TW202102239A - Antiaging agent, antioxidant, antiinflammatory agent and whitening agent, and cosmetic - Google Patents

Abstract

An antiaging agent that comprises, as an active ingredient, at least one of a fermented liquid of an Artemisia plant, a fermented liquid of Thymus vulgaris, a fermented liquid of Melissa officinalis and a fermented liquid of Centaurea cyanus; an antioxidant that comprises, as an active ingredient, at least one of a fermented liquid of an Artemisia plant, a fermented liquid of Thymus vulgaris, a fermented liquid of Melissa officinalis and a fermented liquid of Centaurea cyanus; an antiinflammatory agent that comprises, as an active ingredient, at least one of a fermented liquid of an Artemisia plant and a fermented liquid of Melissa officinalis; a whitening agent that comprises, as an active ingredient, at least one of a fermented liquid of an Artemisia plant, a fermented liquid of Melissa officinalis and a fermented liquid of Centaurea cyanus; and a cosmetic.

Description

Anti-aging agents, antioxidants, anti-inflammatory agents, and whitening agents, and cosmetics

The present invention relates to an anti-aging agent, an antioxidant, an anti-inflammatory agent, and a whitening agent containing the fermentation broth obtained from the koji fungus of a plant as an active ingredient, and the anti-inflammatory agent, the anti-oxidant, and the anti-inflammatory agent , And cosmetics of at least any one of the aforementioned whitening agents.

In recent years, active oxygen has attracted attention as a factor that oxidizes biological components, and its adverse effects on living organisms have become a problem. Energy metabolism within the biological active oxygen produced by cells, super oxide (superoxide) [i.e., a superoxide anion generated electron reduction of oxygen molecules (· O ₂ ^-), hydrogen peroxide ( H ₂ O ₂ ), singlet oxygen ( ¹ O ₂ ), hydroxy radical (・OH)], etc. Such reactive oxygen species are necessary for the sterilization mechanism of phagocytes and play an important role in the removal of viruses and cancer cells.

However, the excessive production of the aforementioned reactive oxygen species will attack the biological molecules constituting the membranes and tissues in the body, and induce various diseases. Generally, superoxide, which is the starting material of other reactive oxygen species produced in the body, is gradually eliminated by the catalytic action of superoxide dismutase (SOD) contained in the cell. However, when the production of superoxide is excessive or the effect of SOD is reduced, the elimination of superoxide becomes insufficient and the concentration of superoxide increases. This is believed to cause tissue damage such as rheumatoid arthritis, Behcet's disease, myocardial infarction, stroke, cataract, spots, freckles, wrinkles, diabetes, arteriosclerosis, stiff shoulders, cold sensitivity, skin One of the causes of aging and so on.

Among these, because the skin is directly stimulated by environmental factors such as ultraviolet rays, it is an organ that easily produces superoxide. As a result of the increase in superoxide concentration, for example, there is the so-called decomposition of collagen and other biological tissues. Denaturation, cross-linking, or oxidation of oils and fats to produce lipid peroxides that damage cells, forming skin wrinkles, causing skin aging, inflammation, and skin pigmentation problems such as decreased elasticity of the skin (see Non-Patent Document 1 ). Therefore, it is generally believed that by inhibiting the generation of reactive oxygen species or free radicals in the organism, it is possible to prevent, treat or improve skin aging such as wrinkle formation, reduced elasticity, and tissue damage such as rheumatoid arthritis or Behcet’s disease. , Myocardial infarction, stroke, cataract, diabetes, arteriosclerosis, shoulder stiffness, cold sensitivity and other injuries involving active oxygen.

Therefore, attempts have been made to obtain active oxygen scavengers, free radical scavengers, hydrogen peroxide scavengers, etc., from the viewpoint of safety, which are advantageous natural products, and have been extracted from Brassicaceae (Brassicaceae) plants. (Refer to Patent Document 1), extracts of Crassulaceae (Crassulaceae) plants of the genus Kalanchoe (refer to Patent Document 2), extracts of P. sylvestris (refer to Patent Document 3), extracts of young sweet potato leaves (refer to Patent Document 4) etc. confirmed the validity.

In addition, inflammatory diseases, such as contact dermatitis (rash), psoriasis, pemphigus vulgaris, and other skin diseases associated with rough skin, have various causes and disease mechanisms. For this reason, it is known that it is mainly caused by the hyperactivity of hyaluronidase (hyaluronidase).

Hyaluronidase is the hydrolytic enzyme of hyaluronic acid. Hyaluronic acid, which retains its affinity for biological tissues, is decomposed by ultraviolet rays, oxygen, etc. in the aqueous system, and the water retention effect is also reduced as the molecular weight decreases. In addition, hyaluronic acid exists as an intercellular tissue in organisms and also participates in vascular permeability. Furthermore, hyaluronidase is present in mast cells, but is freed by degranulation caused by its activation, and acts as a chemical agent of inflammation. Accordingly, by inhibiting the activity of hyaluronidase, the enhancement of moisturizing and the prevention and reduction of inflammation are expected. As for those having such hyaluronidase activity inhibitory effect, for example, an extract of Osbeckia plants (see Patent Document 5), an extract of Ampelopsis grossedentata (see Patent Document 6), and the like are known.

In addition, in the skin, melanin also has the function of protecting organisms from ultraviolet rays, but excessive production or uneven accumulation can cause skin darkening or spots. Generally speaking, melanin changes from tyrosine to dopa and from dopa to dopaquinone by the action of the enzyme tyrosinase produced and synthesized in pigment cells, and then 5 , 6-Dihydroxyindoxyl and other intermediates. Therefore, in order to prevent, treat, or improve the dark color of the skin (skin pigmentation), spots, freckles, etc., it is considered to inhibit the activity of tyrosinase related to the production of melanin, or to inhibit the production of melanin.

Historically, for the prevention, treatment or improvement of skin pigmentation, spots, freckles, etc., whitening agents that use chemical compounds such as hydroquinone as active ingredients have been treated as external use. However, chemical compounds such as hydroquinone may have side effects such as skin irritation and allergies. Therefore, it is hoped that the development of a whitening agent that uses a natural raw material with high safety as an effective ingredient is known. For those that have a tyrosinase activity inhibitory effect, for example, Tengcha extract (refer to Patent Document 7), Polygonum serrata (Polygonum hydropiper) extract (see Patent Document 8) and the like. In addition, for those having a melanin production inhibitory effect, for example, extracts derived from the roots of Ricinus communis (see Patent Document 9) and extracts derived from plants belonging to the genus Saussurea are known. (Refer to Patent Document 10) and so on.

In addition, the epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and extracellular matrix such as collagen located outside these cells to support the skin structure. In young skin, by maintaining the constancy of the interaction of these skin tissues, water retention, softness, elasticity, etc. are ensured, and the skin is maintained in a firm, shiny, fresh and young state in appearance. However, due to the influence of some external factors such as ultraviolet (UV-A, UV-B) radiation, significant dryness of the air, and excessive skin washing, as the aging progresses, it is the main component of the extracellular matrix. While the amount of collagen produced is reduced, it also causes a decrease in elasticity due to cross-linking. As a result, the moisturizing function or elasticity of the skin is reduced, the keratin begins to peel off abnormally, and the skin loses firmness or luster, resulting in aging symptoms such as roughness and wrinkles. Such changes that accompany skin aging include wrinkles, dullness, loss of texture, and reduction in elasticity. Various factors can be cited. However, it is related to extracellular matrix components such as collagen, hyaluronic acid, and elastin. Reduction and degeneration are related. Therefore, it is generally believed that by promoting the production of collagen, hyaluronic acid, etc., skin aging can be improved.

Therefore, in terms of safety, substances with collagen production-promoting effects have been tried to obtain them from beneficial natural products. As for those having a collagen production promotion effect, for example, carambola leaf extract (refer to Patent Document 11), Mallotus philippinensis extract (refer to Patent Document 12) and the like have been confirmed.

In addition, the amino acid, which is the main component of Natural Moisturizing Factors, is produced by decomposing filaggrin derived from keratohyalin granule in the stratum corneum. This filaggrin is expressed as profilaggrin in epidermal keratinocytes existing in the granular layer just below the stratum corneum. After that, it is immediately phosphorylated and accumulated in the transparent keratin granules. After dephosphorylation and hydrolysis, it is decomposed into filaggrin and migrates to the stratum corneum. It improves the agglutination efficiency of keratin filaments and participates in the internal structure of keratin cells. Report (refer to Non-Patent Document 2). In recent years, it has been reported that this filaggrin is very important and indispensable for the moisture retention of the skin, and the synthetic power of filaggrin is reduced due to conditions such as dryness, and the amount of amino acids in the stratum corneum is reduced (see Non-Patent Literature 3). Therefore, it is expected that by promoting the expression of filaggrinogen mRNA in epidermal keratinocytes, and by promoting the synthesis of filaggrin, the amount of amino acids in the stratum corneum can be increased, and the water environment of the stratum corneum can be substantially improved.

Regarding natural-derived filaggrin synthesis accelerators, for example, licorice extract (refer to Patent Document 13), licorice which is known as flavanone glycoside contained in natural plants, has been proposed. Liquiritin (refer to Patent Document 14), and a plant extract or yeast belonging to the genus Citrus as at least one of natural-derived filaggrin and filaggrin production promoter Extract (see Patent Document 15) and the like.

The epidermis continuously produces new keratinocytes through the division and subsequent differentiation of keratinocytes, and has the defense function of protecting the skin from various external stimuli. In particular, in the differentiation process of keratinocytes, from the stratum spinosum to the granular layer, there are protein expressions such as involucrin, which is achieved by the enzyme transglutaminase-1 (transglutaminase-1). It is cross-linked to form a cornified envelope (hereinafter abbreviated as "CE") that encloses the insoluble cell membrane-like structure of keratinocytes, which contributes to the stability of the cytoskeleton and structure of keratinocytes . However, due to various factors, when the production of transglutaminase-1 in the epidermis is reduced, CE is formed in an incomplete state, and keratinization proceeds abnormally. It is generally believed that the result is skin symptoms such as reduced keratinous barrier function and skin moisturizing function, rough skin or dry skin. Therefore, by increasing the production of transglutaminase-1 in the epidermis of keratinocytes and promoting the formation of CE to normalize keratinization, the barrier function of the skin associated with external stimuli such as dryness or ultraviolet rays can be suppressed. Reduce, prevent and improve various skin symptoms such as dry skin and rough skin.

As a natural product-derived transglutaminase-1 production promoter, Morinda citrifolia extract (see Patent Document 16), royal jelly extract (see Patent Document 17), and the like have been proposed.

In addition, in skin cells, aquaporin, which is known as a water channel, is expressed on the cell membrane and is responsible for the task of ingesting low-molecular substances, including water, in the intercellular space. In humans, 13 types of aquaporins (AQP0~AQP12) are known to exist. It is generally believed that aquaporin 3 (AQP3) mainly exists in epidermal cells. In addition to intake of water, it also bears the task of intake of low-molecular compounds such as glycerin or urea that participate in water retention.

However, it has been implied that AQP3 decreases with aging, and it is a factor in the decrease of water retention function. Therefore, it is generally believed that by promoting the performance of AQP3, the water retention function or barrier function caused by aging can be controlled (refer to non- Patent Document 4). As a person having an AQP3 performance promoting effect, for example, an extract derived from the leaf of star fruit (see Patent Document 18) and the like are known.

Traditionally, it is generally believed that the barrier function of the skin is only responsible for the stratum corneum. However, when the protein that constitutes the tight junction (hereinafter abbreviated as "TJ") in the granular layer of the epidermis is missing at the genetic level, the barrier function of the skin The function will collapse, so in recent years, it is considered that TJ also plays an important role in the barrier function of the skin (see Non-Patent Document 5). TJ not only makes adjacent cells close to each other, but also controls the permeability of the substance by sealing the gap between the cells. The components of TJ are cell membrane proteins claudin and occludin. It is generally believed that these proteins constitute the backbone of the TJ strand and control the barrier function of TJ (see Non-Patent Document 6). From the above, the expression of clonectin and clonectin is reduced for some reason, causing the destruction of the TJ structure and losing the function of passing through the barrier as a substance. Therefore, it is expected to become dry skin, rough skin, and ectopic skin. Causes of skin symptoms such as dermatitis and various infections.

Therefore, it is generally believed that by promoting the production of clonectin and fibronectin in the epidermis, the TJ formation of epidermal keratinocytes is promoted, and the barrier function and water retention function of the skin can be improved, and the aforementioned skin symptoms can be prevented or improved. Based on this idea, in terms of enhancing the barrier function of the skin by promoting the formation of TJ, natural extracts of Coptis Rhizome (see Patent Document 19) and hondo spruce have been proposed. Extract (see Patent Document 20) and the like.

In recent years, it has been pointed out that the participation of matrix metalloproteinases (MMPs; Matrix metalloproteinases) is a factor that induces changes with skin aging. Among these MMPs, it is known that matrix metalloproteinase-1 (MMP-1) is an enzyme that decomposes collagen, which is the main component of the dermal extracellular matrix of the skin. However, it is believed that this expression is greatly increased by ultraviolet radiation and becomes collagen. It is a factor of protein reduction and denaturation, and is a major factor in the formation of skin wrinkles and reduction of elasticity. Therefore, blocking the activity of MMP-1 is important in preventing and improving the symptoms of skin aging.

As for those having such MMP-1 blocking effect, for example, extracts from Prunus cerasoides from Yunnan (see Patent Document 21), Zingiber cassumunar from Zingiberaceae (Zingiber cassumunar) are known. ) Or an extract of Ficus neriifolia (Moraceae) forest banyan (Ficus neriifolia) (see Patent Document 22), etc.

On the other hand, although there are known fermentation broths of Artemisia plants fermented with koji bacteria (see Patent Document 23), and fermentation broths of Thymus vulgaris fermented with Koji bacteria (see Patent Document 24), they are not yet known. These plant fermentation broths have anti-aging, anti-oxidant, anti-inflammatory, and whitening effects.

[Prior Technical Literature] [Patent Literature] [Patent Document 1] JP 2003-81848 Bulletin [Patent Document 2] JP 2005-29483 Bulletin [Patent Document 3] JP 2006-321730 Bulletin [Patent Document 4] JP 2007-8902 Bulletin [Patent Document 5] JP 2003-55242 Bulletin [Patent Document 6] JP 2003-12532 Bulletin [Patent Document 7] JP 2002-370962 Bulletin [Patent Document 8] JP 2004-83488 Bulletin [Patent Document 9] JP 2001-213757 Bulletin [Patent Document 10] JP 2002-201122 Bulletin [Patent Document 11] JP 2002-226323 Bulletin [Patent Document 12] JP 2003-146837 Bulletin [Patent Document 13] JP 2002-363054 Bulletin [Patent Document 14] JP 2003-146886 Bulletin [Patent Document 15] JP 2001-261568 No. [Patent Document 16] JP 2010-090093 Bulletin [Patent Document 17] JP 2009-184955 Bulletin [Patent Document 18] JP 2009-191039 Bulletin [Patent Document 19] JP 2007-176830 Bulletin [Patent Document 20] JP 2007-176835 No. 2007-176835 [Patent Document 21] JP 2003-176232 Bulletin [Patent Document 22] JP 2003-176230 Bulletin [Patent Document 23] JP 2011-140453 Bulletin [Patent Document 24] JP 2011-130689 Bulletin

[Non-Patent Literature] [Non-Patent Document 1] "FRAGRANCE JOURNAL" Temporary Supplement No.14, p156, 1995 [Non-Patent Document 2] FRAGRANCE JOURNAL Temporary Supplement, vol.17, pp.14-19(2000) [Non-Patent Document 3] Arch.Dermatol.Res., vol.288, pp.442-446 (1996) [Non-Patent Document 4] "FRAGRANCE JOURNAL", 2006, Vol.34, No.10, p.19-23 [Non-Patent Document 5] J.Cell Biol., vol.156, pp.1099-1111 (2002) [Non-Patent Document 6] Journal of the Japanese Society of Fragrances and Cosmetics, vol.31, pp.296-301 (2007)

[The problem to be solved by the invention] The subject of the present invention is to solve the aforementioned problems in the past and achieve the following objects. That is, the object of the present invention is to provide an anti-aging agent with excellent anti-aging effect and high safety; an antioxidant with excellent anti-oxidation effect and high safety; and an excellent anti-inflammatory effect with high safety. Anti-inflammatory agent; and whitening agent with excellent whitening effect and high safety. In addition, the object of the present invention is to provide cosmetics with at least one effect selected from the group consisting of excellent anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect, and with high safety.

[Means to solve the problem] The means for solving the aforementioned problems are as follows. which is, <1> An anti-aging agent, which is characterized by containing a fermented broth derived from Aspergillus sp. of the genus Artemisia, a fermented broth derived from Aspergillus sp. of Thyme, and a fermented broth derived from Aspergillus sp. At least one of the obtained fermentation broth and the fermentation broth of Centaurea cyanus (Centaurea cyanus) obtained by koji bacteria is used as an active ingredient. <2> An antioxidant characterized by containing a fermented broth of Artemisia spp. plant by aspergillus, a fermented broth of thyme from aspergillus, and a fermentation broth of Cypress balsam by aspergillus , And the fermentation broth of at least any one of the fermentation broth of cornflower obtained by koji bacteria as the active ingredient. <3> An anti-inflammatory agent characterized by containing at least any one of the fermentation broth of Artemisia spp. plant by the aspergillus and the fermentation broth of Cypress balsam by the aspergillus as effective ingredient. <4> A whitening agent characterized by containing a fermented liquid of Artemisia spp. plant by aspergillus, a fermentation liquid of Cypress balm by aspergillus, and a fermentation liquid of Cornflower by aspergillus The fermentation broth of at least any one of the broths is used as the active ingredient. <5> A cosmetic characterized by containing the anti-aging agent described in the above <1>, the antioxidant described in the above <2>, the anti-inflammatory agent described in the above <3>, and the whitening agent described in the above <4> At least one of the formed groups.

[Effects of Invention] According to the present invention, the aforementioned problems can be solved, and the aforementioned objects can be achieved, providing an anti-aging agent with excellent anti-aging effect and high safety, an antioxidant with excellent anti-oxidation effect and high safety, and excellent anti-aging effect. An anti-inflammatory agent with an anti-inflammatory effect and high safety, and a whitening agent with an excellent whitening effect and high safety. In addition, the present invention can provide cosmetics having at least one effect selected from the group consisting of excellent anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect, and with high safety.

(Anti-aging agents, antioxidants, anti-inflammatory agents, and whitening agents) <Anti-aging agent> The anti-aging agent of the present invention is a fermentation broth (hereinafter, sometimes referred to as "Artificial Artemisia fermented broth") of a plant of the genus Artemisia which is obtained from Koji bacteria, and a thyme which is obtained from Koji bacteria The fermentation broth (hereinafter, sometimes referred to as "thyme fermentation broth"), the fermentation broth of Cypress balm (hereinafter, sometimes referred to as "cypress balm fermentation broth") from koji fungus, and the borrowed from cornflower The fermentation broth of at least any one of the fermentation broths (hereinafter, sometimes referred to as "cornflower fermentation broth") obtained from koji bacteria is used as an active ingredient, and further contains other components as necessary.

The aforementioned Artemisia spp. fermentation broth, the aforementioned thyme fermentation broth, the aforementioned cypress balm fermentation broth, and the aforementioned cornflower fermentation broth have an activity inhibition effect selected from matrix metalloproteinase-1 (MMP-1), hyaluronic acid synthase 3 (hyaluronan synthase 3; HAS3) mRNA expression promotion effect, type I collagen production promotion effect, codonin-1 mRNA expression promotion effect, codonin-4 mRNA expression promotion effect, adenectin mRNA expression promotion effect, transformation Transglutaminase-1 (TGM-1) mRNA expression promotion effect, Aquaporin 3 (Aquaporin 3; AQP3) mRNA expression promotion effect, and filaggrin mRNA expression promotion effect At least one of the effects can be used as an effective ingredient of an anti-aging agent by using these effects.

Therefore, the aforementioned anti-aging agent is selected from the group consisting of MMP-1 activity inhibition effect, hyaluronic acid synthase 3 mRNA expression promotion effect, type I collagen production promotion effect, codonin-1 mRNA expression promotion effect, and codonin-4 mRNA At least one of the group consisting of performance promotion effect, adenectin mRNA performance promotion effect, transglutaminase-1 mRNA expression promotion effect, aquaporin 3 mRNA expression promotion effect, and filaggrin mRNA expression promotion effect The role.

Regarding the use of the aforementioned Artemisia spp. fermented broth, the aforementioned thyme fermentation broth, the aforementioned cypress balm fermentation broth, and the aforementioned cornflower fermentation broth have the MMP-1 activity inhibition effect, hyaluronic acid synthase 3 mRNA expression promotion effect, and type I collagen production Promoting effect, promoting effect of clonectin-1 mRNA expression, promoting effect of clonectin-4 mRNA expression, promoting effect of clonectin mRNA expression, promoting effect of transglutaminase-1 mRNA expression, expression of aquaporin 3 mRNA Although the details of the substance of at least one of the promotion effect and the expression promotion effect of filaggrin mRNA are not clear, the aforementioned Artemisia plant fermentation broth, the aforementioned thyme fermentation broth, the aforementioned cypress balm fermentation broth, and the aforementioned cornflower fermentation broth are not clear. It has such an excellent effect, and its use as an anti-aging agent has never been known in the past, and it is a new discovery of the inventors.

＜Antioxidant＞ The antioxidant of the present invention contains the fermented broth of Artemisia spp. (Artemisia spp. fermented broth), the fermentation broth of thyme (Thymus fermented broth), and the cypress balm The fermentation broth of at least one of the fermentation broth (cypress balm fermentation broth) obtained from the koji fungus and the fermentation broth of the cornflower (the cornflower fermentation broth) derived from the koji bacteria as the effective ingredients, and further according to needs, contains Made from other ingredients.

The aforementioned wormwood fermentation broth, the aforementioned thyme fermentation broth, the aforementioned cypress balm fermentation broth, and the aforementioned cornflower fermentation broth have a diphenyl-p-picrylhydrazyl (diphenyl-p-picrylhydrazyl, DPPH) radical elimination effect , Can take advantage of this effect, use as an effective ingredient of antioxidants. Therefore, the aforementioned antioxidants have DPPH radical elimination effects.

Although the details of the aforementioned Artemisia plant fermentation broth, the aforementioned thyme fermentation broth, the aforementioned Cypress balm fermentation broth, and the aforementioned bluebonnet fermentation broth possess the DPPH radical elimination effect, the details of the aforementioned Artemisia plant fermentation broth are unclear. The aforementioned thyme fermentation broth, the aforementioned cypress balm fermentation broth, and the aforementioned cornflower fermentation broth have such excellent effects, and their usefulness as an antioxidant is completely unknown in the past and is a new discovery of the inventors.

＜Anti-inflammatory agent＞ The anti-inflammatory agent of the present invention contains the fermentation broth of Artemisia spp. (Artemisia spp. fermented broth) and the fermentation broth of Aspergillus cypress (Cypress balm fermentation broth). At least any one of the fermentation broth is used as an active ingredient, and further contains other ingredients as needed.

The aforementioned Artemisia spp. fermentation broth and the aforementioned Cypress balm fermentation broth have a hyaluronidase activity inhibition effect, and this effect can be used as an effective ingredient of an anti-inflammatory agent. Therefore, the aforementioned anti-inflammatory agent has a hyaluronidase activity inhibitory effect.

Although the details of the substance that inhibits hyaluronidase activity possessed by the aforementioned Artemisia plant fermentation broth and the aforementioned Cypress balm fermentation broth are not clear, the aforementioned Artemisia plant fermentation broth and the aforementioned Cypress balm fermentation broth have such advantages. The role of and its usefulness as an antioxidant is completely unknown in the past, and it is a new discovery of the inventors.

＜Whitening agent＞ The whitening agent of the present invention contains the fermentation broth of Artemisia spp. (Artemisia spp. fermented broth) from Aspergillus spp., the fermentation broth of Cypress balm (Cypress spp. fermentation broth) obtained from Aspergillus spp., and The fermentation liquid of at least any one of the fermentation broths (Cornflower fermentation broth) of cornflower obtained by koji bacteria is used as an active ingredient, and further contains other ingredients as needed.

The aforementioned wormwood fermentation broth, cypress balm fermentation broth, and the aforementioned cornflower fermentation broth have at least one of tyrosinase activity inhibitory effect and melanin production inhibitory effect. This effect can be used as an effective ingredient of a whitening agent . Therefore, the aforementioned whitening agent has at least one of tyrosinase activity inhibitory effect and melanin production inhibitory effect.

Although the details of the substance that exerts at least one of the tyrosinase activity inhibitory effect and the melanin production inhibitory effect possessed by the aforementioned Artemisia spp. fermentation broth, the aforementioned Cypress balm fermentation broth, and the aforementioned cornflower fermentation broth, are unclear, However, the aforementioned wormwood fermentation broth, the aforementioned cypress balm fermentation broth, and the aforementioned cornflower fermentation broth have such excellent effects, and their use as a whitening agent has never been known in the past, and this is a new discovery of the inventors.

＜＜Artificial Artemisia Fermentation Liquid＞＞ The aforementioned Artemisia plant fermentation broth is a fermentation broth of a plant belonging to the Artemisia genus (hereinafter, sometimes referred to as "Artificial Artemisia") obtained by koji bacteria.

- mugwort - As the use of perennial fermentation feedstock preceding mugwort as belonging to the Asteraceae (Compositae) Artemisia (Artemisia), and since ancient times been used as food and medicinal raw materials. It is widely native or cultivated in Japan such as Hokkaido, Honshu, Shikoku, Kyushu, etc., and can be easily obtained from these areas.

On the Artemisia species of plants, it is not particularly limited and may be suitably selected in response to purposes, include, for example, mountain wormwood (Artemisia montana (Nakai) Pamp. ), Tarragon (Artemisia capillaris Thunbergii), Artemisia (of Artemisia princeps Pampan.), Artemisia japonica Thunb., Artemisia absinthium L., Artemisia lactiflora Wall., Artemisia maritima L., Artemisia scoparia Waldst. et kit.) and so on. One of these may be used alone, or two or more of them may be used in combination. As for the acquisition method of the aforementioned Artemisia plant, there is no particular limitation, and it can be appropriately selected according to the purpose, and it can be taken from nature, or commercially available products can also be used.

The use parts of the Artemisia genus as the fermentation raw material are not particularly limited, and can be appropriately selected according to the purpose. Examples include flowers, buds, fruits, pericarps, seeds, seed coats, stems, leaves, branches, Aboveground parts of branches and leaves; underground parts of roots, rhizomes, etc. One of these may be used alone, or two or more of them may be used in combination. Among these, the above-ground parts of Artemisia plants are preferred.

Regarding the size of the Artemisia plant used as the fermentation raw material, it is not particularly limited as long as it is a size that can be cultivated for the koji fungus, and it can be appropriately selected according to the purpose. For example, the original size and cut can be used. The desired size of the broken, the size of the micronized (powdered) powder, etc.

Regarding the state of using the aforementioned Artemisia plant as the aforementioned fermentation raw material, it is not particularly limited as long as it is a state where the cultivation of the aforementioned Koji mold is possible, and it can be appropriately selected according to the purpose. For example, the original state and the dried state can be used. The state of the powder, the state of crushing, the state of juicing, the state of the extract, etc. Among these, the original state, the crushed state, the squeezed state, and the state of the extract are preferable at the point where the koji bacteria are easy to act, and the original state and the crushed state are more preferable.

The method of putting the aforementioned Artemisia plant in a dried state is not particularly limited, and can be appropriately selected according to the purpose, for example, a method of drying in the sun, a method of drying using a commonly used dryer, and the like.

The method of making the aforementioned Artemisia plants into the aforementioned pulverized state is not particularly limited, and can be appropriately selected according to the purpose. Examples include the use of a mixer, a sugar mill, and a powder mill (Power). mill), jet mill (jet mill), impact pulverizer, etc. for pulverization.

The method for making the aforementioned Artemisia plant into the aforementioned squeezed state is not particularly limited, and can be appropriately selected according to the purpose, and for example, pressing and the like can be mentioned.

The method for preparing the aforementioned Artemisia plant into the state of the aforementioned extract is not particularly limited, and the method generally used for extraction of plants can be appropriately selected according to the purpose.

It is preferable to use the above-mentioned Artemisia plant as the above-mentioned fermentation raw material which is sterilized before the inoculation of the above-mentioned aspergillus. The means for sterilizing the aforementioned Artemisia plants is not particularly limited, and it can be appropriately selected from known methods.

-Koji mold- The koji mold for fermenting the above-mentioned Artemisia plant is not particularly limited, and can be appropriately selected according to the purpose. Examples include Aspergillus oryzae , Aspergillus sojae , etc. Aspergillus yellow; Aspergillus luchuensis (Aspergillus luchuensis) and other black aspergillus; Hanoi white aspergillus (Aspergillus kawauchii ) and other white aspergillus; these variants, etc. One of these may be used alone, or two or more of them may be used in combination. Among these, as for the aforementioned koji bacteria, at least any one of anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect is excellent, and Aspergillus oryzae is preferable . The method for obtaining the aforementioned koji bacteria is not particularly limited, and can be appropriately selected according to the purpose, and can be taken from nature, or commercially available products can also be used. In addition, as the above-mentioned koji mold, a seed koji using rice or the like as a raw material may be used, the below-mentioned Artemisia species koji may be used, or a koji culture cultured on a medium (agar medium, liquid medium, etc.) may also be used. Among these, the effect of at least any one of anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect is excellent, and it is preferable to use the aforementioned Artemisia koji.

Regarding the inoculation amount of the aforementioned koji fungus to the aforementioned Artemisia plant used as the aforementioned fermentation raw material, it is not particularly limited as long as the aforementioned Artemisia plant can be fermented, and can be appropriately selected according to the purpose. However, it is suitable for the aforementioned fermentation raw material In the case of liquid state, preferably 1×10 ³ pieces/mL~1×10 ⁸ pieces/mL, in the case of the aforementioned fermentation raw materials in solid state, preferably 1×10 ³ pieces/g~1×10 ⁸ Pieces/g.

When inoculating the aforementioned Artemisia plants with the aforementioned Koji bacteria, it is preferable to add water. The addition amount of the aforementioned water is not particularly limited and can be appropriately selected according to the purpose. However, relative to 100 parts by mass of the aforementioned Artemisia plant, it is preferable to add 500 parts by mass to 5,000 parts by mass, more preferably 1,000 parts by mass. Parts ~ 4,000 parts by mass, particularly preferably 1,500 parts by mass to 3,000 parts by mass.

Regarding the temperature of the aforementioned fermentation (cultivation), if it is within the range of the temperature at which the aforementioned koji mold can be used, it is not particularly limited and can be appropriately selected according to the purpose, but 20°C to 40°C is preferable, and 25°C ~35℃ is better. When the temperature of the aforementioned fermentation is lower than 20°C, the aforementioned Artemisia plants cannot be sufficiently fermented, and at least any one of the anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect becomes insufficient. In addition, when it exceeds 50°C, the aforementioned koji bacteria cannot proliferate.

The time for the aforementioned fermentation (cultivation) is not particularly limited and can be appropriately selected according to the purpose, but it is preferably 10 hours to 40 hours, and more preferably 20 hours to 30 hours. If the aforementioned fermentation time is less than 10 hours, the aforementioned Artemisia plants cannot be sufficiently fermented, and at least any one of the anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect becomes insufficient.

The method for stopping the aforementioned fermentation (cultivation) is not particularly limited, and can be appropriately selected according to the purpose, and examples thereof include heating methods. The heating temperature for stopping the fermentation is not particularly limited if it is a temperature at which the koji bacteria cannot grow, and can be appropriately selected according to the purpose, but it is preferably 50°C or higher, more preferably 70°C or higher. Preferably it is 100℃~130℃. When the heating temperature is lower than 50°C, the fermentation may not be stopped, and when it exceeds 130°C, at least one of the anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect becomes insufficient. Regarding the heating time for stopping the aforementioned fermentation, if it can be made into a state where the aforementioned koji bacteria cannot grow, it is not particularly limited and can be appropriately selected according to the purpose, but it is preferably 5 minutes or more, more preferably 10 minutes to 20 minutes. minute. When the heating time is less than 5 minutes, the fermentation cannot be stopped, and when it exceeds 20 minutes, at least any one of the anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect becomes insufficient.

Furthermore, the aforementioned Artemisia plant fermentation broth after stopping the aforementioned fermentation is preferably a cooled one. The cooling method is not particularly limited, and can be appropriately selected according to the purpose. For example, a method of standing still at room temperature, a refrigerator, and the like can be mentioned.

The number of times of performing fermentation of the aforementioned Artemisia plant using the aforementioned Koji mold is not particularly limited, and can be appropriately selected according to the purpose, and it may be one time or plural times.

In the case where the fermentation is performed multiple times, the koji bacteria may be inoculated only for the first time, or may be inoculated only a few times, or may be inoculated at all times, but it is preferable to perform the inoculation only for the first time. In the case of performing the aforementioned fermentation multiple times, the fermentation temperature and the fermentation time may be different or the same.

--Artemisia spp. Koji-- The aforementioned Artemisia spp. plant uses the aforementioned Artemisia spp. as a seed koji raw material, and the spores are inoculated with the spores of the genus Artemisia spore. By using it for the aforementioned fermentation, it is advantageous in that a fermentation broth of Artemisia plants with excellent skin affinity can be obtained more efficiently and easily.

The aforementioned Artemisia plant system used as the raw material of the aforementioned species can be the same as those described in the aforementioned -Artemisia plant-, and the aspect, size, state, and the like of the aforementioned Artemisia plant are also the same.

As for the above-mentioned koji mold used in the production of the above-mentioned Artemisia plant species koji, the same as those described in the above-mentioned-koji mold-can be used.

Regarding the inoculation amount of the aforementioned koji fungi to the aforementioned Artemisia spp. used as the aforementioned species of koji raw material, there is no particular limitation, and can be appropriately selected according to the purpose, but it is preferable to inoculate 5 with respect to 100 parts by mass of the aforementioned Artemisia spp. Mass ~ 100 mass parts of Koji bacteria suspended in sterilized water (1×10 ³ cells/mL~1×10 ⁸ cells/mL), more preferably 10 mass parts to 50 mass parts, particularly preferably 20 mass parts Parts ~ 30 parts by mass. When the inoculation amount of the koji fungus relative to 100 parts by mass of the artemisia plant is less than 5 parts by mass, a sufficient amount of spores cannot be attached to the artemisia plant, and when it exceeds 100 parts by mass, it abnormally multiplies due to excessive moisture.

When inoculating the above-mentioned koji bacterium when using the above-mentioned artemisia plant as the raw material of the above-mentioned species of koji, it is preferable to add water. The amount of water added is not particularly limited and can be appropriately selected according to the purpose. However, relative to 100 parts by mass of the aforementioned Artemisia plant, it is preferable to add 10 parts by mass to 250 parts by mass, more preferably 20 parts by mass. Parts to 200 parts by mass, and it is particularly preferable to add 30 parts by mass to 150 parts by mass. When the amount of the water added to 100 parts by mass of the Artemisia plant is less than 10 parts by mass, a sufficient amount of spores may not be attached to the Artemisia plant.

Regarding the aforementioned culture temperature, if it is within the temperature range at which the aforementioned koji bacteria can grow, it is not particularly limited, and can be appropriately selected according to the purpose, but it is preferably 20°C to 40°C, more preferably 25°C to 35°C. ℃. When the temperature of the aforementioned culture is lower than 20°C, sometimes a sufficient amount of spores cannot be attached to the aforementioned Artemisia plant. In addition, when the temperature exceeds 50°C, the aforementioned koji bacteria may not proliferate.

Regarding the aforementioned cultivation time, it is not particularly limited and can be appropriately selected according to the purpose, but is preferably 80 hours to 210 hours, more preferably 100 hours to 190 hours, and particularly preferably 120 hours to 170 hours. If the aforementioned culture time is less than 80 hours, a sufficient amount of spores may not be attached to the aforementioned Artemisia plant, and if it exceeds 210 hours, the germination rate of the spores may decrease.

The aforementioned Artemisia plant fermentation broth may contain the above-mentioned aspergillus cells, or it may be the result of removing the aforementioned aspergillus cells, but it is preferably one that has removed the aforementioned aspergillus cells.

The state of the aforementioned Artemisia plant fermentation broth is not particularly limited and can be appropriately selected according to the purpose. For example, it may be the aforementioned Artemisia plant fermentation broth itself, or the purified product of the aforementioned Artemisia plant fermentation broth, and the aforementioned Artemisia plant fermentation broth. Concentrate of fermentation broth of genus plants, dilution of fermentation broth of genus Artemisia, etc. In addition, the aforementioned Artemisia plant fermentation broth may be obtained by mixing or dissolving the dried product of the Artemisia plant fermentation broth with a solvent such as water or a hydrophilic solvent.

The purified product of the aforementioned Artemisia plant fermentation broth is not particularly limited, and can be appropriately selected according to the purpose. For example, the removal of solid components in the aforementioned Artemisia plant fermentation broth (for example, the aforementioned Artemisia plant body, Bacteria, dregs, etc.) of koji bacteria. The aforementioned removal means is not particularly limited, and can be appropriately selected according to the purpose, and examples thereof include filtration. As for the aforementioned filtering method, there is no special restriction, and it can be selected appropriately according to the purpose among the known methods.

The dilution of the aforementioned Artemisia plant fermentation broth and the aforementioned Concentrate of the Artemisia plant fermentation broth are not particularly limited, and can be appropriately selected according to the purpose. For example, the fermentation broth of the Artemisia plant is prepared to a desired concentration. Wait. As for the aforementioned dilution method, there is no particular limitation, and it can be appropriately selected according to the purpose among the known methods. The aforementioned means of concentration is not particularly limited, and can be appropriately selected according to the purpose, and examples thereof include concentration under reduced pressure.

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

The aforementioned Artemisia plant fermentation broth is not particularly limited as long as it is a fermentation broth of Artemisia plant derived from Koji bacteria, and can be appropriately selected according to the purpose. However, from the viewpoint of good skin-friendliness, the contact angle is preferably The fermentation broth of Artemisia plants with a contact angle of less than 81° is more preferably a fermentation broth of Artemisia plants with a contact angle of 78° or less.

In this manual, the contact angle means the value obtained as follows: Using a dynamic contact angle and surface tension measuring device (FTA1000 Falcon, manufactured by First Ten Angstroms), drop 3 μL of the measurement sample on the sample stage of the aforementioned device ( On the sample stage), under the conditions of a temperature of 22°C and a relative humidity of 20%, the measurement was performed by the droplet method, and the value of the contact angle θ(°) of 1,000 ms was obtained by the θ/2 method. The aforementioned contact angle is used as an indicator of "wetting" and is defined as "the angle formed by the liquid surface and the solid surface (the angle taken from the inside of the liquid) where the free surface of a stationary liquid adjoins the solid wall" (Refer to the 4th edition of the Physics and Chemistry Dictionary, Iwanami Bookstore Co., Ltd.). The aforementioned contact angle depends on the relationship between the cohesive force between the liquid molecules and the adhesion force between the solid walls. It is an obtuse angle when the liquid wets the solid (high adhesion), and it is an obtuse angle when it is not wet. Therefore, the smaller the contact angle, the easier it is to get wet, that is, the better the skin affinity, the lower limit of the contact angle of the aforementioned Artemisia plant fermentation broth is not particularly limited, and can be appropriately selected according to the purpose.

＜＜Thyme fermentation broth＞＞ The aforementioned fermented thyme broth is a fermented broth of thyme from Koji bacteria.

-Thyme- Thymus vulgaris Linne used as the aforementioned fermentation raw material is a perennial woody belonging to the genus Labiatae and Thymus . It is a kind of spice plant (herb). It has been used for food and food since ancient times. Medicinal raw materials. As aliases, there are Common thyme and so on. The place of origin is the Mediterranean coast, but it also grows naturally or is cultivated in Japan and can be easily obtained from such regions. The method for obtaining thyme mentioned above is not particularly limited, and it can be appropriately selected according to the purpose, and it can be taken from nature, or commercially available products can also be used.

Regarding the use part of the thyme as the fermentation raw material, it is not particularly limited, and can be appropriately selected according to the purpose. Examples include flowers, buds, fruits, pericarps, seeds, seed coats, stems, leaves, branches, bark, The aboveground parts of stems, branches and leaves; the underground parts of roots, rhizomes, etc. One of these may be used alone, or two or more of them may be used in combination. Among these, the above-mentioned part of thyme is preferably the upper part of the ground.

Regarding the size of the thyme used as the fermentation raw material, it is not particularly limited as long as the size of the koji fungus can be cultivated, and it can be selected appropriately according to the purpose. Examples include the original size taken and cut Desired size, micronized (powdered) size, etc.

Regarding the state of using the thyme as the fermentation raw material, there is no particular limitation as long as it is a state capable of cultivating the koji fungus, and it can be appropriately selected according to the purpose. Examples include the original state, dried state, The state of crushing, the state of squeezed juice, the state of extract, etc. Among these, at the point where the aforementioned koji bacteria is easy to act, it is preferably the original state taken, the crushed state, the juiced state, and the state of the extract, and more preferably the original taken or crushed state.

The method of making the aforementioned thyme into a dried state is not particularly limited, and can be appropriately selected according to the purpose. For example, a method of drying in the sun, a method of drying using a commonly used dryer, and the like can be mentioned.

The method of making the aforementioned thyme into the aforementioned pulverized state is not particularly limited, and can be appropriately selected according to the purpose. For example, the use of a mixer, a sugar mill, a powder mill, a jet mill, and an impact mill can be mentioned. Machines, etc. and crushing methods, etc.

The method of making the thyme into the squeezed state is not particularly limited, and can be appropriately selected according to the purpose, and for example, squeezing and the like can be mentioned.

The method for preparing the aforementioned thyme into the state of the aforementioned extract is not particularly limited, and the method generally used in the extraction of plants can be appropriately selected according to the purpose.

The thyme used as the fermentation raw material is preferably sterilized before the inoculation of the koji fungus. The means for sterilizing the aforementioned thyme is not particularly limited, and can be appropriately selected from conventional methods.

-Koji mold- The koji mold for fermenting the thyme is not particularly limited, and can be appropriately selected according to the purpose. For example, the ones described in the above-mentioned <<Artemisia plant fermentation broth>> can be mentioned. One of these may be used alone, or two or more of them may be used in combination. Among these, as for the aforementioned koji bacteria, at least one of the anti-aging effect and the anti-oxidation effect is excellent, and Aspergillus oryzae (Aspergillus oryzae) is preferred. The method for obtaining the aforementioned koji bacteria is not particularly limited, and can be appropriately selected according to the purpose, and can be taken from nature, or commercially available products can also be used. In addition, as the aforementioned koji bacteria, seed koji using rice or the like as a raw material may be used, the thyme seed koji described later may also be used, or koji culture cultured on a medium (agar medium, liquid medium, etc.) may also be used. Among these, at least one of the anti-aging effect and the anti-oxidation effect is excellent, and it is preferable to use the aforementioned thyme koji.

Regarding the inoculation amount of the aforementioned koji bacteria to the aforementioned thyme used as the aforementioned fermentation raw material, as long as the aforementioned amount of thyme can be fermented, it is not particularly limited and can be appropriately selected according to the purpose, but when the aforementioned fermentation raw material is in a liquid state In this case, it is preferably 1×10 ³ pieces/mL to 1×10 ⁸ pieces/mL, and when the fermentation raw materials are in a solid state, it is preferably 1×10 ³ pieces/g to 1×10 ⁸ pieces/g.

When inoculating the aforementioned koji bacteria to the aforementioned thyme, it is preferable to add water. The amount of water added is not particularly limited and can be appropriately selected according to the purpose. However, it is preferable to add 500 parts by mass to 5,000 parts by mass, and more preferably to add 1,000 parts by mass to 100 parts by mass of the aforementioned thyme. 4,000 parts by mass, particularly preferably 1,500 to 3,000 parts by mass.

Regarding the aforementioned fermentation (cultivation) temperature, if it is within the fermentable temperature range of the aforementioned koji mold, it is not particularly limited and can be appropriately selected according to the purpose, but it is preferably 20°C to 40°C, more preferably It is 25℃~35℃. When the temperature of the aforementioned fermentation is lower than 20°C, the aforementioned thyme cannot be sufficiently fermented, and at least one of the anti-aging effect and the anti-oxidation effect becomes insufficient.

The time for the aforementioned fermentation (cultivation) is not particularly limited and can be appropriately selected according to the purpose, but it is preferably 10 hours to 40 hours, and more preferably 20 hours to 30 hours. If the aforementioned fermentation time is less than 10 hours, the aforementioned thyme cannot be fully fermented, and at least one of the anti-aging effect and the anti-oxidation effect becomes insufficient.

The method for stopping the aforementioned fermentation (cultivation) is not particularly limited, and can be appropriately selected according to the purpose, and examples thereof include heating methods. The heating temperature for stopping the fermentation is not particularly limited if it is a temperature at which the koji bacteria cannot grow, and can be appropriately selected according to the purpose, but it is preferably 50°C or higher, more preferably 70°C or higher. Preferably it is 100℃~130℃. If the heating temperature is lower than 50°C, the fermentation may not be stopped, and if it exceeds 130°C, at least one of the anti-aging effect and the antioxidant effect becomes insufficient. Regarding the heating time for stopping the aforementioned fermentation, if it can be made into a state where the aforementioned koji bacteria cannot grow, it is not particularly limited, and can be appropriately selected according to the purpose, but it is preferably 5 minutes or more, more preferably 10 minutes to 20 minutes. minute. If the heating time is less than 5 minutes, the fermentation may not be stopped, and when it exceeds 20 minutes, at least one of the anti-aging effect and the anti-oxidation effect becomes insufficient.

In addition, the thyme fermentation broth after stopping the fermentation is preferably cooled. The cooling method is not particularly limited, and can be appropriately selected according to the purpose. For example, a method of standing still at room temperature, a refrigerator, and the like can be mentioned.

The number of times of performing the fermentation of the thyme using the koji fungus is not particularly limited, and may be appropriately selected according to the purpose, and may be one time or multiple times.

In the case where the aforementioned fermentation is performed plural times, the aforementioned koji bacteria may be inoculated only for the first time, or may be inoculated only a few times, or may be inoculated at all times, but it is preferable to perform only the first inoculation. When the aforementioned fermentation is performed multiple times, the fermentation temperature and the fermentation time may be different or the same.

--Thyme seed koji-- The thyme seed koji system uses the thyme as a seed koji raw material, and the koji fungus is inoculated to the koji raw material, so that a sufficient amount of spores are attached to the thyme. By using it in the aforementioned fermentation, it is advantageous in that it is more efficient and easy to obtain a thyme fermentation broth with excellent skin affinity.

The thyme system used as the raw material for the seed koji can be the same as described in the above -Thyme-, and the aspect, size, state, and the like of the thyme are also the same.

Regarding the koji bacterium used in the production of the thyme seed koji, the same ones as described in the above-koji mold-can be used.

Regarding the inoculation amount of the aforementioned koji bacteria to the aforementioned thyme used as the aforementioned seed koji raw material, it is not particularly limited and can be appropriately selected according to the purpose. However, it is preferable to inoculate 5 mass parts of sterilized water with respect to 100 parts by mass of the aforementioned thyme 100 parts by mass of suspended koji bacteria (1×10 ³ /mL~1×10 ⁸ /mL), more preferably 10 parts by mass to 50 parts by mass, particularly preferably 20 parts by mass to 30 parts by mass Copies. When the inoculation amount of the above-mentioned koji bacteria with respect to 100 parts by mass of the above-mentioned thyme is less than 5 parts by mass, a sufficient amount of spores may not be attached to the above-mentioned thyme, and when it exceeds 100 parts by mass, it may abnormally multiply due to excessive moisture.

When inoculating the koji bacterium to the thyme as the raw material of the seed koji, it is preferable to add water. The amount of water added is not particularly limited and can be appropriately selected according to the purpose. However, relative to 100 parts by mass of the aforementioned thyme, it is preferable to add 10 parts by mass to 250 parts by mass, and more preferably to add 20 parts by mass to 200 parts by mass. Parts by mass, it is particularly preferable to add 30 parts by mass to 150 parts by mass. When the amount of the water added to 100 parts by mass of the thyme is less than 10 parts by mass, a sufficient amount of spores may not be attached to the thyme.

Regarding the aforementioned culture temperature, if it is within the temperature range at which the aforementioned koji bacteria can grow, it is not particularly limited, and can be appropriately selected according to the purpose, but it is preferably 20°C to 40°C, more preferably 25°C to 35°C. ℃. When the temperature of the aforementioned culture is lower than 20°C, a sufficient amount of spores sometimes cannot be attached to the aforementioned thyme. In addition, when the temperature exceeds 50°C, the aforementioned koji bacteria may not proliferate.

Regarding the aforementioned cultivation time, it is not particularly limited and can be appropriately selected according to the purpose, but is preferably 80 hours to 210 hours, more preferably 100 hours to 190 hours, and particularly preferably 120 hours to 170 hours. If the above-mentioned culture time is less than 80 hours, sometimes sufficient spores cannot be attached to the above-mentioned thyme. When it exceeds 210 hours, the germination rate of the spores may decrease.

The aforementioned thyme fermentation broth may be one that contains the above-mentioned koji fungus, or may be one that removes the above-mentioned koji fungus, but it is preferably one that removes the above-mentioned koji fungus.

The state of the aforementioned thyme fermentation broth is not particularly limited, and can be appropriately selected according to the purpose. For example, it can be the aforementioned thyme fermentation broth itself, the purified product of the aforementioned thyme fermentation broth, the concentrate of the aforementioned thyme fermentation broth, The dilution of the aforementioned thyme fermentation broth, etc. In addition, the aforementioned thyme fermentation broth may be obtained by mixing or dissolving the dried product of the thyme fermentation broth in a solvent such as water or a hydrophilic solvent.

The purified product of the aforementioned thyme fermentation broth is not particularly limited, and can be appropriately selected according to the purpose. For example, solid components in the aforementioned thyme fermentation broth (for example, the aforementioned thyme plant body, koji fungus body, residue Etc.) What is removed, etc. The aforementioned removal means is not particularly limited, and can be appropriately selected according to the purpose, for example, filtration and the like. As for the aforementioned filtering method, there is no special restriction, and it can be selected appropriately according to the purpose among the known methods.

The dilution of the thyme fermentation broth and the concentrate of the thyme fermentation broth are not particularly limited, and can be appropriately selected according to the purpose. For example, the thyme fermentation broth is adjusted to the desired concentration. As far as the aforementioned dilution method is concerned, there is no special restriction, and it can be selected appropriately according to the purpose among the known methods. The aforementioned means of concentration is not particularly limited, and can be appropriately selected according to the purpose, and examples thereof include concentration under reduced pressure.

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

The aforementioned thyme fermentation broth is not particularly limited as long as it is a fermentation broth obtained from koji bacteria of thyme, and can be appropriately selected according to the purpose. However, in terms of good skin affinity, a contact angle of 87° or less is preferred. The thyme fermentation broth is more preferably a thyme fermentation broth with a contact angle of 81° or less.

＜＜Cypress Bee Grass Fermentation Liquid＞＞ The aforementioned cypress balm fermentation broth is a fermentation broth of cypress balm which is obtained from koji bacteria.

-Cypress balm- As the aforementioned fermentation raw material, Melissa officinalis Linne is a perennial herb belonging to the genus Melissa of the Mint family (Labiatae). It is a kind of spice plant and has been used as edible and medicinal raw materials since ancient times. As aliases, there are lemon balm, Xiyangshan mint and the like. The place of origin is Southern Europe. It has been grown or cultivated naturally in Japan and can be easily obtained from such regions. The method for obtaining the aforementioned Cypress balm is not particularly limited, and it can be appropriately selected according to the purpose, and it can be taken from nature, or commercially available products can also be used.

There are no particular restrictions on the use parts of the cypress balm as the fermentation raw material, and can be appropriately selected according to the purpose. Examples include flowers, buds, fruits, pericarps, seeds, seed coats, stems, leaves, branches, Aboveground parts of branches and leaves; underground parts of roots, rhizomes, etc. One of these may be used alone, or two or more of them may be used in combination. Among these, the above ground part is preferable in terms of the use part of the cypress balm.

Regarding the size of the cypress balm used as the fermentation raw material, it is not particularly limited as long as it is a size that is possible for the cultivation of the koji fungus, and it can be appropriately selected according to the purpose. For example, the original size and cut can be used. The desired size of the broken, the size of the micronized (powdered) powder, etc.

Regarding the state of using the cypress balm as the fermentation raw material, it is not particularly limited as long as the cultivation of the koji fungus is possible, and it can be appropriately selected according to the purpose. For example, the original state and the dried state can be used. The state of the powder, the state of crushing, the state of juicing, the state of the extract, etc. Among these, at the point where the aforementioned koji bacteria is easy to act, it is preferably the original state taken, the crushed state, the juiced state, and the state of the extract, and more preferably the original taken or crushed state.

There is no particular limitation on the method of making the aforementioned Cypress balm in a dried state, and it can be appropriately selected according to the purpose. For example, a method of drying in the sun, a method of drying using a commonly used dryer, etc. .

The method of making the aforementioned cypress balm into the aforementioned pulverized state is not particularly limited, and can be appropriately selected according to the purpose. For example, the use of a mixer, a sugar mill, a powder mill, a jet mill, and a punch Pulverizer, etc. and the method of pulverization.

The method of making the aforementioned cypress balm into the aforementioned squeezed state is not particularly limited, and can be appropriately selected according to the purpose, and for example, squeezing and the like can be mentioned.

The method for preparing the cypress balm in the state of the extract is not particularly limited, and the method generally used in the extraction of plants can be appropriately selected according to the purpose.

The cypress balm used as the fermentation raw material is preferably one that has been sterilized before the inoculation of the koji fungus. The means for sterilizing the aforementioned Cypress balm is not particularly limited, and it can be appropriately selected from conventional methods.

-Koji fungus- The koji fungus fermenting the cypress balm is not particularly limited, and can be appropriately selected according to the purpose, and examples thereof include those described in the above-mentioned <<Artemisia plant fermentation broth>>. One of these may be used alone, or two or more of them may be used in combination. Among these, as for the aforementioned koji bacteria, at least any one of the anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect is excellent, and it is preferably Aspergillus oryzae (Aspergillus oryzae). . The method for obtaining the aforementioned koji bacteria is not particularly limited, and can be appropriately selected according to the purpose, and can be taken from nature, or commercially available products can also be used. In addition, as the above-mentioned koji bacteria, seed koji using rice or the like as a raw material may be used, the cypress melissa seed koji described later may also be used, and koji bacteria cultured on a medium (agar medium, liquid medium, etc.) may also be used. Among these, since at least any one of anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect is excellent, it is preferable to use the aforementioned Cypress balm koji.

Regarding the inoculation amount of the koji fungus to the cypress balm used as the fermentation raw material, the amount is not particularly limited as long as the cypress balm used as the fermentation raw material can be fermented. It can be appropriately selected according to the purpose. In the case of liquid state, preferably 1×10 ³ pieces/mL~1×10 ⁸ pieces/mL, in the case of the aforementioned fermentation raw materials in solid state, preferably 1×10 ³ pieces/g~1×10 ⁸ Pieces/g.

When inoculating the aforementioned koji bacterium to the aforementioned cypress balm, it is preferable to add water. The amount of water to be added is not particularly limited and can be appropriately selected according to the purpose. However, it is preferable to add 500 parts by mass to 5,000 parts by mass, and more preferably to add 1,000 parts by mass relative to 100 parts by mass of cypress balm. ~4,000 parts by mass, particularly preferably 1,500 parts by mass to 3,000 parts by mass.

Regarding the aforementioned fermentation (cultivation) temperature, if it is within the temperature range that can be fermented by the aforementioned koji mold, it is not particularly limited and can be appropriately selected according to the purpose, but it is preferably 20°C to 40°C, more preferably 25℃~35℃. When the temperature of the aforementioned fermentation is lower than 20°C, the aforementioned Cypress balm cannot be sufficiently fermented, and at least any one of the anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect becomes insufficient.

The time for the aforementioned fermentation (cultivation) is not particularly limited and can be appropriately selected according to the purpose, but it is preferably 10 hours to 40 hours, and more preferably 20 hours to 30 hours. If the fermentation time is less than 10 hours, the cypress balm cannot be fully fermented, and at least any one of the anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect becomes insufficient.

The method for stopping the aforementioned fermentation (cultivation) is not particularly limited, and can be appropriately selected according to the purpose, and examples thereof include heating methods. Regarding the heating temperature for stopping the aforementioned fermentation, if it is a temperature at which the aforementioned koji bacteria cannot grow, it is not particularly limited, and can be appropriately selected according to the purpose, but it is preferably 50°C or higher, more preferably 70°C or higher. Preferably it is 100℃~130℃. When the heating temperature is lower than 50°C, the fermentation may not be stopped, and when it exceeds 130°C, at least one of the anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect becomes insufficient. Regarding the heating time for stopping the aforementioned fermentation, if it can be made into a state where the aforementioned koji bacteria cannot grow, it is not particularly limited, and can be appropriately selected according to the purpose, but it is preferably 5 minutes or more, more preferably 10 minutes~ 20 minutes. If the heating time is less than 5 minutes, the fermentation may not be stopped, and when it exceeds 20 minutes, at least any one of the anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect becomes insufficient.

In addition, the fermented cypress balm after the fermentation is stopped is preferably a cooled one. The cooling method is not particularly limited, and can be appropriately selected according to the purpose. For example, a method of standing still at room temperature, a refrigerator, and the like can be mentioned.

The number of times of performing the fermentation of the cypress balm by the koji fungus is not particularly limited, and may be appropriately selected according to the purpose, and may be one time or multiple times.

In the case where the aforementioned fermentation is performed plural times, the aforementioned koji bacteria may be inoculated only for the first time, or may be inoculated only a few times, or may be inoculated at all times, but it is preferable to perform only the first inoculation. When the aforementioned fermentation is performed multiple times, the fermentation temperature and the fermentation time may be different or the same.

--Cypress Bee Grass Seed Koji-- The above-mentioned cypress balm species Koji uses the above-mentioned cypress mellifera as a seed koji raw material, and inoculates the koji bacterium to the raw material of the cypress koji so that a sufficient amount of spores are attached to the cypress balm. By using it in the aforementioned fermentation, it is advantageous in that it is more efficient and easy to obtain a fermentation broth of Cymbidium cypress with excellent skin affinity.

The cypress balm system used as the raw material for the seed koji can be the same as those described in the above-Cypress balm-, and the aspect, size, state, and the like of the cypress balm are also the same.

As for the koji bacterium used in the production of the above-mentioned cypress balm species koji, it is possible to use the same one as described in the above -Koji mold.

The inoculation amount of the koji fungus to the cypress balm used as the raw material of the seed koji is not particularly limited, and can be appropriately selected according to the purpose, but it is preferably 5 mass parts relative to 100 parts by mass of the cypress balm ~100 parts by mass of koji bacteria suspended in sterile water (1×10 ³ /mL~1×10 ⁸ /mL), more preferably 10 parts by mass to 50 parts by mass, particularly preferably 20 parts by mass ~30 parts by mass. When the inoculation amount of the koji fungus relative to 100 parts by mass of the cypress balm is less than 5 parts by mass, a sufficient amount of spores may not be attached to the cypress balm, and when it exceeds 100 parts by mass, the moisture may be excessive. And abnormal reproduction.

When inoculating the koji bacterium to the cypress balm which is the raw material for the seed koji, it is preferable to add water. The amount of water added is not particularly limited and can be appropriately selected according to the purpose. However, relative to 100 parts by mass of the aforementioned cypress balm, it is preferable to add 10 parts by mass to 250 parts by mass, and more preferably to add 20 parts by mass ~200 parts by mass, particularly preferably adding 30 parts by mass to 150 parts by mass. When the amount of the water added to 100 parts by mass of the cypress balm is less than 10 parts by mass, a sufficient amount of spores may not be attached to the cypress balm.

Regarding the aforementioned culture temperature, if it is within the temperature range at which the aforementioned koji bacteria can grow, it is not particularly limited, and can be appropriately selected according to the purpose, but it is preferably 20°C to 40°C, more preferably 25°C to 35°C. ℃. When the temperature of the aforementioned culture is lower than 20°C, a sufficient amount of spores cannot sometimes be attached to the aforementioned Cypress balm. In addition, when the temperature exceeds 50°C, the aforementioned koji bacteria may not proliferate.

Regarding the aforementioned cultivation time, it is not particularly limited and can be appropriately selected according to the purpose, but is preferably 80 hours to 210 hours, more preferably 100 hours to 190 hours, and particularly preferably 120 hours to 170 hours. If the above-mentioned culture time is less than 80 hours, a sufficient amount of spores may not be attached to the above-mentioned cypress balm. If it exceeds 210 hours, the germination rate of the spores may decrease.

The above-mentioned cypress balm fermentation broth may contain the above-mentioned aspergillus spores, or one which removes the aforementioned aspergillus spores, but is preferably one that removes the aforementioned aspergillus spores.

The state of the aforementioned cypress balm fermentation broth is not particularly limited, and can be appropriately selected according to the purpose. For example, it may be the aforementioned cypress balm fermentation broth itself, the purified product of the aforementioned cypress balm fermentation broth, and the aforementioned cypress balm fermentation Concentrate of liquid, the dilution of the aforementioned cypress balm fermentation liquid, etc. In addition, the aforementioned cypress balm fermentation broth may be one that remixes or dissolves the dried product of the cypress balm fermentation broth in a solvent such as water or a hydrophilic solvent.

The purified product of the aforementioned cypress balm fermentation broth is not particularly limited, and can be appropriately selected according to the purpose. For example, solid components in the aforementioned cypress balm fermentation broth (for example, the plant body of the aforementioned cypress balm, koji Bacteria, slag, etc.) to be removed. The aforementioned removal means is not particularly limited, and can be appropriately selected according to the purpose, for example, filtration and the like. As for the aforementioned filtering method, there is no special restriction, and it can be selected appropriately according to the purpose among the known methods.

The dilution of the fermentation broth of the cypress balm and the concentrate of the fermentation broth of the cypress balm are not particularly limited, and can be appropriately selected according to the purpose. For example, the fermentation broth of the cypress balm is prepared to a desired concentration. Wait. As far as the aforementioned dilution method is concerned, there is no special restriction, and it can be selected appropriately according to the purpose among the known methods. The aforementioned means of concentration is not particularly limited, and can be appropriately selected according to the purpose, and examples thereof include concentration under reduced pressure.

The dried substance of the fermentation broth of Cypress balm is not particularly limited, and can be appropriately selected according to the purpose. For example, a dried substance of the fermentation broth of Cypress balm may be mentioned. The aforementioned drying means is not particularly limited, and can be appropriately selected according to the purpose. For example, freeze-drying and the like can be mentioned.

The aforementioned cypress balm fermentation broth is a fermentation broth of cypress balm by koji bacteria, and is not particularly limited, and can be appropriately selected according to the purpose, but in terms of good skin-friendliness, the contact angle is preferred It is a cypress balm fermentation broth with 85° or less, and more preferably a cypress balm fermentation broth with a contact angle of 79° or less.

＜＜Cornflower fermentation broth＞＞ The aforementioned cornflower fermentation broth is a fermentation broth of cornflower obtained from koji bacteria.

- Cornflower - cornflower used as the aforementioned fermentation feedstock (Centaurea cyanus Linne) as belonging to the Asteraceae (Compositae) knapweed (Centaurea) is an annual herb, has since ancient times been used as edible and medicinal raw materials. As aliases, there are cornflower, Centaurea, centaurea, etc. Although the place of origin is Europe, it has been grown or cultivated naturally in Japan and can be easily obtained from such regions. As for the acquisition method of the aforementioned cornflower, there is no particular limitation, and it can be appropriately selected according to the purpose, and it can be taken in nature, or a commercially available product can also be used.

There are no particular restrictions on the use parts of the cornflower as the fermentation raw material, and it can be appropriately selected according to the purpose. Examples include flowers, buds, fruits, pericarps, seeds, seed coats, stems, leaves, branches, branches, etc. The upper part of the ground; the underground part of roots, rhizomes, etc. One of these may be used alone, or two or more of them may be used in combination. Among these, the ground part is preferable in terms of the use part of the aforementioned cornflower.

Regarding the size of the cornflower used as the fermentation raw material, it is not particularly limited as long as it is a size possible for the cultivation of the koji fungus, and it can be appropriately selected according to the purpose. For example, the original size taken and cut Desired size, micronized (powdered) size, etc.

Regarding the state of using the cornflower as the fermentation raw material, if it is a state in which the cultivation of the koji fungus is possible, it is not particularly limited, and can be appropriately selected according to the purpose. For example, the original state and the dried state can be mentioned. , The state of crushing, the state of juicing, the state of extracts, etc. Among these, at the point where the aforementioned koji bacteria is easy to act, it is preferably the original state taken, the crushed state, the juiced state, and the state of the extract, and more preferably the original taken or crushed state.

The method of making the cornflower into a dried state is not particularly limited, and can be appropriately selected according to the purpose. For example, a method of drying in the sun, a method of drying using a commonly used dryer, and the like can be mentioned.

The method of making the cornflower into the pulverized state is not particularly limited, and can be appropriately selected according to the purpose. For example, the use of a mixer, a sugar mill, a powder mill, a jet mill, and an impact mill can be used. Machines, etc. and crushing methods, etc.

The method of making the cornflower into the juice-squeezed state is not particularly limited, and can be appropriately selected according to the purpose, and for example, squeezing and the like can be mentioned.

The method for preparing the cornflower into the state of the extract is not particularly limited, and the method generally used for the extraction of plants can be appropriately selected according to the purpose.

It is preferable to use the cornflower system as the fermentation raw material which has been sterilized before the inoculation of the koji fungus. The method for sterilizing the aforementioned cornflower is not particularly limited, and it can be appropriately selected from among conventional methods.

-Koji mold- The koji mold for fermenting the cornflower is not particularly limited, and can be appropriately selected according to the purpose. For example, the ones described in the above-mentioned <<Artificial plant fermentation broth>> can be mentioned. One of these may be used alone, or two or more of them may be used in combination. Among these, as for the aforementioned koji mold, at least any one of the anti-aging effect, anti-oxidation effect, and whitening effect is excellent, and it is preferably Aspergillus oryzae (Aspergillus oryzae). The method for obtaining the aforementioned koji bacteria is not particularly limited, and can be appropriately selected according to the purpose, and can be taken from nature, or commercially available products can also be used. In addition, as the aforementioned koji bacteria, seed koji using rice or the like as a raw material may be used, the cornflower seed koji described later may be used, or the koji bacteria cultured on a medium (agar medium, liquid medium, etc.) may be used. Among these, at least any one of anti-aging effect, anti-oxidation effect, and whitening effect is excellent, and it is preferable to use the aforementioned cornflower koji.

Regarding the inoculation amount of the aforementioned koji bacteria to the aforementioned cornflower used as the aforementioned fermentation raw material, as long as the aforementioned amount of cornflower can be fermented, it is not particularly limited and can be appropriately selected according to the purpose, but when the aforementioned fermentation raw material is in a liquid state In this case, it is preferably 1×10 ³ pieces/mL to 1×10 ⁸ pieces/mL, and when the fermentation raw materials are in a solid state, it is preferably 1×10 ³ pieces/g to 1×10 ⁸ pieces/g.

When inoculating the aforementioned koji bacteria to the aforementioned cornflower, it is preferable to add water. The amount of water added is not particularly limited and can be appropriately selected according to the purpose. However, it is preferable to add 500 parts by mass to 5,000 parts by mass, and more preferably to add 1,000 parts by mass to 4,000 parts by mass relative to 100 parts by mass of the aforementioned cornflower. Parts by mass, it is particularly preferable to add 1,500 parts by mass to 3,000 parts by mass.

Regarding the aforementioned fermentation (cultivation) temperature, if it is within the temperature range that can be fermented by the aforementioned koji mold, it is not particularly limited and can be appropriately selected according to the purpose, but it is preferably 20°C to 40°C, more preferably 25℃~35℃. When the temperature of the aforementioned fermentation is lower than 20°C, the aforementioned cornflower cannot be sufficiently fermented, and at least any one of the anti-aging effect, anti-oxidation effect, and whitening effect becomes insufficient.

The time for the aforementioned fermentation (cultivation) is not particularly limited and can be appropriately selected according to the purpose, but it is preferably 10 hours to 40 hours, and more preferably 20 hours to 30 hours. If the fermentation time is less than 10 hours, the cornflower cannot be fully fermented, and at least any one of the anti-aging effect, anti-oxidation effect, and whitening effect becomes insufficient.

The method for stopping the aforementioned fermentation (cultivation) is not particularly limited, and can be appropriately selected according to the purpose, and examples thereof include heating methods. Regarding the heating temperature for stopping the aforementioned fermentation, if it is a temperature at which the aforementioned koji bacteria cannot grow, it is not particularly limited, and can be appropriately selected according to the purpose, but it is preferably 50°C or higher, more preferably 70°C or higher. Preferably it is 100℃~130℃. When the heating temperature is lower than 50°C, the fermentation may not be stopped, and when it exceeds 130°C, at least one of the anti-aging effect, anti-oxidation effect, and whitening effect becomes insufficient. Regarding the heating time for stopping the aforementioned fermentation, if it can be made into a state where the aforementioned koji bacteria cannot grow, it is not particularly limited, and can be appropriately selected according to the purpose, but it is preferably 5 minutes or more, more preferably 10 minutes~ 20 minutes. If the heating time is less than 5 minutes, the fermentation may not be stopped, and when it exceeds 20 minutes, at least one of the anti-aging effect, anti-oxidation effect, and whitening effect becomes insufficient.

In addition, the cornflower fermentation liquid after the fermentation is stopped is preferably a cooled one. The cooling method is not particularly limited, and can be appropriately selected according to the purpose. For example, a method of standing still at room temperature, a refrigerator, and the like can be mentioned.

The number of times of performing the fermentation of the cornflower using the koji fungus is not particularly limited, and can be appropriately selected according to the purpose, and it may be one time or plural times.

In the case where the aforementioned fermentation is performed plural times, the aforementioned koji bacteria may be inoculated only for the first time, or may be inoculated only a few times, or may be inoculated at all times, but it is preferable to perform only the first inoculation. When the aforementioned fermentation is performed multiple times, the fermentation temperature and the fermentation time may be different or the same.

--Cornflower seed koji-- The cornflower seed koji uses the cornflower as the seed koji raw material, and the koji is inoculated with the koji raw material, so that a sufficient amount of spores are attached to the cornflower. By using it in the aforementioned fermentation, it is advantageous that a cornflower fermentation broth with excellent skin affinity can be obtained more efficiently and easily.

The cornflower system used as the raw material of the seed koji can be the same as those described in the aforementioned-cornflower-, and the aspect, size, state, etc. of the cornflower are also the same.

As for the koji fungus used in the production of the cornflower seed koji, the same ones as described in the above-koji fungus-can be used.

The inoculation amount of the koji fungus to the cornflower used as the raw material of the seed koji is not particularly limited, and can be appropriately selected according to the purpose, but relative to 100 parts by mass of the cornflower, the inoculation is preferably 5 parts by mass to 100 parts by mass Parts of koji bacteria suspended in sterilized water (1×10 ³ /mL~1×10 ⁸ /mL), more preferably 10 parts by mass to 50 parts by mass, particularly preferably 20 parts by mass to 30 parts by mass . When the inoculation amount of the koji fungus with respect to 100 parts by mass of the cornflower is less than 5 parts by mass, a sufficient amount of spores may not be attached to the cornflower, and when it exceeds 100 parts by mass, it may abnormally multiply due to excessive water content.

When inoculating the koji bacterium, when using the cornflower as the raw material of the seed koji, it is preferable to add water. The amount of water added is not particularly limited and can be appropriately selected according to the purpose. However, relative to 100 parts by mass of the aforementioned cornflower, it is preferable to add 10 parts by mass to 250 parts by mass, and more preferably to add 20 parts by mass to 200 parts by mass. Parts by mass, it is particularly preferable to add 30 parts by mass to 150 parts by mass. When the addition amount of the water with respect to 100 parts by mass of the cornflower is less than 10 parts by mass, a sufficient amount of spores may not be attached to the cornflower in some cases.

Regarding the aforementioned culture temperature, there is no particular limitation as long as it is within the temperature range at which the aforementioned koji bacteria can grow. It can be appropriately selected according to the purpose, but it is preferably 20°C to 40°C, more preferably 25°C to 35°C . When the temperature of the aforementioned culture is lower than 20°C, a sufficient amount of spores may not be attached to the aforementioned cornflower. In addition, when the temperature exceeds 50°C, the aforementioned koji bacteria may not proliferate.

Regarding the aforementioned cultivation time, it is not particularly limited and can be appropriately selected according to the purpose, but is preferably 80 hours to 210 hours, more preferably 100 hours to 190 hours, and particularly preferably 120 hours to 170 hours. If the aforementioned culture time is less than 80 hours, sometimes a sufficient amount of spores cannot be attached to the aforementioned cornflower, and if it exceeds 210 hours, the germination rate of the spores sometimes decreases.

The cornflower fermentation broth may be one that contains the above-mentioned aspergillus spores, or one that removes the aforementioned aspergillus spores, but is preferably one that removes the aforementioned aspergillus spores.

The state of the cornflower fermentation broth is not particularly limited, and can be appropriately selected according to the purpose. For example, it may be the cornflower fermentation broth itself, the purified product of the cornflower fermentation broth, the concentrate of the cornflower fermentation broth, the cornflower fermentation Dilution of liquid, etc. In addition, the aforementioned cornflower fermentation broth may be one that remixes or dissolves the dried product of the cornflower fermentation broth in a solvent such as water or a hydrophilic solvent.

The purified product of the aforementioned cornflower fermentation broth is not particularly limited, and can be appropriately selected according to the purpose. For example, solid components in the aforementioned cornflower fermentation broth (for example, the aforementioned cornflower plant body, koji fungus cell, residue Etc.) Removal, etc. The aforementioned removal means is not particularly limited, and can be appropriately selected according to the purpose, for example, filtration and the like. As for the aforementioned filtering method, there is no special restriction, and it can be selected appropriately according to the purpose among the known methods.

The dilution of the cornflower fermentation broth and the concentrate of the cornflower fermentation broth are not particularly limited, and can be appropriately selected according to the purpose. For example, the cornflower fermentation broth is prepared to a desired concentration. As far as the aforementioned dilution method is concerned, there is no special restriction, and it can be selected appropriately according to the purpose among the known methods. The aforementioned means of concentration is not particularly limited, and can be appropriately selected according to the purpose, and examples thereof include concentration under reduced pressure.

The dried product of the cornflower fermentation broth is not particularly limited, and can be appropriately selected according to the purpose. For example, the dried cornflower fermentation broth can be exemplified. The aforementioned drying means is not particularly limited, and can be appropriately selected according to the purpose. For example, freeze-drying and the like can be mentioned.

The aforementioned cornflower fermentation broth is not particularly limited as long as it is a fermentation broth obtained from koji bacteria of cornflower, and it can be appropriately selected according to the purpose. However, in terms of good skin-friendliness, it is preferably one with a contact angle of 85° or less The cornflower fermentation broth is more preferably a cornflower fermentation broth with a contact angle of 79° or less.

＜＜Other ingredients＞＞ The aforementioned other ingredients in the aforementioned anti-aging agents, antioxidants, anti-inflammatory agents, and whitening agents are not particularly limited, and can be appropriately selected according to the purpose. For example, excipients, anti-humidity agents, preservatives, Strengtheners, thickeners, emulsifiers, antioxidants, sweeteners, sour flavors, seasonings, colorants, fragrances, whitening agents, humectants, oily ingredients, UV absorbers, surfactants, thickeners, alcohols , Powder ingredients, toners, water-based ingredients, water, skin nutrients, etc. One of these may be used alone, or two or more of them may be used in combination. The content of the aforementioned other components is not particularly limited, and can be appropriately selected according to the purpose.

-use- The anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention has excellent anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect at least any one of the effects, so it can be suitably used as For example, pharmaceuticals, quasi-drugs, cosmetics, food and beverages, etc., can be appropriately selected in terms of their blending amount, usage, and dosage form according to the purpose of use.

Regarding the aforementioned blending amount, it can be appropriately adjusted according to the physiological activity of the aforementioned fermentation broth and the like. In addition, the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent may be the fermentation broth itself.

The aforementioned usage is not particularly limited, and can be appropriately selected according to the purpose, and examples thereof include oral, parenteral, and topical usage. Among these, other uses are preferable.

The aforementioned dosage forms are not particularly limited, and can be appropriately selected according to the purpose. Examples include tablets, powders, capsules, granules, extractants, and syrups for oral administration; injections, drips , And suppositories, etc. for parenteral administration; lotions, lotions, creams, ointments, beauty lotions, lotions, masks, gels, lipsticks, lipsticks, foundations, bath agents, soaps, body washes, astringents, scalp External agents such as care lotion, hair cream, hair lotion, hair wax, shampoo, conditioner, etc.

In addition, the anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention can also be used as reagents for research related to the mechanism of anti-aging, antioxidant, anti-inflammatory, or whitening effects.

The anti-aging agents, antioxidants, anti-inflammatory agents, and whitening agents of the present invention are suitable for humans, but as long as they produce their respective effects, they can also be applied to animals other than humans (for example, mice, rats, hamsters). , Dogs, cats, cows, pigs, monkeys, etc.).

(cosmetic) The cosmetic 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 needed.

＜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 cosmetics is not particularly limited, and can be appropriately selected according to the purpose, but It is preferably 5 vol% or more with respect to the total amount of the aforementioned cosmetics, and more preferably 20 vol% or more. When the content of at least one selected from the group consisting of the aforementioned anti-aging agent, the aforementioned antioxidant, the aforementioned anti-inflammatory agent, and the aforementioned whitening agent is less than 5% by volume, anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and At least any one of the whitening effects becomes insufficient. In addition, 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 as large as possible, and the upper limit is not particularly limited, and it can be used according to the purpose. And choose appropriately. In addition, 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.

＜Other ingredients＞ In the aforementioned cosmetics, various main agents, auxiliary agents, and other ingredients commonly used in the manufacture of cosmetics can be added to the extent that the purpose and effects of the present invention are not impaired according to needs. The aforementioned other ingredients are not particularly limited, and can be appropriately selected according to the purpose. Examples include astringents, bactericides, antibacterial agents, ultraviolet absorbers, cell activators, oils and fats, waxes, hydrocarbons, and fatty acids. , Alcohols, Esters, Surfactants, Spices, etc. One of these may be used alone, or two or more of them may be used in combination. When these ingredients are used in combination with at least one selected from the group consisting of the aforementioned anti-aging agent, the aforementioned antioxidant, the aforementioned anti-inflammatory agent, and the aforementioned whitening agent, it may act synergistically, bringing about the above-mentioned expectations. Excellent effect. The content of the aforementioned other ingredients in the aforementioned cosmetics is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected according to the purpose.

＜Use＞ The use of the aforementioned cosmetics is not particularly limited, and can be appropriately selected from general cosmetics. Examples include lotions, lotions, creams, ointments, beauty lotions, lotions, facial masks, gels, lipsticks, lipsticks, Skin cosmetics such as foundation, bathing agent, soap, shower gel; astringent, scalp care lotion, hair cream, hair lotion, wax, shampoo, conditioner and other scalp and hair cosmetics.

The aforementioned cosmetic may be at least one selected from the group consisting of the aforementioned anti-aging agent, the aforementioned antioxidant, the aforementioned anti-inflammatory agent, and the aforementioned whitening agent, in a manner that does not hinder its activity, and can be blended with any cosmetic, or It is a cosmetic containing as a main ingredient at least one selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent. In addition, 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.

The cosmetics of the present invention are suitable for humans, but as long as they produce their respective effects, they can also be applied to animals other than humans (for example, mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.).

The cosmetic of the present invention contains at least one selected from the group consisting of the aforementioned anti-aging agent, the aforementioned antioxidant, the aforementioned anti-inflammatory agent, and the aforementioned whitening agent. Therefore, when it is used on the skin, it exerts an excellent anti-aging effect and anti-aging effect. The point of action of at least any one of oxidative action, anti-inflammatory action, and whitening action is useful.

[Example] Hereinafter, manufacturing examples and test examples are given to specifically explain the present invention, but the present invention is not limited to these test examples at all.

<Production example 1: Preparation of sage fermentation broth 1> -Seed koji preparation step- Take the koji fungus (Aspergillus oryzae , strain name: AOK1714, manufactured by Akita Konno Co., Ltd.) with platinum earrings, and suspend it in 50 mL of sterilized water was used to prepare a koji bacteria solution. As a result of calculating the number of bacteria in the aforementioned koji bacteria solution using a Thoma blood cell counting board (manufactured by EKDS), it was 1.0×10 ⁵ cells/mL. Next, 10 g of sage (manufactured by ALBION Co., Ltd.) cut into 0.5 cm to 5 cm was placed in an Erlenmeyer flask, autoclaved, and 2 mL of the above-mentioned koji bacteria solution was inoculated in the Erlenmeyer flask, followed by static culture at 30°C for 168 hours . After the cultivation, it was dried at 45°C for 24 hours to obtain "sage koji".

-Fermentation step-The sage (manufactured by ALBION Co., Ltd.) was pulverized using a pulverizer (sugar mill) and passed through a 2 mm mesh sieve to obtain a pulverized sage. 1,000 mL of water was added to 50 g of this crushed sage, and after mixing, 20 mL of the sage seed koji obtained in the aforementioned seed koji preparation step (the number of bacteria: about 1.0×10 ⁶ cells/mL) was inoculated. Then, incubate for 22 hours before 25°C. The obtained fermentation broth was filtered using diatomaceous earth to obtain "sage fermentation broth 1".

<Manufacturing example 2: Preparation of sage fermentation broth 2> In addition to the aforementioned manufacturing example 1, the sage seed koji was changed to rice-based seed koji ( Aspergillus oryzae , Shirakami white koji, Akita Konano Co., Ltd.) Co., Ltd.) (hereinafter, sometimes referred to as "rice seed koji"), by the same method as the aforementioned production example 1, "sage fermentation broth 2" was obtained.

<Comparative Manufacturing Example 1: Preparation of Sage Extract> The sage (manufactured by ALBION Co., Ltd.) was pulverized using a pulverizer (sugar mill) and passed through a 2 mm mesh sieve to obtain a pulverized sage. 1,000 mL of water was added to 50 g of this ground sage, and after mixing, the mixture was stirred at 25°C for 22 hours. Next, the obtained mixture was filtered using diatomaceous earth to obtain a "sage extract".

(Test Example A-1: Measurement of Contact Angle) The sage fermentation broth 1 obtained in Production Example 1, the sage fermentation broth 2 obtained in Production Example 2 and the sage extract obtained in Comparative Production Example 1 were used as test samples, and the contact was measured by the following method angle. Specifically, using a dynamic contact angle and surface tension measuring device (FTA1000 Falcon, manufactured by First Ten Angstroms), 3μL of each test sample was dropped into the sample table (made of aluminum) of the aforementioned device, and the temperature was 22°C and relative humidity. Under the condition of 20%, the measurement is carried out by the drop method. Use the θ/2 method to obtain a contact angle θ(°) of 1,000 ms. The measurement of the contact angle was performed 3 times, and the average value was obtained. The results are shown in Table 1 below. In addition, one of the droplets during the measurement of the contact angle of each test sample is shown in FIGS. 1A to 1C.

[Table 1] Test sample Ferment Aspergillus Contact angle θ(°) Manufacturing example 1 Sage fermentation broth 1 Have Sage Koji 77.44 Manufacturing example 2 Sage fermentation broth 2 Have Rice Koji 80.02 Comparative Manufacturing Example 1 Sage Extract no - 81.57

Compared with the sage extract obtained in Comparative Production Example 1, the sage fermentation broth 1 obtained in Production Example 1 and the sage fermentation broth 2 obtained in Production Example 2 all have a small contact angle of 81 Below °, for those with excellent skin affinity. In addition, the sage fermentation broth 1 obtained in Production Example 1 had a contact angle of 78° or less, and had more excellent skin affinity.

(Test Example 1-1: Matrix Metalloproteinase-1 (MMP-1) Activity Inhibition Test) The sage fermentation broth 1 obtained in Production Example 1, the sage fermentation broth 2 obtained in Production Example 2 and the sage extract obtained in Comparative Production Example 1 were used as test samples. The following test method, which was partially modified by the Wunsch and Heidrich method, was used to test the inhibitory effect of matrix metalloproteinase-1 (MMP-1) activity.

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

In addition, as a blank group, the same operation and absorbance measurement as described above were performed except that the MMP-1 solution (enzyme solution) was changed to 0.1 mol/L Tris-HCl buffer (pH 7.1).

Furthermore, as a control group, except that the dissolving solution of the test sample was changed to a 0.1 mol/L Tris-HCl buffer (pH 7.1) containing 20 mmol/L calcium chloride that did not contain the test sample, the same was performed as described above. The same operation and measurement of absorbance.

Based on the obtained measurement value of absorbance, the MMP-1 activity inhibition rate was calculated based on the following formula 1. The results are shown in Table 2 below. ＜Formula 1＞ Inhibition rate of MMP-1 activity (%)={1-(C-D)/(A-B)}×100 In the aforementioned formula 1, A to D each represent the following. A: Absorbance at a wavelength of 320nm with no test material added and enzyme added B: Absorbance at a wavelength of 320nm with no added test material and no enzyme C: Absorbance at 320nm wavelength when the tested sample and enzyme are added D: Absorbance at a wavelength of 320nm when the test sample is added and no enzyme is added

[Table 2] Tested sample Concentration of tested sample (μg/mL) MMP-1 activity inhibition rate (%) Manufacturing example 1 Sage fermentation broth 1 400 7.0 Manufacturing example 2 Sage fermentation broth 2 400 4.2 Comparative Manufacturing Example 1 Sage Extract 400 1.5

(Test example 1-2: Hyaluronic acid synthase 3 (HAS3) mRNA expression promotion test) Using the sage fermentation broth 1 obtained in Production Example 1, the sage fermentation broth 2 obtained in Production Example 2 and the sage extract obtained in Comparative Production Example 1 as test materials, the following Test method, test hyaluronic acid synthase 3 (HAS3) mRNA performance promotion effect.

In a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries Co., Ltd.), each test sample was dissolved so that the final concentration became the concentration shown in Table 3 below, and the test sample medium was prepared. The normal human neonatal epidermal keratinocytes (Normal Human Epidermal Keratinocytes; NHEK, manufactured by Kurabo Industries Co., Ltd.) were used in a medium for proliferation of normal human epidermal keratinocytes (HuMedia-KG2, manufactured by Kurabo Industries Co., Ltd.). After culturing under the conditions of ℃ and 5% CO ₂ until the cells converge, the cells are recovered by trypsin treatment. The normal human epidermal keratinocyte proliferation medium (HuMedia-KG2) was used to adjust it to 1.5×10 ⁵ cells/mL. Next, 2 mL of the aforementioned NHEK (1.5×10 ⁵ cells/mL) was inoculated into a 35 mm dish, and cultured overnight _{at 37° C. and 5% CO 2.} After the cultivation, the basal medium was exchanged for normal human epidermal keratinocyte basal medium (HuMedia-KB2) and cultured for another 24 hours. After the completion of the cultivation, the medium was exchanged with 2 mL of the aforementioned medium with the test sample added, and the medium _{was cultured at 37°C and 5% CO 2} for 24 hours. After the completion of the culture, the culture solution was removed, and total RNA was extracted with RNA extraction reagent (ISOGEN II (Cat. No.: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer. The total RNA was adjusted to 200ng/μL with water.

In addition, as a control group, the same operation and absorbance measurement as described above were performed except that 2 mL of the aforementioned test sample medium was changed to 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) without the test sample. In the same way as above, the total RNA was prepared to 200ng/μL.

Using the aforementioned total RNA as a template, the expression levels of hyaluronic acid synthase 3 (HAS3) mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, which is an internal standard, were measured. mRNA detection is performed by using a real-time PCR (Real-time PCR) device (Thermal Cycler DIce (registered trademark) Real Time System III, manufactured by Takara Bio Co., Ltd.), and SYBR (registered trademark) PrimeScript (registered trademark) RT- PCR Kit (Perfect Real Time (catalog number: RR063A), a 2-step RT-PCR reaction manufactured by Takara Bio Co., Ltd.) was performed. The expression level of HAS3 mRNA without the test material and the test material added is corrected with the expression level of GAPDH mRNA. From this correction value, the HAS3 mRNA expression promotion rate was calculated based on the following Equation 2. The results are shown in Table 3 below. ＜Formula 2＞ HAS3 mRNA expression promotion rate (%) = A/B×100 In the aforementioned formula 2, A and B each represent the following. A: The correction value when adding the tested sample B: The correction value when the tested sample is not added

[table 3] Tested sample Concentration of tested sample (μg/mL) HAS3 mRNA expression promotion rate (%) Manufacturing example 1 Sage fermentation broth 1 5 172.0 Manufacturing example 2 Sage fermentation broth 2 5 127.3 Comparative Manufacturing Example 1 Sage Extract 5 125.3

(Test example 1-3: DPPH free radical elimination effect test) Using the sage fermentation broth 1 obtained in Production Example 1, the sage fermentation broth 2 obtained in Production Example 2 and the sage extract obtained in Comparative Production Example 1 as test materials, the following Test method, test DPPH free radical elimination effect.

Dissolve each test sample in an ethanol solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a test sample solution. Add 3 mL of the aforementioned test sample solution to 3 mL of 150 μmol/L DPPH (diphenyl-p-picric acid hydrazino) ethanol solution. Immediately stop the container and shake it. After standing for 30 minutes, measure the absorbance at a wavelength of 520 nm. In addition, the final concentration of the test sample at this time is the concentration shown in Table 4 below.

In addition, as a blank group, the same operation and absorbance measurement as above were performed except that the DPPH ethanol solution was changed to an ethanol solution not containing DPPH.

In addition, as a control group, the same operation and absorbance measurement as described above were performed 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.

Based on the obtained measurement value of absorbance, the DPPH radical elimination rate was calculated based on the following formula 3. The results are shown in Table 4 below. ＜Formula 3＞ DPPH radical elimination rate (%) = {A-(B-C)}/A×100 In the aforementioned formula 3, A to C each represent the following. A: Absorbance at a wavelength of 520nm with no test material added and DPPH added B: The absorbance in the wavelength of 520nm with the test material added and DPPH added C: Absorbance at a wavelength of 520nm without added test material and without DPPH

[Table 4] Tested sample Concentration of tested sample (μg/mL) DPPH free radical elimination rate (%) Manufacturing example 1 Sage fermentation broth 1 12.5 35.0 Manufacturing example 2 Sage fermentation broth 2 12.5 32.1 Comparative Manufacturing Example 1 Sage Extract 12.5 29.7

(Test example 1-4: Hyaluronidase activity inhibition test) Using the sage fermentation broth 1 obtained in Production Example 1, the sage fermentation broth 2 obtained in Production Example 2 and the sage extract obtained in Comparative Production Example 1 as test materials, the following Test method, test the inhibitory effect of hyaluronidase activity.

Each test sample was dissolved in 0.1 mol/L acetate buffer (pH 3.5) to prepare a test sample solution. 0.1 mL of a hyaluronidase solution (Type IV-S (from bovine testis), 400 NF units/mL, manufactured by SIGMA) was added to 0.2 mL of the aforementioned test sample solution, and reacted at 37°C for 20 minutes. Next, 0.2 mL of 2.5 mmol/L calcium chloride as an activator was added, and the reaction was further carried out at 37°C for 20 minutes. 0.5 mL of 0.8 mg/mL sodium hyaluronate solution (from rooster comb) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added thereto, and reacted at 37°C for 40 minutes. In addition, the final concentration of the test sample at this time is 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 a boric acid solution was added to each reaction solution, and the mixture was boiled for 3 minutes. After ice-cold, add 6mL of p-DABA reagent (dissolve 10g of p-dimethylaminobenzaldehyde in a mixture of 12.5mL of 10N hydrochloric acid and 87.5mL of acetic acid, dilute 10 times with acetic acid), and react at 37°C for 20 minute. Next, the absorbance at a wavelength of 585 nm was measured.

In addition, as a blank group, the same operation and absorbance measurement as described above were performed except that the hyaluronidase solution (enzyme solution) was changed to 0.1 mol/L acetate buffer (pH 3.5).

In addition, as a control group, the same operation and absorbance measurement as described above were performed except that the test sample solution was changed to a 0.1 mol/L acetate buffer solution (pH 3.5) that did not contain the test sample.

Based on the obtained measurement value of absorbance, the hyaluronidase activity inhibition rate was calculated based on the following formula 4. The results are shown in Table 5 below. ＜Formula 4＞ Inhibition rate of hyaluronidase activity (%) = {1-(C-D)/(A-B)}×100 In the aforementioned formula 4, the systems A to D each represent the following. A: Absorbance at a wavelength of 585nm with no test material added and enzyme added B: Absorbance at a wavelength of 585nm with no added test material and no enzyme C: Absorbance at a wavelength of 585nm where the test material and enzyme are added D: Absorbance at a wavelength of 585nm with the test material added and no enzyme added

[table 5] Tested sample Concentration of tested sample (μg/mL) Inhibition rate of hyaluronidase activity (%) IC ₅₀ (µg/mL) Manufacturing example 1 Sage fermentation broth 1 400 67.7 320.6 Manufacturing example 2 Sage fermentation broth 2 400 66.4 317.2 Comparative Manufacturing Example 1 Sage Extract 400 49.8 ＞400

(Test Example 1-5: Tyrosinase activity inhibition test) Using the sage fermentation broth 1 obtained in Production Example 1, the sage fermentation broth 2 obtained in Production Example 2 and the sage extract obtained in Comparative Production Example 1 as test materials, the following Test method, test the inhibitory effect of tyrosinase activity.

Dissolve each test sample in a 25% DMSO solution to prepare a test sample solution. In a 48-well plate, 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 aforementioned test sample solution were added, and the mixture was allowed to stand at 37°C for 10 minutes. To this, 0.02 mL of 800 units/mL tyrosinase solution (manufactured by SIGMA) was added, and the reaction was further carried out at 37°C for 15 minutes. After the reaction, the absorbance at a wavelength of 475 nm was measured. In addition, the final concentration of the test sample at this time is the concentration shown in Table 6 below.

In addition, as a blank group, the same operation and absorbance measurement as described above were performed except that the tyrosinase solution (enzyme solution) was changed to Mcllvaine buffer (pH 6.8).

In addition, as a control group, the same operation and absorbance measurement as described above were performed except that the test sample solution was changed to a 25% DMSO solution that did not contain the test sample.

Based on the obtained measurement value of the absorbance, the tyrosinase activity inhibition rate was calculated based on the following formula 5. The results are shown in Table 6 below. <Formula 5> Tyrosinase activity inhibition rate (%) = {1-(C-D)/(A-B)}×100 In the aforementioned formula 5, the systems A to D each represent the following. A: Absorbance at a wavelength of 475nm with no test material added and enzyme added B: Absorbance at a wavelength of 475nm with no added test material and no enzyme C: The absorbance at the wavelength of 475nm with the test sample and the enzyme added D: Absorbance at a wavelength of 475nm with the test material added and no enzyme added

[Table 6] Tested sample Concentration of tested sample (μg/mL) Tyrosinase activity inhibition rate (%) Manufacturing example 1 Sage fermentation broth 1 400 5.7 Manufacturing example 2 Sage fermentation broth 2 400 9.5 Comparative Manufacturing Example 1 Sage Extract 400 4.2

<Production Example 3: Preparation of Artemisia Artemisia Fermentation Broth 1> In the seed koji preparation step of Production Example 1, except for changing the sage to Artemisia capillaris Thunbergii (manufactured by ALBION Co., Ltd.), The "Artemisia sylvestris seed koji" was prepared in the same way as the seed koji preparation step of the aforementioned production example 1. In addition, in the fermentation step of the aforementioned Production Example 1, except for changing the sage to Artemisia capillaris Thunbergii (manufactured by ALBION Co., Ltd.), the same method as the fermentation step of the aforementioned Production Example 1 was used to obtain the Fermentation broth 1 of Artemisia spp.".

<Manufacturing Example 4: Preparation of Artemisia sphaerocephala Fermentation Liquid 2> In the aforementioned Manufacture Example 3, except for the change of Artemisia sphaerocephala seed koji to rice seed koji ( Aspergillus oryzae , Shirakami white koji, Akita Konano Co., Ltd.), The "Artemisia serrata fermentation broth 2" was obtained by the same method as the aforementioned production example 3.

<Comparative Production Example 2: Preparation of Artemisia Artemisiae Extract> In the aforementioned Comparative Production Example 1, except that sage was changed to Artemisia capillaris Thunbergii (manufactured by ALBION Co., Ltd.), the production was compared with the above In the same way as in Example 1, the "Artemisia sphaerocephala extract" was obtained.

(Test Example A-2: Measurement of Contact Angle) In the aforementioned Test Example A-1, except that the test sample was changed to the Artemisia sphaerocephala fermentation broth 1 obtained in Production Example 3, the Artemisia sphaerocephala fermentation broth 2 obtained from Production Example 4, and the sphacelia serrata obtained from Comparative Production Example 2 Except for the Artemisia extract, the contact angle was measured in the same manner as in the aforementioned Test Example A-1. The results are shown in Table 7 below. In addition, an example of the droplets at the time of measuring the contact angle of each test sample is shown in FIGS. 2A to 2C.

[Table 7] Test sample Ferment Aspergillus Contact angle θ(°) Manufacturing example 3 Artemisia serrata fermentation broth 1 Have Artemisia spp 77.50 Manufacturing example 4 Artemisia serrata fermentation broth 2 Have Rice Koji 79.58 Comparative Manufacturing Example 2 Artemisia serrata extract no - 87.75

Compared with the Artemisia sphaerocephala extract obtained in Comparative Production Example 2, the Artemisia sphaerocephala fermentation broth 1 obtained in Production Example 3 and the Artemisia sphaerocephala fermentation broth 2 obtained in Production Example 4 have a small contact angle of 81° or less , For those with excellent skin-friendliness. Furthermore, the Artemisia sphaerocephala fermentation broth 1 obtained in Production Example 3 has a contact angle of 78° or less and is more excellent in skin affinity.

(Test example 2-1: Hyaluronic acid synthase 3 (HAS3) mRNA expression promotion test) In Test Example 1-2, except that the tested sample was changed to the Artemisia sphaerocephala fermentation broth 1 obtained in Production Example 3, the Artemisia sphaerocephala fermentation broth 2 obtained in Production Example 4, and the saccharomyces cinerariae obtained in Comparative Production Example 2. Artemisia extract, except that the final concentration of the tested sample was changed to the concentration shown in Table 8 below, the same method as in Test Example 1-2 was used to test the hyaluronic acid synthase 3 (HAS3) mRNA expression promotion effect. The results are shown in Table 8 below.

[Table 8] Tested sample Concentration of tested sample (μg/mL) HAS3 mRNA expression promotion rate (%) Manufacturing example 3 Artemisia serrata fermentation broth 1 20 38.7 Manufacturing example 4 Artemisia serrata fermentation broth 2 20 46.1 Comparative Manufacturing Example 2 Artemisia serrata extract 20 28.3

(Test example 2-2: Type I collagen production promotion effect test) Using the Artemisia sphaerocephala fermentation broth obtained in Production Example 3 1, the Artemisia sphaerocephala fermentation broth 2 obtained in Production Example 4, and the Artemisia serrata extract obtained in Comparative Production Example 2 as the test samples, the following The test method is to test the promoting effect of type I collagen.

In Dulbecco MEM containing 0.25% fetal bovine serum (Fetal bovine serum; FBS, manufactured by Biosera, manufactured by Nissui Pharmaceutical Co., Ltd.), dissolve each test sample so that the final concentration becomes the concentration shown in Table 9 below. And prepare and add the test sample medium. Using DMEM containing 10% FBS, normal human fibroblasts (NB1RGB, purchased from RIKEN BRC) _{were cultured at 37°C and 5% CO 2} until the cells met, and then the cells were recovered by trypsin treatment. Adjust it to 1.6×10 ⁵ cells/mL with DMEM containing 10% FBS. Next, in a 96-well microplate, the aforementioned NB1RGB (1.6×10 ⁵ cells/mL) was inoculated with 100 μL per well, and cultured overnight at 37° C. and 5% CO ₂ . After the completion of the culture, the medium was exchanged with 100 μL of the medium added with the test sample _{, and cultured at 37°C and 5% CO 2} for 3 days. After the cultivation, the amount of type I collagen in the culture medium of each well was measured by the ELISA method.

Specifically, transfer 90 μL of the culture supernatant to an ELISA plate, and overnight at 4°C to adsorb to the plate, discard the solution, and wash with phosphate physiological buffer (PBS-T) containing 0.05% Tween-20 net. Afterwards, a blocking operation was performed with a phosphate physiological buffer containing 1% FBS. The solution was discarded and washed with phosphate physiological buffer (PBS-T) containing 0.05% Tween-20 to react with anti-human collagen type I antibody (rabbit IgG, manufactured by Chemi-Con). The solution was discarded and washed with phosphate physiological buffer (PBS-T) containing 0.05% Tween-20. After reacting with the HRP-labeled anti-rabbit IgG antibody, the same washing operation was performed and the color reaction was performed. The amount of type I collagen was calculated by performing the above-mentioned ELISA using a standard product, creating a calibration line.

In addition, as a control group, the test sample solution was changed to 0.25% FBS containing DMEM without the test sample, and the same operation and measurement by the ELISA method as described above were performed.

Based on the obtained measurement values, the type I collagen production promotion rate was calculated based on the following formula 6. The results are shown in Table 9 below. ＜Formula 6＞ Type I collagen production promotion rate (%)=A/B×100 In the aforementioned formula 6, A and B each represent the following. A: The amount of type I collagen when the test sample is added B: The amount of type I collagen when the tested sample is not added

[Table 9] Tested sample Concentration of tested sample (μg/mL) Type I collagen production promotion rate (%) Manufacturing example 3 Artemisia serrata fermentation broth 1 100 72.9 Manufacturing example 4 Artemisia serrata fermentation broth 2 100 65.5 Comparative Manufacturing Example 2 Artemisia serrata extract 100 56.9

(Test example 2-3: Test of promoting effect of clonectin-1 mRNA expression) Using the Artemisia sphaerocephala fermentation broth obtained in Production Example 3 1, the Artemisia sphaerocephala fermentation broth 2 obtained in Production Example 4, and the Artemisia serrata extract obtained in Comparative Production Example 2 as the test samples, the following The test method is to test the expression promoting effect of codonin-1 mRNA.

In a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries Co., Ltd.), each test sample was dissolved so that the final concentration became the concentration shown in Table 10 below, and the test sample was added to the medium. Using normal human epidermal keratinocyte proliferation medium (HuMedia-KG2, manufactured by Kurabo Industries Co., Ltd.), normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurabo Industries Co., Ltd.) were heated at 37°C, 5% CO _{After culturing under the conditions of 2} until the cells meet, the cells are recovered by trypsin treatment. The normal human epidermal keratinocyte growth medium (HuMedia-KG2) was used to adjust it to 1.5×10 ⁵ cells/mL. Next, 2 mL of the aforementioned NHEK (1.5×10 ⁵ cells/mL) was inoculated into a 35 mm dish, and cultured overnight at 37° C. and 5% CO ₂ . After the cultivation, the medium was exchanged with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the completion of the culture, the culture medium was exchanged with 2 mL of the aforementioned test medium supplemented with the test material, and the culture _{was cultured at 37°C and 5% CO 2} for 24 hours. After the incubation, the culture solution was removed, and the total RNA was extracted with a reagent for RNA extraction (ISOGEN II (Cat. No.: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer. The total RNA prepared with water was 200 ng/μL.

In addition, as a control group, the same operation and absorbance measurement as described above were performed except that 2 mL of the aforementioned test sample medium was added to 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) that did not contain the test sample. Prepare the total RNA to 200ng/μL in the same way as above.

Using each of the aforementioned total RNAs as templates, the expression levels of clonectin-1 mRNA and GAPDH mRNA, which is an internal standard, were measured. mRNA detection is performed by using a real-time PCR device (Thermal Cycler DIce (registered trademark) Real Time System III, manufactured by Takara Bio Co., Ltd.), and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (Catalog number: RR063A), Takara Bio Co., Ltd. product) two-step real-time PCR reaction. The expression level of clonectin-1 mRNA without the addition of the test sample and the addition of the test sample was corrected with the expression level of GAPDH mRNA. From this correction value, the rate of promoting the expression of fibronectin-1 mRNA was calculated based on Equation 7 below. The results are shown in Table 10 below. ＜Formula 7＞ Claudin-1 mRNA expression promotion rate (%) = A/B×100 In the aforementioned formula 7, A and B each represent the following. A: The correction value when adding the tested sample B: The correction value when the tested sample is not added

[Table 10] Tested sample Concentration of tested sample (μg/mL) Claudin-1 mRNA expression promotion rate (%) Manufacturing example 3 Artemisia serrata fermentation broth 1 20 739.6 Manufacturing example 4 Artemisia serrata fermentation broth 2 20 690.5 Comparative Manufacturing Example 2 Artemisia serrata extract 20 662.8

(Experiment 2-4: Test of promoting effect of clonectin-4 mRNA expression) Using the Artemisia sphaerocephala fermentation broth obtained in Production Example 3 1, the Artemisia sphaerocephala fermentation broth 2 obtained in Production Example 4, and the Artemisia serrata extract obtained in Comparative Production Example 2 as the test samples, the following The test method is to test the expression-promoting effect of codonin-4 mRNA.

In a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries Co., Ltd.), each test sample was dissolved so that the final concentration became the concentration shown in Table 11 below, and the test sample medium was prepared. Using normal human epidermal keratinocyte proliferation medium (HuMedia-KG2, manufactured by Kurabo Industries Co., Ltd.), normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurabo Industries Co., Ltd.) under 5% CO ₂ conditions After culturing until the cells meet, the cells are recovered by trypsin treatment. The normal human epidermal keratinocyte growth medium (HuMedia-KG2) was used to adjust it to 1.5×10 ⁵ cells/mL. Next, 2 mL of the aforementioned NHEK (1.5×10 ⁵ cells/mL) was inoculated in a 35 mm dish, and cultured overnight at 37° C. and 5% CO ₂ . After the cultivation, the medium was exchanged with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the completion of the culture, the culture medium was exchanged with 2 mL of the aforementioned test medium supplemented with the test material, _{and cultured at 37°C and 5% CO 2} for 24 hours. After the cultivation, the culture solution was removed, and total RNA was extracted with RNA extraction reagent (ISOGEN II (Cat. No.: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer, using pure water The prepared total RNA is 200ng/μL.

In addition, as a control group, the same operation and absorbance measurement as described above were performed except that 2 mL of the aforementioned test sample medium was changed to 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) without the test sample. In the same way as above, the total RNA was prepared to 200ng/μL.

Using each of the aforementioned total RNAs as templates, the expression levels of Claudin-4 mRNA and GAPDH mRNA, which is an internal standard, were measured. mRNA detection is performed by using a real-time PCR device (Thermal Cycler DIce (registered trademark) Real Time System III, manufactured by Takara Bio Co., Ltd.), and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (Catalog number: RR063A), Takara Bio Co., Ltd. product) two-step real-time PCR reaction. The expression level of clonectin-4 mRNA without the addition of the test sample and the addition of the test sample was corrected with the expression of GAPDH mRNA. Based on this correction value, the rate of promoting expression of the fibronectin-4 mRNA was calculated based on the following equation 8. The results are shown in Table 11 below. ＜Formula 8＞ Claudin-4 mRNA expression promotion rate (%) = A/B×100 In the aforementioned formula 8, A and B each represent the following. A: The correction value when adding the tested sample B: The correction value when the tested sample is not added

[Table 11] Tested sample Concentration of tested sample (μg/mL) Claudin-4 mRNA expression promotion rate (%) Manufacturing example 3 Artemisia serrata fermentation broth 1 20 674.0 Manufacturing example 4 Artemisia serrata fermentation broth 2 20 734.6 Comparative Manufacturing Example 2 Artemisia serrata extract 20 487.6

(Experimental example 2-5: agglutinin mRNA expression promoting effect test) Using the Artemisia sphaerocephala fermentation broth obtained in Production Example 3 1, the Artemisia sphaerocephala fermentation broth 2 obtained in Production Example 4, and the Artemisia serrata extract obtained in Comparative Production Example 2 as the test samples, the following Test method, test clinectin mRNA performance promotion effect.

In a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries Co., Ltd.), each test sample was dissolved so that the final concentration became the concentration shown in Table 12 below, and the test sample medium was prepared and added. Using normal human epidermal keratinocyte proliferation medium (HuMedia-KG2, manufactured by Kurabo Industries Co., Ltd.), normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurabo Industries Co., Ltd.) were heated at 37°C, 5% CO _{After culturing under the conditions of 2} until the cells meet, the cells are recovered by trypsin treatment. The normal human epidermal keratinocyte growth medium (HuMedia-KG2) was used to adjust it to 1.5×10 ⁵ cells/mL. Next, 2 mL of the aforementioned NHEK (1.5×10 ⁵ cells/mL) was inoculated into a 35 mm dish, and cultured overnight at 37° C. and 5% CO ₂ . After the cultivation, the medium was exchanged with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the cultivation, the culture solution was removed, and total RNA was extracted with RNA extraction reagent (ISOGEN II (Cat. No.: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer, using pure water The prepared total RNA is 200ng/μL.

In addition, as a control group, the same operation and absorbance measurement as described above were performed except that 2 mL of the aforementioned test sample medium was changed to 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) without the test sample. In the same way as above, the total RNA was prepared to 200ng/μL.

Using each of the aforementioned total RNAs as templates, the expression levels of clinectin mRNA and GAPDH mRNA, which is an internal standard, were measured. mRNA detection is performed by using a real-time PCR device (Thermal Cycler DIce (registered trademark) Real Time System III, manufactured by Takara Bio Co., Ltd.), and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (Catalog number: RR063A), Takara Bio Co., Ltd. product) two-step real-time PCR reaction. The expression level of the untested sample and the addition of the tested sample adenectin mRNA was corrected with the expression level of GAPDH mRNA. Based on this correction value, the clonectin mRNA expression promotion rate was calculated based on the following formula 9. The results are shown in Table 12 below. ＜Formula 9＞ The expression promotion rate of clinectin mRNA (%)=A/B×100 In the aforementioned formula 9, A and B each represent the following. A: The correction value when adding the tested sample B: The correction value when the tested sample is not added

[Table 12] Tested sample Concentration of tested sample (μg/mL) Adenin mRNA performance promotion rate (%) Manufacturing example 3 Artemisia serrata fermentation broth 1 20 707.9 Manufacturing example 4 Artemisia serrata fermentation broth 2 20 635.7 Comparative Manufacturing Example 2 Artemisia serrata extract 20 621.0

<Production Example 5: Preparation of Thyme Fermentation Broth 1> In the step of preparing seed koji in Production Example 1, except that sage was changed to Thymus ( Thymus vulgaris Linne) (manufactured by ALBION Co., Ltd.), the same as the previous production example Prepare "Thyme Koji" in the same way as in the preparation steps of 1 Seed Koji. In addition, in the fermentation step of the aforementioned production example 1, except that sage was changed to thyme ( Thymus vulgaris Linne) (manufactured by ALBION Co., Ltd.), the same method as the fermentation step of the aforementioned production example 1 was used to obtain "thyme fermentation". Liquid 1".

<Manufacturing Example 6: Preparation of Thyme Fermentation Liquid 2> In the above-mentioned Manufacturing Example 5, except that the thyme seed koji was changed to the rice seed koji ( Aspergillus oryzae , Shirakami white koji, Akita Konno Co., Ltd.), the same as the aforementioned manufacturing In the same way as in Example 5, "thyme fermentation broth 2" was obtained.

<Comparative Production Example 3: Preparation of Thyme Extract> In the aforementioned Comparative Production Example 1, except for changing the sage to Thymus ( Thymus vulgaris Linne) (manufactured by ALBION Co., Ltd.), the same as the aforementioned Comparative Production Example 1 Method to obtain "thyme extract".

(Test Example A-3: Measurement of Contact Angle) In the foregoing Test Example A-1, except that the test sample was changed to the thyme fermentation liquid obtained in Production Example 5 1, the thyme fermentation liquid 2 obtained in Production Example 6 and the thyme extract obtained in Comparative Production Example 3, The contact angle was measured in the same manner as in the aforementioned Test Example A-1. The results are shown in Table 13 below. In addition, an example of the droplets at the time of measuring the contact angle of each test sample is shown in FIGS. 3A to 3C.

[Table 13] Test sample Ferment Aspergillus Contact angle θ(°) Manufacturing example 5 Thyme fermentation broth 1 Have Thyme Koji 78.32 Manufacturing example 6 Thyme fermentation broth 2 Have Rice Koji 82.77 Comparative Manufacturing Example 3 Thyme Extract no - 88.59

Compared with the thyme extract obtained in Comparative Production Example 3, the thyme fermentation liquid 1 obtained in Production Example 5 and the thyme fermentation liquid 2 obtained in Production Example 6 all have a small contact angle of 87° or less, which is excellent in skin affinity. By. Furthermore, the thyme fermentation broth 1 obtained in Production Example 5 has a contact angle of 81° or less, which is more excellent in skin affinity.

(Test Example 3-1: Transglutaminase-1 (TGM-1) mRNA expression promotion test) Using the thyme fermentation broth obtained in Production Example 5 1, the thyme fermentation broth 2 obtained in Production Example 6 and the thyme extract obtained in Comparative Production Example 3 as test materials, the following test method was used to test the conversion to bran Glucidase-1 (TGM-1) mRNA shows a promoting effect.

In a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries Co., Ltd.), each test sample was dissolved so that the final concentration became the concentration shown in Table 14 below, and the test sample medium was prepared and added. Using normal human epidermal keratinocyte proliferation medium (HuMedia-KG2, manufactured by Kurabo Industries Co., Ltd.), normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurabo Industries Co., Ltd.) were heated at 37°C, 5% CO _{After culturing under the conditions of 2} until the cells meet, the cells are recovered by trypsin treatment. The normal human epidermal keratinocyte growth medium (HuMedia-KG2) was used to adjust it to 1.5×10 ⁵ cells/mL. Next, 2 mL of the aforementioned NHEK (1.5×10 ⁵ cells/mL) was inoculated in a 35 mm dish, and cultured overnight at 37° C. and 5% CO ₂ . After the cultivation, the medium was exchanged with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the completion of the culture, the culture medium was exchanged with 2 mL of the aforementioned test medium supplemented with the test material, _{and cultured at 37°C and 5% CO 2} for 24 hours. After the cultivation, the culture solution was removed, and total RNA was extracted with RNA extraction reagent (ISOGEN II (Cat. No.: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer, using pure water The prepared total RNA is 200ng/μL.

In addition, as a control group, the same operation and absorbance measurement as described above were performed except that 2 mL of the aforementioned test sample medium was changed to 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) without the test sample. In the same way as above, the total RNA was prepared to 200ng/μL.

Using the aforementioned total RNA as a template, the expression levels of transglutaminase-1 (TGM-1) mRNA and GAPDH mRNA, which is an internal standard, were measured. mRNA detection is performed by using a real-time PCR device (Thermal Cycler DIce (registered trademark) Real Time System III, manufactured by Takara Bio Co., Ltd.), and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (Catalog number: RR063A), Takara Bio Co., Ltd. product) two-step real-time PCR reaction. The expression level of TGM-1 mRNA without the test material and the test material added is corrected with the expression level of GAPDH mRNA. From this correction value, the TGM-1 mRNA expression promotion rate was calculated based on the following formula 10. The results are shown in Table 14 below. ＜Formula 10＞ TGM-1 mRNA expression promotion rate (%) = A/B×100 In the aforementioned formula 10, A and B each represent the following. A: The correction value when adding the tested sample B: The correction value when the tested sample is not added

[Table 14] Tested sample Concentration of tested sample (μg/mL) TMG-1 mRNA expression promotion rate (%) Manufacturing example 5 Thyme fermentation broth 1 20 625.0 Manufacturing example 6 Thyme fermentation broth 2 20 586.4 Comparative Manufacturing Example 3 Thyme Extract 20 515.7

(Test example 3-2: aquaporin 3 (AQP3) mRNA expression promoting effect test) Using the thyme fermentation broth 1 obtained in Production Example 5 1, the thyme fermentation broth 2 obtained in Production Example 6 and the thyme extract obtained in Comparative Production Example 3 as test samples, the water holes were tested by the following test method Protein 3 (AQP3) mRNA shows a promoting effect.

In a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries Co., Ltd.), each test sample was dissolved so that the final concentration became the concentration shown in Table 15 below, and the test sample medium was prepared and added. Using normal human epidermal keratinocyte proliferation medium (HuMedia-KG2, manufactured by Kurabo Industries Co., Ltd.), normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurabo Industries Co., Ltd.) were heated at 37°C, 5% CO _{After culturing under the conditions of 2} until the cells meet, the cells are recovered by trypsin treatment. The normal human epidermal keratinocyte growth medium (HuMedia-KG2) was used to adjust it to 1.5×10 ⁵ cells/mL. Next, 2 mL of the aforementioned NHEK (1.5×10 ⁵ cells/mL) was inoculated into a 35 mm dish, and cultured overnight at 37° C. and 5% CO ₂ . After the cultivation, the medium was exchanged with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the completion of the culture, the culture medium was exchanged with 2 mL of the aforementioned test medium supplemented with the test material, _{and cultured at 37°C and 5% CO 2} for 24 hours. After the cultivation, the culture solution was removed, and total RNA was extracted with RNA extraction reagent (ISOGEN II (Cat. No.: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer, using pure water The prepared total RNA is 200ng/μL.

In addition, as a control group, the same operation and absorbance measurement as described above were performed except that 2 mL of the aforementioned test sample medium was changed to 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) without the test sample. In the same way as above, the total RNA was prepared to 200ng/μL.

Using the aforementioned total RNA as a template, the expression levels of aquaporin 3 (AQP3) mRNA and GAPDH mRNA, which is an internal standard, were measured. mRNA detection is performed by using a real-time PCR device (Thermal Cycler DIce (registered trademark) Real Time System III, manufactured by Takara Bio Co., Ltd.), and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (Catalog number: RR063A), Takara Bio Co., Ltd. product) two-step real-time PCR reaction. The expression level of AQP3 mRNA without the test material and the test material added was corrected with the expression level of GAPDH mRNA. From this correction value, the AQP3 mRNA expression promotion rate was calculated based on the following formula 11. The results are shown in Table 15 below. ＜Formula 11＞ AQP3 mRNA expression promotion rate (%) = A/B×100 In the aforementioned formula 11, A and B each represent the following. A: The correction value when adding the tested sample B: The correction value when the tested sample is not added

[Table 15] Tested sample Concentration of tested sample (μg/mL) AQP3 mRNA expression promotion rate (%) Manufacturing example 5 Thyme fermentation broth 1 20 196.4 Manufacturing example 6 Thyme fermentation broth 2 20 160.5 Comparative Manufacturing Example 3 Thyme Extract 20 145.7

(Test Example 3-3: Test of promoting effect of clonectin mRNA expression) In Test Example 2-5, except that the test sample was changed to the thyme fermentation broth 1 obtained in Manufacturing Example 5, the thyme fermentation broth 2 obtained in Manufacturing Example 6 and the thyme extract obtained in Comparative Manufacturing Example 3, they were The final concentration of the test sample was changed to the concentration shown in Table 16 below, and in the same manner as in Test Example 2-5, the agglutinin mRNA expression promoting effect was tested. The results are shown in Table 16 below.

[Table 16] Tested sample Concentration of tested sample (μg/mL) Adenin mRNA performance promotion rate (%) Manufacturing example 5 Thyme fermentation broth 1 20 795.2 Manufacturing example 6 Thyme fermentation broth 2 20 1023.7 Comparative Manufacturing Example 3 Thyme Extract 20 562.9

(Test example 3-4: DPPH free radical elimination effect test) In Test Example 1-3, except that the test sample was changed to the thyme fermentation broth 1 obtained in Manufacturing Example 5, the thyme fermentation broth 2 obtained in Manufacturing Example 6, and the thyme extract obtained in Comparative Manufacturing Example 3, the thyme extract was The final concentration of the test sample was changed to the concentration shown in Table 17 below, and the DPPH radical elimination effect was tested in the same manner as in Test Example 1-3. The results are shown in Table 17 below.

[Table 17] Tested sample Concentration of tested sample (μg/mL) DPPH free radical elimination rate (%) Manufacturing example 5 Thyme fermentation broth 1 12.5 41.9 Manufacturing example 6 Thyme fermentation broth 2 12.5 39.9 Comparative Manufacturing Example 3 Thyme Extract 12.5 36.4

<Production Example 7: Preparation of Cypress Bee Grass Fermentation Liquid 1> In the seed koji preparation step of Production Example 1, except for changing the sage to Melissa officinalis Linne (manufactured by ALBION Co., Ltd.) "Cypress cypress seed koji" was prepared in the same way as the seed koji preparation step of the aforementioned production example 1. In addition, in the fermentation step of the aforementioned Production Example 1, except for changing the sage to Melissa officinalis Linne (manufactured by ALBION Co., Ltd.), the same method as the fermentation step of the aforementioned Production Example 1 was used to obtain Bee Grass Fermentation Broth 1".

<Manufacturing Example 8: Preparation of Cypress balm fermented liquid 2> In the aforementioned manufacturing example 7, except for the change of Cypress cypress seed koji to rice seed koji ( Aspergillus oryzae , Shirakami white koji, Akita Konno Co., Ltd.), The "cypress balm fermented liquid 2" was obtained in the same manner as in the aforementioned production example 7.

<Comparative Production Example 4: Preparation of Cypress balm extract> In the aforementioned Comparative Production Example 1, except that sage was changed to Melissa officinalis Linne (manufactured by ALBION Co., Ltd.), it was produced in comparison with the above The same method as in Example 1 was used to obtain the "cypress balm extract".

(Test Example A-4: Measurement of Contact Angle) In the foregoing test example A-1, except that the test sample was changed to the cypress balm fermentation broth 1 obtained in Production Example 7, the cypress balm fermentation broth 2 obtained in Production Example 8 and the cypress balm fermentation broth 2 obtained in Comparative Production Example 4 Except for the grass extract, the contact angle was measured in the same manner as in the aforementioned Test Example A-1. The results are shown in Table 18 below. In addition, an example of the droplets at the time of measuring the contact angle of each test sample is shown in FIGS. 4A to 4C.

[Table 18] Test sample Ferment Aspergillus Contact angle θ(°) Manufacturing example 7 Cypress balm fermentation broth 1 Have Cypress balm 77.09 Manufacturing example 8 Cypress balm fermentation broth 2 Have Rice Koji 80.16 Comparative Manufacturing Example 4 Cypress balm extract no - 86.53

Compared with the cypress balm extract obtained in Comparative Production Example 4, the cypress balm fermentation broth 1 obtained in Production Example 7 and the cypress balm fermentation broth 2 obtained in Production Example 8 all have a small contact angle of 85° or less , For those with excellent skin-friendliness. Furthermore, the cypress balm fermentation broth 1 obtained in Production Example 7 has a contact angle of 79° or less, which is more excellent in skin affinity.

(Test Example 4-1: Type I collagen production promotion effect test) In Test Example 2-2, except that the tested sample was changed to the cypress balm fermentation broth 1 obtained in Manufacturing Example 7, the cypress balm fermentation broth 2 obtained in Manufacturing Example 8 and the cypress balm fermentation broth 2 obtained in Comparative Manufacturing Example 4 For the grass extract, the final concentration of the test sample was changed to the concentration shown in Table 19 below, and the same method as in Test Example 2-2 was used to test the type I collagen production promotion effect. The results are shown in Table 19 below.

[Table 19] Tested sample Concentration of tested sample (μg/mL) Type I collagen production promotion rate (%) Manufacturing example 7 Cypress balm fermentation broth 1 1.56 94.4 Manufacturing example 8 Cypress balm fermentation broth 2 1.56 89.2 Comparative Manufacturing Example 4 Cypress balm extract 1.56 77.7

(Test example 4-2: aquaporin 3 (AQP3) mRNA expression promoting effect test) In Test Example 3-2, except that the test sample was changed to the cypress balm fermentation broth 1 obtained in Manufacturing Example 7, the cypress balm fermentation broth 2 obtained in Manufacturing Example 8 and the cypress balm fermentation solution obtained in Comparative Manufacturing Example 4 For the grass extract, the final concentration of the test sample was changed to the concentration shown in Table 20 below, and the aquaporin 3 (AQP3) mRNA expression promotion effect was tested in the same manner as in Test Example 3-2. The results are shown in Table 20 below.

[Table 20] Tested sample Concentration of tested sample (μg/mL) AQP3 mRNA expression promotion rate (%) Manufacturing example 7 Cypress balm fermentation broth 1 20 105.4 Manufacturing example 8 Cypress balm fermentation broth 2 20 135.7 Comparative Manufacturing Example 4 Cypress balm extract 20 97.7

(Test example 4-3: DPPH free radical elimination effect test) In Test Example 1-3, except that the tested sample was changed to the cypress balm fermentation broth 1 obtained in Manufacturing Example 7, the cypress balm fermentation broth 2 obtained in Manufacturing Example 8 and the cypress balm fermentation broth 2 obtained in Comparative Manufacturing Example 4 For the grass extract, the final concentration of the tested sample was changed to the concentration shown in Table 21 below, and the DPPH radical elimination effect was tested in the same way as in Test Example 1-3. The results are shown in Table 21 below.

　 Tested sample Concentration of tested sample (Μg/mL) DPPH free radical elimination rate (%) Manufacturing example 7 Cypress balm fermentation broth 1 12.5 73.2 Manufacturing example 8 Cypress balm fermentation broth 2 12.5 72.2 Comparative Manufacturing Example 4 Cypress balm extract 12.5 69.6

(Test Example 4-4: Hyaluronidase activity inhibition test) In Test Example 1-4, except that the tested sample was changed to the cypress balm fermentation broth 1 obtained in Manufacturing Example 7, the cypress balm fermentation broth 2 obtained in Manufacturing Example 8 and the cypress balm fermentation broth 2 obtained in Comparative Manufacturing Example 4 For the grass extract, the final concentration of the test sample was changed to the concentration shown in Table 22 below, and the hyaluronidase activity inhibition effect was tested in the same manner as in Test Example 1-4. The results are shown in Table 22 below.

[Table 22] Tested sample Concentration of tested sample (μg/mL) Inhibition rate of hyaluronidase activity (%) Manufacturing example 7 Cypress balm fermentation broth 1 100 71.9 Manufacturing example 8 Cypress balm fermentation broth 2 100 67.7 Comparative Manufacturing Example 4 Cypress balm extract 100 63.9

(Test Example 4-5: Tyrosinase Activity Inhibition Test) In Test Example 1-5, except that the test sample was changed to the cypress balm fermentation broth 1 obtained in Manufacturing Example 7, the cypress balm fermentation broth 2 obtained in Manufacturing Example 8 and the cypress balm fermentation broth 2 obtained in Comparative Manufacturing Example 4 For the grass extract, the final concentration of the test sample was changed to the concentration shown in Table 23 below, and the tyrosinase activity inhibition effect was tested in the same manner as in Test Example 1-5. The results are shown in Table 23 below.

[Table 23] Tested sample Concentration of tested sample (μg/mL) Tyrosinase activity inhibition rate (%) Manufacturing example 7 Cypress balm fermentation broth 1 100 3.6 Manufacturing example 8 Cypress balm fermentation broth 2 100 2.6 Comparative Manufacturing Example 4 Cypress balm extract 100 -2

<Manufacturing Example 9: Preparation of Cornflower Fermentation Liquid 1> In the step of preparing the seed koji of Manufacturing Example 1, except that sage was changed to Centaurea cyanus Linne (manufactured by ALBION Co., Ltd.), the same as the previous manufacturing example The same method as the preparation step of 1 seed koji, prepare "cornflower seed koji". In addition, in the fermentation step of Production Example 1 described above, except that sage was changed to Centaurea cyanus Linne (manufactured by ALBION Co., Ltd.), the same method as the fermentation step of Production Example 1 was used to obtain "Centaurea fermentation Liquid 1".

<Manufacturing Example 10: Preparation of Cornflower Fermentation Liquid 2> In the aforementioned manufacturing example 9, except that the cornflower seed koji was changed to the rice seed koji ( Aspergillus oryzae , Shirakami white koji, Akita Konno Co., Ltd.), the same as the aforementioned manufacturing In the same way as in Example 9, "Cornflower fermentation broth 2" was obtained.

<Comparative Production Example 5: Preparation of Cornflower Extract> In the aforementioned Comparative Production Example 1, except that the sage was changed to Centaurea cyanus Linne (manufactured by ALBION Co., Ltd.), the same as the aforementioned Comparative Production Example 1 Method to obtain "Cornflower Extract".

(Test Example A-5: Measurement of Contact Angle) In the aforementioned Test Example A-1, except that the test sample was changed to the cornflower fermentation liquid 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, The contact angle was measured in the same manner as in the aforementioned Test Example A-1. The results are shown in Table 24 below. In addition, an example of the droplets at the time of measuring the contact angle of each test sample is shown in FIGS. 5A to 5C.

[Table 24] Test sample Ferment Aspergillus Contact angle θ(°) Manufacturing example 9 Cornflower fermentation broth 1 Have Cornflower Koji 78.52 Manufacturing example 10 Cornflower fermentation broth 2 Have Rice Koji 80.83 Comparative Manufacturing Example 5 Cornflower Extract no - 86.96

Compared with the cornflower extract obtained in Comparative Production Example 5, the cornflower fermentation broth 1 obtained in Production Example 9 and the cornflower fermentation broth 2 obtained in Production Example 10 have a small contact angle of 85° or less, which is excellent in skin affinity. By. In addition, the cornflower fermentation broth 1 obtained in Production Example 9 had a contact angle of 79° or less, which was more excellent in skin affinity.

(Test Example 5-1: Type I collagen production promotion effect test) In Test Example 2-2, except that the test sample was changed to the cornflower fermentation broth 1 obtained in Manufacturing Example 9, the cornflower fermentation broth 2 obtained in Manufacturing Example 10, and the cornflower extract obtained in Comparative Manufacturing Example 5, they were The final concentration of the test sample was changed to the concentration shown in Table 25 below, and the type I collagen production promotion effect was tested in the same manner as in Test Example 2-2. The results are shown in Table 25 below.

[Table 25] Tested sample Concentration of tested sample (μg/mL) Type I collagen production promotion rate (%) Manufacturing example 9 Cornflower fermentation broth 1 1.56 98.0 Manufacturing example 10 Cornflower fermentation broth 2 1.56 96.8 Comparative Manufacturing Example 5 Cornflower Extract 1.56 85.3

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

[Table 26] Tested sample Concentration of tested sample (μg/mL) TMG-1 mRNA expression promotion rate (%) Manufacturing example 9 Cornflower fermentation broth 1 20 178.0 Manufacturing example 10 Cornflower fermentation broth 2 20 156.3 Comparative Manufacturing Example 5 Cornflower Extract 20 140.6

(Test Example 5-3: Filaggrin mRNA expression promoting effect test) Using the cornflower fermentation broth 1 obtained in Production Example 9, the cornflower fermentation broth 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5 as test samples, the polysilk was tested by the following test method Protein mRNA performance promotion effect.

In a normal human epidermal keratinocyte basal medium (HuMedia-KB2, manufactured by Kurabo Industries Co., Ltd.), each test sample was dissolved so that the final concentration became the concentration shown in Table 27 below, and the test sample medium was prepared and added. Using normal human epidermal keratinocyte proliferation medium (HuMedia-KG2, manufactured by Kurabo Industries Co., Ltd.), normal human neonatal epidermal keratinocytes (NHEK, manufactured by Kurabo Industries Co., Ltd.) were heated at 37°C, 5% CO _{After culturing under the conditions of 2} until the cells meet, the cells are recovered by trypsin treatment. The normal human epidermal keratinocyte growth medium (HuMedia-KG2) was used to adjust it to 1.5×10 ⁵ cells/mL. Next, 2 mL of the aforementioned NHEK (1.5×10 ⁵ cells/mL) was inoculated into a 35 mm dish, and cultured overnight at 37° C. and 5% CO ₂ . After the cultivation, the medium was exchanged with normal human epidermal keratinocyte basal medium (HuMedia-KB2) and further cultured for 24 hours. After the completion of the culture, the culture medium was exchanged with 2 mL of the aforementioned test medium supplemented with the test material, _{and cultured at 37°C and 5% CO 2} for 24 hours. After the cultivation, the culture solution was removed, and total RNA was extracted with RNA extraction reagent (ISOGEN II (Cat. No.: 311-07361), manufactured by Nippon Gene Co., Ltd.), and the amount of each RNA was measured with a spectrophotometer, using pure water The prepared total RNA is 200ng/μL.

In addition, as a control group, the same operation and absorbance measurement as described above were performed except that 2 mL of the aforementioned test sample medium was changed to 2 mL of normal human epidermal keratinocyte basal medium (HuMedia-KB2) without the test sample. In the same way as above, the total RNA was prepared to 200ng/μL.

Using the aforementioned total RNA as a template, the expression levels of filaggrin mRNA and GAPDH mRNA as an internal standard were measured. mRNA detection is performed by using a real-time PCR device (Thermal Cycler DIce (registered trademark) Real Time System III, manufactured by Takara Bio Co., Ltd.), and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR Kit (Perfect Real Time (Catalog number: RR063A), Takara Bio Co., Ltd. product) 2-step real-time PCR reaction was performed. The expression level of filaggrin mRNA without the test material and the test material added is corrected with the expression level of GAPDH mRNA. From this correction value, the filaggrin mRNA expression promotion rate was calculated based on the following formula 12. The results are shown in Table 27 below. ＜Formula 12＞ Filaggrin mRNA performance promotion rate (%) = A/B×100 In the aforementioned formula 12, A and B each represent the following. A: The correction value when adding the tested sample B: The correction value when the tested sample is not added

[Table 27] Tested sample Concentration of tested sample (μg/mL) Filaggrin mRNA performance promotion rate (%) Manufacturing example 9 Cornflower fermentation broth 1 20 159.8 Manufacturing example 10 Cornflower fermentation broth 2 20 136.8 Comparative Manufacturing Example 5 Cornflower Extract 20 122.3

(Test example 5-4: aquaporin 3 (AQP3) mRNA expression promoting effect test) In Test Example 3-2, except that the test sample was changed to the cornflower fermentation broth 1 obtained in Manufacturing Example 9, the cornflower fermentation broth 2 obtained in Manufacturing Example 10, and the cornflower extract obtained in Comparative Manufacturing Example 5, and The final concentration of the test sample was changed to the concentration shown in Table 28 below, and 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 28 below.

[Table 28] Tested sample Concentration of tested sample (μg/mL) AQP3 mRNA expression promotion rate (%) Manufacturing example 9 Cornflower fermentation broth 1 20 119.3 Manufacturing example 10 Cornflower fermentation broth 2 20 98.3 Comparative Manufacturing Example 5 Cornflower Extract 20 78.1

(Test example 5-5: Hyaluronic acid synthase 3 (HAS3) mRNA expression promotion test) In Test Example 1-2, except that the test sample was changed to the cornflower fermentation liquid 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 concentration of the test sample was changed to the concentration shown in Table 29 below, and the hyaluronic acid synthase 3 (HAS3) mRNA expression promotion effect was tested in the same manner as in Test Example 1-2. The results are shown in Table 29 below.

[Table 29] Tested sample Concentration of tested sample (μg/mL) HAS3 mRNA expression promotion rate (%) Manufacturing example 9 Cornflower fermentation broth 1 20 145.0 Manufacturing example 10 Cornflower fermentation broth 2 20 170.0 Comparative Manufacturing Example 5 Cornflower Extract 20 112.3

(Test example 5-6: Test of promoting effect of clonectin-1 mRNA expression) In Test Example 2-3, except that the tested sample was changed to the cornflower fermentation broth 1 obtained in Manufacturing Example 9, the cornflower fermentation broth 2 obtained in Manufacturing Example 10, and the cornflower extract obtained in Comparative Manufacturing Example 5, and The final concentration of the test sample was changed to the concentration shown in Table 30 below, and the effect of promoting the expression of codonin-1 mRNA was tested in the same manner as in Test Example 2-3. The results are shown in Table 30 below.

[Table 30] Tested sample Concentration of tested sample (μg/mL) Claudin-1 mRNA expression promotion rate (%) Manufacturing example 9 Cornflower fermentation broth 1 5 98.2 20 158.3 Manufacturing example 10 Cornflower fermentation broth 2 5 101.5 20 184.5 Comparative Manufacturing Example 5 Cornflower Extract 5 84.0 20 129.2

(Test Example 5-7: Test of promoting effect of clonectin-4 mRNA expression) In Test Example 2-4, except that the test sample was changed to the cornflower fermentation liquid 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 concentration of the test sample was changed to the concentration shown in the following Table 31, and in the same manner as in Test Example 2-4, the effect of promoting the expression of clonectin-4 mRNA was tested. The results are shown in Table 31 below.

[Table 31] Tested sample Concentration of tested sample (μg/mL) Claudin-4 mRNA expression promotion rate (%) Manufacturing example 9 Cornflower fermentation broth 1 5 138.8 20 141.2 Manufacturing example 10 Cornflower fermentation broth 2 5 82.9 20 85.6 Comparative Manufacturing Example 5 Cornflower Extract 5 64.0 20 71.8

(Test example 5-8: agglutinin mRNA expression promoting effect test) In Test Example 2-5, except that the tested sample was changed to the cornflower fermentation broth 1 obtained in Manufacturing Example 9, the cornflower fermentation broth 2 obtained in Manufacturing Example 10, and the cornflower extract obtained in Comparative Manufacturing Example 5, and The final concentration of the test sample was changed to the concentration shown in the following Table 32, and in the same manner as in Test Example 2-5, the expression promoting effect of clonectin mRNA was tested. The results are shown in Table 32 below.

[Table 32] Tested sample Concentration of tested sample (μg/mL) Adenin mRNA performance promotion rate (%) Manufacturing example 9 Cornflower fermentation broth 1 5 137.9 Manufacturing example 10 Cornflower fermentation broth 2 5 113.6 Comparative Manufacturing Example 5 Cornflower Extract 5 108.3

(Test example 5-9: DPPH free radical elimination effect test) In Test Example 1-3, except that the test sample was changed to the cornflower fermentation broth 1 obtained in Manufacturing Example 9, the cornflower fermentation broth 2 obtained in Manufacturing Example 10, and the cornflower extract obtained in Comparative Manufacturing Example 5, and The final concentration of the test sample was changed to the concentration shown in Table 33 below, and the DPPH radical elimination effect was tested in the same manner as in Test Example 1-3. The results are shown in Table 33 below.

[Table 33] Tested sample Concentration of tested sample (μg/mL) DPPH free radical elimination rate (%) IC ₅₀ (µg/mL) Manufacturing example 9 Cornflower fermentation broth 1 200 90.0 107.1 Manufacturing example 10 Cornflower fermentation broth 2 200 89.4 106.6 Comparative Manufacturing Example 5 Cornflower Extract 200 81.3 123.0

(Test example 5-10: Test for inhibiting melanin production in B16 melanoma cells) Using the cornflower fermentation broth 1 obtained in Production Example 9, the cornflower fermentation broth 2 obtained in Production Example 10, and the cornflower extract obtained in Comparative Production Example 5 as the test samples, the test was performed against B16 by the following test method. Melanoma cells have an inhibitory effect on melanin.

Dissolve each test sample in DMEM (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% FBS (manufactured by Biosera) and 1mmol/L theophylline (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and Prepare and add test sample medium. Using DMEM containing 10% FBS, B16 melanoma cells were cultured at 37°C and 5% CO ₂ until the cells met, and then the cells were recovered by trypsin treatment. ^{It was adjusted to 2.4×10 5} cells/mL with DMEM containing 10% FBS and 1 mmol/L theophylline. Then, in a 48-well plate, each well was seeded with 300 ^{μL of the aforementioned B16 melanoma cells (2.4×10 5} cells/mL), and cultured at 37° C. and 5% CO ₂ for 6 hours. After the completion of the culture, the medium was exchanged with 100 μL of the medium added with the test sample _{, and cultured at 37°C and 5% CO 2} for 3 days. After the completion of the culture, add 300μL of the aforementioned test sample medium to each well _{, and incubate for 4 days under the conditions of 37°C and 5% CO 2. In} addition, the final concentration of the aforementioned test sample at this time is the following Table 34 The concentration shown.

After the completion of the culture, the medium was removed from each well, and 200 μL of a 2 mol/L sodium hydroxide solution was added to destroy the cells by an ultrasonic disruptor, and the absorbance at a wavelength of 475 nm was measured. The self-measured absorbance value calculated the amount of melanin based on a calibration line created using synthetic melanin (manufactured by SIGMA).

In addition, in order to measure the cell survival rate, the B16 melanoma cells were cultured with the test medium added as described above in the same method as the above method, the medium was removed, and the medium was washed with 400 μL of PBS buffer. Next, add 200 μL of neutral red to each well and dissolve it in a DMEM solution containing 10% FBS to a final concentration of 0.05 mg/mL, and incubate for 2.5 hours. After the incubation, the neutral red solution was removed, and 200 μL of ethanol and acetic acid solution (ethanol: acetic acid: water = 50:1:49 (volume ratio)) was added to each well, and the pigment was extracted. After extraction, the absorbance at a wavelength of 540 nm was measured.

In addition, as a control group, the test sample solution was changed to DMEM containing 10% FBS and 1 mmol/L theophylline that did not contain the test sample, and the same operation and absorbance measurement as described above were performed.

From the obtained measurement values, the melanin production inhibition rate (%) corrected from the cell survival rate 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 survival rate (%) = (D/C) × 100 In the aforementioned formula 13, the C and D systems each represent the following. C: Absorbance at a wavelength of 540nm without the test material added D: Absorbance in the wavelength of 540nm with the test material added ＜Formula 14＞ Inhibition rate of melanin production (%) = {1-(B/D)/(A/C)}×100 In the aforementioned formula 14, the A to D systems each represent the following. A: The amount of melanin in the tested sample is not added B: Add the amount of melanin in the tested sample C: Absorbance at a wavelength of 540nm without the test material added D: Absorbance in the wavelength of 540nm with the test material added

[Table 34] Tested sample Concentration of tested sample (μg/mL) Inhibition rate of melanin production (%) Manufacturing example 9 Cornflower fermentation broth 1 100 24.3 200 32.7 Manufacturing example 10 Cornflower fermentation broth 2 100 18.8 200 26.0 Comparative Manufacturing Example 5 Cornflower extract 100 15.7 200 20.5

[Industrial Utilization Possibility] The anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention has excellent anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect at least any one of the effects, and is highly safe Natural substances are therefore not limited to the fields of cosmetics, food, pharmaceuticals, etc., but can be widely used. The cosmetic 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, so it can be suitably used in lotions, emulsions, and creams. , Ointment, beauty lotion, lotion, facial mask, gel, lipstick, lipstick, foundation, bathing agent, soap, body wash and other skin cosmetics; astringent, scalp care lotion, hair cream, hair lotion, hair wax, shampoo, Scalp and scalp cosmetics such as conditioner etc.

no

Fig. 1A is a photograph showing an example of droplets when the contact angle of the sage fermentation broth 1 of Production Example 1 is measured. FIG. 1B is a photograph showing an example of droplets when the contact angle of the sage fermentation broth 2 of Production Example 2 was measured. Fig. 1C is a photograph showing an example of droplets when the contact angle of the sage extract of Comparative Production Example 1 was measured. 2A is a photograph showing an example of droplets when the contact angle of Artemisia capillaris fermentation broth 1 of Production Example 3 was measured. FIG. 2B is a photograph showing an example of droplets when the contact angle of the Artemisia sphaerocephala fermentation liquid 2 of Production Example 4 was measured. Fig. 2C is a photograph showing an example of droplets when the contact angle of the Artemisia sphaerocephala extract in Comparative Production Example 2 was measured. FIG. 3A is a photograph showing an example of droplets when the contact angle of the thyme fermentation liquid 1 of Production Example 5 was measured. FIG. 3B is a photograph showing an example of droplets when the contact angle of the thyme fermentation liquid 2 of Production Example 6 was measured. FIG. 3C is a photograph showing an example of droplets when the contact angle of the thyme extract of Comparative Production Example 3 was measured. Fig. 4A is a photograph showing an example of droplets when the contact angle of the cypress balm fermentation broth 1 of Production Example 7 was measured. Fig. 4B is a photograph showing an example of droplets when the contact angle of the cypress balm fermentation broth 2 of Production Example 8 was measured. FIG. 4C is a photograph showing an example of droplets when the contact angle of the cypress balm extract of Comparative Production Example 4 was measured. 5A is a photograph showing an example of droplets when the contact angle of the cornflower fermentation broth 1 of Production Example 9 was measured. FIG. 5B is a photograph showing an example of droplets when the contact angle of the cornflower fermentation broth 2 of Production Example 10 was measured. FIG. 5C is a photograph showing an example of droplets when the contact angle of the cornflower extract of Comparative Production Example 5 was measured.

no

Claims (5)

An anti-aging agent, which is characterized by containing a fermented broth of Artemisia (Artemisia) plant by aspergillus, a fermented broth of Thymus (Thymus vulgaris), a fermented broth obtained by aspergillus, and a mixture of Melissa officinalis (Melissa officinalis) The fermentation broth of at least any one of the fermentation broth obtained from the koji fungus and the fermentation broth of Centaurea cyanus (Centaurea cyanus) which is obtained from the koji fungus is used as an active ingredient. An antioxidant characterized by containing a fermented broth of Artemisia spp. plant by aspergillus, a fermented broth of thyme, a fermented broth of aspergillus, and a fermented broth of cypress balm, and cornflower The fermentation broth of at least any one of the fermentation broths obtained by koji bacteria is used as an active ingredient. An anti-inflammatory agent characterized by containing at least any one of the fermentation broth of Artemisia spp. plant by the aspergillus and the fermentation broth of Cypress balsam by the aspergillus as an active ingredient. A whitening agent characterized by at least any of the fermentation broth of Artemisia spp., the fermentation broth obtained by Aspergillus sp., the fermentation broth of Cypress balm, the fermentation broth obtained by Aspergillus sp., and the fermentation broth of Cornflower, the fermentation broth obtained by aspergillus. One of the fermentation broth is used as the effective ingredient. A cosmetic, characterized by containing at least one selected from the group consisting of the anti-aging agent described in claim 1, the antioxidant described in claim 2, the anti-inflammatory agent described in claim 3, and the whitening agent described in claim 4 One kind.

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