JP7708495B2 - Anti-aging agents, antioxidants, anti-inflammatory agents, skin whitening agents, and cosmetics - Google Patents

Description

The present invention relates to an anti-aging agent, an antioxidant, an anti-inflammatory agent, and a skin whitening agent that contain as an active ingredient a fermentation liquid produced by a plant Aspergillus oryzae, and to a cosmetic product that contains at least one of the anti-inflammatory agent, the antioxidant, the anti-inflammatory agent, and the skin whitening agent.

In recent years, active oxygen has been attracting attention as a factor that oxidizes biological components, and its adverse effects on the living body have become a problem. Active oxygen is generated during the energy metabolism process in living cells, and includes superoxide (i.e., superoxide anion (.O ₂ ^- ), hydrogen peroxide (H ₂ O ₂ ), singlet oxygen ( ¹ O ₂ ), and hydroxyl radical (.OH) generated by one-electron reduction of oxygen molecules). Such active oxygen is essential for the bactericidal mechanism of phagocytes, and plays an important role in removing viruses and cancer cells.

However, excessive production of the above-mentioned reactive oxygen species attacks the biological molecules that compose the membranes and tissues of the body, inducing various diseases. Superoxide, which is produced in the body and is also the starting material for other reactive oxygen species, is usually eliminated sequentially by the catalytic action of superoxide dismutase (SOD) contained in cells. However, when there is excessive production of superoxide or when the function of SOD is reduced, the elimination of superoxide becomes insufficient and the concentration of superoxide increases. This is thought to be one of the causes of tissue disorders such as rheumatoid arthritis and Behcet's disease, myocardial infarction, stroke, cataracts, age spots, freckles, wrinkles, diabetes, arteriosclerosis, stiff shoulders, poor circulation, and skin aging.

Among these, the skin is an organ that is easily stimulated by environmental factors such as ultraviolet rays and is therefore prone to the production of superoxide. Therefore, an increase in the concentration of superoxide can, for example, decompose, denature, or crosslink collagen and other biological tissues, or oxidize fats and oils to produce lipid peroxides that damage cells, resulting in the formation of wrinkles in the skin, aging such as reduced skin elasticity, inflammation, and skin pigmentation (see Non-Patent Document 1). Therefore, by inhibiting or suppressing the production of active oxygen and radicals in the body, it is believed that it is possible to prevent, treat, or improve various disorders involving active oxygen, such as wrinkle formation, reduced elasticity, and other skin aging, tissue disorders such as rheumatoid arthritis and Behcet's disease, myocardial infarction, stroke, cataracts, diabetes, arteriosclerosis, stiff shoulders, and poor circulation.

Therefore, attempts have been made to obtain active oxygen scavengers, radical scavengers, hydrogen peroxide scavengers, etc. from natural products that are advantageous in terms of safety, and the effectiveness of extracts of plants of the genus Brassica in the Brassicaceae family (see Patent Document 1), extracts of plants of the genus Kalanchoe in the Crassulaceae family (see Patent Document 2), extracts of the butterfly hyacinth (see Patent Document 3), and extracts of the water lily (see Patent Document 4) have been confirmed.

Inflammatory diseases, such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, and other skin diseases accompanied by rough skin, have a wide variety of causes and onset mechanisms. It is known that the main cause of these diseases is increased activity of hyaluronidase.

Hyaluronidase is a hydrolase of hyaluronic acid. Hyaluronate, which maintains its affinity to body tissues, is decomposed by ultraviolet light, oxygen, etc. in a water-containing system, and the water-retaining effect decreases with the decrease in molecular weight. Hyaluronic acid also exists as intercellular tissue in the body and is involved in vascular permeability. Furthermore, hyaluronidase exists in mast cells, but is released by degranulation caused by their activation, and acts as an inflammatory chemical mediator. Therefore, by inhibiting the activity of hyaluronidase, it is expected to enhance moisturization and prevent and reduce inflammation.
For example, extracts of plants from the genus Osbeckia (see Patent Document 5) and wisteria tea extract (see Patent Document 6) are known to have such hyaluronidase activity inhibitory effect.

In addition, melanin in the skin also plays a role in protecting the body from ultraviolet rays, but excessive production or uneven accumulation can cause skin darkening and blemishes. In general, melanin is thought to be formed by the action of the enzyme tyrosinase, which is biosynthesized in pigment cells, converting tyrosine to dopa, and then dopa to dopaquinone, and then passing through intermediates such as 5,6-dihydroxyindophenol. Therefore, in order to prevent, treat, or improve dark skin (cutaneous pigmentation), blemishes, freckles, etc., it is thought that the activity of tyrosinase, which is involved in the production of melanin, or the production of melanin can be inhibited.

Conventionally, skin whitening agents containing chemically synthesized products such as hydroquinone as an active ingredient have been applied topically to prevent, treat, or improve skin pigmentation, age spots, freckles, etc. However, chemically synthesized products such as hydroquinone may cause side effects such as skin irritation and allergies.
Therefore, there is a demand for the development of a skin whitening agent containing a highly safe natural raw material as an active ingredient, and examples of agents known to have a tyrosinase activity inhibitory effect include a wisteria tea extract (see Patent Document 7) and a willow knotweed extract (see Patent Document 8). Also, examples of agents known to have a melanin production inhibitory effect include an extract from the root of castor bean (see Patent Document 9) and an extract from a plant belonging to the genus Saussurea (see Patent Document 10).

The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and extracellular matrices such as collagen that are located outside these cells and support the skin structure. In young skin, the interaction of these skin tissues maintains homeostasis, ensuring moisture retention, flexibility, elasticity, etc., and the skin is maintained in a firm, glossy, and moist state in appearance.
However, when exposed to certain external factors such as exposure to ultraviolet rays (UV-A, UV-B), extremely dry air, or excessive skin washing, or as we age, the production of collagen, the main component of the extracellular matrix, decreases and elasticity is reduced due to cross-linking. As a result, the skin's moisturizing function and elasticity decrease, and the keratin begins to peel abnormally, causing the skin to lose firmness and luster, and to show signs of aging such as roughness and wrinkles.
There are various factors that cause the changes accompanying skin aging, such as wrinkles, dullness, loss of texture, and loss of elasticity, but the decrease and denaturation of extracellular matrix components such as collagen, hyaluronic acid, and elastin are involved. Therefore, it is believed that skin aging can be prevented and improved by promoting the production of collagen, hyaluronic acid, and the like.

Therefore, attempts have been made to obtain substances with collagen production promoting properties from natural products that are advantageous in terms of safety. For example, star fruit leaf extract (see Patent Document 11) and camphor tree extract (see Patent Document 12) have been confirmed to have collagen production promoting properties.

In addition, amino acids, which are the main components of natural moisturizing factors, are produced by the decomposition of filaggrin derived from keratohyalin granules in the stratum corneum. This filaggrin is expressed as profilaggrin in epidermal keratinocytes present in the granular layer just below the stratum corneum. It has been reported that it is then immediately phosphorylated, accumulated in keratohyalin granules, decomposed into filaggrin through dephosphorylation and hydrolysis, migrates to the stratum corneum, increases the aggregation efficiency of keratin filaments, and is involved in the internal structure of keratinocytes (see Non-Patent Document 2).
In recent years, it has been reported that filaggrin is extremely important and essential for maintaining moisture in the skin, and that conditions such as dryness reduce the synthetic ability of filaggrin, resulting in a decrease in the amount of amino acids in the stratum corneum (see non-patent document 3).
Therefore, it is expected that promoting the expression of profilaggrin mRNA in epidermal keratinocytes will promote the synthesis of filaggrin, thereby increasing the amount of amino acids in the stratum corneum and essentially improving the moisture environment of the stratum corneum.

As a naturally derived filaggrin synthesis promoter, for example, licorice extract (see Patent Document 13), liquiritin, known as a flavanone glycoside contained in natural plants (see Patent Document 14), and as at least one of naturally derived profilaggrin and filaggrin protein production promoters, a plant extract belonging to the genus Citrus or a yeast extract (see Patent Document 15) have been proposed.

The epidermis has a defense function to protect the skin from various external stimuli by constantly producing new keratinocytes through the division and subsequent differentiation of keratinocytes. In particular, during the differentiation process of keratinocytes, proteins such as involucrin are expressed from the spinous layer to the granular layer, and are crosslinked by the action of the enzyme transglutaminase-1 to form a cornified envelope (hereinafter abbreviated as "CE"), which is an insoluble cell membrane-like structure that encases keratinocytes, and contributes to the stability of the cytoskeleton and structure of keratinocytes.
However, when the production of transglutaminase-1 in the epidermis is reduced due to various factors, CE formation becomes incomplete and keratinization does not proceed normally, which is thought to result in a decrease in the stratum corneum barrier function and the skin's moisturizing function, leading to skin symptoms such as rough skin and dry skin.
Based on these findings, it is believed that by increasing the production of transglutaminase-1 in the epidermis by keratinocytes and promoting the formation of CE to normalize keratinization, it is possible to suppress the decline in skin barrier function associated with external stimuli such as dryness and ultraviolet rays, and prevent and improve various skin symptoms such as dry and rough skin.

As transglutaminase-1 production promoters derived from natural products, noni extract (see Patent Document 16) and royal jelly extract (see Patent Document 17) have been proposed.

In addition, in skin cells, aquaporins, known as water channels, are expressed on the cell membrane and are known to play a role in taking in low molecular weight substances, including water in the intercellular space, into the cells. Thirteen types of aquaporins (AQP0-AQP12) are known to exist in humans. In epidermal cells, aquaporin 3 (AQP3) is mainly present, and it is thought to play a role in taking in not only water, but also low molecular weight compounds such as glycerol and urea, which are involved in the moisture retention function.

However, AQP3 decreases with age, and it has been suggested that this is one of the causes of the decline in water retention function. Therefore, it is believed that it is possible to control water retention function and barrier function caused by aging by promoting the expression of AQP3 (see Non-Patent Document 4). For example, extracts from star fruit leaves (see Patent Document 18) are known to have the effect of promoting AQP3 expression.

Traditionally, it was thought that only the stratum corneum was responsible for the barrier function of the skin. However, since the barrier function of the skin collapses when the constituent proteins of tight junctions (hereinafter abbreviated as "TJ") present in the granular layer of the epidermis are genetically deleted, TJs are now thought to play an important role in the barrier function of the skin (see Non-Patent Document 5). 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. TJs are composed of cell membrane proteins such as claudins and occludins, and these proteins are thought to form the skeleton of the TJ strand and control the barrier function of TJs (see Non-Patent Document 6). From the above, if the expression of claudins or occludins 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 and occludin 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 19) and spruce extract (see Patent Document 20) have been proposed as substances that improve skin barrier function through the action of promoting TJ formation.

In recent years, the involvement of matrix metalloproteinases (MMPs) has been pointed out as a factor inducing changes associated with skin aging. Among these MMPs, matrix metalloproteinase-1 (MMP-1) is known as an enzyme that breaks down collagen, a major component of the skin's dermal extracellular matrix. Its expression is greatly increased by exposure to ultraviolet light, and it is thought to be one of the causes of collagen reduction and denaturation, as well as a major factor in the formation of wrinkles and reduced elasticity in the skin. Therefore, inhibiting the activity of MMP-1 is important in preventing and improving the symptoms of skin aging.

Examples of substances that have such MMP-1 inhibitory activity include extracts from Himalayan cherry (see Patent Document 21), extracts from Zingiber cassamuna of the Zingiberaceae family, and extracts from Ficus nerifolia of the Mulberry family (see Patent Document 22).

On the other hand, although fermented liquids obtained by fermenting Artemisia plants with Aspergillus oryzae (see Patent Document 23) and Thymus vulgare with Aspergillus oryzae (see Patent Document 24) are known, it is not known that these plant fermented liquids have any effects such as anti-aging, antioxidant, anti-inflammatory, or whitening effects.

JP 2003-81848 A JP 2005-29483 A JP 2006-321730 A JP 2007-8902 A JP 2003-55242 A JP 2003-12532 A JP 2002-370962 A JP 2004-83488 A JP 2001-213757 A JP 2002-201122 A JP 2002-226323 A JP 2003-146837 A JP 2002-363054 A JP 2003-146886 A JP 2001-261568 A JP 2010-090093 A JP 2009-184955 A JP 2009-191039 A JP 2007-176830 A JP 2007-176835 A JP 2003-176232 A JP 2003-176230 A JP 2011-140453 A JP 2011-130689 A

"Fragrance Journal" Special Issue No. 14, p. 156, 1995 Fragrance Journal Special Issue, vol. 17, pp. 14-19 (2000) Arch. Dermatol. Res. , vol. 288, pp. 442-446 (1996) "Fragrance Journal", 2006, Vol. 34, No. 10, pp. 19-23 J. Cell Biol. , vol. 156, pp. 1099-1111 (2002) Journal of the Japanese Society of Cosmetic Science, vol. 31, pp. 296-301 (2007)

The present invention aims to solve the above-mentioned problems in the prior art and to achieve the following objects.
That is, an object of the present invention is to provide an anti-aging agent having an excellent anti-aging effect and being highly safe, an antioxidant having an excellent antioxidant effect and being highly safe, an anti-inflammatory agent having an excellent anti-inflammatory effect and being highly safe, and a whitening agent having an excellent whitening effect and being highly safe.
Another object of the present invention is to provide a cosmetic preparation which has at least one effect selected from the group consisting of an excellent anti-aging effect, an antioxidant effect, an anti-inflammatory effect, and a whitening effect, and which is highly safe.

The means for solving the above problems are as follows.
<1> An anti-aging agent characterized by containing, as an active ingredient, at least any one of a fermentation liquid of Aspergillus oryzae from a plant of the genus Artemisia, a fermentation liquid of Aspergillus oryzae from Thymus vulgare, a fermentation liquid of Aspergillus oryzae from Japanese peppermint, and a fermentation liquid of Aspergillus oryzae from centaurea.
<2> An antioxidant characterized by containing, as an active ingredient, at least any one of a fermentation liquid of Aspergillus oryzae from a plant of the genus Artemisia, a fermentation liquid of Aspergillus oryzae from Thymus vulgare, a fermentation liquid of Aspergillus oryzae from Japanese peppermint, and a fermentation liquid of Aspergillus oryzae from centaurea.
<3> An anti-inflammatory agent comprising at least one fermentation liquid of a plant of the genus Artemisia by a koji mold and a fermentation liquid of Japanese peppermint by a koji mold as an active ingredient.
<4> A skin whitening agent comprising at least one fermentation liquid of an Aspergillus oryzae from a plant of the genus Artemisia, an Aspergillus oryzae from Japanese peppermint, or an Aspergillus oryzae from a centaurea cyanus as an active ingredient.
<5> A cosmetic preparation comprising at least one selected from the group consisting of the anti-aging agent described in <1>, the antioxidant described in <2>, the anti-inflammatory agent described in <3>, and the whitening agent described in <4>.

According to the present invention, it is possible to solve the above-mentioned conventional problems and achieve the above-mentioned object, and to provide an anti-aging agent having excellent anti-aging effect and being highly safe, an antioxidant having excellent anti-oxidation effect and being highly safe, an anti-inflammatory agent having excellent anti-inflammatory effect and being highly safe, and a whitening agent having excellent whitening effect and being highly safe.
Furthermore, the present invention can provide a cosmetic preparation that has at least one effect selected from the group consisting of an excellent anti-aging effect, an antioxidant effect, an anti-inflammatory effect, and a whitening effect, and is highly safe.

FIG. 1A is a photograph showing an example of a droplet when measuring the contact angle of Artemisia fermentation liquid 1 of Production Example 1. FIG. 1B is a photograph showing an example of a droplet when measuring the contact angle of Artemisia fermentation solution 2 of Production Example 2. FIG. 1C is a photograph showing an example of a droplet when measuring the contact angle of the Artemisia anguicida extract of Comparative Production Example 1. FIG. 2A is a photograph showing an example of a droplet when measuring the contact angle of Artemisia capillaris fermentation liquid 1 of Production Example 3. FIG. 2B is a photograph showing an example of a droplet when measuring the contact angle of Artemisia capillaris fermentation solution 2 of Production Example 4. FIG. 2C is a photograph showing an example of a droplet when measuring the contact angle of the Artemisia capillaris extract of Comparative Production Example 2. FIG. 3A is a photograph showing an example of a droplet when measuring the contact angle of the thyme fermentation liquid 1 of Production Example 5. FIG. 3B is a photograph showing an example of a droplet when measuring the contact angle of the thyme fermentation liquid 2 of Production Example 6. FIG. 3C is a photograph showing an example of a droplet when measuring the contact angle of the Thyme extract of Comparative Production Example 3. FIG. 4A is a photograph showing an example of a droplet when measuring the contact angle of the peppermint fermentation liquid 1 of Production Example 7. FIG. 4B is a photograph showing an example of a droplet when measuring the contact angle of the peppermint fermentation liquid 2 of Production Example 8. FIG. 4C is a photograph showing an example of a droplet when measuring the contact angle of the peppermint extract of Comparative Production Example 4. FIG. 5A is a photograph showing an example of a droplet when measuring the contact angle of the cornflower fermentation liquor 1 of Production Example 9. FIG. 5B is a photograph showing an example of a droplet when measuring the contact angle of the cornflower fermentation liquor 2 of Production Example 10. FIG. 5C is a photograph showing an example of a droplet when measuring the contact angle of the cornflower extract of Comparative Production Example 5.

(Anti-aging, antioxidant, anti-inflammatory, and skin whitening agent)
<Anti-aging agent>
The anti-aging agent of the present invention contains as an active ingredient at least any one of fermentation broths of Aspergillus oryzae from Artemisia genus plants (hereinafter sometimes referred to as "Artemisia genus fermentation broth"), fermentation broth of Aspergillus oryzae from Thymus vulgaris (hereinafter sometimes referred to as "Thymus vulgaris fermentation broth"), fermentation broth of Aspergillus oryzae from Common Mint (hereinafter sometimes referred to as "Common Mint fermentation broth"), and fermentation broth of Aspergillus oryzae from Cornflower (hereinafter sometimes referred to as "Cornflower fermentation broth"), and further contains other ingredients as necessary.

The Artemisia fermentation liquid, Thymus vulgaris fermentation liquid, Mint fermentation liquid, and Cornflower fermentation liquid have the following effects: Inhibition of matrix metalloproteinase-1 (MMP-1) activity, promotion of hyaluronan synthase 3 (HAS3) mRNA expression, promotion of type I collagen production, promotion of claudin-1 mRNA expression, promotion of claudin-4 mRNA expression, promotion of occludin mRNA expression, promotion of transglutaminase-1 (TGM-1) mRNA expression, promotion of aquaporin 3 (Aquaporin 3; AQP3) mRNA expression promoting action and filaggrin mRNA expression promoting action, and these actions can be utilized to use it as an active ingredient in an anti-aging agent.

The anti-aging agent has at least one action selected from the group consisting of an MMP-1 activity inhibitory action, a hyaluronic acid synthase 3 mRNA expression promoting action, a type I collagen production promoting action, a claudin-1 mRNA expression promoting action, a claudin-4 mRNA expression promoting action, an occludin mRNA expression promoting action, a transglutaminase-1 mRNA expression promoting action, an aquaporin 3 mRNA expression promoting action, and a filaggrin mRNA expression promoting action.

Details of the substances exhibiting at least any one of the following actions, which are possessed by the Artemisia fermentation liquid, the Thymus vulgaris fermentation liquid, the Mentha arvensis fermentation liquid, and the Cornflower fermentation liquid, are unknown: MMP-1 activity inhibitory action, hyaluronic acid synthase 3 mRNA expression promoting action, type I collagen production promoting action, claudin-1 mRNA expression promoting action, claudin-4 mRNA expression promoting action, occludin mRNA expression promoting action, transglutaminase-1 mRNA expression promoting action, aquaporin 3 mRNA expression promoting action, and filaggrin mRNA expression promoting action. However, it was not known at all before that the Artemisia fermentation liquid, the Thymus vulgaris fermentation liquid, the Mentha arvensis fermentation liquid, and the Cornflower fermentation liquid have such excellent actions and are useful as anti-aging agents, and this is a new finding by the present inventors.

<Antioxidants>
The antioxidant of the present invention contains as an active ingredient at least any one of fermentation broths of a plant of the genus Artemisia by Aspergillus oryzae (Artemisia fermentation broth), a fermentation broth of thyme by Aspergillus oryzae (thyme fermentation broth), a fermentation broth of peppermint by Aspergillus oryzae (peppermint fermentation broth), and a fermentation broth of cornflower by Aspergillus oryzae (cornflower fermentation broth), and further contains other ingredients as necessary.

The Artemisia fermentation liquid, the Thyme fermentation liquid, the Japanese peppermint fermentation liquid, and the Cornflower fermentation liquid have a diphenyl-p-picrylhydrazyl (DPPH) radical scavenging effect, and this effect can be utilized to use them as active ingredients of antioxidants.
Therefore, the antioxidant has a DPPH radical scavenging effect.

Details of the substances that exhibit DPPH radical scavenging activity contained in the Artemisia fermentation liquid, the Thyme fermentation liquid, the Mentha arvensis fermentation liquid, and the Cornflower fermentation liquid are unknown, but it was not previously known that the Artemisia fermentation liquid, the Thyme fermentation liquid, the Mentha arvensis fermentation liquid, and the Cornflower fermentation liquid have such excellent activity and are useful as an antioxidant, and this is a new discovery by the present inventors.

<Anti-inflammatory Agents>
The anti-inflammatory agent of the present invention contains at least one fermentation liquid of a plant of the genus Artemisia by Aspergillus oryzae (Artemisia fermentation liquid) and a fermentation liquid of Japanese peppermint by Aspergillus oryzae (Japanese peppermint fermentation liquid) as an active ingredient, and further contains other ingredients as necessary.

The Artemisia fermentation liquid and the Japanese peppermint fermentation liquid have a hyaluronidase activity inhibitory effect, and this effect can be utilized to use them as active ingredients of anti-inflammatory agents.
Therefore, the anti-inflammatory agent has an inhibitory effect on hyaluronidase activity.

The details of the substances that exhibit the hyaluronidase activity inhibitory effect of the fermented liquid of the Artemisia genus plant and the fermented liquid of the Japanese peppermint are unknown, but the fact that the fermented liquid of the Artemisia genus plant and the fermented liquid of the Japanese peppermint have such excellent effects and are useful as anti-inflammatory agents was not previously known, and is a new discovery by the present inventors.

<Skin whitening agent>
The skin whitening agent of the present invention contains at least any one of fermentation liquids of a plant of the genus Artemisia by Aspergillus oryzae (Artemisia fermentation liquid), a fermentation liquid of peppermint by Aspergillus oryzae (peppermint fermentation liquid), and a fermentation liquid of centaurea by Aspergillus oryzae (cornflower fermentation liquid) as an active ingredient, and further contains other ingredients as necessary.

The Artemisia fermentation liquid, the Japanese peppermint fermentation liquid, and the centaurea fermentation liquid have at least one of the effects of inhibiting tyrosinase activity and suppressing melanin production, and these effects can be utilized as active ingredients of skin whitening agents.
Therefore, the skin whitening agent has at least one of the following actions: an inhibitory action on tyrosinase activity and an inhibitory action on melanin production.

Details of the substances that exhibit at least one of the tyrosinase activity inhibitory effect and melanin production inhibitory effect possessed by the Artemisia fermentation liquid, the Japanese peppermint fermentation liquid, and the cornflower fermentation liquid are unknown, but it was not previously known that the Artemisia fermentation liquid and the cornflower fermentation liquid have such excellent effects and are useful as skin whitening agents, and this is a new discovery by the present inventors.

<< Artemisia genus plant fermented liquid >>
The Artemisia fermentation liquid is a fermentation liquid of a plant belonging to the Artemisia genus (hereinafter, sometimes referred to as "Artemisia plant") using an Aspergillus oryzae.

- Artemisia plants -
The mugwort plant used as the fermentation raw material is a perennial herb belonging to the genus Artemisia in the family Compositae , and has been used as a food or medicinal raw material since ancient times. It grows wild or is cultivated widely in Japan, such as Hokkaido, Honshu, Shikoku, and Kyushu, and is easily available from these regions.

The type of Artemisia plant is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include Artemisia montana (Nakai) Pamp., Artemisia capillaris Thunbergii, Artemisia princeps Pampan., Artemisia japonica Thumb., Artemisia absinthium L., Artemisia lactiflora Wall., Artemisia maritima L., and Artemisia scoparia Waldst.et kit. These may be used alone or in combination of two or more.
The method for obtaining the Artemisia plant is not particularly limited and can be appropriately selected depending on the purpose. The plant may be collected from nature or may be a commercially available product.

The part of the Artemisia plant used as the fermentation raw material is not particularly limited and can be appropriately selected depending on the purpose. Examples include above-ground parts such as flowers, buds, fruits, pericarp, seeds, seed coats, stems, leaves, branches, and leaves; and underground parts such as roots and rhizomes. These may be used alone or in combination of two or more. Among these, above-ground parts are preferred as the part of the Artemisia plant.

The size of the Artemisia plant used as the fermentation raw material is not particularly limited as long as it is a size that allows the Aspergillus oryzae to be cultured, and can be appropriately selected depending on the purpose. For example, the size as harvested, the size cut to the desired size, or the size pulverized (powdered) can be mentioned.

The state of the Artemisia plant used as the fermentation raw material is not particularly limited as long as it is in a state that allows the koji mold to be cultured, and can be appropriately selected depending on the purpose. For example, it can be in the state as it is harvested, in a dried state, in a crushed state, in a squeezed state, in an extract state, etc. Among these, in terms of the ease with which the koji mold can act, the state as it is harvested, in a crushed state, in a squeezed state, and in an extract state are preferred, and the state as it is harvested and in a crushed state are more preferred.

There are no particular limitations on the method for drying the Artemisia plant, and it can be appropriately selected depending on the purpose. For example, it can be dried in the sun or using a commonly used dryer.

The method for pulverizing the Artemisia plant is not particularly limited and can be appropriately selected depending on the purpose. For example, the method may include pulverizing the plant using a mixer, sugar mill, power mill, jet mill, impact crusher, etc.

The method for converting the Artemisia plant into the juice is not particularly limited and can be appropriately selected depending on the purpose, and examples include squeezing.

There are no particular limitations on the method for preparing the extract from the Artemisia plant, and any method commonly used for plant extraction can be appropriately selected depending on the purpose.

The Artemisia plant used as the fermentation raw material is preferably sterilized before inoculation with the Aspergillus oryzae. There are no particular limitations on the means for sterilizing the Artemisia plant, and it can be appropriately selected from known methods.

- Aspergillus oryzae -
The koji mold for fermenting the Artemisia plant is not particularly limited and can be appropriately selected depending on the purpose. For example, yellow koji mold such as Aspergillus oryzae and Aspergillus sojae ; black koji mold such as Aspergillus luchuensis ; white koji mold such as Aspergillus kawauchii ; and mutants thereof. These may be used alone or in combination of two or more. Among these, Aspergillus oryzae is preferred as the koji mold because it has excellent at least one of anti-aging, antioxidant, anti-inflammatory, and whitening effects.
The method of obtaining the koji mold is not particularly limited and can be appropriately selected depending on the purpose. It may be collected from nature or a commercially available product. As the koji mold, a seed koji made from rice or the like may be used, or a seed koji from a plant of the genus Artemisia, which will be described later, or a koji mold cultured in a medium (agar medium, liquid medium, etc.) may be used. Among these, it is preferable to use the seed koji from a plant of the genus Artemisia, since it has excellent effects such as anti-aging, antioxidant, anti-inflammatory, and whitening.

The amount of the koji mold to be inoculated into the Artemisia plant used as the fermentation raw material is not particularly limited as long as it is an amount that can ferment the Artemisia plant, and can be appropriately selected depending on the purpose. When the fermentation raw material is in a liquid state, the amount of the koji mold is preferably 1 x 10 ³ cells/mL to 1 x 10 ⁸ cells/mL, and when the fermentation raw material is in a solid state, the amount of the koji mold is preferably 1 x 10 ³ cells/g to 1 x 10 ⁸ cells/g.

When the Aspergillus oryzae is inoculated into the Artemisia plant, it is preferable to add water. The amount of water to be added is not particularly limited and can be appropriately selected depending on the purpose, but it is preferable to add 500 to 5,000 parts by mass of water, more preferably 1,000 to 4,000 parts by mass, and particularly preferably 1,500 to 3,000 parts by mass, per 100 parts by mass of the Artemisia plant.

The temperature of the fermentation (culture) is not particularly limited as long as it is within the range of temperatures at which fermentation by the koji mold can be performed, and can be appropriately selected depending on the purpose, but 20°C to 40°C is preferable, and 25°C to 35°C is more preferable. If the fermentation temperature is below 20°C, the Artemisia plant cannot be fermented sufficiently, and at least one of the anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect may be insufficient. Note that if the temperature exceeds 50°C, the koji mold may not grow.

The fermentation (culture) time is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 10 to 40 hours, and more preferably 20 to 30 hours. If the fermentation time is less than 10 hours, the Artemisia plant cannot be fermented sufficiently, and at least one of the anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect may be insufficient.

The method for stopping the fermentation (culture) is not particularly limited and can be appropriately selected depending on the purpose. For example, a heating method can be used.
The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the koji mold cannot grow, and can be appropriately selected depending on the purpose, but is preferably 50° C. or higher, more preferably 70° C. or higher, and particularly preferably 100° C. to 130° C. If the heating temperature is less than 50° C., the fermentation may not be stopped, and if it exceeds 130° C., at least one of the anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect may become insufficient.
The heating time for stopping the fermentation is not particularly limited as long as it can make the koji mold unable to grow, and can be appropriately selected depending on the purpose, but is preferably 5 minutes or more, and more preferably 10 to 20 minutes. If the heating time is less than 5 minutes, the fermentation may not be stopped, and if it exceeds 20 minutes, at least one of the anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect may become insufficient.

The Artemisia fermentation liquid is preferably cooled after the fermentation is stopped. There are no particular limitations on the cooling method, and it can be appropriately selected depending on the purpose. For example, it can be left at room temperature or in a refrigerator.

The number of times that the Artemisia plant is fermented by the Aspergillus oryzae is not particularly limited, and can be appropriately selected depending on the purpose. The number of times may be one or multiple.

When the fermentation is carried out multiple times, the koji mold may be inoculated only the first time, or only a few times, or may be inoculated every time, but it is preferable to inoculate it only the first time.
When the fermentation is carried out multiple times, the fermentation temperatures and fermentation times may be different or the same for each time.

--Artemisia genus plant seed koji--
The above-mentioned Artemisia genus plant seed koji is prepared by using the above-mentioned Artemisia genus plant as a seed koji material, inoculating the seed koji material with Aspergillus oryzae, and allowing a sufficient amount of spores to adhere to the Artemisia genus plant. By using this in the fermentation, it is advantageous in that a Artemisia genus plant fermentation liquid having excellent skin compatibility can be obtained more efficiently and easily.

The Artemisia plant used as the seed koji raw material can be the same as those described in "Artemisia plants" above, and the part, size, condition, and other aspects of the Artemisia plant used are also the same.

The koji mold used in producing the Artemisia genus plant seed koji can be the same as those described above in "Koji mold."

The amount of the koji mold to be inoculated into the Artemisia plant used as the seed koji material is not particularly limited and can be appropriately selected depending on the purpose, but it is preferable to inoculate 5 to 100 parts by mass of the koji mold (1 x ¹⁰³ /mL to 1 x ¹⁰⁸ /mL) suspended in sterilized water to 100 parts by mass of the Artemisia plant, more preferably 10 to 50 parts by mass, and particularly preferably 20 to 30 parts by mass. If the amount of the koji mold to be inoculated to 100 parts by mass of the Artemisia plant is less than 5 parts by mass, a sufficient number of spores may not attach to the Artemisia plant, and if it exceeds 100 parts by mass, excessive moisture may cause abnormal growth.

When the koji fungus is inoculated into the Artemisia plant used as the seed koji material, it is preferable to add water. There is no particular limit to the amount of water added, and it can be selected appropriately depending on the purpose. However, it is preferable to add 10 to 250 parts by mass, more preferably 20 to 200 parts by mass, and particularly preferably 30 to 150 parts by mass, per 100 parts by mass of the Artemisia plant. If the amount of water added per 100 parts by mass of the Artemisia plant is less than 10 parts by mass, a sufficient number of spores may not adhere to the Artemisia plant.

The temperature for the culture is not particularly limited as long as it is within the range of temperatures at which the koji mold can grow, and can be appropriately selected depending on the purpose, but 20°C to 40°C is preferable, and 25°C to 35°C is more preferable. If the culture temperature is below 20°C, a sufficient number of spores may not attach to the Artemisia plant. If the temperature exceeds 50°C, the koji mold may not grow.

The culture time is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 80 to 210 hours, more preferably 100 to 190 hours, and particularly preferably 120 to 170 hours. If the culture time is less than 80 hours, a sufficient number of spores may not attach to the Artemisia plant, and if it exceeds 210 hours, the germination rate of the spores may decrease.

The Artemisia fermentation liquid may contain the koji mold fungus or may be one from which the koji mold fungus has been removed, but is preferably one from which the koji mold fungus has been removed.

The state of the Artemisia fermentation liquid is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be the Artemisia fermentation liquid itself, a purified product of the Artemisia fermentation liquid, a concentrated product of the Artemisia fermentation liquid, or a diluted product of the Artemisia fermentation liquid. The Artemisia fermentation liquid may also be obtained by mixing or dissolving the dried product of the Artemisia fermentation liquid again in a solvent such as water or a hydrophilic solvent.

The purified product of the Artemisia plant fermentation broth is not particularly limited and can be appropriately selected depending on the purpose, and examples include a product from which solid components in the Artemisia plant fermentation broth (e.g., the Artemisia plant body, the Aspergillus oryzae fungus body, lees, etc.) have been removed.
The removal means is not particularly limited and can be appropriately selected depending on the purpose. For example, filtration and the like can be mentioned.
The filtration method is not particularly limited and can be appropriately selected from known methods depending on the purpose.

The dilution of the Artemisia plant fermentation liquid and the concentrate of the Artemisia plant fermentation liquid are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include Artemisia plant fermentation liquid prepared to a desired concentration.
The dilution means is not particularly limited and can be appropriately selected from known methods depending on the purpose.
The concentration means is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be concentration under reduced pressure.

The dried product of the fermentation broth of a plant of the genus Artemisia is not particularly limited and can be appropriately selected depending on the purpose. For example, a dried product of the fermentation broth of a plant of the genus Artemisia can be mentioned.
The drying means is not particularly limited and can be appropriately selected depending on the purpose. For example, freeze-drying and the like can be mentioned.

The fermented liquid of a plant of the genus Artemisia is not particularly limited as long as it is a fermented liquid of a plant of the genus Artemisia by Aspergillus oryzae, and can be appropriately selected according to the purpose. However, in terms of good compatibility with the skin, a fermented liquid of a plant of the genus Artemisia with a contact angle of 81° or less is preferred, and a fermented liquid of a plant of the genus Artemisia with a contact angle of 78° or less is more preferred.

In this specification, the contact angle refers to a value obtained by measuring a contact angle θ (°) at 1,000 ms using a dynamic contact angle/surface tension measuring device (FTA1000 Falcon, manufactured by First Ten Angstroms) by dropping 3 μL of a measurement sample onto a sample stage of the device and measuring the contact angle θ (°) at 1,000 ms using a θ/2 method under conditions of a temperature of 22° C. and a relative humidity of 20% using a sessile drop method.
The contact angle is used as an index of "wetting" and is defined as "the angle between the liquid surface and the solid surface at the place where the free surface of a stationary liquid contacts a solid wall (the angle inside the liquid)" (see Physics and Chemistry Dictionary, 4th Edition, Iwanami Shoten Co., Ltd.). The contact angle is determined by the magnitude relationship between the cohesive force between liquid molecules and the adhesive force between solid walls, and is an acute angle when the liquid wets the solid (high adhesive force) and an obtuse angle when it does not wet the solid. Therefore, the smaller the contact angle, the easier it is to wet, i.e., the better it blends with the skin. Therefore, there is no particular limit to the lower limit of the contact angle of the Artemisia fermentation liquid, and it can be appropriately selected according to the purpose.

<<Thyme fermented liquid>>
The thyme fermentation liquid is a fermentation liquid of thyme by Aspergillus oryzae.

- Thymus vulgare -
Thymus vulgaris Linne, used as the fermentation raw material, is a perennial woody plant belonging to the genus Thymus in the family Labiatae , a type of herb, and has been used as a food and medicinal raw material since ancient times. It is also known as common thyme. It is native to the Mediterranean coast, but it grows wild or is cultivated in Japan, and is easily available from these areas.
The method for obtaining the thyme is not particularly limited and can be appropriately selected depending on the purpose. The thyme may be collected from nature or may be a commercially available product.

The part of Thymus vulgaris used as the fermentation raw material is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include above-ground parts such as flowers, buds, fruits, pericarp, seeds, seed coats, stems, leaves, branches, bark, trunks, branches and leaves; and underground parts such as roots and rhizomes. These may be used alone or in combination of two or more. Among these, the above-ground parts are preferred as the part of Thymus vulgaris used.

The size of the thyme used as the fermentation raw material is not particularly limited as long as it is a size that allows the Aspergillus oryzae to be cultured, and can be appropriately selected depending on the purpose. For example, the size as it is when harvested, the size cut into the desired size, or the size pulverized (powdered) can be mentioned.

The state of the thyme used as the fermentation raw material is not particularly limited as long as it is in a state that allows the koji mold to be cultured, and can be appropriately selected depending on the purpose. For example, it can be in the state as it is collected, in a dried state, in a crushed state, in a squeezed juice state, in an extract state, etc. Among these, in terms of the ease with which the koji mold can act, the state as it is collected, in a crushed state, in a squeezed juice state, and in an extract state are preferred, and the state as it is collected and in a crushed state are more preferred.

There are no particular limitations on the method for drying the thyme, and it can be appropriately selected depending on the purpose. For example, it can be dried in the sun or in a commonly used dryer.

The method for pulverizing the thyme is not particularly limited and can be appropriately selected depending on the purpose. For example, pulverization can be performed using a mixer, sugar mill, power mill, jet mill, impact crusher, etc.

The method for converting the thyme into the juice is not particularly limited and can be appropriately selected depending on the purpose, and examples include squeezing.

There are no particular limitations on the method for converting the thyme into the extract, and any method commonly used for plant extraction can be appropriately selected depending on the purpose.

The thyme used as the fermentation raw material is preferably sterilized before inoculation with the koji mold. There are no particular limitations on the means for sterilizing the thyme, and it can be appropriately selected from known methods.

- Aspergillus oryzae -
The koji mold for fermenting the thyme is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include those described in the above section "Fermented broth of Artemisia genus plant". These may be used alone or in combination of two or more. Among these, Aspergillus oryzae is preferred as the koji mold, since it has excellent anti-aging and/or antioxidant effects.
The method of obtaining the koji mold is not particularly limited and can be appropriately selected depending on the purpose. It may be collected from nature or a commercially available product. As the koji mold, a seed koji made from rice or the like may be used, or a Thymus vulgaris seed koji described later may be used, or a koji mold cultured in a medium (agar medium, liquid medium, etc.) may be used. Among these, it is preferable to use the Thymus vulgaris seed koji, since it has excellent at least one of anti-aging and antioxidant effects.

The amount of the koji mold to be inoculated into the thyme used as the fermentation raw material is not particularly limited as long as it is an amount that can ferment the thyme, and can be appropriately selected depending on the purpose. When the fermentation raw material is in a liquid state, the amount is preferably 1 x 10 ³ cells/mL to 1 x 10 ⁸ cells/mL, and when the fermentation raw material is in a solid state, the amount is preferably 1 x 10 ³ cells/g to 1 x 10 ⁸ cells/g.

When the koji mold is inoculated into the thyme, it is preferable to add water. There is no particular limit to the amount of water added, and it can be selected appropriately depending on the purpose. However, it is preferable to add 500 to 5,000 parts by mass of water, more preferably 1,000 to 4,000 parts by mass, and particularly preferably 1,500 to 3,000 parts by mass, per 100 parts by mass of the thyme.

The temperature of the fermentation (culture) is not particularly limited as long as it is within the range of temperatures at which fermentation by the koji mold can be performed, and can be appropriately selected depending on the purpose, but 20°C to 40°C is preferable, and 25°C to 35°C is more preferable. If the fermentation temperature is below 20°C, the thyme cannot be sufficiently fermented, and at least one of the anti-aging effect and the antioxidant effect may be insufficient.

The fermentation (culture) time is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 10 to 40 hours, and more preferably 20 to 30 hours. If the fermentation time is less than 10 hours, the thyme cannot be fermented sufficiently, and at least one of the anti-aging effect and the antioxidant effect may be insufficient.

The method for stopping the fermentation (culture) is not particularly limited and can be appropriately selected depending on the purpose. For example, a heating method can be used.
The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the koji mold cannot grow, and can be appropriately selected depending on the purpose, but is preferably 50° C. or higher, more preferably 70° C. or higher, and particularly preferably 100° C. to 130° C. If the heating temperature is less than 50° C., the fermentation may not be able to be stopped, and if it exceeds 130° C., at least one of the anti-aging effect and the antioxidant effect may become insufficient.
The heating time for stopping the fermentation is not particularly limited as long as it can make the koji mold unable to grow, and can be appropriately selected depending on the purpose, but is preferably 5 minutes or more, and more preferably 10 to 20 minutes. If the heating time is less than 5 minutes, the fermentation may not be stopped, and if it exceeds 20 minutes, at least one of the anti-aging effect and the antioxidant effect may be insufficient.

It is preferable that the thyme fermentation liquid is cooled after the fermentation is stopped. There are no particular limitations on the cooling method, and it can be selected appropriately depending on the purpose. For example, it can be left to stand at room temperature or in a refrigerator.

There is no particular limit to the number of times that the thyme is fermented by the koji mold, and the number of times that the fermentation is performed can be appropriately selected depending on the purpose, and may be one or multiple times.

When the fermentation is carried out multiple times, the koji mold may be inoculated only the first time, or only a few times, or may be inoculated every time, but it is preferable to inoculate it only the first time.
When the fermentation is carried out multiple times, the fermentation temperatures and fermentation times may be different or the same for each time.

--Thyme seed koji--
The thyme seed koji is prepared by inoculating the thyme seed koji material with koji mold to allow a sufficient amount of spores to grow on the thyme. By using this in the fermentation, it is advantageous in that a thyme fermentation liquid with excellent skin compatibility can be obtained more efficiently and easily.

The Thymus vulgaris used as the seed koji raw material can be the same as that described in "Thymus vulgaris" above, and the part, size, condition, and other aspects of the Thymus vulgaris used are also the same.

The koji mold used in producing the thyme seed koji can be the same as that described above in "Koji mold."

The amount of the koji mold to be inoculated into the thyme used as the seed koji material is not particularly limited and can be appropriately selected depending on the purpose, but it is preferable to inoculate 5 to 100 parts by mass of the koji mold (1 x ¹⁰³ /mL to 1 x ¹⁰⁸ /mL) suspended in sterilized water to 100 parts by mass of the thyme, more preferably 10 to 50 parts by mass, and particularly preferably 20 to 30 parts by mass. If the amount of the koji mold to be inoculated to 100 parts by mass of the thyme is less than 5 parts by mass, a sufficient amount of spores may not adhere to the thyme, and if it exceeds 100 parts by mass, excessive moisture may cause abnormal growth.

When the koji fungus is inoculated into the thyme used as the seed koji material, it is preferable to add water. There is no particular limit to the amount of water added, and it can be selected appropriately depending on the purpose. However, it is preferable to add 10 to 250 parts by mass, more preferably 20 to 200 parts by mass, and particularly preferably 30 to 150 parts by mass, per 100 parts by mass of the thyme. If the amount of water added is less than 10 parts by mass per 100 parts by mass of the thyme, a sufficient amount of spores may not adhere to the thyme.

The temperature for the culture is not particularly limited as long as it is within the range of temperatures at which the koji mold can grow, and can be appropriately selected depending on the purpose, but 20°C to 40°C is preferable, and 25°C to 35°C is more preferable. If the culture temperature is below 20°C, a sufficient number of spores may not attach to the thyme. If the temperature exceeds 50°C, the koji mold may not grow.

The culture time is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 80 to 210 hours, more preferably 100 to 190 hours, and particularly preferably 120 to 170 hours. If the culture time is less than 80 hours, a sufficient number of spores may not attach to the thyme, and if it exceeds 210 hours, the germination rate of the spores may decrease.

The thyme fermentation liquid may contain the koji mold fungus or may be one from which the koji mold fungus has been removed, but it is preferable that the koji mold fungus has been removed.

The state of the thyme fermentation liquid is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be the thyme fermentation liquid itself, a purified product of the thyme fermentation liquid, a concentrated product of the thyme fermentation liquid, a diluted product of the thyme fermentation liquid, etc. Furthermore, the thyme fermentation liquid may be obtained by mixing or dissolving the dried thyme fermentation liquid again in a solvent such as water or a hydrophilic solvent.

The purified product of the thyme fermentation liquid is not particularly limited and can be appropriately selected depending on the purpose, and examples include a product from which solid components in the thyme fermentation liquid (e.g., the thyme plant body, the koji mold body, the lees, etc.) have been removed.
The removal means is not particularly limited and can be appropriately selected depending on the purpose. For example, filtration and the like can be mentioned.
The filtration method is not particularly limited and can be appropriately selected from known methods depending on the purpose.

The dilution of the thyme fermentation liquid and the concentrate of the thyme fermentation liquid are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a thyme fermentation liquid prepared to a desired concentration.
The dilution means is not particularly limited and can be appropriately selected from known methods depending on the purpose.
The concentration means is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be concentration under reduced pressure.

The dried product of the thyme fermentation liquor is not particularly limited and can be appropriately selected depending on the purpose. For example, a dried product of the thyme fermentation liquor can be mentioned.
The drying means is not particularly limited and can be appropriately selected depending on the purpose. For example, freeze-drying and the like can be mentioned.

The thyme fermentation liquid is not particularly limited as long as it is a fermentation liquid of thyme koji mold and can be appropriately selected depending on the purpose. However, in terms of good compatibility with the skin, a thyme fermentation liquid having a contact angle of 87° or less is preferred, and a thyme fermentation liquid having a contact angle of 81° or less is more preferred.

<< Peppermint fermented liquid >>
The peppermint fermentation liquid is a fermentation liquid of peppermint by the Aspergillus oryzae.

-Japanese mint-
The common mint ( Melissa officinalis Linne) used as the fermentation raw material is a perennial herb belonging to the genus Melissa in the family Labiatae . It is a type of herb and has been used as an edible and medicinal raw material since ancient times. It is also known by other names such as lemon balm and common mint. It is native to southern Europe, but grows wild or is cultivated in Japan, and is easily available from these regions.
The method for obtaining the peppermint is not particularly limited and can be appropriately selected depending on the purpose. The peppermint may be collected from nature or may be a commercially available product.

The part of the peppermint used as the fermentation raw material is not particularly limited and can be appropriately selected depending on the purpose, and examples include above-ground parts such as flowers, buds, fruits, pericarp, seeds, seed coats, stems, leaves, branches, and foliage; and underground parts such as roots and rhizomes. These may be used alone or in combination of two or more. Among these, the above-ground parts are preferred as the part of the peppermint.

The size of the peppermint used as the fermentation raw material is not particularly limited as long as it is a size that allows the Aspergillus oryzae to be cultured, and can be appropriately selected depending on the purpose. For example, the size as it is when harvested, the size cut into the desired size, or the size pulverized (powdered) can be mentioned.

The state of the Japanese peppermint used as the fermentation raw material is not particularly limited as long as it is in a state that allows the koji mold to be cultured, and can be appropriately selected depending on the purpose. For example, it can be in the state as it is collected, in a dried state, in a crushed state, in a squeezed juice state, in an extract state, etc. Among these, in terms of the ease with which the koji mold can act, the state as it is collected, in a crushed state, in a squeezed juice state, and in an extract state are preferred, and the state as it is collected and in a crushed state are more preferred.

There are no particular limitations on the method for drying the peppermint, and it can be appropriately selected depending on the purpose. For example, it can be dried in the sun or in a commonly used dryer.

The method for crushing the peppermint is not particularly limited and can be appropriately selected depending on the purpose. For example, the peppermint can be crushed using a mixer, sugar mill, power mill, jet mill, impact crusher, etc.

There are no particular limitations on the method for converting the peppermint into juice, and it can be appropriately selected depending on the purpose, and examples include squeezing.

There are no particular limitations on the method for converting the peppermint into the extract, and any method commonly used for plant extraction can be appropriately selected depending on the purpose.

The peppermint used as the fermentation raw material is preferably sterilized before inoculation with the koji mold. There are no particular limitations on the means for sterilizing the peppermint, and any method known in the art can be appropriately selected.

- Aspergillus oryzae -
The koji mold for fermenting the Japanese peppermint is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include those described in the above section "Fermented broth of Artemisia genus plant". These may be used alone or in combination of two or more. Among these, Aspergillus oryzae is preferred as the koji mold, since it has excellent at least one of anti-aging, antioxidant, anti-inflammatory, and whitening effects.
The method of obtaining the koji mold is not particularly limited and can be appropriately selected depending on the purpose. It may be collected from nature or a commercially available product. As the koji mold, a seed koji made from rice or the like may be used, or a Japanese peppermint seed koji, which will be described later, or a koji mold cultured in a medium (agar medium, liquid medium, etc.) may be used. Among these, it is preferable to use the Japanese peppermint seed koji, which is excellent in at least one of the following actions: anti-aging action, antioxidant action, anti-inflammatory action, and whitening action.

The amount of the koji mold to be inoculated into the Japanese peppermint used as the fermentation raw material is not particularly limited as long as it is an amount that can ferment the Japanese peppermint, and can be appropriately selected depending on the purpose. When the fermentation raw material is in a liquid state, the amount is preferably 1 x 10 ³ cells/mL to 1 x 10 ⁸ cells/mL, and when the fermentation raw material is in a solid state, the amount is preferably 1 x 10 ³ cells/g to 1 x 10 ⁸ cells/g.

When the koji mold is inoculated into the peppermint, it is preferable to add water. The amount of water to be added is not particularly limited and can be selected appropriately depending on the purpose, but it is preferable to add 500 to 5,000 parts by mass of water, more preferably 1,000 to 4,000 parts by mass, and particularly preferably 1,500 to 3,000 parts by mass, per 100 parts by mass of the peppermint.

The fermentation (cultivation) temperature is not particularly limited as long as it is within the range of temperatures at which fermentation by the koji mold can be performed, and can be appropriately selected depending on the purpose, but is preferably 20°C to 40°C, and more preferably 25°C to 35°C. If the fermentation temperature is below 20°C, the peppermint cannot be fermented sufficiently, and at least one of the anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect may be insufficient.

The fermentation (culture) time is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 10 to 40 hours, and more preferably 20 to 30 hours. If the fermentation time is less than 10 hours, the peppermint cannot be fermented sufficiently, and at least one of the anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect may be insufficient.

The method for stopping the fermentation (culture) is not particularly limited and can be appropriately selected depending on the purpose. For example, a heating method can be used.
The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the koji mold cannot grow, and can be appropriately selected depending on the purpose, but is preferably 50° C. or higher, more preferably 70° C. or higher, and particularly preferably 100° C. to 130° C. If the heating temperature is less than 50° C., the fermentation may not be stopped, and if it exceeds 130° C., at least one of the anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect may become insufficient.
The heating time for stopping the fermentation is not particularly limited as long as it can make the koji mold unable to grow, and can be appropriately selected depending on the purpose, but is preferably 5 minutes or more, and more preferably 10 to 20 minutes. If the heating time is less than 5 minutes, the fermentation may not be stopped, and if it exceeds 20 minutes, at least one of the anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect may become insufficient.

It is preferable that the peppermint fermentation liquid is cooled after the fermentation is stopped. There are no particular limitations on the cooling method, and it can be selected appropriately depending on the purpose. For example, it can be left at room temperature or in a refrigerator.

The number of times that the Japanese peppermint is fermented by the Aspergillus oryzae is not particularly limited, and can be appropriately selected depending on the purpose. The number of times may be one or multiple times.

When the fermentation is carried out multiple times, the koji mold may be inoculated only the first time, or only a few times, or may be inoculated every time, but it is preferable to inoculate it only the first time.
When the fermentation is carried out multiple times, the fermentation temperatures and fermentation times may be different or the same for each time.

--Mint peppermint seed koji--
The mint seed koji is prepared by inoculating the mint seed koji with Aspergillus oryzae to allow a sufficient amount of spores to grow on the mint. The use of this in the fermentation is advantageous in that it allows for a mint fermentation liquid with excellent skin compatibility to be obtained more efficiently and easily.

The Japanese peppermint used as the seed koji raw material can be the same as that described in "Japanese peppermint" above, and the part, size, condition, and other aspects of the Japanese peppermint used are also the same.

The koji mold used in the preparation of the Japanese peppermint seed koji can be the same as that described above in "Koji mold."

The amount of the koji mold to be inoculated into the Japanese peppermint used as the seed koji material is not particularly limited and can be appropriately selected depending on the purpose, but it is preferable to inoculate 5 to 100 parts by mass of the koji mold (1 x ¹⁰³ /mL to 1 x ¹⁰⁸ /mL) suspended in sterilized water to 100 parts by mass of the Japanese peppermint, more preferably 10 to 50 parts by mass, and particularly preferably 20 to 30 parts by mass. If the amount of the koji mold to be inoculated to 100 parts by mass of the Japanese peppermint is less than 5 parts by mass, a sufficient amount of spores may not be able to attach to the Japanese peppermint, and if it exceeds 100 parts by mass, excessive moisture may cause abnormal growth.

When the koji fungus is inoculated into the Japanese peppermint used as the seed koji raw material, it is preferable to add water. There is no particular limit to the amount of water added, and it can be selected appropriately depending on the purpose, but it is preferable to add 10 to 250 parts by mass, more preferably 20 to 200 parts by mass, and particularly preferably 30 to 150 parts by mass, per 100 parts by mass of the Japanese peppermint. If the amount of water added per 100 parts by mass of the Japanese peppermint is less than 10 parts by mass, a sufficient amount of spores may not adhere to the Japanese peppermint.

The temperature for the culture is not particularly limited as long as it is within the range of temperatures at which the koji mold can grow, and can be appropriately selected depending on the purpose, but 20°C to 40°C is preferable, and 25°C to 35°C is more preferable. If the culture temperature is below 20°C, a sufficient number of spores may not attach to the Japanese peppermint. If the temperature exceeds 50°C, the koji mold may not grow.

The culture time is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 80 to 210 hours, more preferably 100 to 190 hours, and particularly preferably 120 to 170 hours. If the culture time is less than 80 hours, a sufficient amount of spores may not attach to the peppermint, and if it exceeds 210 hours, the germination rate of the spores may decrease.

The Japanese peppermint fermentation liquid may contain the koji mold fungus or may be one from which the koji mold fungus has been removed, but it is preferable that the koji mold fungus has been removed.

The state of the peppermint fermentation liquid is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be the peppermint fermentation liquid itself, a purified product of the peppermint fermentation liquid, a concentrated product of the peppermint fermentation liquid, or a diluted product of the peppermint fermentation liquid. The peppermint fermentation liquid may also be a product obtained by mixing or dissolving the dried peppermint fermentation liquid again in a solvent such as water or a hydrophilic solvent.

The purified product of the peppermint fermentation liquid is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a product from which solid components in the peppermint fermentation liquid (e.g., the peppermint plant body, the koji mold fungus body, the lees, etc.) have been removed.
The removal means is not particularly limited and can be appropriately selected depending on the purpose. For example, filtration and the like can be mentioned.
The filtration method is not particularly limited and can be appropriately selected from known methods depending on the purpose.

The dilution of the peppermint fermentation liquid and the concentrate of the peppermint fermentation liquid are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a peppermint fermentation liquid prepared to a desired concentration.
The dilution means is not particularly limited and can be appropriately selected from known methods depending on the purpose.
The concentration means is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be concentration under reduced pressure.

The dried product of the peppermint fermentation broth is not particularly limited and can be appropriately selected depending on the purpose. For example, a dried product of the peppermint fermentation broth can be mentioned.
The drying means is not particularly limited and can be appropriately selected depending on the purpose. For example, freeze-drying and the like can be mentioned.

The peppermint fermentation liquid is not particularly limited as long as it is a fermentation liquid made from the peppermint koji mold, and can be appropriately selected depending on the purpose. However, in terms of good compatibility with the skin, peppermint fermentation liquid with a contact angle of 85° or less is preferred, and peppermint fermentation liquid with a contact angle of 79° or less is more preferred.

<<Cornflower fermented liquid>>
The cornflower fermentation liquid is a fermentation liquid of cornflower by Aspergillus oryzae.

- Cornflower -
Cornflower ( Centaurea cyanus Linne) used as the fermentation raw material is an annual herb belonging to the genus Centaurea in the family Asteraceae ( Compositae ), and has been used as a raw material for food and medicine since ancient times. It is also known by other names such as knapweed, centaurea, and centaurea. It is native to Europe, but grows wild or is cultivated in Japan, and is easily available from these regions.
The method for obtaining the cornflower is not particularly limited and can be appropriately selected depending on the purpose. The cornflower may be collected from nature or may be a commercially available product.

The part of the cornflower used as the fermentation raw material is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include above-ground parts such as flowers, buds, fruits, pericarp, seeds, seed coats, stems, leaves, branches, and foliage; and underground parts such as roots and rhizomes. These may be used alone or in combination of two or more. Among these, the above-ground parts are preferred as the part of the cornflower used.

The size of the cornflowers used as the fermentation raw material is not particularly limited as long as it is a size that allows the koji mold to be cultured, and can be appropriately selected depending on the purpose. For example, the size of the cornflowers as they are when harvested, the size of the cornflowers cut into the desired size, or the size of the cornflowers pulverized (powdered) can be mentioned.

The state of the cornflower used as the fermentation raw material is not particularly limited as long as it is in a state that allows the koji mold to be cultured, and can be appropriately selected depending on the purpose. For example, it can be in the state as it is collected, in a dried state, in a crushed state, in a squeezed state, in an extract state, etc. Among these, in terms of facilitating the action of the koji mold, the state as it is collected, in a crushed state, in a squeezed state, and in an extract state are preferred, and the state as it is collected and in a crushed state are more preferred.

The method for drying the cornflowers is not particularly limited and can be appropriately selected depending on the purpose. For example, the method may be drying in the sun or using a commonly used dryer.

The method for crushing the cornflower is not particularly limited and can be appropriately selected depending on the purpose. For example, the cornflower may be crushed using a mixer, sugar mill, power mill, jet mill, impact crusher, etc.

There are no particular limitations on the method for converting the cornflowers into juice, and it can be appropriately selected depending on the purpose, and examples of the method include squeezing.

There are no particular limitations on the method for converting the cornflower into the extract, and any method commonly used for plant extraction can be appropriately selected depending on the purpose.

The cornflowers used as the fermentation raw material are preferably sterilized before inoculation with the koji mold. There are no particular limitations on the means for sterilizing the cornflowers, and any known method can be appropriately selected.

- Aspergillus oryzae -
The koji mold for fermenting the cornflower is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include those described in the above section "Fermented broth of Artemisia genus plant". These may be used alone or in combination of two or more. Among these, Aspergillus oryzae is preferred as the koji mold, since it has excellent at least one of anti-aging, antioxidant, and whitening effects.
The method of obtaining the koji mold is not particularly limited and can be appropriately selected depending on the purpose. It may be collected from nature or a commercially available product. As the koji mold, a koji seed made from rice or the like may be used, or a koji seed from cornflower (described later) may be used, or a koji mold cultured in a medium (agar medium, liquid medium, etc.) may be used. Among these, it is preferable to use the koji seed from cornflower, since it has excellent effects such as anti-aging, antioxidant, and whitening.

The amount of the koji mold to be inoculated into the cornflower used as the fermentation raw material is not particularly limited as long as it is an amount that can ferment the cornflower, and can be appropriately selected depending on the purpose. When the fermentation raw material is in a liquid state, the amount is preferably 1 x ¹⁰³ cells/mL to 1 x ¹⁰⁸ cells/mL, and when the fermentation raw material is in a solid state, the amount is preferably 1 x ¹⁰³ cells/g to 1 x ¹⁰⁸ cells/g.

When the koji mold is inoculated into the cornflowers, it is preferable to add water. There is no particular limit to the amount of water added, and it can be appropriately selected depending on the purpose. However, it is preferable to add 500 to 5,000 parts by mass of water, more preferably 1,000 to 4,000 parts by mass, and particularly preferably 1,500 to 3,000 parts by mass, per 100 parts by mass of the cornflowers.

The fermentation (cultivation) temperature is not particularly limited as long as it is within the range of temperatures at which fermentation by the koji mold can be performed, and can be appropriately selected depending on the purpose, but is preferably 20°C to 40°C, and more preferably 25°C to 35°C. If the fermentation temperature is below 20°C, the cornflowers cannot be fermented sufficiently, and at least one of the anti-aging effect, antioxidant effect, and whitening effect may be insufficient.

The fermentation (culture) time is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 10 to 40 hours, and more preferably 20 to 30 hours. If the fermentation time is less than 10 hours, the cornflowers cannot be fermented sufficiently, and at least one of the anti-aging effect, antioxidant effect, and whitening effect may be insufficient.

The method for stopping the fermentation (culture) is not particularly limited and can be appropriately selected depending on the purpose. For example, a heating method can be used.
The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the koji mold cannot grow, and can be appropriately selected depending on the purpose, but is preferably 50° C. or higher, more preferably 70° C. or higher, and particularly preferably 100° C. to 130° C. If the heating temperature is less than 50° C., the fermentation may not be able to be stopped, and if it exceeds 130° C., at least one of the anti-aging effect, anti-oxidation effect, and whitening effect may become insufficient.
The heating time for stopping the fermentation is not particularly limited as long as it can make the koji mold unable to grow, and can be appropriately selected depending on the purpose, but is preferably 5 minutes or more, and more preferably 10 to 20 minutes. If the heating time is less than 5 minutes, the fermentation may not be stopped, and if it exceeds 20 minutes, at least one of the anti-aging effect, anti-oxidation effect, and whitening effect may be insufficient.

It is preferable that the cornflower fermentation liquid is cooled after the fermentation is stopped. The cooling method is not particularly limited and can be appropriately selected depending on the purpose, and examples include a method of leaving the liquid at room temperature or in a refrigerator.

The number of times the cornflower is fermented by the koji mold is not particularly limited, and can be appropriately selected depending on the purpose. The number of times may be one or multiple.

When the fermentation is carried out multiple times, the koji mold may be inoculated only the first time, or only a few times, or may be inoculated every time, but it is preferable to inoculate it only the first time.
When the fermentation is carried out multiple times, the fermentation temperatures and fermentation times may be different or the same for each time.

--Cornflower koji starter--
The cornflower seed koji is prepared by inoculating the cornflower seed koji with koji mold to allow a sufficient amount of spores to grow on the cornflower, and is advantageous in that the cornflower fermentation liquid having excellent skin compatibility can be obtained more efficiently and easily by using the koji in the fermentation.

The cornflower used as the seed koji raw material can be the same as that described in "Cornflower" above, and the part, size, condition, and other aspects of the cornflower used are also the same.

The koji mold used in producing the cornflower seed koji can be the same as that described above in "Koji mold."

The amount of the koji mold to be inoculated into the cornflower used as the seed koji raw material is not particularly limited and can be appropriately selected depending on the purpose, but it is preferable to inoculate 5 to 100 parts by mass, more preferably 10 to 50 parts by mass, and particularly preferably 20 to 30 parts by mass of the koji mold suspended in sterilized water (1 x ¹⁰³ cells/mL to 1 x ¹⁰⁸ cells/mL) per 100 parts by mass of the cornflower. If the amount of the koji mold to be inoculated into 100 parts by mass of the cornflower is less than 5 parts by mass, a sufficient number of spores may not adhere to the cornflower, and if it exceeds 100 parts by mass, excessive moisture may cause abnormal growth.

When the koji fungus is inoculated into the cornflowers used as the seed koji raw material, it is preferable to add water. There is no particular limit to the amount of water added, and it can be appropriately selected depending on the purpose. However, it is preferable to add 10 to 250 parts by mass, more preferably 20 to 200 parts by mass, and particularly preferably 30 to 150 parts by mass, per 100 parts by mass of the cornflowers. If the amount of water added is less than 10 parts by mass per 100 parts by mass of the cornflowers, a sufficient number of spores may not adhere to the cornflowers.

The temperature for the culture is not particularly limited as long as it is within the range of temperatures at which the koji mold can grow, and can be appropriately selected depending on the purpose, but 20°C to 40°C is preferable, and 25°C to 35°C is more preferable. If the culture temperature is below 20°C, a sufficient number of spores may not attach to the cornflower. If the temperature exceeds 50°C, the koji mold may not grow.

The culture time is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 80 to 210 hours, more preferably 100 to 190 hours, and particularly preferably 120 to 170 hours. If the culture time is less than 80 hours, a sufficient number of spores may not attach to the cornflower, and if it exceeds 210 hours, the germination rate of the spores may decrease.

The cornflower fermentation liquid may contain the koji mold fungus or may be one from which the koji mold fungus has been removed, but it is preferable that the koji mold fungus has been removed.

The state of the cornflower fermentation liquid is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be the cornflower fermentation liquid itself, a purified product of the cornflower fermentation liquid, a concentrated product of the cornflower fermentation liquid, or a diluted product of the cornflower fermentation liquid. The cornflower fermentation liquid may also be obtained by mixing or dissolving a dried product of the cornflower fermentation liquid again in a solvent such as water or a hydrophilic solvent.

The purified product of the cornflower fermentation liquor is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a product obtained by removing solid components in the cornflower fermentation liquor (e.g., the cornflower plant body, the koji mold body, the sediment, etc.).
The removal means is not particularly limited and can be appropriately selected depending on the purpose. For example, filtration and the like can be mentioned.
The filtration method is not particularly limited and can be appropriately selected from known methods depending on the purpose.

The dilution product of the cornflower fermentation liquor and the concentrate of the cornflower fermentation liquor are not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a cornflower fermentation liquor prepared to a desired concentration.
The dilution means is not particularly limited and can be appropriately selected from known methods depending on the purpose.
The concentration means is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be concentration under reduced pressure.

The dried product of the cornflower fermentation broth is not particularly limited and may be appropriately selected depending on the purpose. For example, a dried product of the cornflower fermentation broth may be mentioned.
The drying means is not particularly limited and can be appropriately selected depending on the purpose. For example, freeze-drying and the like can be mentioned.

The cornflower fermentation liquid is not particularly limited as long as it is a fermentation liquid of cornflowers by Aspergillus oryzae and can be appropriately selected depending on the purpose. However, in terms of good compatibility with the skin, a cornflower fermentation liquid having a contact angle of 85° or less is preferred, and a cornflower fermentation liquid having a contact angle of 79° or less is more preferred.

<<Other ingredients>>
The other components in the anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include excipients, moisture-proofing agents, preservatives, strengthening agents, thickeners, emulsifiers, antioxidants, sweeteners, acidulants, seasonings, colorants, fragrances, whitening agents, moisturizers, oily components, UV absorbers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, skin nutrients, etc. These may be used alone or in combination of two or more.
The content of the other components is not particularly limited and can be appropriately selected depending on the purpose.

-Applications-
The anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention have at least one of excellent anti-aging action, antioxidant action, anti-inflammatory action, and whitening action, and therefore can be suitably used, for example, as medicines, quasi-drugs, cosmetics, foods and beverages, and the blending amount, method of use, and dosage form can be appropriately selected depending on the purpose of use.

The blending amounts can be adjusted appropriately depending on the physiological activity of the fermentation liquid. The anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent may be the fermentation liquid itself.

The method of use is not particularly limited and can be appropriately selected depending on the purpose. Examples of the method of use include oral, parenteral, and external use. Among these, external use is preferred.

The dosage form is not particularly limited and can be appropriately selected depending on the purpose. Examples of the dosage form include oral administration agents such as tablets, powders, capsules, granules, extracts, and syrups; parenteral administration agents such as injections, drips, and suppositories; and external applications such as skin lotions, milky lotions, creams, ointments, beauty essences, lotions, packs, jellies, lip balms, lipsticks, foundations, bath additives, soaps, body shampoos, astringents, hair tonics, hair creams, hair liquids, pomades, shampoos, and rinses.

The anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention can also be used as reagents for research into the mechanism of action of anti-aging action, antioxidant action, anti-inflammatory action, or whitening action.

The anti-aging agent, antioxidant, anti-inflammatory agent, and skin whitening agent of the present invention are suitable for use in humans, but can also be used in animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as the respective action effects are achieved.

(Cosmetics)
The cosmetic preparation of the present invention contains at least one selected from the group consisting of the anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention, and further contains other ingredients as necessary.

<Anti-aging agent, antioxidant, anti-inflammatory agent, whitening agent>
The content of at least one selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent in the cosmetic is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 5% by volume or more, more preferably 20% by volume or more, based on the total amount of the cosmetic. If the content of at least one selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent is less than 5% by volume, at least one of the anti-aging action, the antioxidant action, the anti-inflammatory action, and the whitening action may be insufficient. The content of at least one selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent is preferably as high as possible, and the upper limit is not particularly limited and can be appropriately selected depending on the purpose. The cosmetic may also be at least one selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent itself.

<Other ingredients>
If necessary, the cosmetic may further contain various main ingredients, auxiliaries and other ingredients that are commonly used in the manufacture of cosmetics, provided that the purpose and effects of the present invention are not impaired.
The other components are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include astringents, bactericides, antibacterial agents, ultraviolet absorbers, cell activators, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, and fragrances. These may be used alone or in combination of two or more. When these components are used in combination with at least one selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent, they may act synergistically to provide a superior effect that is greater than that normally expected.
The content of the other components in the cosmetic is not particularly limited as long as it does not impair the effects of the present invention, and can be appropriately selected depending on the purpose.

<Application>
The use of the cosmetic is not particularly limited and can be appropriately selected from common cosmetic products. Examples include skin cosmetics such as lotion, milky lotion, cream, ointment, beauty essence, lotion, pack, jelly, lip balm, lipstick, foundation, bath additives, soap, and body shampoo; scalp and hair cosmetics such as astringent, hair tonic, hair cream, hair liquid, pomade, shampoo, and rinse.

The cosmetic may be at least one selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent blended into any cosmetic so as not to impede its activity, or may be a cosmetic containing at least one selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent as a main component. The cosmetic may also be at least one selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent itself.

The cosmetic composition of the present invention is suitable for use on humans, but can also be used on animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as the respective functional effects are achieved.

The cosmetic composition of the present invention contains at least one selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent, and is therefore useful in that it exerts at least one of the following effects when used on the skin: excellent anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect.

The present invention will be specifically explained below with reference to the following manufacturing examples and test examples, but the present invention is not limited to these test examples.

<Production Example 1: Preparation of Artemisia fermentation liquid 1>
- Seed koji preparation process -
A koji mold ( Aspergillus oryzae , strain name: AOK1714, manufactured by Akita Konno Shoten Co., Ltd.) was taken with a platinum loop and suspended in 50 mL of sterilized water to prepare a koji mold solution. The number of bacteria in the koji mold solution was calculated using a Thoma hemocytometer (manufactured by EKDS) to be 1.0 x ¹⁰⁵ bacteria/mL.
Next, 10 g of Yamayomogi (manufactured by Albion Co., Ltd.) cut into 0.5 cm to 5 cm pieces was placed in an Erlenmeyer flask and autoclaved, and 2 mL of the Aspergillus oryzae solution was inoculated therein and cultured at 30° C. for 168 hours. After completion of the culture, the mixture was dried at 45° C. for 24 hours to obtain "Yamayomogi seed koji."

-Fermentation process-
Artemisia japonica (manufactured by Albion Co., Ltd.) was crushed using a crusher (sugar mill) and passed through a 2 mm mesh screen to obtain Artemisia japonica crushed material. 1,000 mL of water was added to 50 g of the Artemisia japonica crushed material, and after mixing, 20 mL of Artemisia japonica seed koji (number of bacteria: about 1.0 × 10 ⁶ / mL) obtained in Preparation Example 1 was inoculated. Then, it was pre-cultured at 25 ° C. for 22 hours. The obtained fermentation liquid was filtered using diatomaceous earth to obtain "Artemisia japonica fermentation liquid 1".

<Production Example 2: Preparation of Artemisia fermentation liquid 2>
A "Yamayomogi fermented liquid 2" was obtained in the same manner as in Production Example 1, except that in Production Example 1, the Yamayomogi seed koji was changed to a rice-based seed koji ( Aspergillus oryzae , Shirakami, manufactured by Akita Konno Shoten Co., Ltd.) (hereinafter sometimes referred to as "rice seed koji").

Comparative Production Example 1: Preparation of Artemisia anguicida extract
Artemisia japonica (manufactured by Albion Co., Ltd.) was pulverized using a pulverizer (sugar mill) and passed through a 2 mm mesh screen to obtain Artemisia japonica pulverized material. 1,000 mL of water was added to 50 g of the Artemisia japonica pulverized material, mixed, and stirred at 25° C. for 22 hours. The resulting mixture was then filtered using diatomaceous earth to obtain a “Artemisia japonica extract.”

(Test Example A-1: Measurement of Contact Angle)
The contact angles of the test samples, Artemisia fermentation liquid 1 obtained in Production Example 1, Artemisia fermentation liquid 2 obtained in Production Example 2, and Artemisia extract obtained in Comparative Production Example 1, were measured by the following method.
Specifically, using a dynamic contact angle/surface tension measuring device (FTA1000 Falcon, First Ten Angstroms), 3 μL of each test sample was dropped onto the sample stage (made of aluminum) of the device, and measurements were performed by the sessile drop method under conditions of a temperature of 22° C. and a relative humidity of 20%. The contact angle θ (°) at 1,000 ms was determined by the θ/2 method. The contact angle was measured three times, and the average value was calculated. The results are shown in Table 1 below. Also, examples of droplets used when measuring the contact angle of each test sample are shown in Figures 1A to 1C.

Compared to the Artemisia anguicida extract obtained in Comparative Production Example 1, Artemisia anguicida fermentation liquid 1 obtained in Production Example 1 and Artemisia anguicida fermentation liquid 2 obtained in Production Example 2 both had small contact angles of 81° or less, and were excellent in compatibility with the skin. Furthermore, Artemisia anguicida fermentation liquid 1 obtained in Production Example 1 had a contact angle of 78° or less, and was even more excellent in compatibility with the skin.

(Test Example 1-1: Matrix metalloproteinase-1 (MMP-1) activity inhibitory effect test)
Using Artemisia fermentation liquid 1 obtained in Production Example 1, Artemisia fermentation liquid 2 obtained in Production Example 2, and Artemisia extract obtained in Comparative Production Example 1 as test samples, the inhibitory effect on matrix metalloproteinase-1 (MMP-1) activity was tested by the following test method, which is a partial modification of the Wunsch and Heidrich method.

In a capped test tube, each test sample was dissolved in 0.1 mol/L Tris-HCl buffer (pH 7.1) containing 20 mmol/L calcium chloride. Next, 50 μL of the test sample solution was mixed with 50 μL of MMP-1 (COLLAGENASE Type IV from Clostridium histolyticum, manufactured by Sigma) solution and 400 μL of Pz-peptide (Pz-Pro-Leu-Gly-Pro-D-Arg-OH, manufactured by BACHEM Feinchemikalien AG) solution, and the mixture was reacted at 3°C for 30 minutes, after which 1 mL of 25 mmol/L citric acid solution was added to stop the reaction. The final concentration of the test sample at this time was the concentration shown in Table 2 below. Next, 5 mL of ethyl acetate was added and vigorously shaken. This was centrifuged at 1,600 x g for 10 minutes, and the absorbance of the ethyl acetate layer was measured at a wavelength of 320 nm.

As a blank, the same operations and absorbance measurements were performed as above, except that the MMP-1 solution (enzyme solution) was replaced with 0.1 mol/L Tris-HCl buffer (pH 7.1).

Furthermore, as a control, the same operations and absorbance measurements were performed as above, except that the dissolution solution of the test sample was changed to 0.1 mol/L Tris-HCl buffer (pH 7.1) containing 20 mmol/L calcium chloride without the test sample.

From the obtained absorbance measurements, the MMP-1 activity inhibition rate was calculated according to the following formula 1. The results are shown in Table 2 below.
<Formula 1>
MMP-1 activity inhibition rate (%) = {1-(CD)/(AB)}×100
In the formula 1, A to D respectively represent the following.
A: Absorbance at 320 nm without adding test sample and adding enzyme. B: Absorbance at 320 nm without adding test sample and adding enzyme. C: Absorbance at 320 nm with adding test sample and adding enzyme. D: Absorbance at 320 nm with adding test sample and adding no enzyme.

(Test Example 1-2: Hyaluronic acid synthase 3 (HAS3) mRNA expression promoting effect test)
Using Artemisia fermentation liquid 1 obtained in Production Example 1, Artemisia fermentation liquid 2 obtained in Production Example 2, and Artemisia extract obtained in Comparative Production Example 1 as test samples, the effect of promoting hyaluronic acid synthase 3 (HAS3) mRNA expression was tested using the test method described below.

Each test sample was dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries, Ltd.) to give a final concentration shown in Table 3 below, to prepare a medium containing the test sample.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurashiki Boseki Co., Ltd.) were cultured in a normal human epidermal keratinocyte growth medium (HuMedia-KG2, manufactured by Kurashiki Boseki Co., Ltd.) at 37°C and 5% _CO2 until confluent, and the cells were then harvested by trypsin treatment. The cells were then adjusted to 1.5 x ¹⁰⁵ cells/mL using normal human epidermal keratinocyte growth medium (HuMedia-KG2).
Next, 2 mL of the NHEK (1.5 x ¹⁰⁵ cells/mL) was seeded in a 35 mm petri dish and cultured overnight at ₃₇ ° C. and 5% CO2. After the culture was completed, the medium was replaced with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the culture was completed, the medium was replaced with 2 mL of the test sample-added medium and cultured for 24 hours at 37 ° C. and 5% _CO2 . After the culture was completed, the culture medium was removed, and total RNA was extracted with an RNA extraction reagent (ISOGEN II (catalog number: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared to be 200 ng / μL using purified water.

As a control, the same procedures and absorbance measurements were carried out as above, except that 2 mL of the medium containing the test sample was replaced with 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) not containing the test sample, and total RNA was prepared to 200 ng/μL using the same method as above.

Using each of the total RNAs as a template, the expression levels of hyaluronic acid synthase 3 (HAS3) mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal standard were measured. The mRNAs were detected by a two-step RT-PCR reaction using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (catalog number: RR063A), manufactured by Takara Bio Inc.).
The expression level of HAS3 mRNA in the absence and presence of the test sample was corrected by the expression level of GAPDH mRNA. From this corrected value, the promotion rate of HAS3 mRNA expression was calculated according to the following formula 2. The results are shown in Table 3.
<Formula 2>
HAS3 mRNA expression promotion rate (%) = A/B x 100
In the formula 2, A and B respectively represent the following.
A: Corrected value when test sample was added B: Corrected value when test sample was not added

(Test Example 1-3: DPPH radical scavenging effect test)
The Artemisia fermentation liquid 1 obtained in Production Example 1, the Artemisia fermentation liquid 2 obtained in Production Example 2, and the Artemisia extract obtained in Comparative Production Example 1 were used as test samples to test the DPPH radical scavenging activity according to the following test method.

Each test sample was dissolved in an ethanol solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a test sample solution.
3 mL of the test sample solution was added to 3 mL of 150 μmol/L DPPH (diphenyl-p-picrylhydrazyl) ethanol solution, and the container was immediately sealed and shaken, and the mixture was allowed to stand for 30 minutes, after which the absorbance at a wavelength of 520 nm was measured. The final concentration of the test sample was as shown in Table 4 below.

As a blank, the same procedure and absorbance measurement were performed as above, except that the DPPH ethanol solution was replaced with an ethanol solution that did not contain DPPH.

Furthermore, as a control, the same operations and absorbance measurements were performed as above, except that the test sample solution was changed to an ethanol solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) that did not contain the test sample.

From the obtained absorbance measurement values, the DPPH radical scavenging rate was calculated according to the following formula 3. The results are shown in Table 4 below.
<Formula 3>
DPPH radical scavenging rate (%) = {A - (B - C)} / A x 100
In the formula 3, A to C respectively represent the following.
A: Absorbance at a wavelength of 520 nm without the addition of test sample and with the addition of DPPH. B: Absorbance at a wavelength of 520 nm with the addition of test sample and with the addition of DPPH. C: Absorbance at a wavelength of 520 nm without the addition of test sample and without the addition of DPPH.

(Test Example 1-4: Hyaluronidase activity inhibitory effect test)
The Artemisia fermentation liquid 1 obtained in Production Example 1, the Artemisia fermentation liquid 2 obtained in Production Example 2, and the Artemisia extract obtained in Comparative Production Example 1 were used as test samples to test the hyaluronidase activity inhibitory effect according to the following test method.

Each test sample was dissolved in 0.1 mol/L acetate buffer (pH 3.5) to prepare a test sample solution.
To 0.2 mL of the test sample solution, 0.1 mL of hyaluronidase solution (Type IV-S (from bovine tests), 400 NF units / mL, manufactured by SIGMA) was added, and the mixture was reacted at 37 ° C. for 20 minutes. Next, 0.2 mL of 2.5 mmol / L calcium chloride was added as an activator, and the mixture was further reacted at 37 ° C. for 20 minutes. 0.5 mL of 0.8 mg / mL sodium hyaluronate solution (from rooster comb) (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added, and the mixture was reacted at 37 ° C. for 40 minutes. The final concentration of the test sample at this time was the concentration shown in Table 5 below. Next, 0.2 mL of 0.4 mol / L sodium hydroxide was added to stop the reaction, and after cooling, 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 (10 g of p-dimethylaminobenzaldehyde was dissolved in a mixture of 12.5 mL of 10N hydrochloric acid and 87.5 mL of acetic acid, and diluted 10-fold with acetic acid) was added and reacted at 37° C. for 20 minutes. Then, the absorbance at a wavelength of 585 nm was measured.

As a blank, the same operations and absorbance measurements were performed as above, except that the hyaluronidase solution (enzyme solution) was replaced with 0.1 mol/L acetate buffer (pH 3.5).

Furthermore, as a control, the same operations and absorbance measurements were performed as above, except that the test sample solution was changed to 0.1 mol/L acetate buffer (pH 3.5) that did not contain the test sample.

The hyaluronidase activity inhibition rate was calculated from the obtained absorbance measurement value according to the following formula 4. The results are shown in Table 5 below.
<Formula 4>
Hyaluronidase activity inhibition rate (%) = {1 - (C - D) / (A - B)} x 100
In the formula 4, A to D respectively represent the following.
A: Absorbance at 585 nm without adding test sample and adding enzyme. B: Absorbance at 585 nm without adding test sample and adding enzyme. C: Absorbance at 585 nm with adding test sample and adding enzyme. D: Absorbance at 585 nm with adding test sample and adding no enzyme.

(Test Example 1-5: Tyrosinase activity inhibitory effect test)
The Artemisia fermentation liquid 1 obtained in Production Example 1, the Artemisia fermentation liquid 2 obtained in Production Example 2, and the Artemisia extract obtained in Comparative Production Example 1 were used as test samples to test the tyrosinase activity inhibitory effect by the following test method.

Each test sample was dissolved in a 25% DMSO solution to prepare a test sample solution.
0.2 mL of Mcllvaine buffer (pH 6.8), 0.06 mL of 0.3 mg/mL tyrosine solution, and 0.18 mL of the test sample solution were added to a 48-well plate, and the plate was left to stand at 37° C. for 10 minutes. 0.02 mL of 800 unit/mL tyrosinase solution (manufactured by SIGMA) was added thereto, and the plate was further reacted at 37° C. for 15 minutes. After the reaction, the absorbance at a wavelength of 475 nm was measured. The final concentration of the test sample was the concentration shown in Table 6 below.

As a blank, the same procedure and absorbance measurement were performed as above, except that the tyrosinase solution (enzyme solution) was replaced with Mcllvaine buffer (pH 6.8).

Furthermore, as a control, the same procedures and absorbance measurements were performed as above, except that the test sample solution was replaced with a 25% DMSO solution that did not contain the test sample.

From the obtained absorbance measurements, the tyrosinase activity inhibition rate was calculated according to the following formula 5. The results are shown in Table 6 below.
<Formula 5>
Tyrosinase activity inhibition rate (%) = {1 - (C - D) / (A - B)} x 100
In the formula 5, A to D respectively represent the following.
A: Absorbance at 475 nm without adding test sample and adding enzyme. B: Absorbance at 475 nm without adding test sample and adding enzyme. C: Absorbance at 475 nm with adding test sample and adding enzyme. D: Absorbance at 475 nm with adding test sample and adding no enzyme.

<Production Example 3: Preparation of Artemisia capillaris fermentation liquid 1>
" Artemisia capillaris Thunbergii seed koji" was prepared in the same manner as in the seed koji preparation step of Production Example 1, except that Artemisia capillaris Thunbergii (manufactured by Albion Co., Ltd.) was used in place of Artemisia oryzae in the seed koji preparation step of Production Example 1.
In addition, " Artemisia capillaris fermented liquid 1" was obtained in the same manner as in the fermentation process of Production Example 1, except that Artemisia capillaris Thunbergii (manufactured by Albion Co., Ltd.) was used in place of Artemisia mountainus in the fermentation process of Production Example 1.

<Production Example 4: Preparation of Artemisia capillaris fermentation liquid 2>
A "Mugwort fermentation liquid 2" was obtained in the same manner as in Production Example 3, except that the mugwort seed koji in Production Example 3 was changed to rice seed koji ( Aspergillus oryzae , Shirakami, manufactured by Akita Konno Shoten Co., Ltd.).

Comparative Production Example 2: Preparation of Artemisia capillaris extract
A " Artemisia capillaris extract" was obtained in the same manner as in Comparative Production Example 1, except that Artemisia capillaris Thunbergii (manufactured by Albion Co., Ltd.) was used instead of Artemisia oryzae in Comparative Production Example 1.

(Test Example A-2: Measurement of Contact Angle)
In the above-mentioned Test Example A-1, the contact angle was measured in the same manner as in the above-mentioned Test Example A-1, except that the test samples were changed to Artemisia capillaris fermentation liquid 1 obtained in Production Example 3, Artemisia capillaris fermentation liquid 2 obtained in Production Example 2, and Artemisia capillaris extract obtained in Comparative Production Example 2. The results are shown in the following Table 7. In addition, an example of the droplet when measuring the contact angle of each test sample is shown in Figures 2A to 2C.

Compared to the Artemisia capillaris extract obtained in Comparative Production Example 2, Artemisia capillaris fermentation liquid 1 obtained in Production Example 3 and Artemisia capillaris fermentation liquid 2 obtained in Production Example 4 both had small contact angles of 81° or less, and were excellent in compatibility with the skin. Furthermore, Artemisia capillaris fermentation liquid 1 obtained in Production Example 3 had a contact angle of 78° or less, and was even more excellent in compatibility with the skin.

(Test Example 2-1: Hyaluronic acid synthase 3 (HAS3) mRNA expression promoting effect test)
In Test Example 1-2, the test samples were changed to Artemisia capillaris fermentation liquid 1 obtained in Production Example 3, Artemisia capillaris fermentation liquid 2 obtained in Production Example 4, and Artemisia capillaris extract obtained in Comparative Production Example 2, and the final concentrations of the test samples were changed to the concentrations shown in Table 8 below, but the same method as Test Example 1-2 was used to test the hyaluronic acid synthase 3 (HAS3) mRNA expression promoting effect. The results are shown in Table 8 below.

(Test Example 2-2: Type I collagen production promoting effect test)
The Artemisia capillaris fermentation liquid 1 obtained in Production Example 3, the Artemisia capillaris fermentation liquid 2 obtained in Production Example 4, and the Artemisia capillaris extract obtained in Comparative Production Example 2 were used as test samples to test the type I collagen production promoting effect by the following test method.

Each test sample was dissolved in Dulbecco's MEM (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 0.25% fetal bovine serum (FBS, manufactured by Biosera) to the final concentration shown in Table 9 below, to prepare a medium containing the test sample.
Normal human fibroblasts (NB1RGB, purchased from RIKEN BRC) were cultured in 10% FBS-containing DMEM at 37°C and 5% _CO2 until confluent, and then the cells were harvested by trypsinization. The cells were adjusted to 1.6 x ¹⁰⁵ cells/mL with 10% FBS-containing DMEM.
Next, 100 μL of the NB1RGB (1.6×10 ⁵ cells/mL) was seeded in a 96-well microplate and cultured overnight at 37° C. and 5% CO _2. After the culture was completed, the medium was replaced with 100 μL of medium containing the test sample, and cultured for 3 days at 37° C. and 5% CO _2. After the culture was completed, the amount of type I collagen in the medium of each well was measured by ELISA.

Specifically, 90 μL of the culture supernatant was transferred to an ELISA plate, and the plate was adsorbed at 4° C. overnight. The solution was then discarded, and washing was performed with phosphate buffered saline (PBS-T) containing 0.05% Tween-20. Thereafter, blocking was performed with phosphate buffered saline containing 1% FBS. The solution was discarded, washing was performed with phosphate buffered saline (PBS-T) containing 0.05% Tween-20, and anti-human collagen type I antibody (rabbit IgG, manufactured by Chemi-Con Corporation) was reacted. The solution was discarded, washing was performed with phosphate buffered saline (PBS-T) containing 0.05% Tween-20, and reaction with HRP-labeled anti-rabbit IgG antibody was performed, and the same washing operation was performed to carry out a color reaction.
The type I collagen production promotion rate was calculated by carrying out the above-mentioned ELISA using a standard sample and preparing a calibration curve.

As a control, the same procedure and ELISA method were performed as above, except that the test sample solution was changed to DMEM containing 0.25% FBS without the test sample.

From the obtained measured values, the type I collagen production promotion rate was calculated according to the following formula 6. The results are shown in Table 9 below.
<Formula 6>
Type I collagen production promotion rate (%) = A/B x 100
In the formula 6, A and B respectively represent the following.
A: Amount of type I collagen when test sample was added B: Amount of type I collagen when test sample was not added

(Test Example 2-3: Claudin-1 mRNA expression promoting effect test)
Using Artemisia capillaris fermentation liquid 1 obtained in Production Example 3, Artemisia capillaris fermentation liquid 2 obtained in Production Example 4, and Artemisia capillaris extract obtained in Comparative Production Example 2 as test samples, the promoting effect of claudin-1 mRNA expression was tested by the following test method.

Each test sample was dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries, Ltd.) to give a final concentration shown in Table 10 below, to prepare a medium containing the test sample.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurashiki Boseki Co., Ltd.) were cultured in a normal human epidermal keratinocyte growth medium (HuMedia-KG2, manufactured by Kurashiki Boseki Co., Ltd.) at 37°C and 5% _CO2 until confluent, and the cells were then harvested by trypsin treatment. The cells were then adjusted to 1.5 x ¹⁰⁵ cells/mL using normal human epidermal keratinocyte growth medium (HuMedia-KG2).
Next, 2 mL of the NHEK (1.5 x ¹⁰⁵ cells/mL) was seeded in a 35 mm petri dish and cultured overnight at ₃₇ ° C. and 5% CO2. After the culture was completed, the medium was replaced with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the culture was completed, the medium was replaced with 2 mL of the test sample-added medium and cultured for 24 hours at 37 ° C. and 5% _CO2 . After the culture was completed, the culture medium was removed, and total RNA was extracted with an RNA extraction reagent (ISOGEN II (catalog number: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared to be 200 ng / μL using purified water.

As a control, the same procedures and absorbance measurements were carried out as above, except that 2 mL of the medium containing the test sample was replaced with 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) not containing the test sample, and total RNA was prepared to 200 ng/μL using the same method as above.

Using each of the total RNAs as a template, the expression levels of claudin-1 mRNA and GAPDH mRNA as an internal standard were measured. The mRNA was detected by a two-step real-time PCR reaction using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (catalog number: RR063A), manufactured by Takara Bio Inc.).
The expression levels of claudin-1 mRNA in the absence and presence of the test sample were corrected with the expression level of GAPDH mRNA. From this corrected value, the claudin-1 mRNA expression promotion rate was calculated according to the following formula 7. The results are shown in Table 10 below.
<Formula 7>
Claudin-1 mRNA expression promotion rate (%) = A/B x 100
In the formula 7, A and B respectively represent the following.
A: Corrected value when test sample was added B: Corrected value when test sample was not added

(Test Example 2-4: Claudin-4 mRNA expression promoting effect test)
Using Artemisia capillaris fermentation liquid 1 obtained in Production Example 3, Artemisia capillaris fermentation liquid 2 obtained in Production Example 4, and Artemisia capillaris extract obtained in Comparative Production Example 2 as test samples, the promoting effect of claudin-4 mRNA expression was tested by the following test method.

Each test sample was dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries, Ltd.) to give a final concentration shown in Table 11 below, to prepare a medium containing the test sample.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurashiki Boseki Co., Ltd.) were cultured in a normal human epidermal keratinocyte growth medium (HuMedia-KG2, manufactured by Kurashiki Boseki Co., Ltd.) at 37°C and 5% _CO2 until confluent, and the cells were then harvested by trypsin treatment. The cells were then adjusted to 1.5 x ¹⁰⁵ cells/mL using normal human epidermal keratinocyte growth medium (HuMedia-KG2).
Next, 2 mL of the NHEK (1.5 x ¹⁰⁵ cells/mL) was seeded in a 35 mm petri dish and cultured overnight at ₃₇ ° C. and 5% CO2. After the culture was completed, the medium was replaced with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the culture was completed, the medium was replaced with 2 mL of the test sample-added medium and cultured for 24 hours at 37 ° C. and 5% _CO2 . After the culture was completed, the culture medium was removed, and total RNA was extracted with an RNA extraction reagent (ISOGEN II (catalog number: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared to be 200 ng / μL using purified water.

As a control, the same procedures and absorbance measurements were carried out as above, except that 2 mL of the medium containing the test sample was replaced with 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) not containing the test sample, and total RNA was prepared to 200 ng/μL using the same method as above.

Using each of the total RNAs as a template, the expression levels of claudin-4 mRNA and GAPDH mRNA as an internal standard were measured. The mRNA was detected by a two-step real-time PCR reaction using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (catalog number: RR063A), manufactured by Takara Bio Inc.).
The expression levels of claudin-4 mRNA in the absence and presence of the test sample were corrected with the expression level of GAPDH mRNA. From this corrected value, the claudin-4 mRNA expression promotion rate was calculated according to the following formula 8. The results are shown in Table 11 below.
<Formula 8>
Claudin-4 mRNA expression promotion rate (%) = A/B x 100
In the formula 8, A and B respectively represent the following.
A: Corrected value when test sample was added B: Corrected value when test sample was not added

(Test Example 2-5: Test of promoting effect of occludin mRNA expression)
The Artemisia capillaris fermentation liquid 1 obtained in Production Example 3, the Artemisia capillaris fermentation liquid 2 obtained in Production Example 4, and the Artemisia capillaris extract obtained in Comparative Production Example 2 were used as test samples to test the occludin mRNA expression promoting effect by the test method described below.

Each test sample was dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries, Ltd.) to give a final concentration shown in Table 12 below, to prepare a medium containing the test sample.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurashiki Boseki Co., Ltd.) were cultured in a normal human epidermal keratinocyte growth medium (HuMedia-KG2, manufactured by Kurashiki Boseki Co., Ltd.) at 37°C and 5% _CO2 until confluent, and the cells were then harvested by trypsin treatment. The cells were then adjusted to 1.5 x ¹⁰⁵ cells/mL using normal human epidermal keratinocyte growth medium (HuMedia-KG2).
Next, 2 mL of the NHEK (1.5 x ¹⁰⁵ cells/mL) was seeded in a 35 mm petri dish and cultured overnight under conditions _of 37 ° C. and 5% CO2. After completion of the culture, the medium was replaced with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After completion of the culture, the culture medium was removed, and total RNA was extracted with an RNA extraction reagent (ISOGEN II (catalog number: 311-07361), manufactured by Nippon Gene Co., Ltd.), the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared to 200 ng/μL using purified water.

As a control, the same procedures and absorbance measurements were carried out as above, except that 2 mL of the medium containing the test sample was replaced with 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) not containing the test sample, and total RNA was prepared to 200 ng/μL using the same method as above.

Using each of the total RNAs as a template, the expression levels of occludin mRNA and GAPDH mRNA as an internal standard were measured. The mRNAs were detected by a two-step real-time PCR reaction using a real-time PCR device (Thermal Cycler Dice® Real Time System III, manufactured by Takara Bio Inc.) and a SYBR® PrimeScript® RT-PCR Kit (Perfect Real Time (catalog number: RR063A), manufactured by Takara Bio Inc.).
The expression levels of occludin mRNA in the absence and presence of the test sample were corrected with the expression level of GAPDH mRNA. From this corrected value, the promotion rate of occludin mRNA expression was calculated according to the following formula 9. The results are shown in Table 12 below.
<Formula 9>
Occludin mRNA expression promotion rate (%) = A/B x 100
In the formula 9, A and B respectively represent the following.
A: Corrected value when test sample was added B: Corrected value when test sample was not added

<Production Example 5: Preparation of Thyme Fermentation Liquid 1>
" Thymus vulgaris seed koji" was prepared in the same manner as in the seed koji preparation step of Production Example 1, except that in the seed koji preparation step of Production Example 1, mugwort was changed to Thymus vulgaris Linne (manufactured by Albion Co., Ltd.).
In addition, " Thymus vulgaris fermented liquid 1" was obtained in the same manner as in the fermentation process of Production Example 1, except that in the fermentation process of Production Example 1, mugwort was replaced with Thymus vulgaris Linne (manufactured by Albion Co., Ltd.).

<Production Example 6: Preparation of Thyme Fermentation Liquid 2>
A "thyme fermented liquid 2" was obtained in the same manner as in Production Example 5, except that the thyme seed koji in Production Example 5 was changed to rice seed koji ( Aspergillus oryzae , Shirakami, manufactured by Akita Konno Shoten Co., Ltd.).

Comparative Production Example 3: Preparation of Thyme Extract
A "thyme extract" was obtained in the same manner as in Comparative Production Example 1, except that mugwort in Comparative Production Example 1 was replaced with thyme ( Thymus vulgaris Linne) (manufactured by Albion Co., Ltd.).

(Test Example A-3: Measurement of Contact Angle)
The contact angles were measured in the same manner as in Test Example A-1, except that the test samples in Test Example A-1 were changed to Thymus fermented liquid 1 obtained in Production Example 5, Thymus fermented liquid 2 obtained in Production Example 6, and Thymus extract obtained in Comparative Production Example 3. The results are shown in Table 13 below. Examples of droplets when measuring the contact angles of each test sample are shown in Figures 3A to 3C.

Compared to the thyme extract obtained in Comparative Production Example 3, the thyme fermented liquid 1 obtained in Production Example 5 and the thyme fermented liquid 2 obtained in Production Example 6 both had small contact angles of 87° or less, and were excellent in skin compatibility. Furthermore, the thyme fermented liquid 1 obtained in Production Example 5 had a contact angle of 81° or less, and was even more excellent in skin compatibility.

(Test Example 3-1: Transglutaminase-1 (TGM-1) mRNA expression promoting effect test)
The thyme fermented liquid 1 obtained in Production Example 5, the thyme fermented liquid 2 obtained in Production Example 6, and the thyme extract obtained in Comparative Production Example 3 were used as test samples to test the transglutaminase-1 (TGM-1) mRNA expression promoting effect by the following test method.

Each test sample was dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries, Ltd.) to give a final concentration shown in Table 14 below, to prepare a medium containing the test sample.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurashiki Boseki Co., Ltd.) were cultured in a normal human epidermal keratinocyte growth medium (HuMedia-KG2, manufactured by Kurashiki Boseki Co., Ltd.) at 37°C and 5% _CO2 until confluent, and the cells were then harvested by trypsin treatment. The cells were then adjusted to 1.5 x ¹⁰⁵ cells/mL using normal human epidermal keratinocyte growth medium (HuMedia-KG2).
Next, 2 mL of the NHEK (1.5 x ¹⁰⁵ cells/mL) was seeded in a 35 mm petri dish and cultured overnight at ₃₇ ° C. and 5% CO2. After the culture was completed, the medium was replaced with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the culture was completed, the medium was replaced with 2 mL of the test sample-added medium and cultured for 24 hours at 37 ° C. and 5% _CO2 . After the culture was completed, the culture medium was removed, and total RNA was extracted with an RNA extraction reagent (ISOGEN II (catalog number: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared to be 200 ng / μL using purified water.

As a control, the same procedures and absorbance measurements were carried out as above, except that 2 mL of the medium containing the test sample was replaced with 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) not containing the test sample, and total RNA was prepared to 200 ng/μL using the same method as above.

Using each of the total RNAs as a template, the expression levels of transglutaminase-1 (TGM-1) mRNA and GAPDH mRNA as an internal standard were measured. The mRNAs were detected by a two-step real-time PCR reaction using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (catalog number: RR063A), manufactured by Takara Bio Inc.).
The expression level of TGM-1 mRNA in the absence of test sample and the presence of test sample was corrected by the expression level of GAPDH mRNA. From this corrected value, the TGM-1 mRNA expression promotion rate was calculated according to the following formula 10. The results are shown in Table 14 below.
<Formula 10>
TGM-1 mRNA expression promotion rate (%) = A/B x 100
In the formula 10, A and B respectively represent the following.
A: Corrected value when test sample was added B: Corrected value when test sample was not added

(Test Example 3-2: Aquaporin 3 (AQP3) mRNA expression promoting effect test)
The thyme fermented liquid 1 obtained in Production Example 5, the thyme fermented liquid 2 obtained in Production Example 6, and the thyme extract obtained in Comparative Production Example 3 were used as test samples to test the aquaporin 3 (AQP3) mRNA expression promoting effect by the following test method.

Each test sample was dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries, Ltd.) to give a final concentration shown in Table 15 below, to prepare a medium containing the test sample.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurashiki Boseki Co., Ltd.) were cultured in a normal human epidermal keratinocyte growth medium (HuMedia-KG2, manufactured by Kurashiki Boseki Co., Ltd.) at 37°C and 5% _CO2 until confluent, and the cells were then harvested by trypsin treatment. The cells were then adjusted to 1.5 x ¹⁰⁵ cells/mL using normal human epidermal keratinocyte growth medium (HuMedia-KG2).
Next, 2 mL of the NHEK (1.5 x ¹⁰⁵ cells/mL) was seeded in a 35 mm petri dish and cultured overnight at ₃₇ ° C. and 5% CO2. After the culture was completed, the medium was replaced with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the culture was completed, the medium was replaced with 2 mL of the test sample-added medium and cultured for 24 hours at 37 ° C. and 5% _CO2 . After the culture was completed, the culture medium was removed, and total RNA was extracted with an RNA extraction reagent (ISOGEN II (catalog number: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared to be 200 ng / μL using purified water.

As a control, the same procedures and absorbance measurements were carried out as above, except that 2 mL of the medium containing the test sample was replaced with 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) not containing the test sample, and total RNA was prepared to 200 ng/μL using the same method as above.

Using each of the total RNAs as a template, the expression levels of aquaporin 3 (AQP3) mRNA and GAPDH mRNA as an internal standard were measured. The mRNAs were detected by a two-step real-time PCR reaction using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (catalog number: RR063A), manufactured by Takara Bio Inc.).
The expression level of AQP3 mRNA in the absence and presence of the test sample was corrected by the expression level of GAPDH mRNA. The AQP3 mRNA expression promotion rate was calculated from this corrected value according to the following formula 11. The results are shown in Table 15 below.
<Formula 11>
AQP3 mRNA expression promotion rate (%) = A / B × 100
In the formula 11, A and B respectively represent the following.
A: Corrected value when test sample was added B: Corrected value when test sample was not added

(Test Example 3-3: Test of promoting effect of occludin mRNA expression)
In Test Example 2-5, the thyme fermented liquid 1 obtained in Production Example 5, the thyme fermented liquid 2 obtained in Production Example 6, and the thyme extract obtained in Comparative Production Example 3 were used as the test samples, and the final concentrations of the test samples were changed to the concentrations shown in Table 16 below, and the occludin mRNA expression promoting effect was tested in the same manner as in Test Example 2-5. The results are shown in Table 16 below.

(Test Example 3-4: DPPH radical scavenging effect test)
In Test Example 1-3, the DPPH radical scavenging activity was tested in the same manner as in Test Example 1-3, except that the test samples were changed to the thyme fermented liquid 1 obtained in Production Example 5, the thyme fermented liquid 2 obtained in Production Example 6, and the thyme extract obtained in Comparative Production Example 3, and the final concentrations of the test samples were changed to the concentrations shown in Table 17 below. The results are shown in Table 17 below.

<Production Example 7: Preparation of peppermint fermentation liquid 1>
A "peppermint seed koji" was prepared in the same manner as in the seed koji preparation step of Production Example 1, except that in the seed koji preparation step of Production Example 1, mugwort was changed to peppermint ( Melissa officinalis Linne) (manufactured by Albion Co., Ltd.).
In addition, "peppermint fermented liquid 1" was obtained in the same manner as in the fermentation process of Production Example 1, except that in the fermentation process of Production Example 1, mugwort was replaced with peppermint ( Melissa officinalis Linne) (manufactured by Albion Co., Ltd.).

<Production Example 8: Preparation of peppermint fermentation liquid 2>
A "Japanese peppermint fermented liquid 2" was obtained in the same manner as in Production Example 7, except that the Japanese peppermint seed koji in Production Example 7 was changed to rice seed koji ( Aspergillus oryzae , Shirakami, manufactured by Akita Konno Shoten Co., Ltd.).

Comparative Production Example 4: Preparation of peppermint extract
A "peppermint extract" was obtained in the same manner as in Comparative Production Example 1, except that the mugwort in Comparative Production Example 1 was replaced with peppermint ( Melissa officinalis Linne) (manufactured by Albion Co., Ltd.).

(Test Example A-4: Measurement of Contact Angle)
The contact angles were measured in the same manner as in Test Example A-1, except that the test samples in Test Example A-1 were changed to the peppermint fermentation liquid 1 obtained in Production Example 7, the peppermint fermentation liquid 2 obtained in Production Example 8, and the peppermint extract obtained in Comparative Production Example 4. The results are shown in Table 18 below. Examples of droplets when measuring the contact angles of each test sample are shown in Figures 4A to 4C.

Compared to the peppermint extract obtained in Comparative Production Example 4, peppermint fermentation liquid 1 obtained in Production Example 7 and peppermint fermentation liquid 2 obtained in Production Example 8 both had small contact angles of 85° or less, and were excellent in skin compatibility. Furthermore, peppermint fermentation liquid 1 obtained in Production Example 7 had a contact angle of 79° or less, and was even more excellent in skin compatibility.

(Test Example 4-1: Type I collagen production promoting effect test)
In Test Example 2-2, the type I collagen production promoting effect was tested in the same manner as in Test Example 2-2, except that the test samples were changed to the peppermint fermentation liquid 1 obtained in Production Example 7, the peppermint fermentation liquid 2 obtained in Production Example 8, and the peppermint extract obtained in Comparative Production Example 4, and the final concentrations of the test samples were changed to the concentrations shown in Table 19 below. The results are shown in Table 19 below.

(Test Example 4-2: Aquaporin 3 (AQP3) mRNA expression promoting effect test)
In Test Example 3-2, the test samples were changed to the peppermint fermentation liquid 1 obtained in Production Example 7, the peppermint fermentation liquid 2 obtained in Production Example 8, and the peppermint extract obtained in Comparative Production Example 4, and the final concentrations of the test samples were changed to the concentrations shown in Table 20 below, but the aquaporin 3 (AQP3) mRNA expression promoting effect was tested in the same manner as in Test Example 3-2. The results are shown in Table 20 below.

(Test Example 4-3: DPPH radical scavenging effect test)
In Test Example 1-3, the test samples were changed to the peppermint fermentation liquid 1 obtained in Production Example 7, the peppermint fermentation liquid 2 obtained in Production Example 8, and the peppermint extract obtained in Comparative Production Example 4, and the final concentrations of the test samples were changed to the concentrations shown in Table 21 below. The DPPH radical scavenging activity was tested in the same manner as in Test Example 1-3. The results are shown in Table 21 below.

(Test Example 4-4: Hyaluronidase activity inhibitory effect test)
In Test Example 1-4, the test samples were changed to the peppermint fermentation liquid 1 obtained in Production Example 7, the peppermint fermentation liquid 2 obtained in Production Example 8, and the peppermint extract obtained in Comparative Production Example 4, and the final concentrations of the test samples were changed to the concentrations shown in Table 22 below, but the hyaluronidase activity inhibitory effect was tested in the same manner as in Test Example 1-4. The results are shown in Table 22 below.

(Test Example 4-5: Tyrosinase activity inhibitory effect test)
In Test Example 1-5, the test samples were changed to the peppermint fermentation liquid 1 obtained in Production Example 7, the peppermint fermentation liquid 2 obtained in Production Example 8, and the peppermint extract obtained in Comparative Production Example 4, and the final concentrations of the test samples were changed to the concentrations shown in Table 22 below, but the tyrosinase activity inhibitory effect was tested in the same manner as in Test Example 1-5. The results are shown in Table 23 below.

<Production Example 9: Preparation of cornflower fermented liquid 1>
A "cornflower seed koji" was prepared in the same manner as in the seed koji preparation step of Production Example 1, except that in the seed koji preparation step of Production Example 1, artemisia was changed to cornflower ( Centaurea cyanus Linne) (manufactured by Albion Co., Ltd.).
In addition, a "cornflower fermented liquid 1" was obtained in the same manner as in the fermentation step of Production Example 1, except that in the fermentation step of Production Example 1, artemisanthemum was replaced with cornflower ( Centaurea cyanus Linne) (manufactured by Albion Co., Ltd.).

<Production Example 10: Preparation of cornflower fermentation liquid 2>
A "cornflower fermented liquid 2" was obtained in the same manner as in Production Example 9, except that the cornflower seed koji in Production Example 9 was changed to rice seed koji ( Aspergillus oryzae , Shirakami, manufactured by Akita Konno Shoten Co., Ltd.).

Comparative Production Example 5: Preparation of Cornflower Extract
A "cornflower extract" was obtained in the same manner as in Comparative Production Example 1, except that artemisia was replaced with cornflower ( Centaurea cyanus Linne) (manufactured by Albion Co., Ltd.).

(Test Example A-5: Measurement of Contact Angle)
The contact angles were measured in the same manner as in Test Example A-1, except that the test samples in Test Example A-1 were changed to the cornflower fermented liquor 1 obtained in Production Example 9, the cornflower fermented liquor 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5. The results are shown in Table 24 below. Examples of droplets when measuring the contact angles of each test sample are shown in Figures 5A to 5C.

Compared to the cornflower extract obtained in Comparative Production Example 5, the cornflower fermented liquid 1 obtained in Production Example 9 and the cornflower fermented liquid 2 obtained in Production Example 10 both had small contact angles of 85° or less, and were excellent in compatibility with the skin. Furthermore, the cornflower fermented liquid 1 obtained in Production Example 9 had a contact angle of 79° or less, and was even more excellent in compatibility with the skin.

(Test Example 5-1: Type I collagen production promoting effect test)
In Test Example 2-2, the type I collagen production promoting effect was tested in the same manner as in Test Example 2-2, except that the test samples were changed to the cornflower fermented liquid 1 obtained in Production Example 9, the cornflower fermented liquid 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5, and the final concentrations of the test samples were changed to the concentrations shown in Table 25 below. The results are shown in Table 25 below.

(Test Example 5-2: Transglutaminase-1 mRNA expression promoting effect test)
The transglutaminase-1 mRNA expression promoting effect was tested in the same manner as in Test Example 3-1, except that in Test Example 3-1, the test samples were changed to the cornflower fermented broth 1 obtained in Production Example 9, the cornflower fermented broth 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5, and the final concentrations of the test samples were changed to the concentrations shown in Table 26 below. The results are shown in Table 26 below.

(Test Example 5-3: Filaggrin mRNA expression promoting effect test)
The cornflower fermentation liquid 1 obtained in Production Example 9, the cornflower fermentation liquid 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5 were used as test samples to test the filaggrin mRNA expression promoting effect by the test method described below.

Each test sample was dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries, Ltd.) to give a final concentration shown in Table 27 below, to prepare a medium containing the test sample.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurashiki Boseki Co., Ltd.) were cultured in a normal human epidermal keratinocyte growth medium (HuMedia-KG2, manufactured by Kurashiki Boseki Co., Ltd.) at 37°C and 5% _CO2 until confluent, and the cells were then harvested by trypsin treatment. The cells were then adjusted to 1.5 x ¹⁰⁵ cells/mL using normal human epidermal keratinocyte growth medium (HuMedia-KG2).
Next, 2 mL of the NHEK (1.5 x ¹⁰⁵ cells/mL) was seeded in a 35 mm petri dish and cultured overnight at ₃₇ ° C. and 5% CO2. After the culture was completed, the medium was replaced with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the culture was completed, the medium was replaced with 2 mL of the test sample-added medium and cultured for 24 hours at 37 ° C. and 5% _CO2 . After the culture was completed, the culture medium was removed, and total RNA was extracted with an RNA extraction reagent (ISOGEN II (catalog number: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared to be 200 ng / μL using purified water.

As a control, the same procedures and absorbance measurements were carried out as above, except that 2 mL of the medium containing the test sample was replaced with 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) not containing the test sample, and total RNA was prepared to 200 ng/μL using the same method as above.

Using each of the total RNAs as a template, the expression levels of filaggrin mRNA and GAPDH mRNA as an internal standard were measured. The mRNA was detected by a two-step real-time PCR reaction using a real-time PCR device (Thermal Cycler Dice (registered trademark) Real Time System III, manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (catalog number: RR063A), manufactured by Takara Bio Inc.).
The expression levels of filaggrin mRNA in the absence and presence of the test sample were corrected with the expression level of GAPDH mRNA. From this corrected value, the filaggrin mRNA expression promotion rate was calculated according to the following formula 12. The results are shown in Table 27 below.
<Formula 12>
Filaggrin mRNA expression promotion rate (%) = A/B x 100
In the formula 12, A and B respectively represent the following.
A: Corrected value when test sample was added B: Corrected value when test sample was not added

(Test Example 5-4: Aquaporin 3 (AQP3) mRNA expression promoting effect test)
In Test Example 3-2, the aquaporin 3 (AQP3) mRNA expression promoting effect was tested in the same manner as in Test Example 3-2, except that the test samples were changed to the cornflower fermentation liquid 1 obtained in Production Example 9, the cornflower fermentation liquid 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5, and the final concentrations of the test samples were changed to the concentrations shown in Table 28 below. The results are shown in Table 28 below.

(Test Example 5-5: Hyaluronic acid synthase 3 (HAS3) mRNA expression promoting effect test)
In Test Example 1-2, the test samples were changed to the cornflower fermentation liquid 1 obtained in Production Example 9, the cornflower fermentation liquid 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5, and the final concentrations of the test samples were changed to the concentrations shown in Table 29 below, but the hyaluronic acid synthase 3 (HAS3) mRNA expression promoting effect was tested in the same manner as in Test Example 1-2. The results are shown in Table 29 below.

(Test Example 5-6: Claudin-1 mRNA expression promoting effect test)
In Test Example 2-3, the promoting effect of claudin-1 mRNA expression was tested in the same manner as in Test Example 2-3, except that the test samples were changed to the cornflower fermented broth 1 obtained in Production Example 9, the cornflower fermented broth 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5, and the final concentrations of the test samples were changed to the concentrations shown in Table 30 below. The results are shown in Table 30 below.

(Test Example 5-7: Claudin-4 mRNA expression promoting effect test)
In Test Example 2-4, the promoting effect of claudin-4 mRNA expression was tested in the same manner as in Test Example 2-4, except that the test samples were changed to the cornflower fermented broth 1 obtained in Production Example 9, the cornflower fermented broth 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5, and the final concentrations of the test samples were changed to the concentrations shown in the following Table 31. The results are shown in the following Table 31.

(Test Example 5-8: Test of occludin mRNA expression promoting effect)
In Test Example 2-5, the occludin mRNA expression promoting effect was tested in the same manner as in Test Example 2-5, except that the test samples were changed to the cornflower fermented broth 1 obtained in Production Example 9, the cornflower fermented broth 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5, and the final concentrations of the test samples were changed to the concentrations shown in Table 32 below. The results are shown in Table 32 below.

(Test Example 5-9: DPPH radical scavenging effect test)
In Test Example 1-3, the DPPH radical scavenging activity was tested in the same manner as in Test Example 1-3, except that the test samples were changed to the cornflower fermentation liquid 1 obtained in Production Example 9, the cornflower fermentation liquid 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5, and the final concentrations of the test samples were changed to the concentrations shown in Table 33 below. The results are shown in Table 33 below.

(Test Example 5-10: Test for melanin production inhibitory effect on B16 melanoma cells)
The cornflower fermented liquid 1 obtained in Production Example 9, the cornflower fermented liquid 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5 were used as test samples to test their inhibitory effect on melanin production in B16 melanoma cells by the following test method.

Each test sample was dissolved in DMEM (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% FBS (manufactured by Biosera) and 1 mmol/L theophylline (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a medium containing the test sample.
B16 melanoma cells were cultured in 10% FBS-containing DMEM under conditions of 37°C and 5% _CO2 until they became confluent, and then the cells were harvested by trypsinization. The cells were then adjusted to 2.4 x ¹⁰⁵ cells/mL with DMEM containing 10% FBS and 1 mmol/L theophylline.
Next, the B16 melanoma cells (2.4 x ¹⁰⁵ cells/mL) were seeded in a 48-well plate at 300 μL per well and cultured at 37°C and 5% _CO2 for 6 hours. After the culture was completed, the medium was replaced with 100 μL of medium containing the test sample, and cultured at 37°C and 5% _CO2 for 3 days. After the culture was completed, 300 μL of the medium containing the test sample was added per well, and cultured at 37°C and 5% _CO2 for 4 days. The final concentration of the test sample at this time was the concentration shown in Table 34 below.

After the incubation, the medium was removed from each well, 200 μL of 2 mol/L sodium hydroxide solution was added, the cells were disrupted using an ultrasonic disrupter, 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).

To measure cell viability, B16 melanoma cells were cultured in the test sample-added medium in the same manner as above, and then the medium was removed and washed with 400 μL of PBS buffer. Next, 200 μL of a solution of neutral red dissolved in 10% FBS-containing DMEM at a final concentration of 0.05 mg/mL was added to each well and cultured for 2.5 hours. After the culture was completed, the neutral red solution was removed, and 200 μL of an ethanol-acetic acid solution (ethanol:acetic acid:water = 50:1:49 (volume ratio)) was added to each well to extract the pigment. After extraction, the absorbance at a wavelength of 540 nm was measured.

As a control, the same procedures and absorbance measurements were performed as above, except that the test sample solution was changed to DMEM containing 10% FBS and 1 mmol/L theophylline without the test sample.

From the obtained measured values, the melanin production inhibition rate (%) corrected by the cell viability calculated based on the following formula 13 was calculated based on the following formula 14. The results are shown in Table 34 below.
<Formula 13>
Cell viability (%) = (C/D) x 100
In the formula 13, C and D respectively represent the following.
C: Absorbance at a wavelength of 540 nm when no test sample is added D: Absorbance at a wavelength of 540 nm when a test sample is added <Equation 14>
Melanin production inhibition rate (%) = {1 - (B/D)/(A/C)} x 100
In the formula 14, A to D respectively represent the following.
A: Amount of melanin without test sample added B: Amount of melanin with test sample added C: Absorbance at a wavelength of 540 nm without test sample added D: Absorbance at a wavelength of 540 nm with test sample added

The anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention have at least one of an excellent anti-aging effect, an antioxidant effect, an anti-inflammatory effect, and a whitening effect, and are highly safe natural product-based agents, and therefore can be widely used in a wide range of fields, including cosmetics, foods, and pharmaceuticals.
The cosmetic preparation of the present invention contains at least one selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent of the present invention, and therefore can be suitably used for skin cosmetics such as lotions, milky lotions, creams, ointments, beauty essences, lotions, packs, jelly, lip balm, lipstick, foundations, bath additives, soaps, and body shampoos; and scalp and hair cosmetics such as astringents, hair tonics, hair creams, hair liquids, pomades, shampoos, and rinses.

Claims (10)

The present invention contains, as an active ingredient, a fermented liquid obtained by a seed koji preparation step of inoculating 5 to 100 parts by mass of Aspergillus oryzae at 1 x ¹⁰³ /mL to 1 x ¹⁰⁸ /mL per 100 parts by mass of Japanese Artemisia ananasima and culturing at 20°C to 40°C for 80 to 210 hours to obtain Japanese Artemisia seed koji, and a fermentation step of adding 500 to 5,000 parts by mass of water per 100 parts by mass of Japanese Artemisia ananasima, inoculating 1 x ¹⁰³ /mL to 1 x ¹⁰⁸ /mL of the Japanese Artemisia seed koji, and fermenting at 20°C to 40°C for 10 to 40 hours ,
An anti-aging agent characterized by having at least one effect selected from the group consisting of an inhibitory effect on matrix metalloproteinase-1 activity and an effect of promoting expression of hyaluronic acid synthase 3 mRNA. For 100 parts by mass of Artemisia capillaris, 1 x 10 ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation process in which 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) having a concentration of 100 to 100 cells/mL is inoculated and cultured at 20° C. to 40° C. for 80 to 210 hours to obtain a seed koji of Artemisia kawara. ³ pieces/mL ~ 1×10 ⁸ The present invention contains, as an active ingredient, a mugwort fermentation liquid obtained by inoculating the mugwort seed koji at a concentration of 100/mL and fermenting the koji at 20°C to 40°C for 10 to 40 hours,
An anti-aging agent characterized by having at least one action selected from the group consisting of an action of promoting hyaluronic acid synthase 3 mRNA expression, an action of promoting type I collagen production, an action of promoting claudin-1 mRNA expression, an action of promoting claudin-4 mRNA expression, and an action of promoting occludin mRNA expression. 1 x 10 for 100 parts by mass of Thymus vulgare ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation step of inoculating 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) at a concentration of 100 to 150 cells/mL and culturing the mixture at 20° C. to 40° C. for 80 to 210 hours to obtain thyme seed koji; and a step of adding 500 to 5,000 parts by mass of water to 100 parts by mass of thyme, and then culturing the mixture at 1×10 ³ pieces/mL ~ 1×10 ⁸ a fermentation step of inoculating the Thymus vulgaris seed koji at a concentration of 100/mL and fermenting the koji at 20°C to 40°C for 10 to 40 hours; and
An anti-aging agent characterized by having at least one action selected from the group consisting of an action of promoting occludin mRNA expression, an action of promoting transglutaminase-1 mRNA expression, and an action of promoting aquaporin 3 mRNA expression. 1 x 10 for 100 parts by mass of peppermint ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation process is a process in which 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) having a concentration of 100 to 150 cells/mL is inoculated and cultured at 20° C. to 40° C. for 80 to 210 hours to obtain Japanese peppermint seed koji; and a process in which 500 to 5,000 parts by mass of water is added to 100 parts by mass of Japanese peppermint, and then 1×10 ³ pieces/mL ~ 1×10 ⁸ The method comprises the steps of inoculating the Japanese peppermint seed koji at a concentration of 1000/mL and fermenting the mixture at 20°C to 40°C for 10 to 40 hours, and the step of fermenting the mixture at 20°C to 40°C for 10 to 40 hours.
An anti-aging agent characterized by having at least one action selected from the group consisting of an action of promoting type I collagen production and an action of promoting transglutaminase-1 mRNA expression. 1 x 10 for 100 parts by mass of cornflower ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation step of inoculating 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) at 100 parts by mass of cornflowers/mL and culturing the mixture at 20° C. to 40° C. for 80 to 210 hours to obtain cornflower seed koji; and a step of adding 500 to 5,000 parts by mass of water per 100 parts by mass of cornflowers and then culturing the mixture at 1×10 ³ pieces/mL ~ 1×10 ⁸ and a fermentation step of inoculating the cornflower seed koji at a concentration of cornflower koji seeds/mL and fermenting the koji at 20° C. to 40° C. for 10 to 40 hours.
An anti-aging agent characterized by having at least one action selected from the group consisting of hyaluronic acid synthase 3 mRNA expression promoting action, type I collagen production promoting action, claudin-1 mRNA expression promoting action, claudin-4 mRNA expression promoting action, occludin mRNA expression promoting action, transglutaminase-1 mRNA expression promoting action, aquaporin 3 mRNA expression promoting action, and filaggrin mRNA expression promoting action. For 100 parts by mass of mugwort, 1 x 10 ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation process is performed by inoculating 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) at a concentration of 100 to 150 cells/mL and culturing the mixture at 20 to 40° C. for 80 to 210 hours to obtain a seed koji of Yamayomogi. A seed koji preparation process is performed by adding 500 to 5,000 parts by mass of water to 100 parts by mass of Yamayomogi, and then culturing the mixture at 1×10 ³ pieces/mL ~ 1×10 ⁸ A fermentation step of inoculating the Yamayomogi seed koji at a concentration of 100/mL and fermenting at 20°C to 40°C for 10 to 40 hours; and
1 x 10 for 100 parts by mass of thyme ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation step of inoculating 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) at a concentration of 100 to 150 cells/mL and culturing the mixture at 20° C. to 40° C. for 80 to 210 hours to obtain thyme seed koji; and a step of adding 500 to 5,000 parts by mass of water to 100 parts by mass of thyme, and then culturing the mixture at 1×10 ³ pieces/mL ~ 1×10 ⁸ a fermentation step of inoculating the thyme seed koji at a concentration of 1000/mL and fermenting the koji at 20°C to 40°C for 10 to 40 hours;
1 x 10 for 100 parts by mass of peppermint ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation process is a process in which 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) having a concentration of 100 to 150 cells/mL is inoculated and cultured at 20° C. to 40° C. for 80 to 210 hours to obtain Japanese peppermint seed koji; and a process in which 500 to 5,000 parts by mass of water is added to 100 parts by mass of Japanese peppermint, and then 1×10 ³ pieces/mL ~ 1×10 ⁸ A fermentation step of inoculating the Japanese peppermint seed koji at a concentration of 100/mL and fermenting the mixture at 20°C to 40°C for 10 to 40 hours; and
1 x 10 for 100 parts by mass of cornflower ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation step of inoculating 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) at 100 parts by mass of cornflowers/mL and culturing the mixture at 20° C. to 40° C. for 80 to 210 hours to obtain cornflower seed koji; and a step of adding 500 to 5,000 parts by mass of water per 100 parts by mass of cornflowers and then culturing the mixture at 1×10 ³ pieces/mL ~ 1×10 ⁸ a cornflower fermentation liquid obtained by inoculating the cornflower seed koji at a concentration of 100/mL and fermenting the koji at 20°C to 40°C for 10 to 40 hours;
Contains at least one fermentation liquid selected from the group consisting of:
An antioxidant characterized by having a diphenyl-p-picrylhydrazyl radical scavenging effect. For 100 parts by mass of mugwort, 1 x 10 ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation process is performed by inoculating 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) at a concentration of 100 to 150 cells/mL and culturing the mixture at 20 to 40° C. for 80 to 210 hours to obtain a seed koji of Yamayomogi. A seed koji preparation process is performed by adding 500 to 5,000 parts by mass of water to 100 parts by mass of Yamayomogi, and then culturing the mixture at 1×10 ³ pieces/mL ~ 1×10 ⁸ A fermentation step of inoculating the Yamayomogi seed koji at a concentration of 100/mL and fermenting at 20°C to 40°C for 10 to 40 hours; and
1 x 10 for 100 parts by mass of peppermint ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation process is a process in which 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) having a concentration of 100 to 150 cells/mL is inoculated and cultured at 20° C. to 40° C. for 80 to 210 hours to obtain Japanese peppermint seed koji; and a process in which 500 to 5,000 parts by mass of water is added to 100 parts by mass of Japanese peppermint, and then 1×10 ³ pieces/mL ~ 1×10 ⁸ a fermentation step of inoculating the Japanese peppermint seed koji at a concentration of 100/mL and fermenting the mixture at 20°C to 40°C for 10 to 40 hours; and
Contains at least one fermentation liquid selected from the group consisting of:
An anti-inflammatory agent characterized by having an inhibitory effect on hyaluronidase activity. 1 x 10 for 100 parts by mass of peppermint ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation process is a process in which 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) having a concentration of 100 to 1000 cells/mL is inoculated and cultured at 20° C. to 40° C. for 80 to 210 hours to obtain Japanese peppermint seed koji; and a process in which 500 to 5,000 parts by mass of water is added to 100 parts by mass of Japanese peppermint, and then 1×10 ³ pieces/mL ~ 1×10 ⁸ and a fermentation step of inoculating the Japanese peppermint seed koji at a concentration of 100/mL and fermenting the mixture at 20°C to 40°C for 10 to 40 hours.
A skin whitening agent characterized by having a tyrosinase activity inhibitory effect. 1 x 10 for 100 parts by mass of cornflower ³ pieces/mL ~ 1×10 ⁸ A seed koji preparation step of inoculating 5 to 100 parts by mass of Aspergillus oryzae (Aspergillus oryzae) at 100 parts by mass of cornflowers/mL and culturing the mixture at 20° C. to 40° C. for 80 to 210 hours to obtain cornflower seed koji; and a step of adding 500 to 5,000 parts by mass of water per 100 parts by mass of cornflowers and then culturing the mixture at 1×10 ³ pieces/mL ~ 1×10 ⁸ and a fermentation step of inoculating the cornflower seed koji at a concentration of cornflower koji seeds/mL and fermenting the koji at 20° C. to 40° C. for 10 to 40 hours.
A skin whitening agent characterized by having at least one melanin production inhibitory effect. A cosmetic preparation comprising at least one selected from the group consisting of the anti-aging agent according to claim 1, 2 or 4, the antioxidant according to claim 6, the anti-inflammatory agent according to claim 7 and the whitening agent according to claim 8 (excluding those containing thyme fermentation liquor or cornflower fermentation liquor).