1 production promoting action, occludin production promoting action, epidermal tight junction constituent protein production promoting action in human skin three-dimensional model, skin barrier function decline suppressing action, testosterone 5α- It has at least one of a reductase activity inhibitory action and a hair papilla cell proliferation action, and based on these actions, the hyaluronidase activity inhibitor, hydrogen peroxide scavenger, whitening agent, antiaging agent, and hair growth agent of the present invention. It is suitably available as at least one of the active ingredients.
Based on each of the above-mentioned actions, the hibiscus extract contains a melanin production inhibitor, an endoserin-1 mRNA expression inhibitor, a stem cell growth factor mRNA expression inhibitor, and a basic fibroblast growth factor mRNA. Expression increase inhibitor, proopiomelanocortin mRNA expression increase inhibitor, epidermal hyaluronic acid production promoter, glutathione production promoter, celympulmityltransferase mRNA expression promoter, mailard reaction inhibitor, final saccharified product formation inhibitor, final saccharified product degradation Accelerator, claudin-1 production promoter, occludin production promoter, epidermal tight junction constituent protein production promoter in human skin three-dimensional model, skin barrier function decline inhibitor, testosterone 5α-reductase activity inhibitor, and hair papilla cells It can also be suitably used as a proliferation agent.

The hyaluronidase activity inhibitor of the present invention is exerted based on the hyaluronidase activity inhibitory action.
The hydrogen peroxide scavenging agent of the present invention is exhibited based on the hydrogen peroxide scavenging action.
The whitening action of the whitening agent of the present invention includes a melanin production inhibitory effect on B16 melanoma cells, an endoserin-1 mRNA expression increase inhibitory effect, a stem cell growth factor mRNA expression increase inhibitory effect, a basic fibroblast growth factor mRNA expression increase inhibitory effect, and an effect. It is exerted based on at least one of the effects of suppressing the increase in proopiomelanocortin mRNA expression.
The anti-aging action of the anti-aging agent of the present invention is an epidermal hyaluronic acid production promoting action, a glutathione production promoting action, a seryllumitoyltransferase mRNA expression promoting action, a Maillard reaction inhibitory action, an advanced glycation end product formation inhibitory action, and an advanced glycation end product decomposition promoting action. , Claudin-1 production promoting action, occludin production promoting action, epidermal tight junction constituent protein production promoting action in human skin three-dimensional model, and skin barrier function deterioration inhibitory action are exerted based on at least one of them.
The hair-growth action of the hair-growth agent of the present invention is exerted based on at least one of the testosterone 5α-reductase activity inhibitory action and the dermal papilla cell proliferation action.

The content of the hyaluronidase activity inhibitor, the hydrogen peroxide scavenger, the whitening agent, the anti-aging agent, and the hibiscus extract in the hair restorer is not particularly limited and may be appropriately selected depending on the intended purpose. Further, the hyaluronidase activity inhibitor, the hydrogen peroxide scavenger, the whitening agent, the anti-aging agent, and the hair restorer may be the extract of the hibiscus itself.

Further, the present invention can also be used as other components other than the hibiscus extract that can be contained in the hyaluronidase activity inhibitor, the hydrogen peroxide scavenger, the whitening agent, the anti-aging agent, and the hair restorer. As long as the effect is not impaired, there is no particular limitation, and the hibiscus extract can be appropriately selected depending on the intended purpose. For example, a physiological saline solution for diluting the hibiscus extract to a desired concentration may be mentioned. Be done. Further, the content of the hyaluronidase activity inhibitor, the hydrogen peroxide scavenger, the whitening agent, the anti-aging agent, and the other components in the hair restorer is not particularly limited, and is appropriately used according to the purpose. You can choose.
Further, the hyaluronidase activity inhibitor, the hydrogen peroxide scavenger, the whitening agent, the anti-aging agent, and the hair restorer can be formulated as necessary to form powders, granules, tablets, etc. It can be in any dosage form.

The hyaluronidase activity inhibitor, hydrogen peroxide scavenger, whitening agent, anti-aging agent, and hair restorer of the present invention have excellent hyaluronidase activity inhibitory action, hydrogen peroxide scavenging action, whitening action, anti-aging action, and hair growth action. It is suitable for use in various cosmetics, foods and drinks, for example, because it has and is excellent in safety.

The hyaluronidase activity inhibitor, hydrogen peroxide scavenger, whitening agent, anti-aging agent, and hair restorer of the present invention are suitably applied to humans, but as long as their respective actions and effects are exhibited. It can also be applied to animals other than humans.

Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples.

<Extract of hibiscus>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample.

(Example 1)
<Hyaluronidase activity inhibitory effect test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the hyaluronidase activity inhibitory effect was tested by the following test method.

Hyaluronidase solution (Type IV-S (derived from bovine testis), 400 NF units / mL; Sigma-Aldrich Japan) in 0.2 mL of 0.1 mol / L acetate buffer (pH 3.5) in which the test sample was dissolved. 1 mL was added and the reaction was carried out at 37 ° C. for 20 minutes. Further, 2.5 mmol / L calcium chloride 0.2 mL was added as an activator, and the reaction was carried out at 37 ° C. for 20 minutes. To this, 0.5 mL of a 0.8 mg / mL sodium hyaluronate solution (sodium hyaluronate derived from chicken comb, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was reacted at 37 ° C. for 40 minutes. Then, 0.2 mL of 0.4 mol / L sodium hydroxide was added to stop the reaction and the mixture was cooled, and then 0.2 mL of a boric acid solution was added to each reaction solution and boiled for 3 minutes. After cooling with ice, 6 mL of p-DABA reagent (p-dimethylaminobenzaldehyde, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the reaction was carried out at 37 ° C. for 20 minutes. Then, the absorbance at a wavelength of 585 nm was measured. A blank test was performed in the same manner to correct the problem.
The calculation method of the hyaluronidase activity inhibition rate is as follows. The results are shown in Table 1.
Hyaluronidase activity inhibition rate (%) =
1- (St-Sb) / (Ct-Cb)} x 100
However, in the above formula, St is the absorbance of the test sample solution at a wavelength of 585 nm, Sb is the absorbance of the test sample solution blank at a wavelength of 585 nm, Ct is the absorbance of the control solution at a wavelength of 585 nm, and Cb is the wavelength of the control solution blank at 585 nm. Represents the absorbance in.

表１の結果から、ハイビスカスの抽出物が、ヒアルロニダーゼ活性阻害作用を有することが認められた。

From the results in Table 1, it was confirmed that the extract of hibiscus has a hyaluronidase activity inhibitory action.

(Example 2)
<Hydrogen peroxide scavenging action test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the hydrogen peroxide scavenging action was tested by the following test method.

Place 25 μL of the test sample solution in a 96-well plate, add _{10 μL of 0.15 mM H 2} O ₂ , 0.1 mol / L PIPES buffer (pH 7.0) (0.5% Triton X-100, 100 unit / mL). 25 μL of peroxidase (containing 1 mL) was added, and the mixture was reacted at 37 ° C. for 20 minutes. After the reaction, 180 μL of 100 μM DA-67 was immediately added, then 10 μL of ethanol was added, and a color development reaction was carried out at 37 ° C. for 5 minutes. After the color reaction was completed, the absorbance at a wavelength of 650 nm was measured. In addition, a blank test was performed in the same manner to make corrections.
The calculation method of the hydrogen peroxide elimination rate is as follows. In addition, a 50% inhibitory activity concentration (IC ₅₀ : μg / mL) was calculated. These results are shown in Table 2.
Hydrogen peroxide elimination rate (%) = {1- (St-Sb) / (Ct-Cb)} × 100
However, in the above formula, St is the absorbance of the test sample solution at a wavelength of 650 nm, Sb is the absorbance of the test sample solution blank at a wavelength of 650 nm, Ct is the absorbance of the control solution at a wavelength of 650 nm, and Cb is the wavelength of the control solution blank. Represents the absorbance at 650 nm, respectively.

表２の結果から、ハイビスカスの抽出物が、過酸化水素消去作用を有することが認められた。

From the results in Table 2, it was confirmed that the hibiscus extract had a hydrogen peroxide scavenging effect.

(Example 3)
<Test of melanin production inhibitory effect on B16 melanoma cells>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the melanin production inhibitory effect on B16 melanoma cells was tested by the following test method.

B16 melanoma cells were cultured in Dulbecco MEM (Dulbecco modified Eagle's medium (1), manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% by volume FBS (STANDARD FETAL BOVINE SERUM, manufactured by HyClone), and then the cells were treated with trypsin. Collected. ^{The collected cells were diluted to a concentration of 24.0 × 10 4} cells / mL with Dalveco MEM containing 10% by volume FBS and 1 mmol / L theophylline (manufactured by Wako Pure Chemical Industries, Ltd.), and then 1 well in a 48-well plate. 300 μL per seed was sown and cultured for 6 hours. After completion of the culture, 300 μL of the test sample dissolved in Dulbecco MEM containing 10% by volume FBS and 1 mmol / L theophylline was added to each well and cultured for 4 days. After completion of the culture, the medium was removed from each well, 200 μL of 2 mol / L NaOH solution was added, the cells were destroyed by an ultrasonic crusher, and the absorbance at a wavelength of 475 nm was measured. The amount of melanin was calculated from the measured absorbance value based on the calibration curve prepared using synthetic melanin (manufactured by SIGMA).
In addition, for the measurement of cell viability, the cells were similarly cultured, washed with 400 μL of PBS (-) phosphate buffer, and dissolved in 10% by volume FBS-containing Dalveco MEM at a final concentration of 0.05 mg / mL. 200 μL of 8 mmol / L neutral red was added to each well. After culturing for 2.5 hours, the neutral red solution was discarded, and 200 μL of an ethanol / acetic acid solution (ethanol: acetic acid: water = 50: 1: 49) was added to each well to extract the dye. After extraction, the absorbance at a wavelength of 540 nm was measured.
As a blank test, cells cultured only with 10% by volume FBS and Dulbecco MEM containing 1 mmol / L theophylline were tested in the same manner.
The calculation method of the melanin production suppression rate is as follows. The results are shown in Table 3.
Melanin production inhibition rate (%) = {1- (B / D) / (A / C)} × 100
However, in the above formula, A is the absorbance at a wavelength of 475 nm in the cell to which the test sample is not added, B is the absorbance at the wavelength of 475 nm in the cell to which the test sample is added, and C is the wavelength in the cell to which the test sample is not added. The absorbance at 540 nm and D represent the absorbance at a wavelength of 540 nm in the cell to which the test sample was added.

表３の結果から、ハイビスカスの抽出物が、メラニン産生抑制作用を有することが認められた。

From the results in Table 3, it was confirmed that the hibiscus extract has an inhibitory effect on melanin production.

(Example 4)
<Endothelin-1 mRNA expression inhibitory effect test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the effect of suppressing the increase in endothelin-1 mRNA expression was tested by the following test method.

Normal human neonatal epidermal keratinocytes (NHEK) were ^{precultured in 75 cm 2} flasks in normal human epidermal keratinocyte proliferation medium (KGM) at 37 ° C. _{under 5% CO 2} and treated with trypsin. Collected cells.
^{Using KGM, 40 × 10 4} cells / 2 mL / petri dish was sown in a 35 mm petri dish (manufactured by FALCON) and cultured overnight at _{37 ° C. and 5% CO 2.} After 24 hours, discard the culture medium, add 1 mL of HEPES buffer ^{, perform UV-B irradiation (50 mJ / cm 2} ), and then add 2 mL of the test sample dissolved in the required concentration in KGM to each petri dish at 37 ° C. The cells were cultured under 5% CO _{2 for 24 hours.} After culturing, discard the culture broth, extract total RNA with ISOGEN II (NIPPON GENE; Cat. No. 311-07361), measure the amount of each RNA with a spectrophotometer, and adjust to 200 ng / μL. Total RNA was prepared.
Using this total RNA as a template, the expression levels of endothelin-1 and GAPDH mRNA, which is an internal standard, were measured. ^{Detection is performed by a real-time 2-step RT-PCR reaction by TaKaRa SYBR (R)} PrimeScript ^TM RT-PCR Kit (Perfect Real Time) (code No. RR063A) using a real-time PCR device Smart Cycler ^(R) (manufactured by Cepheid). went.
The expression level of the mRNA of endoserin-1 is the total RNA prepared from the cells cultured by "ultraviolet irradiation, no test sample added", "ultraviolet irradiation, no test sample added", and "ultraviolet irradiation, no test sample added", respectively. Based on the standard, the correction value is obtained from the GAPDH value, and when the correction value of "ultraviolet ray not irradiated, no test sample added" is set to 100, "ultraviolet ray irradiation, no test sample added", and "ultraviolet ray irradiation". , Test sample addition ”was calculated.
Then, from these results, the rate of inhibition of the increase in endothelin-1 mRNA expression was calculated by the following formula. The results are shown in Table 4.
Endothelin-1 mRNA expression increase inhibition rate (%)
= {(AB)-(AC)} / (AB) × 100
However, in the above formula, A is the correction value at the time of "ultraviolet irradiation, no test sample added", B is the correction value at the time of "ultraviolet irradiation, no test sample added", and C is "ultraviolet irradiation, test sample". This is the correction value at the time of "addition".

表４の結果から、ハイビスカスの抽出物が、エンドセリン−１ｍＲＮＡ発現上昇抑制作用を有することが認められた。

From the results in Table 4, it was confirmed that the hibiscus extract has an effect of suppressing the increase in endothelin-1 mRNA expression.

(Example 5)
<Stem cell growth factor (SCF) mRNA expression increase inhibitory effect test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the effect of suppressing the increase in stem cell proliferation factor (SCF) mRNA expression was tested by the following test method.

Normal human neonatal epidermal keratinocytes (NHEK) were ^{pre-cultured in 80 cm 2} flasks in normal human epidermal keratinocyte growth medium (KGM) at 37 ° C. _{under 5% CO 2} and treated with trypsin. Collected cells.
^{Using KGM, 40 × 10 4} cells / 2 mL petri dishes were sown in 35 mm petri dishes (manufactured by FALCON) and cultured overnight at _{37 ° C. and 5% CO 2.} After 24 hours, the culture solution was discarded, 1 mL of HEPES buffer was added, and UV-B irradiation (50 mJ / cm ² ) was performed. Then, 2 mL of the test sample dissolved in KGM to the required concentration was added to each petri dish, and the mixture _{was cultured at 37 ° C. under 5% CO 2} for 24 hours. After culturing, discard the culture broth, extract total RNA with ISOGEN II (NIPPON GENE; Cat. No. 311-07361), measure the amount of each RNA with a spectrophotometer, and adjust to 200 ng / μL. Total RNA was prepared.
Using this total RNA as a template, the expression levels of SCF (Stem Cell Factor) and GAPDH mRNA, which is an internal standard, were measured. ^{Detection is performed by a real-time 2-step RT-PCR reaction by TaKaRa SYBR (R)} PrimeScript ^TM RT-PCR Kit (Perfect Real Time) (code No. RR063A) using a real-time PCR device Smart Cycler ^(R) (manufactured by Cepheid). went.
The expression level of SCF mRNA is a total RNA standard prepared from cells cultured by "ultraviolet irradiation, no test sample added", "ultraviolet irradiation, no test sample added", and "ultraviolet irradiation, no test sample added", respectively. Based on the above, the correction value is obtained from the GAPDH value, and when the correction value of "ultraviolet ray not irradiated, test sample not added" is set to 100, "ultraviolet ray irradiation, test sample not added", and "ultraviolet ray irradiation, test". The correction value of "sample addition" was calculated.
Then, from these results, the rate of inhibition of the increase in stem cell growth factor (SCF) mRNA expression was calculated by the following formula. The results are shown in Table 5.
Inhibition rate of increased expression of stem cell growth factor (SCF) mRNA (%)
= {(AB)-(AC)} / (AB) × 100
However, in the above formula, A is the correction value at the time of "ultraviolet irradiation, no test sample added", B is the correction value at the time of "ultraviolet irradiation, no test sample added", and C is the time of "ultraviolet irradiation, test sample addition". Represents each correction value of.

表５の結果から、ハイビスカスの抽出物が、幹細胞増殖因子（ＳＣＦ）ｍＲＮＡ発現上昇抑制作用を有することが認められた。

From the results in Table 5, it was confirmed that the hibiscus extract has an inhibitory effect on the expression of stem cell growth factor (SCF) mRNA.

(Example 6)
<Test of inhibitory effect on increased expression of basic fibroblast growth factor (bFGF) mRNA>
A freeze-dried product of Hibiscus Hanabe Extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the effect of suppressing the increase in basic fibroblast growth factor (bFGF) mRNA expression was tested by the following test method.

Normal human neonatal epidermal keratinocytes (NHEK) were ^{precultured in a 75 cm 2} flask in a growth medium for normal human epidermal keratinocyte culture (KGM) at 37 ° C. _{under 5% CO 2} and trypsin. Cells were collected by treatment.
^{Using KGM, 40 × 10 4} cells / 2 mL petri dishes were sown in 35 mm petri dishes (manufactured by FALCON) and cultured overnight at _{37 ° C. and 5% CO 2.} After 24 hours, the culture solution was discarded, 1 mL of HEPES buffer was added, and UV-B irradiation (50 mJ / cm ² ) was performed. Then, 2 mL of the test sample dissolved in KGM to the required concentration was added to each petri dish, and the mixture _{was cultured at 37 ° C. under 5% CO 2} for 24 hours. After culturing, discard the culture broth, extract total RNA with ISOGEN II (NIPPON GENE; Cat. No. 311-07361), measure the amount of each RNA with a spectrophotometer, and adjust to 200 ng / μL. Total RNA was prepared.
Using this total RNA as a template, the expression levels of bFGF (basic Fibroblast Growth Factor) and the internal standard GAPDH mRNA were measured. ^{Detection is performed by a real-time 2-step RT-PCR reaction by TaKaRa SYBR (R)} PrimeScript ^TM RT-PCR Kit (Perfect Real Time) (code No. RR063A) using a real-time PCR device Smart Cycler ^(R) (manufactured by Cepheid). went.
The expression level of bFGF mRNA is a total RNA standard prepared from cells cultured by "ultraviolet irradiation, no test sample added", "ultraviolet irradiation, no test sample added", and "ultraviolet irradiation, no test sample added", respectively. Based on the above, the correction value is obtained from the GAPDH value, and when the correction value of "ultraviolet ray not irradiated, test sample not added" is set to 100, "ultraviolet ray irradiation, test sample not added", and "ultraviolet ray irradiation, test". The correction value of "sample addition" was calculated.
From these results, the suppression rate of increase in bFGF mRNA expression was calculated by the following formula. The results are shown in Table 6.
bFGF mRNA expression increase suppression rate (%)
= {(AB)-(AC)} / (AB) × 100
However, in the above formula, A is the correction value when the ultraviolet irradiation is not applied and the test sample is not added, B is the correction value when the ultraviolet irradiation is applied and the test sample is not added, and C is the correction value when the ultraviolet irradiation is applied and the test sample is not added. Represents.

表６の結果から、ハイビスカスの抽出物が、強いｂＦＧＦｍＲＮＡ発現上昇抑制作用を有することが認められた。

From the results in Table 6, it was confirmed that the hibiscus extract has a strong inhibitory effect on the expression of bFGF mRNA.

(Example 7)
<Proopiomelanocortin (POMC) mRNA expression inhibitory effect test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the effect of suppressing the increase in proopiomelanocortin (POMC) mRNA expression was tested by the following test method.

Normal human neonatal epidermal keratinocytes (NHEK) were ^{precultured in a 75 cm 2} flask in a growth medium for normal human epidermal keratinocyte culture (KGM) at 37 ° C. _{under 5% CO 2} and trypsin. Cells were collected by treatment.
^{Using KGM, 40 × 10 4} cells / 2 mL petri dishes were sown in 35 mm petri dishes (manufactured by FALCON) and cultured overnight at _{37 ° C. and 5% CO 2.} After 24 hours, the culture solution was discarded, 1 mL of HEPES buffer was added, and UV-B irradiation (50 mJ / cm ² ) was performed. Then, 2 mL of the test sample dissolved in KGM to the required concentration was added to each petri dish, and the mixture _{was cultured at 37 ° C. under 5% CO 2} for 24 hours. After culturing, discard the culture broth, extract total RNA with ISOGEN II (NIPPON GENE; Cat. No. 311-07361), measure the amount of each RNA with a spectrophotometer, and adjust to 200 ng / μL. Total RNA was prepared.
Using this total RNA as a template, the expression levels of POMC (proopiomelanocortin) and the internal standard GAPDH mRNA were measured. ^{Detection is performed by a real-time 2-step RT-PCR reaction by TaKaRa SYBR (R)} PrimeScript ^TM RT-PCR Kit (Perfect Real Time) (code No. RR063A) using a real-time PCR device Smart Cycler ^(R) (manufactured by Cepheid). went.
The expression level of POMC mRNA is a total RNA standard prepared from cells cultured by "ultraviolet irradiation, no test sample added", "ultraviolet irradiation, no test sample added", and "ultraviolet irradiation, no test sample added", respectively. Based on the above, the correction value is obtained from the GAPDH value, and when the correction value of "ultraviolet ray not irradiated, test sample not added" is set to 100, "ultraviolet ray irradiation, test sample not added", and "ultraviolet ray irradiation, test". The correction value of "sample addition" was calculated.
From these results, the rate of suppression of POMC mRNA expression increase was calculated by the following formula. The results are shown in Table 7.
Rate of suppression of increase in POMC mRNA expression (%)
= {(AB)-(AC)} / (AB) × 100
However, in the above formula, A is the correction value at the time of "ultraviolet irradiation, no test sample added", B is the correction value at the time of "ultraviolet irradiation, no test sample added", and C is "ultraviolet irradiation, test sample". Represents the correction value at the time of "addition".

表７の結果から、ハイビスカスの抽出物が、ＰＯＭＣｍＲＮＡ発現上昇抑制作用を有することが認められた。

From the results in Table 7, it was confirmed that the hibiscus extract has an effect of suppressing the increase in POMC mRNA expression.

(Example 8)
<Epidermal hyaluronic acid production promoting action test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the effect of promoting epidermal hyaluronic acid production was tested by the following test method.

Human normal neonatal skin epidermal keratinocytes (NHEK) were cultured in human normal neonatal epidermal keratinocyte proliferation medium (KGM), and then the cells were recovered by tripsin treatment. The collected cells were ^{diluted with KGM to a concentration of 1 × 10 5} cells / mL, then 100 μL per well was seeded on a 96-well plate and cultured for 24 hours. After completion of the culture, 100 μL of the test sample dissolved in KGM was added to each well and cultured for 7 days. After culturing, the amount of hyaluronic acid in the medium of each well was measured by a sandwich method using a hyaluronic acid-binding protein (HABP, manufactured by Biochemical Biobusiness Co., Ltd.).

The calculation method of the hyaluronic acid production promotion rate is as follows. The results are shown in Table 8.
Hyaluronic acid production promotion rate (%) = A / B × 100
However, in the above formula, A represents the amount of hyaluronic acid when the test sample is added, and B represents the amount of hyaluronic acid when the test sample is not added.

表８の結果から、ハイビスカスの抽出物が、表皮ヒアルロン酸産生促進作用を有することが認められた。

From the results in Table 8, it was confirmed that the hibiscus extract has an epidermal hyaluronic acid production promoting action.

(Example 9)
<Glutathione production promoting action test (human normal skin fibroblasts)>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and a glutathione production promoting action test (human normal skin fibroblasts) was tested by the following test method.

Human normal skin fibroblasts (NB1RGB) were cultured in α-MEM medium containing 10% by mass FBS, and then the cells were recovered by trypsin treatment. The collected cells were ^{diluted with α-MEM medium containing 10% by mass FBS to a concentration of 2.0 × 10 5} cells / mL, and then 200 μL per well was inoculated on a 48-well plate and cultured overnight. After culturing, 200 μL of the test sample dissolved in α-MEM medium containing 1% by mass FBS was added to each well and cultured for 24 hours. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS (−), and the cells were lysed with ^{150 μL of M-PER (R) (PIERCE).} Total glutathione was quantified using 100 μL of this.
That is, 100 μL of cell extract dissolved in a 96-well plate, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH and 25 μL of glutathione reductase (final concentration 17.5 unit / mL) were added, and the mixture was heated at 37 ° C. for 10 minutes. After that, 25 μL of 10 mM 5,5'-dithiobis (2-nitronobenzoic acid) was added, and the absorbance at a wavelength of 412 nm up to 5 minutes later was measured to determine ΔOD / min. The total glutathione concentration was calculated based on the calibration curve prepared using oxidized glutathione.
The obtained value was corrected to the amount of glutathione per total protein amount, and then the glutathione production promotion rate was calculated by the following formula. The results at sample concentrations of 12.5 μg / mL, 50 μg / mL, and 200 μg / mL are shown in Table 9.
Glutathione production promotion rate (%) = (B / A) x 100
However, in the above formula, A represents the amount of glutathione per total protein amount in cells to which the test sample is not added (control), and B represents the amount of glutathione per total protein amount in cells to which the test sample is added.

表９の結果から、ハイビスカスの抽出物が、グルタチオン産生促進作用を有することが認められた。

From the results in Table 9, it was confirmed that the hibiscus extract has a glutathione production promoting action.

(Example 10)
<Test for promoting expression of serine palmitoyltransferase (SPT) mRNA>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the serine lumintoyl transferase (SPT) mRNA expression promoting action was tested by the following test method.

Normal human neonatal epidermal keratinocytes (NHEK) were ^{precultured in a 75 cm 2} flask in normal human epidermal keratinocyte proliferation medium (KGM) at 37 ° C. _{under 5% CO 2} and treated with trypsin. Collected cells.
^{Using KGM, 40 × 10 4} cells / 2 mL / petri dish was sown in a 35 mm petri dish (manufactured by FALCON) and cultured overnight at _{37 ° C. and 5% CO 2.} After 24 hours, the culture solution was discarded, 2 mL of the test sample dissolved in KGM to the required concentration was added to each petri dish, and the cells were _{cultured at 37 ° C. under 5% CO 2} for 24 hours. After culturing, discard the culture broth, extract total RNA with ISOGEN II (NIPPON GENE; Cat.no. 311-07361), measure the amount of each RNA with a spectrophotometer, and adjust to 200 ng / μL. Total RNA was prepared.
Using this total RNA as a template, the expression levels of SPT and GAPDH mRNA, which is an internal standard, were measured. Detection is performed using a real-time PCR device Smart Cycler (registered trademark) ( ^{Cephyid), and a real-time 2 Step RT reaction by TaKaRa SYBR (registered trademark) PrimeScript TM} RT-PCR Kit (Perfect Real Time) (code No. RR063A). Was done by. For the expression level of SPT, the correction value was obtained from the GAPDH value based on the total RNA standard prepared from the cells cultured with the test sample not added and the test sample was added, respectively, and the correction value without the test sample was 100. The correction value for the addition of the test sample was calculated.
The calculation method of the SPT mRNA expression promotion rate is as follows. The results are shown in Table 10.
SPT mRNA expression promotion rate (%) = A / B × 100
However, in the above formula, A represents the correction value when the test sample is added, and B represents the correction value when the test sample is not added.

表１０の結果から、ハイビスカスの抽出物が、高いセリンパルミトイルトランスフェラーゼ（ＳＰＴ）産生促進作用を有することが認められた。

From the results in Table 10, it was confirmed that the hibiscus extract has a high serine palmitoyltransferase (SPT) production promoting effect.

(Example 11)
<Maillard reaction inhibitory effect test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the Maillard reaction inhibitory effect was tested by the following test method.

50 μL of test sample solution in which a lyophilized product of the test sample was dissolved in distilled water, 200 μL of D (-)-ribose at 100 mmol / L, 200 μL of lysoteam at 25 mg / mL, and 500 μL of sodium hydrogen phosphate (pH 7.4) at 100 mmol / L. , And 50 μL of sterile distilled water (total volume 1,000 μL) were mixed and allowed to stand at 37 ° C. The control was prepared in the same manner as above, except that distilled water was used instead of the test sample solution. The blank was prepared in the same manner as described above except that distilled water was used instead of the test sample solution and the mixture was allowed to stand at 4 ° C instead of 37 ° C.

After 7 days, the mixture was stirred with vortex, 40 μL of sample buffer for SDS-PAGE was mixed with 40 μL of the reaction solution, and then heated in a boiling bath for 3 minutes to prepare an analytical sample. An analysis sample of 12 μL was applied to a polyacrylamide gel prepared to have an acrylamide concentration of 15% on a separation gel and 4% on a concentrated gel, and electrophoresis was performed.
The electrophoresed gel was stained with Coomassie Brilliant Blue and then decolorized, detected using an imaging device ChemiDocXRS Plus (Bio-Rad Laboratories), and the band was quantified by Image Lab Software version 2.0 (Bio-Rad Labor) Quantitative. Measured.
As a result, the rate of inhibition of lysozyme dimer and trimer formation was calculated from the following formula using the Net intensity (band intensity) of each band. The results are shown in Table 11.
Maillard reaction inhibition rate (%) = {1- (AC) / (BC)} × 100
However, in the above formula, A is the sum of the net integrity of the dimer and the trimer when the test sample is added, and B is the Net intensity of the dimer and the trimer when the test sample is not added (control). The sum and C represent the sum of the net integrity of the dimer and the trimer that have been allowed to stand (blank) at 4 ° C. when the test sample is not added, respectively.

表１１の結果から、ハイビスカスの抽出物が、メイラード反応阻害作用を有することが認められた。

From the results in Table 11, it was confirmed that the hibiscus extract had a Maillard reaction inhibitory effect.

(Example 12)
<Advanced glycation end product (AGEs) formation inhibitory action test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the action of suppressing the formation of advanced glycation end products (AGEs) was tested by the following test method.

0.2M D (-)-ribose prepared with PBS (-) and test sample (sample concentration: 6.25 μg / mL, 25 μg / mL, 100 μg / mL or 400 μg /) on a 96-well type I collagen coated plate. 100 μL of the mixture of mL) was added and allowed to stand at 37 ° C. for 2 weeks to form AGEs. At this time, those to which only PBS (−) was added as a negative control and those to which only D (−) -ribose was added as a positive control were similarly allowed to stand. After 17 days, the amount of AGEs was measured by the ELISA method using an anti-AGEs antibody (manufactured by Transgenic), and the AGEs formation inhibitory effect was evaluated.
The calculation method of the AGEs formation suppression rate is as follows. The results are shown in Table 12.
AGEs formation inhibition rate (%) = {(BC) / (BA)} × 100
However, in the above formula, A represents the absorbance at the wavelength of 405 nm of the negative control, B represents the absorbance of the positive control at the wavelength of 405 nm, and C represents the absorbance at the wavelength of 405 nm when the test sample is added.

表１２の結果から、ハイビスカスの抽出物が、極めて強い最終糖化産物形成抑制作用を有することが認められた。

From the results in Table 12, it was confirmed that the hibiscus extract has an extremely strong advanced glycation end product formation inhibitory effect.

(Example 13)
<Advanced glycation end product (AGEs) decomposition promoting action test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the action of promoting the decomposition of advanced glycation end products (AGEs) was tested by the following test method.

To a 96-well type I collagen coated plate, 100 μL of 0.2 M D (-)-ribose prepared with PBS (-) was added, and the mixture was allowed to stand at 37 ° C. for 2 weeks to form AGEs. As a negative control, the one to which PBS (−) was added was similarly allowed to stand. After 2 weeks, 100 μL of a test sample (sample concentration: 6.25 μg / mL, 25 μg / mL, 100 μg / mL or 400 μg / mL) prepared with PBS (−) was added, and the mixture was allowed to stand for another 16 days. At this time, as a positive control, a sample to which PBS (-) was added instead of the test sample after D (-)-ribose treatment was similarly allowed to stand. In addition, the negative control was subsequently treated with PBS (-). After 16 days, the amount of AGEs was measured by the ELISA method using an anti-AGEs antibody (manufactured by Transgenic), and the AGEs decomposition promoting action was evaluated.
The calculation method of the AGEs decomposition promotion rate is as follows. The results are shown in Table 13.
AGEs decomposition promotion rate (%) = {(BC) / (BA)} × 100
However, in the above formula, A represents the absorbance at the wavelength of 405 nm of the negative control, B represents the absorbance of the positive control at the wavelength of 405 nm, and C represents the absorbance at the wavelength of 405 nm when the test sample is added.

表１３の結果から、ハイビスカスの抽出物が、最終糖化産物分解促進作用を有することが認められた。

From the results in Table 13, it was confirmed that the extract of hibiscus has an action of promoting the decomposition of advanced glycation end products.

(Example 14)
<Claudin-1 production promoting action test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the effect of promoting claudin-1 production was tested by the following test method.

Normal human skin epidermal keratinocytes (NHEK) were cultured in a normal human epidermal keratinocyte proliferation medium (KGM ^{) in an 80 cm 2} _{flask at 37 ° C. under 5% CO 2} , and the cells were recovered by tripsin treatment. After diluting the collected cells with KGM to a cell density of 2.0 × 10 ⁵ cells / mL, seed 100 μL per well on a 96-well plate and incubate at 37 ° C. under _{5% CO 2 for 1 day.} bottom.
After completion of the culture, 100 μL of the solution of the test sample dissolved in KGM was added to each well, and the cells were _{cultured at 37 ° C. under 5% CO 2} for 24 hours. After completion of the culture, the medium was removed, the cells were immobilized on a plate, and the amount of claudin-1 expressed on the cell surface was measured by the ELISA method using a polyclonal claudin-1 antibody.
From the obtained measurement results, the claudin-1 production promotion rate (%) was calculated by the following formula. The results are shown in Table 14.
Claudin-1 production promotion rate (%) = A / B × 100
However, in the above formula, A represents the absorbance at a wavelength of 405 nm when the test sample is added, and B represents the absorbance at a wavelength of 405 nm when the test sample is not added.

表１４の結果から、ハイビスカスの抽出物が、有意なクローディン−１産生促進作用を有することが認められた。

From the results in Table 14, it was confirmed that the hibiscus extract had a significant claudin-1 production promoting effect.

(Example 15)
<Occludin production promoting action test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the occludin production promoting action was tested by the following test method.

Normal human skin epidermal keratinocytes (NHEK) were cultured in a normal human epidermal keratinocyte proliferation medium (KGM) in a flask of ^{80 cm 3} _{at 37 ° C. under 5% CO 2} , and the cells were recovered by tripsin treatment. After diluting the collected cells with KGM to a cell density of 2.0 × 10 ⁵ cells / mL, seed 100 μL per hole on a 96-well plate and incubate overnight at 37 ° C. under _{5% CO 2.} bottom.
After completion of the culture, 100 μL of the test sample (sample concentration: 3.13 μg / mL, 12.5 μg / mL, or 50 μg / mL) dissolved in KGM was added to each well, and 24 at _{37 ° C. under 5% CO 2.} Cultured for hours. After completion of the culture, the medium was removed, the cells were fixed on a plate, and the amount of occludin expressed on the cell surface was measured by the ELISA method using a polyclonal anti-human occludin antibody.
The calculation method of the occludin production promotion rate is as follows. The results are shown in Table 15.
Occludin production promotion rate (%) = A / B x 100
However, in the above formula, A represents the absorbance at the wavelength of 405 nm when the test sample is added, and B represents the absorbance at the wavelength of 405 nm when the test sample is not added.

表１５の結果から、ハイビスカスの抽出物が、オクルディン産生促進作用を有することが認められた。

From the results in Table 15, it was confirmed that the hibiscus extract has an occludin production promoting action.

(Example 16)
<Epidermal tight junction constituent protein production promoting action test in a three-dimensional model of human skin>
Using a freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) as a test sample, the effect of promoting the production of epidermal tight junction constituent proteins in a human skin three-dimensional model was tested by the following test method.

The test was performed using a normal human skin three-dimensional model (EPI-200, manufactured by KURABO Ltd.).
After purchasing the three-dimensional skin model, the cells were cultured on a 6-well plate using assay medium (EPI-NMM Maintenance Medium, manufactured by KURABO) _{under the conditions of 37 ° C. and 5% CO 2 for 1 hour.} After culturing, 100 μL of the assay medium containing or not containing the test sample dissolved in 1% DMSO was applied to the surface of the skin model, and the assay medium (maintenance medium) was applied to the bottom surface of the skin model at 37 ° C. and 5% CO. _The cells were cultured under 2 conditions for 7 days. During the culture period, the test sample was constantly exposed. On the 4th day of culture, the maintenance medium and the assay medium containing the test sample were exchanged. On the 4th day of culturing and after the end of culturing, the cells were cut out using a 6 mm biopsi punch, the embedding agent was placed in a plastic embedding dish, and the cells were frozen in dry ice and liquid nitrogen. Then, with a cryostat HM550 (manufactured by MICROM), the sections were cut to a thickness of 4 μm, the sections were attached to a slide glass, and the sections were dried.

<Claudin-4 immunofluorescent staining>
The slide glass to which the section was attached was placed in a staining vat, fixed with 4% paraformaldehyde, and the embedding agent around the section was thoroughly washed away with PBS (−). After blocking with 1% BSA, the solution was discarded, and the primary antibody, mouse-derived anti-human claudin-4 monoclonal antibody (ZYMED ^(R) Laboratories), was poured onto each slide glass and incubated at room temperature for 1 hour. .. The antibody solution was discarded, washed with PBS (−), treated with a secondary antibody, Alexa-Fluor 488-labeled goat-derived anti-mouse IgG antibody (manufactured by Invitrogen), and incubated in the dark at 4 ° C. for 1 hour. Further, the cells were washed with PBS (-) and stained with DAPI solution. The target claudin-4 was analyzed with a fluorescence microscope. The results are shown in FIGS. 1A to 1F.
The images of FIGS. 1A to 1F show the part from the lower part of the stratum corneum to the basal layer, FIG. 1A is a control on the 4th day of culture, and FIG. 1B is an extraction of hibiscus having a sample concentration of 100 μg / mL on the 4th day of culture. 1C is an extract of hibiscus having a sample concentration of 500 μg / mL on the 4th day of culture, FIG. 1D is a control on the 7th day of culture, and FIG. 1E is a hibiscus having a sample concentration of 100 μg / mL on the 7th day of culture. The extract, FIG. 1F, shows the result of the hibiscus extract having a sample concentration of 500 μg / mL on the 7th day of culture, and Clodin-4 was stained green (near the cell membrane of the granular layer).
In addition, on both the 4th day and the 7th day of the culture, the linear fluorescence near the cell membrane of the stratum granulosum was stronger in the hibiscus extract than in the control, so that the claudin-4 production promoting action was exhibited. It was confirmed in the skin three-dimensional model.

<ZO-1, ZO-2 immunofluorescent staining>
The slide glass to which the section was attached was placed in a staining vat, fixed with 4% paraformaldehyde, and the embedding agent around the section was thoroughly washed away with PBS (−). After blocking with 1% BSA, discard the solution and use the primary antibody, mouse-derived anti-human ZO-1 monoclonal antibody (manufactured by invitrogen) and rabbit-derived anti-human ZO-2 polyclonal antibody (manufactured by invitrogen). It was poured onto a slide glass and incubated at room temperature for 1 hour. The antibody solution was discarded, washed with PBS (-), and the secondary antibody Alexa-Fluor 488-labeled goat-derived anti-mouse IgG antibody (manufactured by invitrogen) and Alexa-Fluor 594-labeled goat-derived anti-rabbit IgG antibody (invitrogen). ) Was treated and incubated for 1 hour in the dark at 4 ° C. Further, the cells were washed with PBS (-) and stained with DAPI solution. The target ZO-1 and ZO-2 were analyzed by a fluorescence microscope. The results are shown in FIGS. 2A to 2F and FIGS. 3A to 3F.
The photographs of FIGS. 2A to 2F show the portion from the lower part of the stratum corneum to the basal layer.
2A is a control on the 4th day of culture, FIG. 2B is an extract of hibiscus having a sample concentration of 100 μg / mL on the 4th day of culture, and FIG. 2C is an extract of hibiscus having a sample concentration of 500 μg / mL on the 4th day of culture. 2D is a control on the 7th day of culture, FIG. 2E is an extract of hibiscus having a sample concentration of 100 μg / mL on the 7th day of culture, and FIG. 2F is an extraction of hibiscus having a sample concentration of 500 μg / mL on the 7th day of culture. The results of the substances are shown, and ZO-1 is stained green (near the cell membrane of the granular layer to the basal layer) from FIGS. 2A to 2F.
The photographs of FIGS. 3A to 3F show the lower part of the stratum corneum to the basal layer.
FIG. 3A is a control on the 4th day of culture, FIG. 3B is an extract of hibiscus having a sample concentration of 100 μg / mL on the 4th day of culture, and FIG. 3C is an extract of hibiscus having a sample concentration of 500 μg / mL on the 4th day of culture. 3D is a control on the 7th day of culture, FIG. 3E is an extract of hibiscus having a sample concentration of 100 μg / mL on the 7th day of culture, and FIG. 3F is an extraction of hibiscus having a sample concentration of 500 μg / mL on the 7th day of culture. The results of the product are shown, and ZO-2 is stained in red (near the cell membrane of the granular layer to the basal layer) from FIGS. 3A to 3F.
On the 4th day of culture, the hibiscus extract had stronger linear fluorescence near the cell membrane at a concentration of 100 μg / mL as compared with the control, so that the production promoting action of ZO-1 and ZO-2 was tertiary. It was confirmed in the original skin model.

(Example 17)
<Skin barrier function deterioration inhibitory effect test (electrical resistance value TER measurement and permeability evaluation by FITC-Dex>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the skin barrier function deterioration inhibitory effect was tested by the following test method.

Human normal neonatal skin epidermal keratinocytes (NHEK) were cultured using a medium for human normal neonatal epidermal keratinocytes (KGM), and then the cells were recovered by tripsin treatment. After diluting the collected cells with ^{KGM to a concentration of 2.2 × 10 5} cells / mL, 0.5 mL per well is placed on the upper layer of a 12-well transwell (Corning, 12 mm in diameter, 0.4 μm pore). The seeds were sown, and 0.5 mL each of KGM was further added to the lower layer and cultured for 3 days. After completion of the culture _{, 0.5 mL of CaCl 2} (final concentration 1.8 mM) dissolved in KGM was added to the upper and lower layers of each well, and the cells were cultured for 3 days to induce tight junction formation. After completion of the culture, the high CaCl ₂ medium was removed, and 0.5 mL of the test sample dissolved in KGM alone or in KGM was added to the upper and lower layers of each well, and the culture was started in a _{low CaCl 2 state.} At the same time, a well that maintained the barrier function in a _{high CaCl 2 medium was also set as a control.} Three days after the start of culturing, the electrical resistance value (TER) was measured using a Millicell-ERS resistance value measurement system (manufactured by Millipore), and the barrier reduction suppression rate (%) of the test sample was calculated as compared with the control.
After TER measurement, the upper and lower layers are washed with PBS (-), and P buffer (10 mM HEPES, pH 7.4, 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl ₂ , 145 mM NaCl) is applied to the upper layer to 1 mg / mL. 0.5 mL of 4 kDa FITC-Dextran (FITC-Dex, manufactured by Sigma) dissolved in was added, and 0.5 mL of P buffer was added to the lower layer, and the mixture was cultured at 37 ° C. for 90 minutes. After completion of the culture, 100 μL was collected from each lower layer, the fluorescence intensity at an excitation wavelength of 485 nm and a fluorescence wavelength of 545 nm was measured, and the amount of FITC-Dex transmitted from the upper layer to the lower layer was determined based on the calibration curve and compared with the control. The permeation suppression rate (%) of the test sample was calculated and the permeation barrier function was evaluated.
The calculation method of the barrier reduction suppression rate is as follows. The results are shown in Table 16.
Barrier reduction suppression rate (%) = {1- (CA) / (CB)} × 100
However, in the above formula, A is the electrical resistance value (TER) in the cells to which the test sample is added, B is the electrical resistance value (TER) in the cells to which the test sample is not added, and C is the treatment with a _{high CaCl 2 medium.} It represents the electric resistance value (TER) in each of the cells.

The calculation method of the permeation suppression rate is as follows. The results are shown in Table 17.
Permeation suppression rate (%) = {1- (CA) / (CB)} × 100
However, in the above formula, A is the amount of FITC-Dex permeated in the cells to which the test sample was added, B is the amount of FITC-Dex permeated in the cells to which the test sample was not added, and C is the high CaCl ₂ medium. The amount of FITC-Dex permeated in the treated cells is represented respectively.

From the results in Tables 16 and 17, it was confirmed that the hibiscus extract has an effect of suppressing a decrease in electrical resistance value and an effect of suppressing FITC-Dex permeation.

(Example 18)
<Testosterone 5α-reductase activity inhibitory action test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the testosterone 5α-reductase activity inhibitory action was tested by the following test method.

First, in a V-bottomed test tube with a lid, 4.2 mg / mL teststerone 20 μL prepared with propylene glycol, 1 mg / mL NADPH (reduced nicotinamide adenine dinucleotide phosphate) -containing 5 mmol / mL Tris-HCl buffer ( pH 7.13) 825 μL was mixed. To this, 80 μL of a test sample prepared with ethanol, 50% ethanol or purified water and 75 μL of S-9 (Oriental Yeast Co., Ltd.) were added, mixed again, reacted at 37 ° C. for 30 minutes, and then 1 mL of methylene chloride was added. In addition, the reaction was stopped. This was centrifuged (1,600 × g, 10 minutes), and the methylene chloride layer was analyzed by gas chromatography. The conditions of the gas chromatography are as follows. In addition, a blank test was conducted by the same method.
The S-9 is a supernatant obtained by intraperitoneally administering an enzyme inducer (phenobarbital, 5,6-benzoflavone) to male SD rats, grinding the liver, and centrifuging at 9,000 × g. be.

<Conditions for gas chromatography>
Equipment used: Shimadzu GC-7A
Column: DB-1701 (diameter 0.53 mm × 30 m, film thickness; 1.0 μm)
Column / injection temperature: 240 ° C / 300 ° C
Detector: FID
Carrier gas: Nitrogen gas

In advance, standard methylene chloride solutions of 3α-androstandiol (SIGMA), dihydrotestosterone (DHT, Tokyo Kasei Kogyo Co., Ltd.) and testosterone (Tokyo Kasei Kogyo Co., Ltd.) were analyzed by gas chromatography, and these three compounds were analyzed. The amount of compound per peak area was calculated from the exact amount and peak area of.
Then, 3α-androstandiol, dihydrotestosterone (DHT) and testosterone after the reaction with S-9 were analyzed by gas chromatography, and the concentration per peak area of each was calculated according to the following formula (2). Next, the conversion rate of the test sample was calculated according to the following equation (3). Then, based on the conversion rate, the testosterone 5α-reductase activity inhibition rate was calculated according to the following formula (4). The results are shown in Table 18.

Concentration (%) = (peak area of test sample x concentration of standard product) / peak area of standard product ... (2)
Conversion rate (%) = (A + B) / (A + B + C) ... (3)
However, in the above formula (3), A represents the concentration of 3α-androstandiol, B represents the concentration of dihydrotestosterone (DHT), and C represents the concentration of testosterone.

Testosterone 5α-reductase activity inhibition rate (%) = (1-E / D) × 100 ... (4)
However, in the above equation (4), D represents the conversion rate in the blank test, and E represents the conversion rate in the addition of the test sample.

表１８の結果から、ハイビスカスの抽出物が、テストステロン５α−リダクターゼ活性阻害作用を有することが認められた。

From the results in Table 18, it was confirmed that the extract of hibiscus has an inhibitory effect on testosterone 5α-reductase activity.

(Example 19)
<Hair papilla cell proliferation action test>
A freeze-dried product of Hibiscus flower part extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the hair papillary cell proliferation effect was tested by the following test method.

Normal human hair papilla cells (manufactured by Toyo Spinning Co., Ltd.) are cultured using a hair papilla cell proliferation medium (manufactured by Toyo Spinning Co., Ltd.) containing 1% FCS and a proliferation additive, and then the cells are recovered by trypsin treatment. bottom. The collected cells were diluted with Dulbecco MEM containing 10% by volume FBS (Dulvecco's modified Eagle's medium (1), manufactured by Nissui Pharmaceutical Co., Ltd.) to ^{a concentration of 1.0 × 10 4} cells / mL, and then coated with collagen. 200 μL per well was seeded in the 96-well plate and cultured for 3 days. After culturing, the medium was removed, 200 μL of the test sample dissolved in serum-free DMEM (Dulbecco's modified Eagle's medium (1), manufactured by Nissui Pharmaceutical Co., Ltd.) was added to each well, and the cells were further cultured for 4 days. The dermal papilla cell proliferation effect was measured using the MTT assay. After the culture was completed, the medium was removed and dissolved in serum-free DMEM at a final concentration of 0.4 mg / mL 3- (4,5-dimethyl-thiazole-2-yl) -2,5-diphenyltetrazolium bromide (MTT, stock). (Manufactured by Dojin Chemical Laboratory) was added to each well in an amount of 100 μL. After culturing for 2 hours, the intracellularly produced blue formazan was extracted with 100 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. At the same time, the absorbance at a wavelength of 650 nm was measured as the turbidity, and the difference between the two was used as the amount of blue formazan produced.

The method for calculating the proliferation rate of dermal papilla cells is as follows. The results are shown in Table 19.
Hair papilla cell proliferation rate (%) = A / B × 100
However, in the above formula, A represents the amount of blue formazan produced when the test sample is added, and B represents the amount of blue formazan produced when the test sample is not added.

表１９の結果から、ハイビスカスの抽出物が、毛乳頭細胞増殖作用を有することが認められた。

From the results in Table 19, it was confirmed that the extract of Shoeblackplant had a hair papillary cell proliferation effect.

The hyaluronidase activity inhibitor, hydrogen peroxide scavenger, whitening agent, anti-aging agent, and hair growth agent of the present invention are excellent in safety, can be taken on a daily basis, and are inexpensive, yet have an excellent hyaluronidase activity inhibitory action. , Hydrogen peroxide scavenging effect, melanin production inhibitory effect on B16 melanoma cells, endoserin-1 mRNA expression increase inhibitory effect, stem cell growth factor mRNA expression increase inhibitory effect, basic fibroblast growth factor mRNA expression increase inhibitory effect, proopiomelanocortin mRNA Expression increase inhibitory action, epidermal hyaluronic acid production promoting action, glutathione production promoting action, serylluminoyltransferase mRNA expression promoting action, mailard reaction inhibitory action, final saccharified product formation inhibitory action, final saccharified product degradation promoting action, claudin-1 production promoting action Since it has at least one of action, occludin production promoting action, epidermal tight junction constituent protein production promoting action in human skin three-dimensional model, skin barrier function decline inhibitory action, testosterone 5α-reductase activity inhibitory action, and hair papilla cell proliferation action. It can be suitably used as an ingredient in cosmetics, foods and drinks, and as a research reagent.

Claims (2)

Claudin-1 production promoter, occludin production promoter, epidermal tight junction constituent protein production promoter in a human skin three-dimensional model, characterized by containing an extract of the flower part of Hibiscus sabdarifa as an active ingredient. , Or an agent that suppresses the deterioration of skin barrier function (excluding those used to prevent skin aging) . The extract of the flower part of Hibiscus subdarifa is a water extract of the flower part of Hibiscus subdarifa, a hydrophilic organic solvent extract, or a mixed solvent extract of water and a hydrophilic organic solvent. The clodin-1 production promoter, the occludin production promoter, the epidermal tight junction constituent protein production promoter in a human skin three-dimensional model, or the skin barrier function decline inhibitor according to claim 1.

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