JP7465589B2 - Serine palmitoyltransferase mRNA expression promoter - Google Patents

Description

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

Hyaluronate, which maintains its affinity for body tissues, is decomposed by ultraviolet rays, enzymes, etc. in aqueous systems, and the water-retaining effect decreases as the molecular weight decreases. Hyaluronic acid also exists as intercellular tissue and is involved in vascular permeability. Furthermore, hyaluronidase is thought to be involved in degranulation from mast cells through activation in mast cells. Therefore, by inhibiting the activity of hyaluronidase, which is a hydrolase of hyaluronic acid, hyaluronic acid can be stabilized, the release of various chemical mediators from mast cells can be prevented, and anti-inflammatory effects can be expected. Examples of herbal medicines that have such hyaluronidase activity inhibitory effects include extracts of plants from the genus Osbeckia (see Patent Document 1), wisteria tea extracts (see Patent Document 2), rosemary extracts, thyme extracts, and melissa extracts (see Patent Document 3).

Until now, the development of skin whitening agents has focused on tyrosinase, the rate-limiting enzyme in melanin production. Recently, however, it has been reported that cytokines such as α-melanocyte-stimulating hormone (α-MSH), endothelin-1 (ET-1), nitric oxide (NO), basic fibroblast growth factor (bFGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) are produced in increased amounts by epidermal keratinocytes after exposure to ultraviolet UVB light, activating pigment cells (melanocytes). There has been active development of substances that inhibit melanin production and achieve skin whitening effects by blocking the signal transduction systems in which these are involved. For example, chamomile extract and althea extract have been reported as herbal extracts that inhibit the action of endothelin-1 (ET-1) on pigment cells (melanocytes) (see Non-Patent Document 1).

In addition, it was previously believed that only the stratum corneum was responsible for the barrier function of the skin, but since the barrier function of the skin collapses when the constituent proteins of tight junctions (hereinafter sometimes abbreviated as TJ) present in the granular layer of the epidermis are genetically deleted, TJs are now believed to play an important role in the barrier function of the skin (see Non-Patent Document 2). TJs are binding devices that not only bring adjacent cells into close contact with each other, but also control the permeation of substances by sealing the gaps between cells. Proteins that make up this TJ include claudins, occludin, ZO-1, and ZO-2, and these proteins are thought to form the skeleton of the TJ strand and control the barrier function of the TJ (see Non-Patent Document 3). From the above, if the expression of claudins, occludin, ZO-1, and ZO-2 is reduced for some reason, structural destruction of TJs occurs, and they no longer function as a barrier to permeation of substances, which is expected to cause skin symptoms such as dry skin, rough skin, atopic dermatitis, and various infectious diseases.

Therefore, it is believed that by promoting the production of claudins, occludin, ZO-1, and ZO-2 in the epidermis and thereby promoting TJ formation in epidermal keratinocytes, the barrier function and moisture retention function of the skin can be enhanced, and the above-mentioned skin symptoms can be prevented or improved. Based on this idea, natural products such as coptis jasmine extract (see Patent Document 4) and spruce extract (see Patent Document 5) have been disclosed as substances that improve skin barrier function through the action of promoting TJ formation.

The reaction in which the amino groups of amino acids, peptides, and proteins react with ketones, aldehydes, and especially reducing sugars such as glucose to produce brown pigments is called the Maillard reaction. The substances produced as the final products of this Maillard reaction are called advanced glycation end products (hereinafter sometimes referred to as "AGEs"). The Maillard reaction consists of an early reaction in which an amino group reacts nonenzymatically with glucose to form a Schiff base, which then undergoes Amadori rearrangement, a middle reaction that further produces an active intermediate having a dicarbonyl group such as 3-deoxyglucosone (3-DG), and a late reaction in which the active intermediate further reacts nonenzymatically with an amino group, repeating dehydration and condensation reactions to form AGEs.
Identified AGEs include, for example, imidazolone (see Non-Patent Document 4), N ^ε -carboxymethyllysine (CML) (see Non-Patent Document 5), pentosidine, pyrraline, crosslin, N ^ε -carboxyethyllysine, methylglyoxallysine dimer, glyoxallysine dimer, etc. Imidazolone has been reported to be produced by the reaction of 3-DG with arginine (see Non-Patent Document 4).

One of the pathological conditions in which AGEs are involved in the onset and progression is the symptom of aging. The progression of the Maillard reaction in living tissues can lead to aging (loss of elasticity) in skin tissues due to cross-linking of skin elastic fibers, and it is also said that deposition of AGEs in blood vessel wall tissues and neurofibril can lead to arteriosclerosis and Alzheimer's disease.

As an example of a natural product having an AGEs production inhibitory effect, an extract from the pericarp of Diarium indum, a member of the legume family, has been disclosed (see Patent Document 6).
In addition, compounds such as aminoguanidine, OPB-9195, and pyridoxamine are known to have an effect of inhibiting the production of AGEs, but these compounds have problems such as side effects (see Non-Patent Documents 4 to 6).

Many steroid hormones exert their effects by binding to receptors in the molecular form secreted from the organs that produce them, but in the case of male hormones collectively known as androgens, for example, testosterone enters the cells of the target organ and is reduced to 5α-dihydrotestosterone (5α-DHT) by testosterone 5α-reductase before binding to a receptor and exerting its androgen effect.
The androgen is an important hormone, but when it acts excessively, it induces various undesirable symptoms such as male pattern baldness, hirsutism, seborrhea, acne (e.g., acne), prostatic hyperplasia, prostatic tumor, and precocious puberty in boys. Therefore, in order to improve these various symptoms, a method for suppressing the action of excess androgen has been disclosed, specifically, a method for suppressing the generation of active 5α-DHT by inhibiting the action of testosterone 5α-reductase that reduces testosterone to active 5α-DHT, and a method for preventing the expression of androgenic activity by inhibiting the binding of 5α-DHT generated from testosterone to a receptor. As such a plant extract having an action of inhibiting the binding of 5α-DHT to its receptor, for example, at least one extract of Majito and Catcha has been disclosed (see Patent Document 7).

Hair grows and falls out repeatedly according to a periodic hair cycle consisting of the growth phase, regression phase, and resting phase. In this hair cycle, the stage in which new hair follicles are formed from the resting phase to the growth phase is considered to be the most important for hair growth, and it is believed that hair papilla cells play an important role in the proliferation and differentiation of hair follicle epithelial cells in this stage. Hair papilla cells are cells located inside hair follicle epithelial cells consisting of outer root sheath cells and matrix cells near the hair root, and are located in the basement membrane-encased part of the hair root. They act on hair follicle epithelial cells to promote their proliferation, and play an important role in the proliferation and differentiation of hair follicle epithelial cells and hair formation. The hair papilla cells play the most important role in the proliferation and differentiation of hair follicle epithelial cells and hair formation, and a method has been proposed in which a target substance is brought into contact with cultured hair papilla cells, and the presence or absence or strength of the proliferation activity of the cells is identified to test the hair growth effect of the target substance. Examples of herbal medicines that have the effect of promoting hair papilla cell proliferation include extracts of Astragalus chinensis, extracts of Coptis japonica, and extracts of Anemone anguicida (see Patent Documents 8 and 9).

However, to date, there has been no excellent substance that has at least one of the above-mentioned effects and is also highly safe, and therefore can be widely used as an ingredient in cosmetics, food and beverages, research reagents, and the like, and there is a strong demand for such a substance to be provided as soon as possible.

JP 2003-55242 A JP 2003-12532 A Japanese Patent Application Laid-Open No. 8-333267 JP 2007-176830 A JP 2007-176835 A JP 2010-111615 A JP 2002-241297 A Japanese Patent Application Laid-Open No. 9-208431 Japanese Patent Application Laid-Open No. 11-12134

"Fragrance Journal", Vol. 28, No. 9, p. 65-71, 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)

The present invention aims to solve the above-mentioned problems in the prior art and to achieve the following objectives. That is, the present invention aims to provide a hyaluronidase activity inhibitor having excellent hyaluronidase activity inhibitory activity and being highly safe, a hydrogen peroxide eliminator having excellent hydrogen peroxide elimination activity and being highly safe, a whitening agent having excellent whitening activity and being highly safe, an anti-aging agent having excellent anti-aging activity and being highly safe, and a hair growth agent having excellent hair growth activity and being highly safe.

The present inventors conducted extensive research to solve the above problems and discovered that hibiscus extract has excellent hyaluronidase activity inhibitory effects, hydrogen peroxide elimination effects, skin whitening effects, anti-aging effects, and hair growth effects.

The present invention is based on the findings of the present inventors, and the means for solving the above problems are as follows.
<1> A hyaluronidase activity inhibitor characterized by containing an extract of hibiscus.
<2> A hydrogen peroxide eliminator characterized by containing an extract of hibiscus.
<3> A skin whitening agent containing an extract of hibiscus.
<4> The skin whitening agent according to <3>, which has at least any one of an inhibitory effect on melanin production in B16 melanoma cells, an inhibitory effect on increased expression of endothelin-1 mRNA, an inhibitory effect on increased expression of stem cell growth factor mRNA, an inhibitory effect on increased expression of basic fibroblast growth factor mRNA, and an inhibitory effect on increased expression of proopiomelanocortin mRNA.
<5> An anti-aging agent characterized by containing an extract of hibiscus.
<6> The anti-aging agent according to <5>, which has at least any one of an effect of promoting epidermal hyaluronic acid production, an effect of promoting glutathione production, an effect of promoting serine palmitoyltransferase mRNA expression, an effect of inhibiting the Maillard reaction, an effect of suppressing the formation of advanced glycation end products, an effect of promoting the decomposition of advanced glycation end products, an effect of promoting claudin-1 production, an effect of promoting occludin production, an effect of promoting the production of epidermal tight junction constituent proteins in a three-dimensional human skin model, and an effect of suppressing a decrease in skin barrier function.
<7> A hair growth agent comprising an extract of hibiscus.
<8> The hair growth agent according to <7>, which has at least one of a testosterone 5α-reductase activity inhibitory effect and a hair papilla cell proliferation effect.

The present invention can solve the above-mentioned problems of the prior art and achieve the above-mentioned object, and can provide a hyaluronidase activity inhibitor having excellent hyaluronidase activity inhibitory activity and high safety, a hydrogen peroxide eliminator having excellent hydrogen peroxide elimination activity and high safety, a whitening agent having excellent whitening activity and high safety, an anti-aging agent having excellent anti-aging activity and high safety, and a hair restorer having excellent hair growth activity and high safety.

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

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

The hyaluronidase activity inhibitor has an inhibitory effect on hyaluronidase activity.
The hydrogen peroxide eliminator has a hydrogen peroxide elimination effect.
The whitening agent has a whitening effect based on at least any one of the following actions: an inhibitory effect on melanin production in B16 melanoma cells, an inhibitory effect on increased expression of endothelin-1 mRNA, an inhibitory effect on increased expression of stem cell growth factor mRNA, an inhibitory effect on increased expression of basic fibroblast growth factor mRNA, and an inhibitory effect on increased expression of proopiomelanocortin mRNA.
The anti-aging agent has an anti-aging effect based on at least any one of the following actions: promoting the production of epidermal hyaluronic acid, promoting the production of glutathione, promoting the expression of serine palmitoyltransferase mRNA, inhibiting the Maillard reaction, suppressing the formation of advanced glycation end products, promoting the decomposition of advanced glycation end products, promoting the production of claudin-1, promoting the production of occludin, promoting the production of epidermal tight junction constituent proteins in a three-dimensional human skin model, and inhibiting a decline in skin barrier function.
The hair growth agent has a hair growth effect based on at least one of an inhibitory effect on testosterone 5α-reductase activity and an effect on proliferation of hair papilla cells.

Details of the substances contained in the hibiscus extract that exert at least one of the following effects: hyaluronidase activity inhibitory action, hydrogen peroxide elimination action, skin whitening action, anti-aging action, and hair growth action are unknown, but the fact that the hibiscus extract has such excellent effects and is useful as a hyaluronidase activity inhibitor, hydrogen peroxide elimination agent, skin whitening agent, anti-aging agent, and hair growth agent was not previously known, and is a new discovery by the present inventors.

The hibiscus is an evergreen shrub belonging to the genus Hibiscus of the family Malvaceae, with the scientific name Hibiscus sabdarifa , native to Africa, and known as Roselle in Japanese. The hibiscus has a refreshing sour taste and is used as a sauce in French and Italian cuisine, and is also used as an herb.

There are no particular limitations on the parts of the hibiscus used as the extraction raw material, and they can be appropriately selected depending on the purpose. Examples include flowers, calyx, buds, fruit, pericarp, seeds, seed coats, stems, leaves, branches, branches and leaves, trunks, bark, roots, rhizomes, root bark, and mixtures of these. Among these, flower parts such as flowers, calyx, and buds are preferred.

The hibiscus, which is the extraction raw material, can be subjected to solvent extraction, for example, after drying, either as is or after being crushed using a crusher or the like. In particular, it is preferable that the extraction raw material is dried and crushed immediately after collection. The drying may be performed, for example, in the sun or using a commonly used dryer. The hibiscus may be used as the extraction raw material after being subjected to pretreatment such as degreasing with a nonpolar solvent such as hexane or benzene. By performing pretreatment such as degreasing, the extraction process of the hibiscus with a polar solvent can be performed efficiently.

The hibiscus extract can be easily obtained by utilizing a method generally used for plant extraction. In addition, a commercially available product may be used as the hibiscus extract. The hibiscus extract includes the hibiscus extract, a diluted or concentrated solution of the extract, a dried product of the extract, or any of the crude or purified products thereof.

The solvent used for the extraction is not particularly limited and can be appropriately selected depending on the purpose. For example, it is preferable to use water, a hydrophilic organic solvent, or a mixed solvent of these at room temperature or at a temperature below the boiling point of the solvent. The components contained in the hibiscus that exhibit at least one of hyaluronidase activity inhibitory effect, hydrogen peroxide elimination effect, whitening effect, anti-aging effect, and hair growth effect can be easily extracted by an extraction process using a polar solvent as the extraction solvent.

There are no particular limitations on the water that can be used as the extraction solvent, and it can be appropriately selected depending on the purpose. Examples of the water include pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, and water that has been subjected to various treatments. Treatments that can be applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, etc. Therefore, water that can be used as the extraction solvent also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered physiological saline, etc.

The hydrophilic organic solvent that can be used as the extraction solvent is not particularly limited and can be appropriately selected depending on the purpose. For example, lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; 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 can be used as a mixed solvent of the hydrophilic organic solvent and water. When using a mixed solvent of water and the hydrophilic organic solvent, it is preferable to use a mixture of 1 to 90 parts by weight of the lower alcohol per 10 parts by weight of water, and 1 to 40 parts by weight of the lower aliphatic ketone per 10 parts by weight of water. In addition, it is preferable to use a mixture of 1 to 90 parts by weight of the polyhydric alcohol per 10 parts by weight of water.

The method for extracting the hibiscus extract is not particularly limited as long as it is possible to dissolve the fat-soluble components contained in the hibiscus extract raw material in the solvent, and can be performed according to a conventional method. In addition, there is no need to adopt a special extraction method during the extraction process, and any device can be used at room temperature or under reflux heating.
Specifically, the method for extracting the hibiscus extract may include, for example, placing the extraction raw material, such as hibiscus flowers, in a treatment tank filled with the solvent, such as an aqueous ethanol solution, and extracting the raw material by heating at 80°C for 2 hours in a reflux extractor while stirring appropriately as necessary. The extract is then filtered while hot to elute the fat-soluble components, and then concentrated under reduced pressure using an evaporator, and the same filtration process is then performed.
In this case, the extraction conditions can be appropriately adjusted depending on the extraction raw material, etc., but the amount of the extraction solvent is preferably 5 to 20 times (mass ratio) the amount of the extraction raw material, the extraction time is preferably 1 to 3 hours, and the extraction temperature is preferably 20°C to 95°C.

The obtained hibiscus extract may be subjected to treatments such as dilution, concentration, drying, purification, etc. according to conventional methods to obtain a diluted product, concentrate, dried product, crude product, purified product, etc. of the hibiscus extract.
The obtained hibiscus extract can be used as it is as any one of the hyaluronidase activity inhibitor, hydrogen peroxide scavenger, skin whitening agent, anti-aging agent, and hair growth agent, but the concentrated liquid and the dried product are preferred in terms of ease of use. In obtaining the dried product, a carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity.

The other ingredients are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected according to the purpose. Examples of the other ingredients include excipients, binders, disintegrants, lubricants, stabilizers, flavorings, odorants, etc.

Examples of the excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binders include water, ethanol, propanol, simple syrup, glucose liquid, starch liquid, gelatin liquid, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylstarch, methylcellulose, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone. Examples of the disintegrants include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose. Examples of the lubricants include purified talc, stearates, borax, and polyethylene glycol. Examples of the stabilizers include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid. Examples of the flavorings and odorants include sucrose, orange peel, citric acid, and tartaric acid.

The hibiscus extract thus obtained has the following effects: inhibiting hyaluronidase activity, eliminating hydrogen peroxide, suppressing melanin production in B16 melanoma cells, suppressing increases in endothelin-1 mRNA expression, suppressing increases in stem cell growth factor mRNA expression, suppressing increases in basic fibroblast growth factor mRNA expression, suppressing increases in proopiomelanocortin mRNA expression, promoting epidermal hyaluronic acid production, promoting glutathione production, promoting serine palmitoyltransferase mRNA expression, inhibiting the Maillard reaction, and most importantly, The compound has at least any one of the following effects: an inhibitory effect on the formation of advanced glycation end products, an inhibitory effect on the degradation of advanced glycation end products, an inhibitory effect on the production of claudin-1, an inhibitory effect on the production of occludin, an inhibitory effect on the production of epidermal tight junction component proteins in a three-dimensional human skin model, an inhibitory effect on the decline of skin barrier function, an inhibitory effect on testosterone 5α-reductase activity, and an inhibitory effect on the proliferation of hair papilla cells. Based on these effects, the compound can be suitably used as at least any one of the active ingredients of the hyaluronidase activity inhibitor, hydrogen peroxide scavenger, skin whitening agent, anti-aging agent, and hair growth agent of the present invention.
Based on the above-mentioned actions, the hibiscus extract can also be suitably used as an inhibitor of melanin production in B16 melanoma cells, an inhibitor of increased endothelin-1 mRNA expression, an inhibitor of increased stem cell growth factor mRNA expression, an inhibitor of increased basic fibroblast growth factor mRNA expression, an inhibitor of increased proopiomelanocortin mRNA expression, an epidermal hyaluronic acid production promoter, a glutathione production promoter, a serine palmitoyltransferase mRNA expression promoter, a Maillard reaction inhibitor, an inhibitor of advanced glycation end product formation, an advanced glycation end product degradation promoter, a claudin-1 production promoter, an occludin production promoter, an epidermal tight junction constituent protein production promoter in a three-dimensional human skin model, an inhibitor of decreased skin barrier function, a testosterone 5α-reductase activity inhibitor, and a hair papilla cell proliferation agent, respectively.

The hyaluronidase activity inhibitor of the present invention exerts its inhibitory effect on hyaluronidase activity.
The hydrogen peroxide scavenger of the present invention exerts its effect based on its hydrogen peroxide scavenging action.
The whitening effect of the whitening agent of the present invention is exerted based on at least any one of the following actions: an inhibitory effect on melanin production in B16 melanoma cells, an inhibitory effect on increased endothelin-1 mRNA expression, an inhibitory effect on increased stem cell growth factor mRNA expression, an inhibitory effect on increased basic fibroblast growth factor mRNA expression, and an inhibitory effect on increased proopiomelanocortin mRNA expression.
The anti-aging effect of the anti-aging agent of the present invention is exerted based on at least any one of the following effects: promoting epidermal hyaluronic acid production, promoting glutathione production, promoting serine palmitoyltransferase mRNA expression, inhibiting the Maillard reaction, inhibiting the formation of advanced glycation end products, promoting the decomposition of advanced glycation end products, promoting claudin-1 production, promoting occludin production, promoting the production of epidermal tight junction component proteins in a three-dimensional human skin model, and inhibiting a decline in skin barrier function.
The hair growth effect of the hair growth agent of the present invention is exerted based on at least one of the effect of inhibiting testosterone 5α-reductase activity and the effect of proliferating hair papilla cells.

The contents of the hyaluronidase activity inhibitor, the hydrogen peroxide eliminator, the whitening agent, the anti-aging agent, and the hibiscus extract in the hair growth agent are not particularly limited and can be appropriately selected depending on the purpose. Furthermore, the hyaluronidase activity inhibitor, the hydrogen peroxide eliminator, the whitening agent, the anti-aging agent, and the hair growth agent may be the hibiscus extract itself.

In addition, the other components other than the hibiscus extract that may be contained in the hyaluronidase activity inhibitor, the hydrogen peroxide eliminator, the whitening agent, the anti-aging agent, and the hair growth agent are not particularly limited and can be appropriately selected according to the purpose as long as they are within a range that does not impair the effects of the present invention, and examples thereof include physiological saline for diluting the hibiscus extract to a desired concentration. In addition, the contents of the hyaluronidase activity inhibitor, the hydrogen peroxide eliminator, the whitening agent, the anti-aging agent, and the other components in the hair growth agent are not particularly limited and can be appropriately selected according to the purpose.
In addition, the hyaluronidase activity inhibitor, the hydrogen peroxide eliminator, the whitening agent, the anti-aging agent, and the hair growth agent can be formulated as necessary to be in any dosage form, such as powder, granules, or tablets.

The hyaluronidase activity inhibitor, hydrogen peroxide scavenger, skin whitening agent, anti-aging agent, and hair growth agent of the present invention have excellent hyaluronidase activity inhibitory activity, hydrogen peroxide scavenging activity, skin whitening activity, anti-aging activity, and hair growth activity, and are also highly safe, making them suitable for use in, for example, various cosmetics, foods and beverages, etc.

The hyaluronidase activity inhibitor, hydrogen peroxide scavenger, skin whitening agent, anti-aging agent, and hair growth agent of the present invention are preferably applied to humans, but can also be applied to animals other than humans as long as their respective action effects are exerted.

The following describes examples of the present invention, but the present invention is not limited to these examples.

<Hibiscus extract>
A freeze-dried extract of hibiscus flowers (Maruzen Pharmaceutical Co., Ltd.) was used as the test sample.

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

A hyaluronidase solution (Type IV-S (derived from bovine testis), 400 NF units/mL; manufactured by Sigma-Aldrich Japan) of 0.1 mL was added to 0.2 mL of 0.1 mol/L acetate buffer (pH 3.5) in which the test sample was dissolved, and the mixture was reacted at 37 ° C. for 20 minutes. Furthermore, 0.2 mL of 2.5 mmol/L calcium chloride was added as an activator, and the mixture was reacted at 37 ° C. for 20 minutes. 0.5 mL of 0.8 mg/mL sodium hyaluronate solution (chicken comb-derived sodium hyaluronate, manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto, and the mixture was reacted at 37 ° C. for 40 minutes. Thereafter, 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 boric acid solution was added to each reaction solution, and the mixture was boiled for 3 minutes. After cooling on ice, 6 mL of p-DABA reagent (p-dimethylaminobenzaldehyde, manufactured by Wako Pure Chemical Industries, Ltd.) was added and reacted at 37° C. for 20 minutes. Thereafter, the absorbance at a wavelength of 585 nm was measured. A blank test was performed in the same manner to perform correction.
The hyaluronidase activity inhibition rate was calculated as follows. The results are shown in Table 1.
Hyaluronidase activity inhibition rate (%) =
1-(St-Sb)/(Ct-Cb) × 100
In the above formula, St represents the absorbance of the test sample solution at a wavelength of 585 nm, Sb represents the absorbance of the test sample solution blank at a wavelength of 585 nm, Ct represents the absorbance of the control solution at a wavelength of 585 nm, and Cb represents the absorbance of the control solution blank at a wavelength of 585 nm.

The results in Table 1 demonstrate that the hibiscus extract has an inhibitory effect on hyaluronidase activity.

Example 2
<Hydrogen peroxide elimination test>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and its hydrogen peroxide scavenging activity was tested by the following test method.

25 μL of the test sample solution was placed in a 96-well plate, 10 μL of 0.15 mM H ₂ O ₂ , and 25 μL of 0.1 mol/L PIPES buffer (pH 7.0) (containing 0.5% Triton X-100 and 1 mL of 100 unit/mL peroxidase) were added, and the mixture was reacted at 37° C. for 20 minutes. After the reaction, 180 μL of 100 μM DA-67 was added immediately, followed by addition of 10 μL of ethanol, and a color 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. A blank test was also carried out in the same manner to perform correction.
The hydrogen peroxide scavenging rate was calculated as follows. The 50% inhibitory activity concentration (IC ₅₀ : μg/mL) was also calculated. These results are shown in Table 2.
Hydrogen peroxide elimination rate (%) = {1 - (St - Sb) / (Ct - Cb)} x 100
In the above formula, St represents the absorbance of the test sample solution at a wavelength of 650 nm, Sb represents the absorbance of the test sample solution blank at a wavelength of 650 nm, Ct represents the absorbance of the control solution at a wavelength of 650 nm, and Cb represents the absorbance of the control solution blank at a wavelength of 650 nm.

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

Example 3
<Test for melanin production inhibition in B16 melanoma cells>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and its inhibitory effect on melanin production in B16 melanoma cells was tested by the following test method.

B16 melanoma cells were cultured using Dulbecco's MEM (Dulbecco's modified Eagle's medium (1), manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% by volume FBS (Standard Fetal Bovine Serum, manufactured by HyClone Co., Ltd.), and then the cells were harvested by trypsin treatment. The harvested cells were diluted to a concentration of 24.0 x ¹⁰⁴ cells/mL with Dulbecco's MEM containing 10% by volume FBS and 1 mmol/L theophylline (Theophylline, manufactured by Wako Pure Chemical Industries, Ltd.), and then seeded in a 48-well plate at 300 μL per well and cultured for 6 hours. After completion of the culture, 300 μL of the test sample dissolved in Dulbecco's MEM containing 10% by volume FBS and 1 mmol/L theophylline was added to each well and cultured for 4 days. After the incubation, the medium was removed from each well, 200 μL of 2 mol/L NaOH solution was added, the cells were disrupted by an ultrasonicator, and the absorbance at a wavelength of 475 nm was measured. The amount of melanin was calculated from the measured absorbance value based on a calibration curve prepared using synthetic melanin (manufactured by SIGMA).
In addition, to measure cell viability, after culturing in the same manner, the wells were washed with 400 μL of PBS (-) phosphate physiological buffer, and 200 μL of 13.8 mmol/L neutral red dissolved in 10% by volume FBS-containing Dulbecco's MEM at a final concentration of 0.05 mg/mL 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 in Dulbecco's MEM containing 10% by volume FBS and 1 mmol/L theophylline were tested in the same manner.
The melanin production inhibition rate was calculated as follows: The results are shown in Table 3.
Melanin production inhibition rate (%) = {1 - (B/D)/(A/C)} x 100
In the above formula, A represents the absorbance at a wavelength of 475 nm in cells to which a test sample has not been added, B represents the absorbance at a wavelength of 475 nm in cells to which a test sample has been added, C represents the absorbance at a wavelength of 540 nm in cells to which a test sample has not been added, and D represents the absorbance at a wavelength of 540 nm in cells to which a test sample has been added.

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

Example 4
<Test for inhibitory effect on increased expression of endothelin-1 mRNA>
A freeze-dried extract of hibiscus flowers (Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and the inhibitory effect on the increase in endothelin-1 mRNA expression was tested by the following test method.

Normal human neonatal foreskin epidermal keratinocytes (NHEK) were precultured in a 75 cm ² flask in normal human epidermal keratinocyte growth medium (KGM) at 37° C. under 5% CO ₂ , and the cells were collected by trypsinization.
Using KGM, 40 x 10 ⁴ cells/2 mL/dish were seeded on 35 mm dishes (manufactured by FALCON), and cultured overnight at 37 ° C. and 5% CO _2. After 24 hours, the culture medium was discarded, 1 mL of HEPES buffer was added, and UV-B irradiation (50 mJ/cm ² ) was performed, and then 2 mL of the test sample dissolved in KGM at the required concentration was added to each dish, and cultured for 24 hours at 37 ° C. and 5% CO _2. After culture, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE; Cat. No. 311-07361), and the amount of each RNA was measured using a spectrophotometer, and total RNA was prepared to be 200 ng/μL.
Using this total RNA as a template, the expression levels of endothelin-1 and GAPDH mRNA as an internal standard were measured. Detection was performed by real-time two-step RT-PCR reaction using a real-time PCR device Smart Cycler® ⁽ manufactured by Cepheid) and TaKaRa ^SYBR® PrimeScript ^™ RT-PCR Kit (Perfect Real Time) (code No. RR063A).
The expression level of endothelin-1 mRNA was calculated based on the total RNA samples prepared from cells cultured under the conditions "not exposed to UV light and no test sample added,""exposed to UV light and no test sample added," and "exposed to UV light and test sample added," and a corrected value was calculated using the GAPDH value. Furthermore, the corrected value for "not exposed to UV light and no test sample added" was set to 100, and the corrected values for "exposed to UV light and no test sample added" and "exposed to UV light and test sample added" were calculated.
From these results, the inhibition rate of increase in endothelin-1 mRNA expression was calculated using the following formula. The results are shown in Table 4.
Inhibition rate of increased expression of endothelin-1 mRNA (%)
= {(A-B)-(A-C)}/(A-B) x 100
In the above formula, A is the correction value when "no UV irradiation and no test sample added", B is the correction value when "UV irradiation and no test sample added", and C is the correction value when "UV irradiation and test sample added".

From the results in Table 4, it was found that the hibiscus extract has an inhibitory effect on the increase in endothelin-1 mRNA expression.

Example 5
<Test for suppression of increased expression of stem cell factor (SCF) mRNA>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and the inhibitory effect on increased expression of stem cell factor (SCF) mRNA was tested by the following test method.

Normal human neonatal foreskin epidermal keratinocytes (NHEK) were precultured in a 80 cm ² flask in normal human epidermal keratinocyte growth medium (KGM) at 37° C. under 5% CO ₂ , and the cells were collected by trypsinization.
Using KGM, 40 x 10 ⁴ cells/2 mL were seeded on 35 mm petri dishes (manufactured by FALCON) and cultured overnight at 37 ° C. and 5% CO _2. After 24 hours, the culture medium 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 at the required concentration was added to each petri dish, and cultured at 37 ° C. and 5% CO ₂ for 24 hours. After culture, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE; Cat. No. 311-07361), and the amount of each RNA was measured using a spectrophotometer, and total RNA was prepared to be 200 ng/μL.
Using this total RNA as a template, the expression levels of SCF (Stem Cell Factor) and GAPDH mRNA as an internal standard were measured. Detection was performed by real-time two-step RT-PCR reaction using TaKaRa SYBR PrimeScript ^™ RT-PCR Kit (Perfect Real Time) (code No. RR063A) using a real-time PCR device Smart Cycler® ⁽ Cepheid ⁾ .
The expression level of SCF mRNA was calculated based on the total RNA samples prepared from cells cultured under the conditions "not exposed to UV light and no test sample added,""exposed to UV light and no test sample added," and "exposed to UV light and test sample added," and a corrected value was calculated using the GAPDH value. Furthermore, the corrected value for "not exposed to UV light and no test sample added" was set to 100, and the corrected values for "exposed to UV light and no test sample added" and "exposed to UV light and test sample added" were calculated.
From these results, the stem cell factor (SCF) mRNA expression increase inhibition rate was calculated using the following formula. The results are shown in Table 5.
Stem cell factor (SCF) mRNA expression upregulation inhibition rate (%)
= {(A-B)-(A-C)}/(A-B) x 100
In the above formula, A represents the correction value when "no UV irradiation and no test sample added", B represents the correction value when "UV irradiation and no test sample added", and C represents the correction value when "UV irradiation and test sample added".

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

Example 6
<Test for inhibitory effect on increased expression of basic fibroblast growth factor (bFGF) mRNA>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and the inhibitory effect on the increase in basic fibroblast growth factor (bFGF) mRNA expression was tested by the following test method.

Normal human neonatal foreskin 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 the cells were collected by trypsinization.
Using KGM, 40 x 10 ⁴ cells/2 mL were seeded on 35 mm petri dishes (manufactured by FALCON) and cultured overnight at 37 ° C. and 5% CO _2. After 24 hours, the culture medium 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 cultured at 37 ° C. and 5% CO ₂ for 24 hours. After culture, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE; Cat. No. 311-07361), and the amount of each RNA was measured using a spectrophotometer, and total RNA was prepared to be 200 ng/μL.
Using this total RNA as a template, the expression levels of bFGF (basic fibroblast growth factor) and GAPDH mRNA as an internal standard were measured. Detection was performed by real-time two-step RT-PCR reaction using a real-time PCR device Smart Cycler (Cepheid ⁾ and TaKaRa SYBR PrimeScript ^{TM RT} -PCR Kit (Perfect Real Time) (code No. RR063A).
The expression level of bFGF mRNA was calculated based on the total RNA samples prepared from cells cultured under the conditions "not exposed to UV light and no test sample added,""exposed to UV light and no test sample added," and "exposed to UV light and test sample added," and a corrected value was calculated using the GAPDH value. Furthermore, the corrected value for "not exposed to UV light and no test sample added" was set to 100, and the corrected values for "exposed to UV light and no test sample added" and "exposed to UV light and test sample added" were calculated.
From these results, the inhibition rate of increase in bFGF mRNA expression was calculated by the following formula. The results are shown in Table 6.
Inhibition rate of increased bFGF mRNA expression (%)
= {(A-B)-(A-C)}/(A-B) x 100
In the above formula, A represents the correction value when there is no UV irradiation and no test sample is added, B represents the correction value when there is UV irradiation and no test sample is added, and C represents the correction value when there is UV irradiation and the test sample is added.

From the results in Table 6, it was found that the hibiscus extract had a strong inhibitory effect on the increase in bFGF mRNA expression.

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

Normal human neonatal foreskin 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 the cells were collected by trypsinization.
Using KGM, 40 x 10 ⁴ cells/2 mL were seeded on 35 mm petri dishes (manufactured by FALCON) and cultured overnight at 37 ° C. and 5% CO _2. After 24 hours, the culture medium 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 cultured at 37 ° C. and 5% CO ₂ for 24 hours. After culture, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE; Cat. No. 311-07361), and the amount of each RNA was measured using a spectrophotometer, and total RNA was prepared to be 200 ng/μL.
Using this total RNA as a template, the expression levels of POMC (proopiomelanocortin) and GAPDH mRNA as an internal standard were measured. Detection was performed by real-time two-step RT-PCR reaction using a real-time PCR device Smart Cycler ⁽ Cepheid) and TaKaRa SYBR PrimeScript ^{™ RT} -PCR Kit (Perfect Real Time) (code No. RR063A).
The expression level of POMC mRNA was calculated based on the total RNA samples prepared from cells cultured under the conditions "not exposed to UV light and no test sample added,""exposed to UV light and no test sample added," and "exposed to UV light and test sample added," and a corrected value was calculated using the GAPDH value. Furthermore, the corrected value for "not exposed to UV light and no test sample added" was set to 100, and the corrected values for "exposed to UV light and no test sample added" and "exposed to UV light and test sample added" were calculated.
From these results, the inhibition rate of increase in POMC mRNA expression was calculated by the following formula. The results are shown in Table 7.
POMC mRNA expression upregulation suppression rate (%)
= {(A-B)-(A-C)}/(A-B) x 100
In the above formula, A represents the correction value when "no ultraviolet light irradiation and no test sample added", B represents the correction value when "ultraviolet light irradiation and no test sample added", and C represents the correction value when "ultraviolet light irradiation and test sample added".

From the results in Table 7, it was found that the hibiscus extract has an inhibitory effect on the increase in POMC mRNA expression.

(Example 8)
<Epidermal hyaluronic acid production promotion test>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical 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 using human normal neonatal epidermal keratinocyte growth medium (KGM), and then the cells were harvested by trypsin treatment. The harvested cells were diluted with KGM to a concentration of 1 x ¹⁰⁵ cells/mL, and then seeded in a 96-well plate at 100 μL per well and cultured for 24 hours. After the culture was completed, 100 μL of the test sample dissolved in KGM was added to each well and cultured for 7 days. After the culture, the amount of hyaluronic acid in the medium of each well was measured by the sandwich method using hyaluronic acid binding protein (HABP, manufactured by Seikagaku Biobusiness Co., Ltd.).

The hyaluronic acid production promotion rate was calculated as follows. The results are shown in Table 8.
Hyaluronic acid production promotion rate (%) = A/B x 100
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.

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

Example 9
<Glutathione production promotion test (human normal skin fibroblasts)>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample and a glutathione production promoting effect test (human normal skin fibroblasts) was conducted by the following test method.

Human normal skin fibroblasts (NB1RGB) were cultured in α-MEM medium containing 10% by mass FBS, and the cells were harvested by trypsin treatment. The harvested cells were diluted with α-MEM medium containing 10% by mass FBS to a concentration of 2.0 x 10 ⁵ cells/mL, and then seeded in a 48-well plate at 200 μL per well and cultured overnight. After the culture, 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 the culture was completed, the medium was removed from each well, washed with 400 μL of PBS (-), and the cells were lysed using 150 μL of M-PER ^(R) (PIERCE). Quantification of total glutathione was performed using 100 μL of the diluted medium.
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 which 25 μL of 10 mM 5,5'-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm was measured for 5 minutes, and ΔOD/min was calculated. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione.
The obtained values were corrected to the amount of glutathione per total protein amount, and 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
In the above formula, A represents the amount of glutathione per total protein amount in cells to which the test sample has not been added (control), and B represents the amount of glutathione per total protein amount in cells to which the test sample has been added.

From the results in Table 9, it was found that the hibiscus extract has an effect of promoting glutathione production.

Example 10
<Serine palmitoyltransferase (SPT) mRNA expression promoting effect test>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and the serine palmitoyltransferase (SPT) mRNA expression promoting effect was tested by the following test method.

Normal human neonatal foreskin epidermal keratinocytes (NHEK) were precultured in a 75 cm ² flask in normal human epidermal keratinocyte growth medium (KGM) at 37° C. under 5% CO ₂ , and the cells were collected by trypsinization.
Using KGM, 40 x 10 ⁴ cells/2 mL/dish were seeded on 35 mm dishes (manufactured by FALCON), and cultured overnight at 37 ° C. and 5% CO _2. After 24 hours, the culture medium was discarded, and 2 mL of the test sample dissolved in KGM at the required concentration was added to each dish, and cultured at 37 ° C. and 5% CO ₂ for 24 hours. After culture, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE; Cat. no. 311-07361), and the amount of each RNA was measured using a spectrophotometer, and total RNA was prepared to be 200 ng / μL.
Using this total RNA as a template, the expression levels of SPT and GAPDH mRNA, which is an internal standard, were measured. Detection was performed by real-time 2-Step RT-PCR reaction using TaKaRa SYBR PrimeScript ^TM RT-PCR Kit (Perfect Real Time) (code No. RR063A) using a real-time PCR device Smart Cycler (registered trademark) (manufactured by Cepheid). The expression level of SPT was calculated by obtaining a correction value with the value of GAPDH based on total RNA samples prepared from cells cultured without and with the addition of the test sample, and further calculating the correction value with the addition of the test sample when the correction value without the addition of the test sample was set to 100.
The method for calculating the SPT mRNA expression promotion rate is as follows. The results are shown in Table 10.
SPT mRNA expression promotion rate (%) = A/B x 100
In the above formula, A represents the corrected value when the test sample is added, and B represents the corrected value when the test sample is not added.

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

(Example 11)
<Maillard reaction inhibition test>
A freeze-dried extract of hibiscus flower (manufactured by Maruzen Pharmaceutical 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 prepared by dissolving freeze-dried test sample in distilled water, 200 μL of 100 mmol/L D(-)-ribose, 200 μL of 25 mg/mL lysozyme, 500 μL of 100 mmol/L sodium hydrogen phosphate (pH 7.4), and 50 μL of sterile distilled water were mixed (total volume 1,000 μL) and allowed to stand at 37°C. A control was prepared in the same manner as above, except that distilled water was used instead of the test sample solution. A blank was prepared in the same manner as above, except that distilled water was used instead of the test sample solution and the blank was allowed to stand at 4°C instead of 37°C.

After 7 days, the mixture was stirred with a vortex mixer, and 40 μL of the reaction solution was mixed with 40 μL of SDS-PAGE sample buffer, which was then heated in a boiling bath for 3 minutes to prepare an analysis sample. 12 μL of the analysis sample was applied to a polyacrylamide gel in which the acrylamide concentration was adjusted to 15% for the separation gel and 4% for the concentration gel, and electrophoresis was performed.
The electrophoresed gel was stained with Coomassie Brilliant Blue and destained, and detected using an imaging device ChemiDocXRS Plus (Bio-Rad Laboratories), and the bands were quantitatively measured using Image Lab Software version 2.0 (Bio-Rad Laboratories).
The inhibition rate of lysozyme dimer and trimer formation was calculated from the net intensity of each band according to the following formula: The results are shown in Table 11.
Maillard reaction inhibition rate (%) = {1 - (A - C) / (B - C)} x 100
In the above formula, A represents the sum of the net intensities of the dimer and trimer when the test sample is added, B represents the sum of the net intensities of the dimer and trimer when the test sample is not added (control), and C represents the sum of the net intensities of the dimer and trimer when left standing at 4°C when the test sample is not added (blank).

The results in Table 11 demonstrate that the hibiscus extract has an inhibitory effect on the Maillard reaction.

Example 12
<Test for inhibitory effect on the formation of advanced glycation end products (AGEs)>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample to test the inhibitory effect on the formation of advanced glycation end products (AGEs) by the following test method.

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

From the results in Table 12, it was found that the hibiscus extract has an extremely strong inhibitory effect on the formation of advanced glycation end products.

(Example 13)
<Test for promoting the decomposition of advanced glycation end products (AGEs)>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and its effect of promoting the decomposition of advanced glycation end products (AGEs) was tested by the following test method.

100 μL of 0.2 M D(-)-ribose prepared in PBS(-) was added to a 96-well type I collagen-coated plate, and the plate was left to stand at 37°C for 2 weeks to form AGEs. As a negative control, a plate to which PBS(-) was added was left to stand in the same manner. After 2 weeks, 100 μL of test samples (sample concentrations: 6.25 μg/mL, 25 μg/mL, 100 μg/mL, or 400 μg/mL) prepared in PBS(-) were added and the plate was left to stand for another 16 days. At this time, as a positive control, a plate to which PBS(-) was added instead of the test sample after D(-)-ribose treatment was left to stand in the same manner. In addition, the negative control was continued to be treated with PBS(-). After 16 days, the amount of AGEs was measured by ELISA using an anti-AGEs antibody (manufactured by Transgenic Co., Ltd.) to evaluate the AGEs decomposition promoting effect.
The AGEs decomposition promotion rate was calculated as follows. The results are shown in Table 13.
AGEs decomposition promotion rate (%) = {(B - C) / (B - A)} x 100
In the above formula, A represents the absorbance at a wavelength of 405 nm of the negative control, B represents the absorbance at a wavelength of 405 nm of the positive control, and C represents the absorbance at a wavelength of 405 nm when the test sample is added.

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

(Example 14)
<Claudin-1 production promotion test>
A freeze-dried extract of hibiscus flowers (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and the promoting effect of claudin-1 production was tested by the following test method.

Normal human skin epidermal keratinocytes (NHEK) were cultured in a ⁸⁰ cm2 flask in normal human epidermal keratinocyte growth medium (KGM) at 37°C under 5% _CO2 , and the cells were harvested by trypsinization. The harvested cells were diluted with KGM to a cell density of 2.0 x ¹⁰⁵ cells/mL, and then seeded in a 96-well plate at 100 μL per well and cultured at 37°C under 5% _CO2 for 1 day.
After the incubation, 100 μL of the test sample solution dissolved in KGM was added to each well and incubated at 37° C. under 5% CO ₂ for 24 hours. After the incubation, the medium was removed, the cells were fixed on the plate, and the amount of claudin-1 expressed on the cell surface was measured by ELISA using polyclonal claudin-1 antibody.
From the measurement results, the claudin-1 production promotion rate (%) was calculated according to the following formula. The results are shown in Table 14.
Claudin-1 production promotion rate (%) = A/B x 100
In the above formula, A represents the absorbance at a wavelength of 405 nm when a test sample is added, and B represents the absorbance at a wavelength of 405 nm when no test sample is added.

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

(Example 15)
<Occludin production promotion test>
A freeze-dried extract of hibiscus flowers (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and the occludin production promoting effect was tested by the following test method.

Normal human skin epidermal keratinocytes (NHEK) were cultured in a ⁸⁰ cm3 flask in normal human epidermal keratinocyte growth medium (KGM) at 37°C under 5% _CO2 , and the cells were harvested by trypsinization. The harvested cells were diluted with KGM to a cell density of 2.0 x ¹⁰⁵ cells/mL, and then seeded in a 96-well plate at 100 μL per well and cultured overnight at 37°C under 5% _CO2 .
After the incubation, 100 μL of the test sample dissolved in KGM (sample concentration: 3.13 μg/mL, 12.5 μg/mL, or 50 μg/mL) was added to each well and incubated at 37° C. under 5% CO ₂ for 24 hours. After the incubation, the medium was removed, the cells were fixed to the plate, and the amount of occludin expressed on the cell surface was measured by ELISA using a polyclonal anti-human occludin antibody.
The method for calculating the promotion rate of occludin production is as follows. The results are shown in Table 15.
Occludin production promotion rate (%) = A/B x 100
In the above formula, A represents the absorbance at a wavelength of 405 nm when a test sample is added, and B represents the absorbance at a wavelength of 405 nm when no test sample is added.

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

(Example 16)
<Test on the effect of promoting the production of epidermal tight junction component proteins in a three-dimensional human skin model>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and the promoting effect of the production of epidermal tight junction component proteins in a three-dimensional human skin model was tested by the test method described below.

The test was carried out using a three-dimensional normal human skin model (EPI-200, manufactured by KURABO).
After purchasing the three-dimensional skin model, it was cultured in a 6-well plate using an assay medium (EPI-NMM Maintenance Medium, manufactured by KURABO) at 37 ° C. and 5% CO ₂ for 1 hour. After the culture, 100 μL of assay medium containing or not containing the test sample dissolved in 1% DMSO (control) was provided on the surface of the skin model, and assay medium (maintenance medium) was provided on the bottom of the skin model, and the model was cultured for 7 days under conditions of 37 ° C. and 5% CO _2. During the culture period, the model was constantly exposed to the test sample. On the fourth day of culture, the maintenance medium and the assay medium containing the test sample were replaced. On the fourth day of culture and after the end of culture, the model was cut out using a 6 mm biopsy punch, the embedding medium was placed in a plastic embedding dish, and the embedding medium was frozen with dry ice and liquid nitrogen. Then, the slices were cut to a thickness of 4 μm using a cryostat HM550 (manufactured by MICROM), the slices were attached to a slide glass, and the slices were dried.

<Claudin-4 immunofluorescence staining>
The slide glass with the section attached was placed in a staining tray, fixed with 4% paraformaldehyde, and the embedding agent around the section was thoroughly washed off with PBS(-). After blocking with 1% BSA, the liquid 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 liquid was discarded, washed with PBS(-), treated with the 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 washed with PBS(-), nuclear staining was performed with DAPI solution. The target claudin-4 was analyzed using a fluorescent microscope. The results are shown in Figures 1A to 1F.
The images in Figures 1A to 1F show the portion from the lower part of the stratum corneum to the basal layer, where Figure 1A shows the control on day 4 of culture, Figure 1B shows the hibiscus extract at a sample concentration of 100 μg/mL on day 4 of culture, Figure 1C shows the hibiscus extract at a sample concentration of 500 μg/mL on day 4 of culture, Figure 1D shows the control on day 7 of culture, Figure 1E shows the hibiscus extract at a sample concentration of 100 μg/mL on day 7 of culture, and Figure 1F shows the hibiscus extract at a sample concentration of 500 μg/mL on day 7 of culture, in which claudin-4 is stained green (near the cell membrane of the granular layer).
Furthermore, on both the fourth and seventh day of culture, the hibiscus extract showed stronger linear fluorescence near the granular layer cell membrane compared to the control, confirming its effect of promoting claudin-4 production in the three-dimensional skin model.

<ZO-1, ZO-2 Immunofluorescence Staining>
The slide glass with the section attached was placed in a staining tray, fixed with 4% paraformaldehyde, and the embedding agent around the section was thoroughly washed off with PBS(-). After blocking with 1% BSA, the liquid was discarded, and the primary antibodies, mouse-derived anti-human ZO-1 monoclonal antibody (manufactured by Invitrogen) and rabbit-derived anti-human ZO-2 polyclonal antibody (manufactured by Invitrogen), were poured onto each slide glass and incubated at room temperature for 1 hour. The antibody liquid was discarded, washed with PBS(-), and treated with the secondary antibodies, Alexa-Fluor 488-labeled goat-derived anti-mouse IgG antibody (manufactured by Invitrogen) and Alexa-Fluor 594-labeled goat-derived anti-rabbit IgG antibody (manufactured by Invitrogen), and incubated in the dark at 4°C for 1 hour. Furthermore, it was washed with PBS(-) and nuclear stained with DAPI solution. The target ZO-1 and ZO-2 were analyzed by a fluorescence microscope, and the results are shown in Figures 2A to 2F and 3A to 3F.
The photographs in Figures 2A-2F show the lower stratum corneum to basal layer.
FIG. 2A shows the results for the control on day 4 of culture, FIG. 2B shows the results for the hibiscus extract at a sample concentration of 100 μg/mL on day 4 of culture, FIG. 2C shows the results for the hibiscus extract at a sample concentration of 500 μg/mL on day 4 of culture, FIG. 2D shows the results for the control on day 7 of culture, FIG. 2E shows the results for the hibiscus extract at a sample concentration of 100 μg/mL on day 7 of culture, and FIG. 2F shows the results for the hibiscus extract at a sample concentration of 500 μg/mL on day 7 of culture. In FIGS. 2A to 2F, ZO-1 is stained green (near the cell membranes from the granular layer to the basal layer).
The photographs in Figures 3A-3F show the lower stratum corneum to basal layer.
FIG. 3A shows the results for the control on day 4 of culture, FIG. 3B shows the results for the hibiscus extract at a sample concentration of 100 μg/mL on day 4 of culture, FIG. 3C shows the results for the hibiscus extract at a sample concentration of 500 μg/mL on day 4 of culture, FIG. 3D shows the results for the control on day 7 of culture, FIG. 3E shows the results for the hibiscus extract at a sample concentration of 100 μg/mL on day 7 of culture, and FIG. 3F shows the results for the hibiscus extract at a sample concentration of 500 μg/mL on day 7 of culture. In FIGS. 3A to 3F, ZO-2 is stained red (near the cell membranes from the granular layer to the basal layer).
On the fourth day of culture, the hibiscus extract showed stronger linear fluorescence near the cell membrane at a concentration of 100 μg/mL compared to the control, confirming its effect of promoting the production of ZO-1 and ZO-2 in the three-dimensional skin model.

(Example 17)
<Test for inhibiting the decline of skin barrier function (electrical resistance value TER measurement and permeability evaluation using FITC-Dex)>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and the inhibitory effect on the decline of skin barrier function 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 harvested by trypsinization. The harvested cells were diluted with KGM to a concentration of 2.2 x 10 ⁵ cells/mL, and then seeded in the upper layer of a 12-well transwell (Corning, diameter 12 mm, 0.4 μm pore) at 0.5 mL per well, and then KGM was added to the lower layer at 0.5 mL each, and the cells were cultured for 3 days. After the culture was completed, 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 the culture was completed, the high CaCl ₂ medium was removed, and 0.5 mL of KGM alone or the test sample dissolved 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, wells in which the barrier function was maintained in high _CaCl2 medium were also set up as controls. Three days after the start of culture, electrical resistance (TER) was measured using a Millicell-ERS resistance measurement system (Millipore), and the barrier degradation inhibition rate (%) of the test sample was calculated in comparison with the control.
After the TER measurement, the upper and lower layers were washed with PBS(-), and 0.5 mL of 4 kDa FITC-Dextran (FITC-Dex, Sigma) dissolved in P buffer (10 mM HEPES, pH 7.4, 1 mM sodium pyruvate, 10 mM glucose, ₃ mM CaCl2, 145 mM NaCl) to a concentration of 1 mg/mL was added to the upper layer, and 0.5 mL of P buffer was added to the lower layer, followed by incubation at 37°C for 90 minutes. After the incubation, 100 μL of each sample was taken from the lower layer, and the fluorescence intensity at an excitation wavelength of 485 nm and a fluorescence wavelength of 545 nm was measured. The amount of FITC-Dex that had permeated from the upper layer to the lower layer was determined based on the calibration curve, and the permeation inhibition rate (%) of the test sample was calculated in comparison with the control to evaluate the permeation barrier function.
The method for calculating the rate of inhibition of barrier degradation is as follows. The results are shown in Table 16.
Barrier degradation inhibition rate (%)={1-(C-A)/(C-B)}×100
In the above formula, A represents the electrical resistance value (TER) in cells to which a test sample was added, B represents the electrical resistance value (TER) in cells to which a test sample was not added, and C represents the electrical resistance value (TER) in cells treated with a high _CaCl2 medium.

The method for calculating the permeation inhibition rate is as follows. The results are shown in Table 17.
Permeation inhibition rate (%)={1-(C-A)/(C-B)}×100
In the above formula, A represents the amount of FITC-Dex permeated through the cells to which a test sample was added, B represents the amount of FITC-Dex permeated through the cells to which a test sample was not added, and C represents the amount of FITC-Dex permeated through the cells treated with a high _-CaCl2 medium.

The results in Tables 16 and 17 show that hibiscus extract has the effect of inhibiting the decrease in electrical resistance and inhibiting the permeation of FITC-Dex.

(Example 18)
<Testosterone 5α-reductase activity inhibitory effect test>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample and its testosterone 5α-reductase activity inhibitory effect was tested by the following test method.

First, 20 μL of 4.2 mg/mL testosterone prepared with propylene glycol and 825 μL of 5 mmol/mL Tris-HCl buffer (pH 7.13) containing 1 mg/mL NADPH (reduced nicotinamide adenine dinucleotide phosphate) were mixed in a V-bottom test tube with a lid. 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 to this and mixed again, and the reaction was allowed to proceed at 37° C. for 30 minutes, after which 1 mL of methylene chloride was added to stop the reaction. This was centrifuged (1,600×g, 10 minutes), and the methylene chloride layer was analyzed by gas chromatography. The gas chromatography conditions were as follows. A blank test was also conducted in the same manner.
The S-9 sample was obtained by intraperitoneally administering an enzyme inducer (phenobarbital, 5,6-benzoflavone) to male SD rats, grinding the liver, and centrifuging the resulting mixture at 9,000×g to obtain a supernatant.

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

In advance, methylene chloride solutions of standard samples of 3α-androstanediol (Sigma), dihydrotestosterone (DHT, Tokyo Chemical Industry Co., Ltd.), and testosterone (Tokyo Chemical Industry Co., Ltd.) were analyzed by gas chromatography, and the amounts of these three compounds per peak area were calculated from their exact weights and peak areas.
After the reaction with S-9, 3α-androstanediol, dihydrotestosterone (DHT), and testosterone 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 formula (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 × concentration of standard) / peak area of standard (2)
Conversion rate (%) = (A + B) / (A + B + C) ... (3)
In the above formula (3), A represents the concentration of 3α-androstanediol, B represents the concentration of dihydrotestosterone (DHT), and C represents the concentration of testosterone.

Testosterone 5α-reductase activity inhibition rate (%) = (1-E/D) x 100 (4)
In the above formula (4), D represents the conversion rate in the blank test, and E represents the conversion rate when the test sample is added.

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

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

Normal human hair dermal papilla cells (manufactured by Toyobo Co., Ltd.) were cultured using a dermal papilla cell growth medium (manufactured by Toyobo Co., Ltd.) containing 1% FCS and a growth additive, and the cells were then harvested by trypsin treatment. The harvested cells were diluted to a concentration of 1.0 x ¹⁰⁴ cells/mL using Dulbecco's MEM (Dulbecco's modified Eagle's medium (1), manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% by volume FBS, and then seeded at 200 μL per well on a collagen-coated 96-well plate and cultured for 3 days. After culturing, the medium was removed, and 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 cultured for another 4 days. The dermal papilla cell proliferation activity was measured using an MTT assay. After the incubation, the medium was removed, and 100 μL of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Dojindo Laboratories, Inc.) dissolved in serum-free DMEM at a final concentration of 0.4 mg/mL was added to each well. After incubation for 2 hours, blue formazan produced within the cells was extracted with 100 μL of 2-propanol. After extraction, absorbance at a wavelength of 570 nm was measured. At the same time, absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was taken as the amount of blue formazan produced.

The method for calculating the proliferation rate of hair follicle papilla cells is as follows. The results are shown in Table 19.
Hair papilla cell proliferation rate (%) = A/B x 100
In the above formula, A represents the amount of blue formazan produced when a test sample is added, and B represents the amount of blue formazan produced when no test sample is added.

From the results in Table 19, it was found that the hibiscus extract has a hair papilla cell proliferation effect.

The hyaluronidase activity inhibitor, hydrogen peroxide elimination agent, skin whitening agent, anti-aging agent, and hair growth agent of the present invention are safe, can be taken daily, and are inexpensive, while having excellent hyaluronidase activity inhibitory effect, hydrogen peroxide elimination effect, melanin production inhibitory effect in B16 melanoma cells, endothelin-1 mRNA expression inhibitory effect, stem cell growth factor mRNA expression inhibitory effect, basic fibroblast growth factor mRNA expression inhibitory effect, proopiomelanocortin mRNA expression inhibitory effect, epidermal hyaluronic acid production promoting effect, and glutathione Since it has at least one of the following effects: promoting the production of glycated end products, promoting serine palmitoyltransferase mRNA expression, inhibiting the Maillard reaction, inhibiting the formation of advanced glycation end products, promoting the decomposition of advanced glycation end products, promoting the production of claudin-1, promoting the production of occludin, promoting the production of epidermal tight junction component proteins in a three-dimensional human skin model, suppressing the decline of skin barrier function, inhibiting testosterone 5α-reductase activity, and promoting hair papilla cell proliferation, it can be suitably used as an ingredient in cosmetics, food and beverages, and as a research reagent.

Claims (2)

A serine palmitoyltransferase mRNA expression promoter (excluding those used for anti-aging), characterized by containing an extract of the flower parts of Hibiscus sabdariffa as an active ingredient. The serine palmitoyltransferase mRNA expression promoter according to claim 1, wherein the extract of the flower parts of Hibiscus sabdariffa is a water extract, a hydrophilic organic solvent extract, or a mixed solvent extract of water and a hydrophilic organic solvent of the flower parts of Hibiscus sabdariffa.