JP7736340B2 - Advanced glycation end products degradation 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 degraded by ultraviolet light, enzymes, etc. in aqueous systems, and its moisture-retaining effect decreases as its molecular weight decreases. Hyaluronic acid also exists in intercellular tissues and is involved in vascular permeability. Hyaluronidase is thought to be involved in degranulation from mast cells upon activation. Therefore, inhibiting the activity of hyaluronidase, the hydrolase of hyaluronic acid, stabilizes hyaluronic acid, prevents the release of various chemical mediators from mast cells, and is expected to have anti-inflammatory effects. Herbal medicines that have been reported to have hyaluronidase activity inhibitory effects include extracts of plants from the genus Osbeckia (see Patent Document 1), wisteria tea extract (see Patent Document 2), rosemary extract, thyme extract, and melissa extract (see Patent Document 3).

To date, development of skin whitening agents has focused on tyrosinase, the rate-limiting enzyme in melanin production. However, recently, 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) have been reported to be produced more efficiently by epidermal keratinocytes after UVB irradiation, activating melanocytes. Active efforts are underway to develop substances that inhibit melanin production and achieve skin whitening effects by blocking the signaling pathways involved in these cytokines. Herbal extracts that inhibit the action of endothelin-1 (ET-1) on melanocytes include chamomile extract and althea extract (see Non-Patent Document 1).

It was previously thought that the skin's barrier function was solely the responsibility of the stratum corneum. However, genetic deletion of the constituent proteins of tight junctions (hereinafter sometimes abbreviated as TJs) present in the granular layer of the epidermis disrupts the skin's barrier function. In recent years, TJs are also thought to play an important role in the skin's barrier function (see Non-Patent Document 2). TJs are junctions 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 TJs include claudins, occludin, ZO-1, and ZO-2. These proteins are thought to form the skeleton of TJ strands and control the barrier function of TJs (see Non-Patent Document 3). Based on the above, if the expression of claudins, occludin, ZO-1, or ZO-2 is reduced for some reason, structural destruction of TJs occurs, and they no longer function as a permeation barrier to 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, TJ formation in epidermal keratinocytes can be promoted, thereby enhancing the skin's barrier function and moisture retention function, and preventing or improving the above-mentioned skin symptoms. Based on this idea, natural products such as Coptis japonica extract (see Patent Document 4) and Picea abies extract (see Patent Document 5) have been disclosed as substances that improve skin barrier function by promoting TJ formation.

The Maillard reaction is a 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. The substances produced as the end 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 non-enzymatically with glucose to form a Schiff base, followed by an Amadori rearrangement; a middle reaction in which an activated intermediate with a dicarbonyl group, such as 3-deoxyglucosone (3-DG), is produced; and a late reaction in which the activated intermediate further reacts non-enzymatically with an amino group, undergoing repeated 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 pathologies in which AGEs are involved in the onset and progression is the symptom of aging. The progression of the Maillard reaction in biological tissues leads to aging (loss of elasticity) in skin tissue due to cross-linking of skin elastic fibers, and it is also said that the deposition of AGEs in blood vessel wall tissue and neurofibrils can lead to arteriosclerosis and Alzheimer's disease.

As a natural product having an AGE production inhibitory effect, for example, an extract of 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 AGE production inhibitory effect, 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 receptors and exerting its androgen effects.
Androgens are important hormones, but excessive activity can induce various undesirable symptoms, such as male pattern baldness, hirsutism, seborrheic dermatitis, acne, benign prostatic hyperplasia, prostatic tumors, and precocious puberty in boys. To alleviate these symptoms, methods for suppressing the action of excess androgens have been disclosed. Specifically, methods for suppressing the production of active 5α-DHT by inhibiting the action of testosterone 5α-reductase, which reduces testosterone to active 5α-DHT, and methods for preventing the expression of androgenic activity by inhibiting the binding of 5α-DHT generated from testosterone to its receptor have been disclosed. Examples of plant extracts that inhibit the binding of 5α-DHT to its receptor include extracts of at least one of majito and catcha (see Patent Document 7).

Hair grows and falls out repeatedly according to a cyclical hair cycle consisting of the anagen, catagen, and telogen phases. The stage of this hair cycle, from telogen to anagen, during which new hair follicles are formed, is considered to be the most important for hair growth. Dermal papilla cells are believed to play a key role in the proliferation and differentiation of hair follicle epithelial cells during this stage. Dermal papilla cells are located inside the hair follicle epithelial cells, which are composed of outer root sheath cells and matrix cells near the hair root, in the basement membrane-enclosed portion of the hair root. They act on hair follicle epithelial cells to promote their proliferation, thereby playing an important role in the proliferation and differentiation of hair follicle epithelial cells and hair formation. Dermal papilla cells play a key role in the proliferation and differentiation of hair follicle epithelial cells and hair formation. A method has been proposed for testing the hair growth effects of a substance by contacting cultured dermal papilla cells with the substance and determining the presence or absence or strength of the cell's proliferation activity. Examples of herbal medicines that have the effect of promoting hair papilla cell proliferation include Astragalus Root extract, Coptis Rhizome extract, and Anemone ternata extract (see Patent Documents 8 and 9).

However, to date, no excellent substance has been found that possesses at least one of the above-mentioned effects, is highly safe, and therefore can be widely used as an ingredient in cosmetics, foods and beverages, research reagents, and the like, and there is a strong demand for its prompt provision.

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

"Fragrance Journal", Vol. 28, No. 9, pp. 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 of the prior art and achieve the following objectives. Specifically, the present invention aims to provide a hyaluronidase inhibitor that has excellent hyaluronidase activity inhibitory activity and is highly safe, a hydrogen peroxide scavenger that has excellent hydrogen peroxide scavenging activity and is highly safe, a whitening agent that has excellent whitening activity and is highly safe, an anti-aging agent that has excellent anti-aging activity and is highly safe, and a hair growth agent that has excellent hair growth activity and is highly safe.

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

The present invention is based on the above findings of the present inventors, and the means for solving the above problems are as follows:
<1> A hyaluronidase activity inhibitor characterized by containing a hibiscus extract.
<2> A hydrogen peroxide scavenger containing a hibiscus extract.
<3> A skin whitening agent characterized by containing an extract of hibiscus.
<4> The skin whitening agent according to <3>, which has at least 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 a hibiscus extract.
<6> The anti-aging agent according to <5>, which has at least one of the following effects: 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 degradation 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-constituting proteins in a three-dimensional human skin model, and an effect of suppressing a decline in skin barrier function.
<7> A hair growth agent characterized by containing an extract of hibiscus.
<8> The hair growth agent according to <7>, which has at least one of an inhibitory effect on testosterone 5α-reductase activity and an effect on proliferation of hair papilla cells.

The present invention solves the above-mentioned problems of the prior art and achieves the above-mentioned objectives, thereby providing a hyaluronidase inhibitor that has excellent hyaluronidase activity inhibitory activity and is highly safe, a hydrogen peroxide scavenger that has excellent hydrogen peroxide scavenging activity and is highly safe, a whitening agent that has excellent whitening activity and is highly safe, an anti-aging agent that has excellent anti-aging activity and is highly safe, and a hair growth agent that has excellent hair growth activity and is highly safe.

FIG. 1A is a photograph showing an image of claudin-4 immunofluorescence staining in Example 16, showing the control results 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 results 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 control results on day 4 of culture. FIG. 2B is a photograph showing an image of ZO-1 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. 2C is a photograph showing an image of ZO-1 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. 2D is a photograph showing an image of ZO-1 immunofluorescence staining in Example 16, showing the control results on day 7 of culture. FIG. 2E is a photograph showing an image of ZO-1 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. 2F is a photograph showing an image of ZO-1 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. 3A is a photograph showing an image of ZO-2 immunofluorescence staining in Example 16, showing the control results 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 day 4 of culture. FIG. 3C 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 500 μg/mL on day 4 of culture. FIG. 3D is a photograph showing an image of ZO-2 immunofluorescence staining in Example 16, showing the control results 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 an image of ZO-2 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.

(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 scavenger has a hydrogen peroxide scavenging action.
The whitening agent has a whitening effect based on at least 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 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 degradation of advanced glycation end products, promoting the production of claudin-1, promoting the production of occludin, promoting the production of epidermal tight junction-constituting proteins in a three-dimensional human skin model, and suppressing 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.

The details of the substances contained in the hibiscus extract that exhibit at least one of the following effects: hyaluronidase activity inhibitory activity, hydrogen peroxide scavenging activity, whitening activity, anti-aging activity, and hair growth activity are unknown. However, it was not previously known that the hibiscus extract possesses such excellent effects and is useful as a hyaluronidase activity inhibitor, hydrogen peroxide scavenger, whitening agent, anti-aging agent, and hair growth agent, and this is a new discovery made by the inventors.

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

The parts of the hibiscus used as the extraction raw material are not particularly limited and can be selected appropriately depending on the purpose. Examples include flowers, calyx, buds, fruit, pericarp, seeds, seed coat, stems, leaves, branches, foliage, trunks, bark, roots, rhizomes, root bark, and mixtures thereof. Of these, flower parts such as flowers, calyx, and buds are preferred.

The hibiscus used as the extraction raw material can be subjected to solvent extraction either as is or after being crushed using a crusher or the like after drying. In particular, the extraction raw material is preferably dried and crushed immediately after harvesting. The drying can be carried out, for example, in the sun or using a commonly used dryer. The hibiscus may also be used as the extraction raw material after being pretreated, such as by degreasing, using a non-polar solvent such as hexane or benzene. Pretreatment, such as degreasing, allows for efficient extraction of the hibiscus using a polar solvent.

The hibiscus extract can be easily obtained using methods commonly used for plant extraction. Alternatively, commercially available products may be used as the hibiscus extract. The hibiscus extract includes the hibiscus extract, diluted or concentrated solutions of the extract, dried products of the extract, and crude or purified products thereof.

The solvent used for the extraction is not particularly limited and can be selected appropriately 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 a temperature below the boiling point of the solvent. The components contained in the hibiscus that exhibit at least one of the following effects: hyaluronidase activity inhibition, hydrogen peroxide scavenging, whitening, anti-aging, and hair growth. These can be easily extracted by an extraction process using a polar solvent as the extraction solvent.

The water that can be used as the extraction solvent is not particularly limited and can be selected appropriately depending on the purpose. Examples include pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, and water that has undergone 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, saline, phosphate buffer, phosphate-buffered saline, etc.

The hydrophilic organic solvent that can be used as the extraction solvent is not particularly limited and can be selected appropriately depending on the purpose. Examples include 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; and polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin. Mixed solvents of such hydrophilic organic solvents with water can also be used. When using mixed solvents of water and the hydrophilic organic solvent, it is preferable to use a mixture of 1 to 90 parts by weight of lower alcohols per 10 parts by weight of water, and 1 to 40 parts by weight of lower aliphatic ketones per 10 parts by weight of water. It is also preferable to use a mixture of 1 to 90 parts by weight of polyhydric alcohols per 10 parts by weight of water.

The extraction method for the hibiscus extract is not particularly limited, and can be performed according to a conventional method, as long as it is possible to dissolve the fat-soluble components contained in the hibiscus extract raw material in the solvent. Furthermore, there is no need to employ a special extraction method during the extraction process, and any device can be used at room temperature or under reflux heating.
Specifically, the hibiscus extract may be obtained by, for example, placing the extract 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 hot extract is then filtered to elute the fat-soluble components, and the extract is then concentrated under reduced pressure using an evaporator, followed by a similar filtration process.
In this case, the extraction conditions can be adjusted appropriately 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, roughly purified product, purified product, etc. of the hibiscus extract.
The obtained hibiscus extract can be used as it is as any of the hyaluronidase activity inhibitor, hydrogen peroxide scavenger, whitening agent, anti-aging agent, and hair growth agent, but the concentrate and dried product are preferred in terms of ease of use. When 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 selected appropriately depending on the purpose. Examples include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, odorants, etc.

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

The hibiscus extract obtained as described above 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 The compound has at least one of the following effects: an inhibitory effect on the formation of advanced glycation end products, an accelerator effect on the degradation of advanced glycation end products, an accelerator effect on the production of claudin-1, an accelerator effect on the production of occludin, an accelerator 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 accelerator effect on the proliferation of hair papilla cells; and based on these effects, the compound can be suitably used as an active ingredient in at least one of the hyaluronidase activity inhibitors, hydrogen peroxide scavengers, whitening agents, anti-aging agents, and hair growth agents 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 component protein production promoter in a three-dimensional human skin model, an inhibitor of skin barrier function decline, a testosterone 5α-reductase activity inhibitor, and a hair papilla cell proliferation agent, respectively.

The hyaluronidase activity inhibitor of the present invention exhibits its inhibitory effect on hyaluronidase activity.
The hydrogen peroxide scavenger of the present invention exhibits its hydrogen peroxide scavenging activity.
The whitening effect of the whitening agent of the present invention is exerted based on at least 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 effect of the anti-aging agent of the present invention is exerted based on at least one of the following effects: promoting epidermal hyaluronic acid production, promoting glutathione production, promoting serine palmitoyltransferase mRNA expression, inhibiting the Maillard reaction, suppressing the formation of advanced glycation end products, promoting the degradation of advanced glycation end products, promoting claudin-1 production, promoting occludin production, promoting the production of epidermal tight junction-constituting proteins in a three-dimensional human skin model, and suppressing 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 testosterone 5α-reductase activity inhibitory effect and the hair papilla cell proliferation effect.

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

Furthermore, the other components other than the hibiscus extract that may be contained in the hyaluronidase activity inhibitor, the hydrogen peroxide scavenger, the whitening agent, the anti-aging agent, and the hair growth agent are not particularly limited and can be selected appropriately depending on the purpose, as long as they do not impair the effects of the present invention, and examples thereof include physiological saline for diluting the hibiscus extract to a desired concentration. Furthermore, the contents of the hyaluronidase activity inhibitor, the hydrogen peroxide scavenger, the whitening agent, the anti-aging agent, and the other components in the hair growth agent are not particularly limited and can be selected appropriately depending on the purpose.
Furthermore, the hyaluronidase activity inhibitor, the hydrogen peroxide scavenger, the whitening agent, the anti-aging agent, and the hair growth agent can be formulated as needed 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 used in humans, but can also be used in animals other than humans as long as their respective effects are achieved.

Examples of the present invention are described below, but the present invention is not limited to these examples in any way.

<Hibiscus extract>
A freeze-dried extract of hibiscus flower (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 its inhibitory effect on hyaluronidase activity was tested by the following test method.

To 0.2 mL of 0.1 mol/L acetate buffer (pH 3.5) in which the test sample was dissolved, 0.1 mL of hyaluronidase solution (Type IV-S (derived from bovine testis), 400 NF units/mL; manufactured by Sigma-Aldrich Japan) was added, and the mixture was allowed to react 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 allowed to react at 37 ° C for 20 minutes. To this was added 0.5 mL of 0.8 mg/mL sodium hyaluronate solution (chicken comb-derived sodium hyaluronate, manufactured by Wako Pure Chemical Industries, Ltd.), and the mixture was allowed to react 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. After that, 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 and correction was performed.
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 scavenging activity 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 test sample solution was placed in a 96-well plate, and 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, followed by a 20-minute reaction at 37°C. After the reaction, 180 μL of 100 μM DA-67 was quickly added, followed by 10 μL of ethanol, and a color reaction was carried out at 37°C for 5 minutes. After completion of the color reaction, the absorbance at a wavelength of 650 nm was measured. A blank test was also performed in the same manner for 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 scavenging 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 confirmed that the hibiscus extract has a hydrogen peroxide scavenging effect.

Example 3
<Test for melanin production inhibitory effect on 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 in Dulbecco's MEM (Dulbecco's Modified Eagle's Medium (1), Nissui Pharmaceutical Co., Ltd.) containing 10% FBS (Standard Fetal Bovine Serum, HyClone), and then harvested by trypsinization. The harvested cells were diluted to a concentration of 24.0 x ¹⁰⁴ cells/mL with Dulbecco's MEM containing 10% FBS and 1 mmol/L theophylline (Theophylline, Wako Pure Chemical Industries, Ltd.), then seeded at 300 μL per well in a 48-well plate and cultured for 6 hours. After incubation, 300 μL of the test sample dissolved in Dulbecco's MEM containing 10% FBS and 1 mmol/L theophylline was added to each well and cultured for 4 days. After incubation, the medium was removed from each well, 200 μL of 2 mol/L NaOH solution was added, and the cells were disrupted using an ultrasonic disrupter. 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).
To measure cell viability, the plates were incubated in the same manner, washed with 400 μL of PBS(-) phosphate buffer, and 200 μL of 13.8 mmol/L Neutral Red dissolved in 10% (vol/vol) FBS-containing Dulbecco's MEM (FBS) at a final concentration of 0.05 mg/mL was added to each well. After 2.5 hours of incubation, 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 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 confirmed that the hibiscus extract has an inhibitory effect on melanin production.

Example 4
<Test for inhibitory effect on increased endothelin-1 mRNA expression>
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 endothelin-1 mRNA expression was tested by the following test method.

Normal human neonatal foreskin epidermal keratinocytes (NHEK) were pre-cultured 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 into 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). 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 (an internal standard) were measured by real-time two-step RT-PCR using a Smart Cycler® real-time PCR device ⁽ Cepheid) and a TaKaRa ^SYBR® PrimeScript ^™ RT-PCR Kit (Perfect Real Time) (code No. RR063A).
The expression level of endothelin-1 mRNA was determined by calculating a corrected value using the GAPDH value based on total RNA samples prepared from cells cultured under the conditions "not irradiated with UV light and no test sample added,""irradiated with UV light and no test sample added," and "irradiated with UV light and test sample added." Furthermore, the corrected value for "irradiated with UV light and no test sample added" was set at 100, and the corrected values for "irradiated with UV light and no test sample added" and "irradiated with UV light and test sample added" were calculated.
From these results, the rate of inhibition of increased endothelin-1 mRNA expression was calculated using the following formula. The results are shown in Table 4.
Inhibition rate of increased endothelin-1 mRNA expression (%)
={(A-B)-(A-C)}/(A-B)×100
In the above formula, A is the correction value when "no ultraviolet irradiation and no test sample added", B is the correction value when "ultraviolet irradiation and no test sample added", and C is the correction value when "ultraviolet irradiation and test sample added".

The results in Table 4 demonstrate that the hibiscus extract has an inhibitory effect on the increase in endothelin-1 mRNA expression.

Example 5
<Test for inhibitory effect on increased stem cell factor (SCF) mRNA expression>
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 stem cell factor (SCF) mRNA expression was tested by the following test method.

Normal human neonatal foreskin epidermal keratinocytes (NHEK) were pre-cultured in an 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 into 35 mm Petri dishes (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 the cultured for 24 hours at 37°C and 5% CO _2. After culturing, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE; Cat. No. 311-07361). The amount of each RNA was measured using a spectrophotometer, and total RNA was prepared to 200 ng/μL.
Using this total RNA as a template, the expression levels of stem cell factor (SCF) and GAPDH mRNA, an internal standard, were measured by real-time two-step RT-PCR using a Smart Cycler® real-time PCR system ⁽ Cepheid) and the TaKaRa ^SYBR® PrimeScript ^™ RT-PCR Kit (Perfect Real Time) (code No. RR063A).
The expression level of SCF mRNA was determined by calculating a corrected value using the GAPDH value based on total RNA samples prepared from cells cultured under the conditions "not irradiated with UV light and no test sample added,""irradiated with UV light and no test sample added," and "irradiated with UV light and test sample added." Furthermore, the corrected value for "irradiated with UV light and no test sample added" was set at 100, and the corrected values for "irradiated with UV light and no test sample added" and "irradiated with UV light and test sample added" were calculated.
From these results, the rate of inhibition of increased expression of stem cell factor (SCF) mRNA was calculated using the following formula. The results are shown in Table 5.
Stem cell growth factor (SCF) mRNA expression increase suppression rate (%)
={(A-B)-(A-C)}/(A-B)×100
In the above formula, A represents the correction value when "no ultraviolet irradiation and no test sample added", B represents the correction value when "ultraviolet irradiation and no test sample added", and C represents the correction value when "ultraviolet irradiation and test sample added".

The results in Table 5 demonstrate 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 basic fibroblast growth factor (bFGF) mRNA expression>
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 pre-cultured in a 75 cm ² flask in 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 into 35 mm Petri dishes (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 the cultured for 24 hours at 37°C and 5% CO _2. After culturing, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE; Cat. No. 311-07361). The amount of each RNA was measured using a spectrophotometer, and total RNA was prepared to 200 ng/μL.
Using this total RNA as a template, the expression levels of bFGF (basic fibroblast growth factor) and GAPDH mRNA, an internal standard, were measured. Detection was performed by real-time two-step RT-PCR using a real-time PCR device, Smart Cycler® ⁽ manufactured by Cepheid), and the TaKaRa ^SYBR® PrimeScript ^™ RT-PCR Kit (Perfect Real Time) (code No. RR063A).
The expression level of bFGF mRNA was determined based on the total RNA samples prepared from cells cultured under the conditions "not irradiated with UV light and no test sample added,""irradiated with UV light and no test sample added," and "irradiated with UV light and test sample added," and a corrected value was calculated using the GAPDH value.Furthermore, the corrected value for "not irradiated with UV light and no test sample added" was set to 100, and the corrected values for "irradiated with UV light and no test sample added" and "irradiated with UV light and test sample added" were calculated.
From these results, the rate of inhibition of increase in bFGF mRNA expression was calculated using the following formula: The results are shown in Table 6.
bFGF mRNA expression increase suppression rate (%)
={(A-B)-(A-C)}/(A-B)×100
In the above formula, A represents the corrected value when no UV irradiation is performed and no test sample is added, B represents the corrected value when UV irradiation is performed and no test sample is added, and C represents the corrected value when UV irradiation is performed and the test sample is added.

The results in Table 6 demonstrate that the hibiscus extract has a strong inhibitory effect on the increase in bFGF mRNA expression.

Example 7
<Test for inhibitory effect on increased proopiomelanocortin (POMC) mRNA expression>
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 pre-cultured in a 75 cm ² flask in 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 into 35 mm Petri dishes (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 the cultured for 24 hours at 37°C and 5% CO _2. After culturing, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE; Cat. No. 311-07361). The amount of each RNA was measured using a spectrophotometer, and total RNA was prepared to 200 ng/μL.
Using this total RNA as a template, the expression levels of POMC (proopiomelanocortin) and GAPDH mRNA, an internal standard, were measured by real-time two-step RT-PCR using a Smart Cycler® real-time PCR system ^(Cepheid) and the TaKaRa ^SYBR® PrimeScript ^™ RT-PCR Kit (Perfect Real Time) (code No. RR063A).
The expression level of POMC mRNA was determined by calculating a corrected value using the GAPDH value based on total RNA samples prepared from cells cultured under the conditions "not irradiated with UV light and no test sample added,""irradiated with UV light and no test sample added," and "irradiated with UV light and test sample added." Furthermore, the corrected value for "irradiated with UV light and no test sample added" was set to 100, and the corrected values for "irradiated with UV light and no test sample added" and "irradiated with UV light and test sample added" were calculated.
From these results, the rate of inhibition of increased POMC mRNA expression was calculated using the following formula. The results are shown in Table 7.
POMC mRNA expression increase suppression rate (%)
={(A-B)-(A-C)}/(A-B)×100
In the above formula, A represents the correction value when "no ultraviolet irradiation and no test sample added", B represents the correction value when "ultraviolet irradiation and no test sample added", and C represents the correction value when "ultraviolet irradiation and test sample added".

The results in Table 7 demonstrate that the hibiscus extract has an inhibitory effect on the increase in POMC mRNA expression.

(Example 8)
<Test for promoting epidermal hyaluronic acid production>
A freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and its effect of promoting epidermal hyaluronic acid production was tested using the following test method.

Human normal neonatal skin epidermal keratinocytes (NHEK) were cultured in human normal neonatal epidermal keratinocyte growth medium (KGM) and then harvested by trypsinization. The harvested cells were diluted with KGM to a concentration of 1 x ¹⁰⁵ cells/mL, then seeded in a 96-well plate at 100 μL per well and cultured for 24 hours. After incubation, 100 μL of the test sample dissolved in KGM was added to each well and cultured for 7 days. After incubation, 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.

The results in Table 8 demonstrate that hibiscus extract has the 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 activity 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% FBS by mass, and then the cells were harvested by trypsinization. The harvested cells were diluted with α-MEM medium containing 10% FBS by mass to a concentration of 2.0 x 10 ⁵ cells/mL, then seeded in a 48-well plate at 200 μL per well and cultured overnight. After culture, 200 μL of the test sample dissolved in α-MEM medium containing 1% FBS by mass was added to each well and cultured for 24 hours. After culture, the medium was removed from each well, washed with 400 μL of PBS(-), and the cells were lysed using 150 μL of M- ^PER® (PIERCE). Total glutathione was quantified using 100 μL of the diluted solution.
Specifically, 100 μL of cell extract dissolved in 96-well plates, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH, and 25 μL of glutathione reductase (final concentration 17.5 units/mL) were added and heated at 37°C for 10 minutes. After that, 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 to determine ΔOD/min. 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 using the following formula: The results for 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.

The results in Table 9 demonstrate that the hibiscus extract has the 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 promoting effect on serine palmitoyltransferase (SPT) mRNA expression was tested by the following test method.

Normal human neonatal foreskin epidermal keratinocytes (NHEK) were pre-cultured 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 into 35 mm Petri dishes (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 to the required concentration was added to each Petri dish, followed by 24 hours of culture 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). The amount of each RNA was measured using a spectrophotometer, and total RNA was prepared to 200 ng/μL.
Using this total RNA as a template, the expression levels of SPT and GAPDH mRNA, an internal standard, were measured. Detection was performed by real-time 2-step RT-PCR using a real-time PCR device, Smart Cycler (registered trademark) (manufactured by Cepheid), with a TaKaRa SYBR (registered trademark) PrimeScript ^™ RT-PCR Kit (Perfect Real Time) (code No. RR063A). The expression level of SPT was determined based on total RNA samples prepared from cells cultured with and without the test sample, and a corrected value was calculated using the GAPDH value. Furthermore, the corrected value for the test sample was calculated by setting the corrected value for the test sample without the test sample to 100.
The SPT mRNA expression promotion rate was calculated 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.

The results in Table 10 demonstrate that the hibiscus extract has a high promoting effect on the production of serine palmitoyltransferase (SPT).

Example 11
<Maillard reaction inhibitory effect test>
A freeze-dried product of hibiscus flower extract (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 lyophilized 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 mixture was mixed with 40 μL of SDS-PAGE sample buffer, followed by heating in a boiling bath for 3 minutes to prepare an analytical sample. 12 μL of the analytical sample was applied to a polyacrylamide gel prepared with an acrylamide concentration of 15% for the separating gel and 4% for the stacking gel, and electrophoresis was performed.
The electrophoresed gel was stained with Coomassie Brilliant Blue and then destained. Detection was performed using an imaging device ChemiDocXRS Plus (manufactured by Bio-Rad Laboratories), and bands were quantitatively measured using Image Lab Software version 2.0 (manufactured by Bio-Rad Laboratories).
The net intensity of each band was used to calculate the inhibition rate of lysozyme dimer and trimer formation 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 the sample is left standing at 4°C without the addition of the test sample (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 advanced glycation end products (AGEs) formation>
A freeze-dried extract of hibiscus flower (manufactured by Maruzen Pharmaceutical Co., Ltd.) was used as a test sample, and its inhibitory effect on the formation of advanced glycation end products (AGEs) was tested 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 allow AGEs to form. 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) to evaluate the inhibitory effect on AGEs formation.
The AGE formation inhibition rate was calculated as follows: The results are shown in Table 12.
AGEs formation inhibition rate (%) = {(B-C)/(B-A)}×100
In the above formula, A represents the absorbance of the negative control at a wavelength of 405 nm, B represents the absorbance of the positive control at a wavelength of 405 nm, and C represents the absorbance at a wavelength of 405 nm when the test sample is added.

The results in Table 12 demonstrate 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 allowed to stand at 37°C for two weeks to allow AGEs to form. As a negative control, a plate containing PBS(-) was also allowed to stand in the same manner. After two 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(-) was added to each well and allowed to stand for an additional 16 days. As a positive control, a plate containing PBS(-) instead of the test sample after D(-)-ribose treatment was also allowed to stand in the same manner. Furthermore, 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) to evaluate the effect of promoting AGE degradation.
The AGEs decomposition promotion rate was calculated as follows: The results are shown in Table 13.
AGEs degradation promotion rate (%) = {(B-C)/(B-A)}×100
In the above formula, A represents the absorbance of the negative control at a wavelength of 405 nm, B represents the absorbance of the positive control at a wavelength of 405 nm, and C represents the absorbance at a wavelength of 405 nm when the test sample is added.

The results in Table 13 demonstrate that the hibiscus extract has an effect of promoting the decomposition of advanced glycation end products.

Example 14
<Claudin-1 production promoting effect test>
A freeze-dried extract of hibiscus flower (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 epidermal keratinocytes (NHEK) were cultured in an 80 ^cm 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 incubation, 100 μL of a test sample solution dissolved in KGM was added to each well and incubated at 37°C under 5% _CO2 for 24 hours. After incubation, the medium was removed, the cells were fixed to the plate, and the amount of claudin-1 expressed on the cell surface was measured by ELISA using a polyclonal claudin-1 antibody.
From the measurement results obtained, the claudin-1 production promotion rate (%) was calculated using 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.

The results in Table 14 demonstrate that the hibiscus extract has a significant effect of promoting claudin-1 production.

Example 15
<Occludin production promoting effect test>
A freeze-dried extract of hibiscus flower (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 epidermal keratinocytes (NHEK) were cultured in an 80 ^cm3 flask in normal human epidermal keratinocyte growth medium (KGM) at 37°C under 5% _CO2 , and then harvested by trypsinization. The harvested cells were diluted with KGM to a cell density of 2.0 x ¹⁰⁵ cells/mL, then seeded in a 96-well plate at 100 µL per well and cultured overnight at 37°C under 5% CO2 _.
After incubation, 100 μL of 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 in ₅ % CO for 24 hours. After 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 occludin production promotion rate was calculated 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.

The results in Table 15 demonstrate that the hibiscus extract has an occludin production promoting effect.

Example 16
<Test for promoting the production of epidermal tight junction component proteins in a three-dimensional human skin model>
Using a freeze-dried extract of hibiscus flower (Maruzen Pharmaceutical Co., Ltd.) as a test sample, the promoting effect on the production of epidermal tight junction component proteins in a three-dimensional human skin model was tested using 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 assay medium (EPI-NMM Maintenance Medium, manufactured by KURABO) at 37°C and 5% _CO2 for 1 hour. After culturing, 100 μL of assay medium containing or not containing the test sample dissolved in 1% DMSO (control) was applied to the surface of the skin model, and assay medium (maintenance medium) was applied to the bottom of the skin model. The skin model was cultured for 7 days at 37°C and 5% _CO2 . Exposure to the test sample was constant during the culture period. On the fourth day of culture, the maintenance medium was replaced with the assay medium containing the test sample. On the fourth day of culture and after the end of culture, the skin was cut using a 6 mm biopsy punch, embedded in a plastic embedding dish, and frozen with dry ice and liquid nitrogen. Subsequently, sections were cut to a thickness of 4 μm using a cryostat HM550 (manufactured by MICROM), attached to glass slides, and dried.

<Claudin-4 immunofluorescence staining>
The slides with the sections attached were placed in a staining tray and fixed with 4% paraformaldehyde. The embedding medium around the sections 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® Laboratories), was poured onto each slide and incubated at room temperature for 1 hour. The antibody solution was discarded, and the slides were washed with PBS(-). The secondary antibody, Alexa-Fluor 488-labeled goat-derived anti-mouse IgG antibody (Invitrogen), was treated, and incubated in the dark at 4°C for 1 hour. The slides were further washed with PBS(-), and nuclear staining was performed with DAPI solution. The target claudin-4 was analyzed using a fluorescence microscope. The results are shown in Figures 1A to 1F.
The images in Figures 1A to 1F show the region from the lower stratum corneum to the basal layer, with Figure 1A showing the control on day 4 of culture, Figure 1B showing the hibiscus extract at a sample concentration of 100 μg/mL on day 4 of culture, Figure 1C showing the hibiscus extract at a sample concentration of 500 μg/mL on day 4 of culture, Figure 1D showing the control on day 7 of culture, Figure 1E showing the hibiscus extract at a sample concentration of 100 μg/mL on day 7 of culture, and Figure 1F showing 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 granulosa cell membrane compared to the control, confirming its ability to promote claudin-4 production in the three-dimensional skin model.

<ZO-1 and ZO-2 immunofluorescence staining>
The slides with the sections attached were placed in a staining tray and fixed with 4% paraformaldehyde. The embedding agent around the sections was thoroughly washed off with PBS(-). After blocking with 1% BSA, the solution was discarded, and the primary antibodies, mouse anti-human ZO-1 monoclonal antibody (Invitrogen) and rabbit anti-human ZO-2 polyclonal antibody (Invitrogen), were poured onto each slide and incubated at room temperature for 1 hour. The antibody solution was discarded, and the slides were washed with PBS(-). They were then treated with the secondary antibodies, Alexa-Fluor 488-labeled goat anti-mouse IgG antibody (Invitrogen) and Alexa-Fluor 594-labeled goat anti-rabbit IgG antibody (Invitrogen), and incubated in the dark at 4°C for 1 hour. The slides were then washed with PBS(-) and nuclear stained with DAPI solution. The target ZO-1 and ZO-2 were analyzed using a fluorescence microscope, and the results are shown in Figures 2A to 2F and 3A to 3F.
The photographs in Figures 2A to 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 hibiscus extract at a sample concentration of 100 μg/mL on day 4 of culture, FIG. 2C shows the results for 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 hibiscus extract at a sample concentration of 100 μg/mL on day 7 of culture, and FIG. 2F shows the results for 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 of the granular layer to the basal layer).
The photographs in Figures 3A to 3F show the lower stratum corneum to basal layer.
Figure 3A shows the results for the control on day 4 of culture, Figure 3B shows the results for hibiscus extract at a sample concentration of 100 μg/mL on day 4 of culture, Figure 3C shows the results for hibiscus extract at a sample concentration of 500 μg/mL on day 4 of culture, Figure 3D shows the results for the control on day 7 of culture, Figure 3E shows the results for hibiscus extract at a sample concentration of 100 μg/mL on day 7 of culture, and Figure 3F shows the results for hibiscus extract at a sample concentration of 500 μg/mL on day 7 of culture. Figures 3A to 3F show that ZO-2 was stained red (near the cell membranes of the granular layer to the basal layer).
On the fourth day of culture, the linear fluorescence near the cell membrane was stronger at a concentration of 100 μg/mL compared to the control, confirming the ZO-1 and ZO-2 production-promoting effect 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 hibiscus flower extract (manufactured by Maruzen Pharmaceuticals Co., Ltd.) was used as a test sample, and its inhibitory effect on the decline of skin barrier function was tested using the following test method.

Human normal neonatal skin epidermal keratinocytes (NHEK) were cultured in human normal neonatal epidermal keratinocyte medium (KGM) and then harvested by trypsinization. The harvested cells were diluted with KGM to a concentration of 2.2 x ¹⁰ cells/mL and seeded in 0.5 mL of KGM per well in the upper layer of a 12-well transwell (Corning, 12 mm diameter, 0.4 μm pores). 0.5 mL of KGM was added to the lower layer and cultured for 3 days. After incubation, 0.5 mL of CaCl ₂ dissolved in KGM (final concentration 1.8 mM) was added to the upper and lower layers of each well and cultured for 3 days to induce tight junction formation. After incubation, 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 culture was initiated under low-CaCl ₂ conditions. At the same time, wells in which barrier function was maintained in a 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 by comparing 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 CaCl , 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 each lower layer, and the fluorescence intensity was measured at an excitation wavelength of 485 nm and a fluorescence wavelength of 545 nm. 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 by comparing with the control, thereby evaluating 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)} x 100
In the above formula, A represents the electrical resistance (TER) of cells to which a test sample was added, B represents the electrical resistance (TER) of cells to which a test sample was not added, and C represents the electrical resistance (TER) of cells treated with a high _CaCl2 medium.

The permeation inhibition rate was calculated as follows: The results are shown in Table 17.
Transmission suppression rate (%) = {1-(CA)/(CB)}×100
In the above formula, A represents the amount of FITC-Dex that has permeated through the cells to which the test sample has been added, B represents the amount of FITC-Dex that has permeated through the cells to which the test sample has not been added, and C represents the amount of FITC-Dex that has permeated through the cells treated with a high- _CaCl2 medium.

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

(Example 18)
<Testosterone 5α-reductase activity inhibitory effect test>
A freeze-dried extract of hibiscus flower (manufactured by Maruzen Pharmaceutical Co., Ltd.) was used as a test sample and its testosterone 5α-reductase activity inhibitory effect was tested according to 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 capped V-bottom test tube. 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 and mixed again. The mixture was reacted at 37°C for 30 minutes, and then 1 mL of methylene chloride was added to terminate the reaction. This mixture 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 using the same method.
The S-9 sample was obtained by intraperitoneally administering enzyme inducers (phenobarbital, 5,6-benzoflavone) to male SD rats, then mashing the liver and centrifuging it 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

Solutions of standard samples of 3α-androstanediol (Sigma), dihydrotestosterone (DHT, Tokyo Chemical Industry Co., Ltd.), and testosterone (Tokyo Chemical Industry Co., Ltd.) in methylene chloride were analyzed by gas chromatography in advance, and the amounts of these three compounds per peak area were calculated from their precise weighing 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). The conversion rate of the test sample was then calculated according to the following formula (3). 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 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) × 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.

The results in Table 18 demonstrate 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 activity was tested according to the following test method.

Normal human hair dermal papilla cells (manufactured by Toyobo Co., Ltd.) were cultured in a dermal papilla cell growth medium (manufactured by Toyobo Co., Ltd.) containing 1% FCS and growth additives, and then the cells were harvested by trypsinization. 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 an additional 4 days. The dermal papilla cell proliferation activity was measured using an MTT assay. After 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, the blue formazan produced within the cells 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 turbidity, and the difference between the two values 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.
Dermal papilla cell proliferation rate (%) = A/B x 100
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.

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 scavenger, skin whitening agent, anti-aging agent, and hair growth agent of the present invention are highly safe, can be taken daily, and are inexpensive, while exhibiting excellent hyaluronidase activity inhibitory effects, hydrogen peroxide scavenging effects, melanin production inhibitory effects in B16 melanoma cells, endothelin-1 mRNA expression inhibitory effects, stem cell growth factor mRNA expression inhibitory effects, basic fibroblast growth factor mRNA expression inhibitory effects, proopiomelanocortin mRNA expression inhibitory effects, epidermal hyaluronic acid production promoting effects, and glutathione It has at least one of the following effects: promoting the production of epidermal tight junction proteins, promoting serine palmitoyltransferase mRNA expression, inhibiting the Maillard reaction, inhibiting the formation of advanced glycation end products, promoting the degradation 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, suppressing a decline in skin barrier function, inhibiting testosterone 5α-reductase activity, and promoting hair papilla cell proliferation, making it suitable for use as an ingredient in cosmetics, foods and beverages, and as a research reagent.

Claims (2)

An agent for promoting the decomposition of advanced glycation end products (excluding those used for anti-aging), characterized by containing an extract of the flower parts of Hibiscus sabdariffa as an active ingredient. 2. The advanced glycation end products degradation accelerator according to claim 1, wherein the extract of Hibiscus sabdariffa flower parts is a water extract, a hydrophilic organic solvent extract, or a mixed solvent extract of water and a hydrophilic organic solvent of Hibiscus sabdariffa flower parts.