阅读说明：本技术 抗老化剂、抗氧化剂、抗炎剂、及美白剂、以及化妆品 (Antiaging agent, antioxidant, antiinflammatory agent, whitening agent, and cosmetic ) 是由 岩竹美和 桑原浩诚 大户信明 染谷高士 萩谷薰 川野边弘子 佐藤勉 高桥昭仁 于 2020-03-27 设计创作，主要内容包括：本申请公开一种抗老化剂、抗氧化剂、抗炎剂、美白剂、以及化妆品；所述抗老化剂含有蒿属植物发酵液、百里香发酵液、香蜂草发酵液、及矢车菊发酵液中的至少任一种发酵液作为有效成分；所述抗氧化剂含有蒿属植物发酵液、百里香发酵液、香蜂草发酵液、及矢车菊发酵液中的至少任一种发酵液作为有效成分；所述抗炎剂含有蒿属植物发酵液、及香蜂草发酵液中的至少任一种发酵液作为有效成分；所述美白剂含有蒿属植物发酵液、香蜂草发酵液、及矢车菊发酵液中的至少任一种发酵液作为有效成分。(Disclosed are an anti-aging agent, an antioxidant, an anti-inflammatory agent, a whitening agent, and a cosmetic; the anti-aging agent contains at least one of Artemisia plant fermentation broth, thyme fermentation broth, Melissa officinalis fermentation broth, and cornflower fermentation broth as effective component; the antioxidant contains at least one of Artemisia fermentation broth, thyme fermentation broth, Melissa officinalis fermentation broth, and cornflower fermentation broth as effective component; the anti-inflammatory agent contains at least one fermentation liquid of Artemisia fermentation liquid and Melissa officinalis fermentation liquid as effective component; the whitening agent contains at least one of fermentation broth of Artemisia plant, Melissa officinalis and cornflower as effective component.)

1. An anti-aging agent, characterized in that: contains at least one of fermentation broth of Artemisia plant fermented by Aspergillus, fermentation broth of herba Thymi, Melissa officinalis, and cornflower.

2. An antioxidant, characterized by: contains at least one of fermentation broth of Artemisia plant fermented by Aspergillus, fermentation broth of herba Thymi, Melissa officinalis, and cornflower.

3. An anti-inflammatory agent characterized by: contains at least one of fermentation broth of Artemisia plant fermented by Aspergillus and fermentation broth of Melissa officinalis fermented by Aspergillus as effective components.

4. A whitening agent characterized by: contains at least one of fermentation broth of Artemisia plant fermented by Aspergillus, fermentation broth of Melissa officinalis fermented by Aspergillus, and fermentation broth of cornflower fermented by Aspergillus as effective component.

5. A cosmetic characterized by: containing at least 1 selected from the group consisting of the anti-aging agent according to claim 1, the antioxidant according to claim 2, the anti-inflammatory agent according to claim 3, and the whitening agent according to claim 4.

Technical Field

The present invention relates to an anti-aging agent, an antioxidant, an anti-inflammatory agent, and a whitening agent containing a fermentation broth of a plant fermented by aspergillus as an active ingredient, and a cosmetic containing at least any one of the anti-inflammatory agent, the antioxidant, the anti-inflammatory agent, and the whitening agent.

Background

In recent years, active oxygen has attracted attention as a factor causing oxidation of a biological component, and its adverse effect on a living body has become a problem. Reactive oxygen species are produced during energy metabolism in biological cells and include superoxide (i.e., superoxide anion (. O) produced by the one-electron reduction of oxygen molecules₂ ^-) Hydrogen peroxide (H)₂O₂) Singlet oxygen (a)¹O₂) Hydroxyl radical (. OH)]And the like. This active oxygen is essential for the sterilization mechanism of phagocytes, and plays an important role in the clearance of viruses or cancer cells.

However, the excessive production of active oxygen attacks molecules in the living body constituting membranes and tissues in the living body, and induces various diseases. Superoxide, which is produced in the organism and becomes a starting material of other active oxygen, is generally gradually eliminated by the catalytic action of superoxide Dismutase (SOD) contained in the cells. However, when superoxide is produced in excess or the effect of SOD is reduced, the removal of superoxide is insufficient, resulting in an increase in the concentration of superoxide. The above-mentioned condition is considered to be one of the causes of tissue diseases such as rheumatoid arthritis and behcet's disease, myocardial infarction, cerebral apoplexy, cataract, chloasma, freckle, wrinkle, diabetes, arteriosclerosis, shoulder soreness, cold-feeling constitution, skin aging, and the like.

Among these disadvantages, the skin is an organ that is likely to produce superoxide because it is directly stimulated by environmental factors such as ultraviolet rays, and therefore, as the concentration of superoxide increases, for example, the following problems occur: it is known that it decomposes, modifies, or crosslinks biological tissues such as collagen, oxidizes fats and oils to produce lipid peroxides which damage cells, forms skin wrinkles, or causes aging, inflammation, or skin pigmentation such as reduction in skin elasticity (see non-patent document 1). Therefore, it is considered that various active oxygen-related disorders such as skin aging, e.g., wrinkle formation and elasticity reduction, tissue diseases, e.g., rheumatoid arthritis and behcet's disease, myocardial infarction, cerebral apoplexy, cataract, diabetes, arteriosclerosis, shoulder soreness, and cold-feeling constitution can be prevented, treated or improved by inhibiting or suppressing the production of active oxygen and free radicals in the living body.

Therefore, it has been found that an extract of brassica plant of brassicaceae (see patent document 1), an extract of kalanchoe plant of crassulaceae (see patent document 2), an extract of oroxylum indicum (see patent document 3), an extract of sweet potato vine (see patent document 4) and the like are effective by attempting to obtain an active oxygen scavenger, a radical scavenger, a hydrogen peroxide scavenger and the like from natural substances advantageous in terms of safety.

In addition, the causes and pathogenesis of inflammatory diseases such as contact dermatitis (macula), psoriasis, pemphigus vulgaris, various other skin diseases accompanied by rough skin, and the like are various. It is known that the cause is mainly due to increased hyaluronidase activity.

Hyaluronidase is a hydrolase of hyaluronic acid. The hyaluronic acid salt, which retains affinity with body tissues, is decomposed by ultraviolet rays, oxygen, etc. in a water-containing system, so that the molecular weight is reduced and the water-retaining effect is also reduced. In addition, hyaluronic acid exists in vivo in the form of intercellular tissue, and is also associated with vascular permeability. Further, hyaluronidase is present in obesity cells, and its activation causes degranulation and release, and thus acts as an inflammatory chemical mediator. Therefore, by inhibiting the activity of hyaluronidase, it is expected to enhance moisture retention and prevent and reduce inflammation.

As a substance having such a hyaluronidase activity inhibitory action, for example, an extract of a plant of the genus chrysomele (see patent document 5), an extract of ampelopsis grossedentata (see patent document 6), and the like are known.

In addition, melanin in the skin also plays a role in protecting living things from ultraviolet rays, but excessive production or uneven accumulation causes skin blackening or chloasma generation. Generally, melanin is considered to be formed by converting tyrosine into dopa and also from dopa into dopaquinone through the action of tyrosinase biosynthesized in pigment cells, and then passing through an intermediate such as 5, 6-dihydroxyindophenol. Therefore, it is considered that the inhibition of the activity of tyrosinase related to the production of melanin or the inhibition of the production of melanin is required for the prevention, treatment or improvement of skin darkening (skin pigmentation), chloasma, freckles and the like.

Conventionally, in order to prevent, treat or improve skin pigmentation diseases, chloasma, freckles and the like, a whitening agent containing a synthetic product such as hydroquinone as an active ingredient has been taken for external use. However, it is concerned that synthetic products such as hydroquinone may cause side effects such as skin irritation and allergy.

Therefore, it is desired to develop a whitening agent containing highly safe natural materials as active ingredients, and examples of substances having tyrosinase activity inhibitory activity include ampelopsis grossedentata extract (see patent document 7), polygonum hydropiper extract (see patent document 8), and the like. Further, as a substance having a melanin production inhibitory action, for example, an extract derived from the root of castor bean (see patent document 9), an extract derived from a plant belonging to the genus Saussurea (sauussurea) (see patent document 10), and the like are known.

The epidermis and dermis of the skin are composed of extracellular matrices such as epidermal cells, fibroblasts, and collagen that is located outside these cells to support the skin structure. In young skin, the interaction of these skin tissues is kept constant, so that water retention, softness, elasticity, and the like are ensured, and the skin is also firm, strong, and glossy in appearance, and is maintained in a moist state.

However, if the skin is affected by some external factors such as ultraviolet (UV-A, UV-B) irradiation, extreme dryness of air, excessive skin cleansing, etc., or the skin becomes older, the amount of collagen produced, which is a main component of the extracellular matrix, decreases, and the elasticity decreases due to crosslinking. As a result, the moisturizing function and elasticity of the skin are reduced, and the keratin begins to fall off abnormally, so that the skin may have aging symptoms such as sagging, loss of luster, roughness, wrinkles, and the like.

Such changes accompanying skin aging, i.e., wrinkles, dullness, loss of texture, decreased elasticity, and the like, are caused by various factors, for example, reduction and modification of extracellular matrix components such as collagen, hyaluronic acid, and elastin. Therefore, it is considered that aging of the skin can be prevented and improved by promoting the production of collagen, hyaluronic acid, or the like.

Therefore, in view of safety, attempts have been made to obtain a substance having a collagen production promoting effect from a favorable natural substance. Examples of the substance having a collagen production promoting effect include an extract of carambola leaves (see patent document 11), an extract of chaetomium globosum (see patent document 12), and the like.

The following is also reported: amino acids, which are main components of Natural Moisturizing Factors (Natural Moisturizing Factors), are produced by decomposition of filaggrin derived from transparent keratin particles in the stratum corneum. The filaggrin appears as a filaggrin pro-protein in epidermal keratinocytes present in the stratum granulosum just below the stratum corneum. Immediately thereafter, the protein is phosphorylated, deposited on transparent horny particles, degraded into filaggrin by dephosphorylation and hydrolysis, and moved to the horny layer, thereby increasing the aggregation efficiency of keratin fibers and participating in the internal construction of horny cells (see non-patent document 2).

In recent years, it has been reported that this filaggrin is very important and indispensable for the retention of water in the skin, and conditions such as drying lower the synthesis of filaggrin and lower the amount of amino acids in the stratum corneum (see non-patent document 3).

Therefore, it is expected that the expression of profilaggrin mRNA in epidermal keratinocytes is promoted to promote the synthesis of filaggrin, thereby increasing the amount of amino acids in the stratum corneum and fundamentally improving the moisture environment of the stratum corneum.

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

The epidermis continuously forms new keratinocytes through the division of keratinocytes and the subsequent differentiation, thereby having a defensive function of protecting the skin from various stimuli from the outside. In particular, in the process of keratinocyte differentiation, proteins such as outer membrane proteins are expressed from the stratum spinosum to the stratum granulosum, and are crosslinked by the action of transglutaminase-1 to form a keratinocyte envelope (hereinafter, abbreviated as "CE") which is an insoluble cell membrane-like structure covering keratinocytes, contributing to the stabilization of the cytoskeleton and structure of keratinocytes.

However, if the amount of transglutaminase-1 produced in the epidermis is reduced by various factors, the formation of CE is incomplete and normal keratinization does not proceed. As a result, the keratin barrier function and the skin moisturizing function are reduced, and skin symptoms such as rough skin and dry skin are manifested.

In view of the above, it is considered that by increasing the production of transglutaminase-1 in the epidermis of keratinocytes, the formation of CE is promoted to normalize keratinization, and the decrease in skin barrier function accompanying external stimulation such as dryness or ultraviolet rays can be suppressed, thereby preventing and improving various skin symptoms such as dry skin and rough skin.

As a glutamine transaminase-1 production promoter derived from a natural product, noni fruit extract (see patent document 16), royal jelly extract (see patent document 17) and the like have been proposed.

In addition, it is known that, in skin cells, aquaporins, which are known as water channels, are expressed on cell membranes and play a role in absorbing low-molecular substances, such as water in intercellular spaces, into cells. It is known that 13 aquaporins (AQP 0-AQP 12) exist in humans. In epidermal cells, Aquaporin 3(Aquaporin 3, AQP3) is predominantly present, which is thought to play a role: in addition to absorbing water, low molecular weight compounds such as glycerin and urea, which are involved in water retention, are also absorbed.

However, AQP3 decreased with age, suggesting that this decrease in AQP3 is one of the causes of the decrease in water retention function, and therefore, it is considered that the decrease in water retention function, barrier function, and the like with age could be controlled by promoting the expression of AQP3 (see non-patent document 4). As a substance having an action of promoting expression of AQP3, for example, an extract derived from the leaf of carambola (see patent document 18) is known.

Conventionally, it has been considered that only the stratum corneum plays a role in the barrier function of the skin, but if a structural protein of tight junction (hereinafter, abbreviated as "TJ") present in the stratum corneum layer is lost at the gene level, the barrier function of the skin is broken down, and therefore, in recent years, TJ has also been considered to play an important role in the barrier function of the skin (see non-patent document 5). TJ is a connecting device that not only seals adjacent cells, but also seals the gap between cells, thereby controlling the permeation of substances. The TJ is composed of claudin (claudin) and claudin (claudin) which are cell membrane proteins, and these proteins are considered to constitute the backbone of the TJ chain and control the barrier function of TJ (see non-patent document 6). In view of the above, it is predicted that when the expression of claudin or claudin is reduced for some reason, the TJ structure is destroyed and loses the function as a substance permeation barrier, and thus, it becomes a cause of skin symptoms such as dry skin, rough skin, atopic dermatitis, various infectious diseases, and the like.

Therefore, it is thought that the formation of TJ of epidermal keratinocytes is promoted by promoting the production of claudin or claudin in the epidermis, thereby improving the barrier function and water retention function of the skin and preventing or improving the skin symptoms. Based on this idea, as a substance for improving the skin barrier function by the TJ formation promoting action, coptis chinensis extracts (see patent document 19), spruce extracts (see patent document 20), and the like, which are derived from natural substances, have been proposed.

In addition, in recent years, the involvement of Matrix Metalloproteinases (MMPs) has been pointed out as a factor in inducing changes accompanied by skin aging. Among these MMPs, matrix metalloproteinase-1 (MMP-1) is known to be an enzyme that decomposes collagen, which is a major constituent of the extracellular matrix of dermal skin, but its expression is greatly increased by ultraviolet irradiation, and this is considered to be one of the causes of reduction and modification of collagen, and is an important cause of wrinkles, decreased elasticity, and the like in the skin. Therefore, inhibition of MMP-1 activity is important in preventing and ameliorating the symptoms of skin aging.

As a substance having such MMP-1 inhibitory action, for example, an extract of acerola cherry (see patent document 21), an extract of zingiber cassumunar of zingiberaceae, or ficus microcarpa of moraceae (see patent document 22), and the like are known.

On the other hand, fermentation broth obtained by fermenting artemisia plants with aspergillus (see patent document 23) or fermentation broth obtained by fermenting thyme with aspergillus (see patent document 24) is known, but it is not known that these plant fermentation broths have anti-aging, anti-oxidation, anti-inflammatory, whitening effects, and the like.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open No. 2003-81848

Patent document 2: japanese patent laid-open No. 2005-29483

Patent document 3: japanese patent laid-open No. 2006-321730

Patent document 4: japanese patent laid-open No. 2007-8902

Patent document 5: japanese patent laid-open No. 2003-55242

Patent document 6: japanese patent laid-open No. 2003-12532

Patent document 7: japanese patent application laid-open No. 2002-370962

Patent document 8: japanese patent application laid-open No. 2004-83488

Patent document 9: japanese patent laid-open No. 2001-213757

Patent document 10: japanese patent laid-open publication No. 2002-201122

Patent document 11: japanese patent laid-open No. 2002-226323

Patent document 12: japanese patent laid-open No. 2003-146837

Patent document 13: japanese patent laid-open publication No. 2002-363054

Patent document 14: japanese patent laid-open No. 2003-146886

Patent document 15: japanese patent laid-open No. 2001-261568

Patent document 16: japanese patent laid-open publication No. 2010-090093

Patent document 17: japanese patent laid-open publication No. 2009-184955

Patent document 18: japanese patent laid-open publication No. 2009-191039

Patent document 19: japanese patent laid-open No. 2007-176830

Patent document 20: japanese patent laid-open No. 2007-176835

Patent document 21: japanese patent laid-open No. 2003-176232

Patent document 22: japanese patent laid-open No. 2003-176230

Patent document 23: japanese patent application laid-open No. 2011-

Patent document 24: japanese patent laid-open publication No. 2011-130689

Non-patent document

Non-patent document 1: "temporary Journal of Fragrance" supplement No.14, p156, 1995

Non-patent document 2: temporary Journal of Fragrance, 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", Vol.34, No.10, p.19-23, 2006

Non-patent document 5: j.cell biol., vol.156, pp.1099-1111(2002)

Non-patent document 6: japanese society of fragrance and cosmetics science, vol.31, pp.296-301(2007)

Disclosure of Invention

[ problems to be solved by the invention ]

The present invention has been made to solve the above-described problems, and has an object to achieve the following.

That is, an object of the present invention is to provide an anti-aging agent having excellent anti-aging action and high safety, an antioxidant having excellent anti-oxidation action and high safety, an anti-inflammatory agent having excellent anti-inflammatory action and high safety, and a whitening agent having excellent whitening action and high safety.

It is another object of the present invention to provide a cosmetic having at least 1 excellent effect selected from the group consisting of an anti-aging effect, an antioxidant effect, an anti-inflammatory effect, and a whitening effect, and having high safety.

[ means for solving problems ]

The means for solving the problems are as follows. That is to say that the first and second electrodes,

the aging inhibitor is more than 1, and is characterized in that: contains at least one of fermentation broth of Artemisia plant fermented by Aspergillus, fermentation broth of herba Thymi, Melissa officinalis, and cornflower.

Less than 2 is more than one antioxidant, which is characterized in that: contains at least one of fermentation broth of Artemisia plant fermented by Aspergillus, fermentation broth of herba Thymi, Melissa officinalis, and cornflower.

< 3 > an anti-inflammatory agent characterized by: contains at least one of fermentation broth of Artemisia plant fermented by Aspergillus and fermentation broth of Melissa officinalis fermented by Aspergillus as effective components.

Less than 4 is more than one whitening agent, which is characterized in that: contains at least one of fermentation broth of Artemisia plant fermented by Aspergillus, fermentation broth of Melissa officinalis fermented by Aspergillus, and fermentation broth of cornflower fermented by Aspergillus as effective component.

Less than 5 is more than one kind of cosmetics, which is characterized in that: contains at least 1 selected from the group consisting of the anti-aging agent according to the above-mentioned item < 1 >, the antioxidant according to the above-mentioned item < 2 >, the anti-inflammatory agent according to the above-mentioned item < 3 > and the whitening agent according to the above-mentioned item < 4 >.

[ Effect of the invention ]

The present invention can solve the above problems of the prior art, and can provide an anti-aging agent having excellent anti-aging activity and high safety, an antioxidant having excellent anti-oxidation activity and high safety, an anti-inflammatory agent having excellent anti-inflammatory activity and high safety, and a whitening agent having excellent whitening activity and high safety.

The present invention can also provide a cosmetic having at least 1 excellent effect selected from the group consisting of an anti-aging effect, an antioxidant effect, an anti-inflammatory effect, and a whitening effect, and having high safety.

Drawings

FIG. 1A is a photograph showing an example of a droplet when the contact angle of the Artemisia tridentate fermentation broth 1 of production example 1 is measured.

FIG. 1B is a photograph showing an example of a droplet when the contact angle of the Artemisia tridentate fermentation broth 2 of production example 2 is measured.

FIG. 1C is a photograph showing an example of a droplet when the contact angle of the Artemisia tridentate extract liquid of comparative production example 1 is measured.

FIG. 2A is a photograph showing an example of a droplet obtained by measuring the contact angle of the Artemisia capillaris Thunb fermentation broth 1 of production example 3.

FIG. 2B is a photograph showing an example of a droplet obtained by measuring the contact angle of the Artemisia capillaris Thunb fermentation broth 2 of production example 4.

FIG. 2C is a photograph showing an example of a droplet when the contact angle of the Artemisia capillaris Thunb extract liquid of comparative production example 2 was measured.

Fig. 3A is a photograph showing an example of a droplet when the contact angle of thyme fermentation liquid 1 of production example 5 was measured.

Fig. 3B is a photograph showing an example of a droplet when the contact angle of thyme fermentation liquid 2 of production example 6 was measured.

FIG. 3C is a photograph showing an example of a droplet when the contact angle of the thyme extract of comparative production example 3 was measured.

FIG. 4A is a photograph showing an example of a droplet obtained by measuring the contact angle of the Melissa officinalis fermentation broth 1 of production example 7.

FIG. 4B is a photograph showing an example of a droplet obtained by measuring the contact angle of the Melissa officinalis fermentation broth 2 of production example 8.

FIG. 4C is a photograph showing an example of a droplet when the contact angle of the extract liquid of Melissa officinalis of comparative production example 4 was measured.

FIG. 5A is a photograph showing an example of a droplet obtained by measuring the contact angle of the cornflower fermentation broth 1 of production example 9.

FIG. 5B is a photograph showing an example of a droplet obtained by measuring the contact angle of the cornflower fermentation broth 2 of production example 10.

FIG. 5C is a photograph showing an example of a droplet obtained by measuring the contact angle of the cornflower extract liquid of comparative production example 5.

Detailed Description

(anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent)

< anti-aging agent >

The antiaging agent of the present invention contains, as an active ingredient, at least one of a fermentation broth of Artemisia plants by Aspergillus fermentation (hereinafter sometimes referred to as "Artemisia fermentation broth"), a fermentation broth of Thymus vulgaris by Aspergillus fermentation (hereinafter sometimes referred to as "Thymus fermentation broth"), a fermentation broth of Melissa officinalis by Aspergillus fermentation (hereinafter sometimes referred to as "Melissa fermentation broth"), and a fermentation broth of cornflower by Aspergillus fermentation (hereinafter sometimes referred to as "cornflower fermentation broth"), and optionally contains other ingredients.

The Artemisia plant fermentation broth, the thyme fermentation broth, the Melissa officinalis fermentation broth, and the cornflower fermentation broth have at least 1 action selected from the group consisting of matrix metalloproteinase-1 (MMP-1) activity inhibitory action, hyaluronic acid synthase 3(hyaluronan synthase 3, HAS3) mRNA expression promoting action, type I collagen production promoting action, claudin-1 mRNA expression promoting action, claudin-4 mRNA expression promoting action, claudin mRNA expression promoting action, glutamine transaminase-1 (TGM-1) mRNA expression promoting action, Aquaporin 3 (AQP 3) mRNA expression promoting action, and filaggrin mRNA expression promoting action, these effects can be utilized as an active ingredient of an antiaging agent.

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

Although details of substances that exhibit at least any one of an MMP-1 activity inhibitory effect, a hyaluronic acid synthase 3 mRNA expression promoting effect, a collagen type I production promoting effect, a claudin-1 mRNA expression promoting effect, a claudin-4 mRNA expression promoting effect, a claudin mRNA expression promoting effect, a glutamine transaminase-1 mRNA expression promoting effect, an aquaporin 3 mRNA expression promoting effect, and a filaggrin mRNA expression promoting effect possessed by the artemisia plant fermentation broth, the thyme fermentation broth, the melissa fermentation broth, and the cornflower fermentation broth have not been known, the artemisia plant fermentation broth, the thyme fermentation broth, the melissa fermentation broth, and the cornflower fermentation broth have been hitherto known to exhibit such excellent effects and are useful as an anti-aging agent, this is a new finding of the present inventors.

< antioxidant >

The antioxidant of the present invention contains at least one of a fermentation broth of artemisia plants by aspergillus fermentation (artemisia fermentation broth), a fermentation broth of thyme by aspergillus fermentation (thyme fermentation broth), a fermentation broth of melissa by aspergillus fermentation (melissa fermentation broth), and a fermentation broth of cornflower by aspergillus fermentation (cornflower fermentation broth) as an active ingredient, and further contains other ingredients as required.

The artemisia fermentation broth, the thyme fermentation broth, the melissa fermentation broth, and the cornflower fermentation broth have a diphenyl-p-cinnamyl hydrazine (DPPH) radical scavenging action, and can be used as an active ingredient of an antioxidant by utilizing this action.

Thus, the antioxidant has DPPH radical scavenging action.

Although details of the substances having a DPPH radical scavenging action of the artemisia plant fermentation broth, the thyme fermentation broth, the melissa fermentation broth, and the cornflower fermentation broth are not known, it has not been known at all that the artemisia plant fermentation broth, the thyme fermentation broth, the melissa fermentation broth, and the cornflower fermentation broth have such excellent effects and can be used as an antioxidant, which is a novel finding of the present inventors.

< anti-inflammatory agent >

The anti-inflammatory agent of the present invention contains at least one of a fermentation broth of Artemisia plants by Aspergillus fermentation (Artemisia fermentation broth) and a fermentation broth of Melissa officinalis by Aspergillus fermentation (Melissa officinalis fermentation broth) as an active ingredient, and optionally other ingredients.

The Artemisia fermentation broth and Melissa officinalis fermentation broth have hyaluronidase activity inhibiting effect, and can be used as effective component of antiinflammatory agent.

Therefore, the anti-inflammatory agent has hyaluronidase activity inhibitory effect.

Although details of the substances having hyaluronidase activity inhibitory activity possessed by the above artemisia plant fermentation broth and melissa officinalis fermentation broth are not known, it has not been known at all that the artemisia plant fermentation broth and melissa officinalis fermentation broth have such excellent effects and are useful as an anti-inflammatory agent, which is a new finding of the present inventors.

< whitening agent >

The whitening agent of the invention contains at least one of fermentation liquor of artemisia plants fermented by aspergillus (artemisia plant fermentation liquor), melissa officinalis fermented by aspergillus (melissa officinalis fermentation liquor) and cornflower fermented by aspergillus (cornflower fermentation liquor) as an active ingredient, and optionally contains other ingredients.

The Artemisia plant fermentation broth, Melissa officinalis fermentation broth, and cornflower fermentation broth have at least 1 of tyrosinase activity inhibiting effect and melanin production inhibiting effect, and can be used as effective components of whitening agent.

Therefore, the whitening agent has at least 1 of tyrosinase activity inhibitory action and melanin production inhibitory action.

Although details of the substances having at least any one of the tyrosinase activity inhibitory effect and the melanin production inhibitory effect of the artemisia plant fermentation broth, the melissa officinalis fermentation broth, and the cornflower fermentation broth are not known, the present inventors have found that the artemisia plant fermentation broth, the melissa officinalis fermentation broth, and the cornflower fermentation broth have such excellent effects and can be used as a whitening agent.

Artemisia plant fermentation liquor

The artemisia fermentation broth is a fermentation broth obtained by fermenting a plant belonging to the genus artemisia (hereinafter, sometimes referred to as "artemisia") with aspergillus.

Artemisia plant-

The plant of Artemisia used as the fermentation raw material belongs to Compositae (Asteraceae)Compositae) Artemisia genus (Artemisia) The perennial herbs of (a) have been used as raw materials for food or medicine since ancient times. It is widely grown or cultivated in Japan such as Hokkaido, Homonodo, four countries, and Kyushu, and can be easily obtained from these areas.

The species of the above artemisia plant is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include: mountain wormwood (Haw) Artemisia montana(Nakai) Pamp.), Artemisia capillaris (herba Artemisiae Scopariae: (Nakai)Artemisia capillarisThunbergii), kukohao (Artemisia princepsPampan, Artemisia japonica (herba Artemisiae Japonicae) ((herba Artemisiae Japonicae))Artemisia japonicaThunb.), middle Asia wormwood (artemisia scoparia (L.), (Artemisia absinthiumL. and Artemisia rupestris (L.) KraschArtemisia lactifloraWall.), and artemisia maritime (Artemisia maritimaL. and Artemisia scoparia (L.) KraschArtemisia scopariaWaldst. et kit.), and the like. These may be used alone in 1 kind, or may be used in combination of 2 or more kinds.

The method for obtaining the above-mentioned Artemisia plant is not particularly limited, and may be appropriately selected according to the purpose, collected from the natural world, or commercially available.

The site of the artemisia plant used as the fermentation raw material is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include: flowers, buds, fruits, pericarps, seeds, seed coats, stems, leaves, stalks, branches and leaves and other overground parts; root, rhizome, etc. in the lower part. These may be used alone in 1 kind, or may be used in combination of 2 or more kinds. Among these parts, the use part of the artemisia plant is preferably an aerial part.

The size of the artemisia plant used as the fermentation raw material is not particularly limited as long as the size is sufficient for culturing the aspergillus, and may be appropriately selected according to the purpose, and examples thereof include: collected original size, cut into desired size, size converted into fine powder (powder), and the like.

The state of the artemisia plant used as the fermentation raw material is not particularly limited as long as the aspergillus can be cultured, and may be appropriately selected according to the purpose, and examples thereof include: the collected original state, dried state, pulverized state, squeezed state, and extract state. Among these conditions, the collected original condition, crushed condition, squeezed condition, and extract condition are preferable, and the collected original condition and crushed condition are more preferable, from the viewpoint that the aspergillus easily acts.

The method for allowing the above-mentioned artemisia plant to be in a dry state is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include: a method of drying by sun, a method of drying by a conventional dryer, and the like.

The method for pulverizing the above-mentioned Artemisia plant is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of pulverizing with a blender, a sugar mill, an electric mill, a jet mill, an impact mill, and the like.

The method for bringing the above-mentioned artemisia plant into the squeezed juice state is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include squeezing.

The method for making the above-mentioned artemisia plant into the extract state is not particularly limited, and a method commonly used for plant extraction may be appropriately selected according to the purpose.

The Artemisia plant used as the fermentation feedstock is preferably sterilized before inoculation with the Aspergillus. The method for sterilizing the above-mentioned artemisia plant is not particularly limited, and may be appropriately selected from known methods.

Aspergillus-

The aspergillus used for fermenting the artemisia plant is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include: aspergillus oryzae (Aspergillus oryzae) Aspergillus sojae (A. sojae) ((B))Aspergillus sojae) Aspergillus flavus and the like; aspergillus lucidus (A. lucidus), (B. sp.), (BAspergillus luchuensis) Aspergillus niger and the like; aspergillus kawachii (A. kawachii) ((A. kawachii))Aspergillus kawauchii) Aspergillus niger and the like; such mutant strains. These may be used alone in 1 kind, or may be used in combination of 2 or more kinds. Of these aspergillus, aspergillus oryzae (aspergillus oryzae) is preferable as the aspergillus having excellent at least one of anti-aging effect, anti-oxidation effect, anti-inflammatory effect and whitening effectAspergillus oryzae)。

The method for obtaining the aspergillus is not particularly limited, and may be appropriately selected according to the purpose, collected from the nature, or a commercially available product may be used. The aspergillus may be a seed koji made from rice or the like, or a seed koji made from an artemisia plant described below, or an aspergillus cultured in a medium (e.g., an agar medium or a liquid medium). Among these aspergillus, the above-mentioned artemisia mother starter is preferably used in view of its excellent at least one of anti-aging effect, anti-oxidation effect, anti-inflammatory effect, and whitening effect.

The amount of the Aspergillus to be inoculated to the Artemisia plant used as the fermentation raw material is not particularly limited as long as the amount is sufficient to ferment the Artemisia plant, and may be appropriately selected according to the purpose, and when the fermentation raw material is in a liquid state, it is preferably 1X 10³1X 10 to one/mL⁸The amount per mL is preferably 1X 10 in the case where the fermentation raw material is in a solid state³Per g to 1X 10⁸Per gram.

When the Artemisia plant is inoculated with the Aspergillus, water is preferably added. The amount of the water to be added is not particularly limited and may be appropriately selected according to the purpose, and is preferably 500 to 5,000 parts by mass, more preferably 1,000 to 4,000 parts by mass, and particularly preferably 1,500 to 3,000 parts by mass, based on 100 parts by mass of the above-mentioned artemisia plant.

The fermentation (culture) temperature is not particularly limited as long as it is within a temperature range in which the fermentation by the aspergillus is possible, and may be appropriately selected according to the purpose, and is preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the fermentation temperature is lower than 20 ℃, there is a case where at least one of the anti-aging, anti-oxidant, anti-inflammatory, and whitening effects of the artemisia plant is insufficient by the method 1. Further, if it exceeds 50 ℃, the Aspergillus cannot be proliferated in some cases.

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

The method for stopping the fermentation (culture) is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of heating and the like.

The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the aspergillus cannot grow any more, and may be appropriately selected according to the purpose, and is preferably 50 ℃ or more, more preferably 70 ℃ or more, and particularly preferably 100 to 130 ℃. If the heating temperature is less than 50 ℃, the fermentation may not be stopped, and if it exceeds 30 ℃, at least one of the anti-aging effect, the antioxidant effect, the anti-inflammatory effect, and the whitening effect may be insufficient.

The heating time for stopping the fermentation is not particularly limited as long as the aspergillus can be prevented from growing, and may be appropriately selected according to the purpose, and is preferably 5 minutes or more, and more preferably 10 minutes to 20 minutes. If the heating time is less than 5 minutes, the fermentation may not be stopped, and if it exceeds 20 minutes, at least one of the anti-aging effect, the antioxidant effect, the anti-inflammatory effect, and the whitening effect may be insufficient.

Further, the artemisia fermentation liquid after the fermentation is stopped is preferably cooled. The cooling method is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of standing at room temperature, a method of standing in a refrigerator, and the like.

The number of times the Artemisia plant is fermented by the Aspergillus is not particularly limited, and may be appropriately selected according to the purpose, and may be 1 time or more.

In the case where the fermentation is carried out a plurality of times, the Aspergillus may be inoculated only at the first inoculation, may be inoculated only a plurality of times, may be inoculated at each fermentation, and is preferably inoculated only at the first inoculation.

In the case where the fermentation is performed a plurality of times, the fermentation temperature and the fermentation time may be different from each other or may be the same.

- - -Artemisia seed-

The Artemisia mother culture is obtained by using the Artemisia as mother culture raw material, inoculating Aspergillus to the mother culture raw material, and attaching sufficient spores to the Artemisia for growth. The use of the koji for the fermentation is advantageous in that it is possible to obtain a fermentation solution of an artemisia plant with excellent skin compatibility more efficiently and easily.

The plant of the above-described Artemisia-Artemisia can be used as the above-described Artemisia starting material, and the plant of the above-described Artemisia can be used in the same manner, and the plant can be used in the same form such as the site, size, state, etc. of the above-described Artemisia.

As the Aspergillus used for producing the koji of Artemisia, the same bacteria as described in the above-Aspergillus can be used.

The amount of the Aspergillus to be inoculated to the above-mentioned Artemisia plant used as the starting material for the koji is not particularly limited, and may be appropriately selected depending on the purpose, and it is preferable to inoculate 5 to 100 parts by mass of Aspergillus (1X 10) suspended in sterilized water to 100 parts by mass of the above-mentioned Artemisia plant³1X 10 to one/mL⁸one/mL), more preferably from 10 to 50 parts by mass, and particularly preferably from 20 to 30 parts by mass. If the inoculation amount of the aspergillus is less than 5 parts by mass with respect to 100 parts by mass of the artemisia plant, a sufficient amount of spores may not be attached to the artemisia plant and grow, and if the inoculation amount exceeds 100 parts by mass, abnormal propagation may occur due to excessive moisture.

When the Aspergillus is inoculated to the Artemisia plant used as the koji material, water is preferably added. The amount of the water to be added is not particularly limited and may be appropriately selected according to the purpose, and is preferably 10 to 250 parts by mass, more preferably 20 to 200 parts by mass, and particularly preferably 30 to 150 parts by mass, based on 100 parts by mass of the above-mentioned artemisia plant. If the amount of water added is less than 10 parts by mass per 100 parts by mass of the artemisia plant, a sufficient amount of spores may not be attached to the artemisia plant and grow.

The culture temperature is not particularly limited as long as it is within a temperature range in which the aspergillus can grow, and may be appropriately selected according to the purpose, and is preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the cultivation temperature is lower than 20 ℃, there is a case where sufficient spores cannot be attached to the artemisia plant to grow. Further, if it exceeds 50 ℃, the Aspergillus cannot be proliferated in some cases.

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

The fermentation broth of Artemisia may contain the cells of Aspergillus, or may be a fermentation broth from which the cells of Aspergillus have been removed, preferably a fermentation broth from which the cells of Aspergillus have been removed.

The state of the fermentation broth of the artemisia plant is not particularly limited, and may be appropriately selected according to the purpose, and for example, the fermentation broth of the artemisia plant itself, a purified product of the fermentation broth of the artemisia plant, a concentrate of the fermentation broth of the artemisia plant, a diluted product of the fermentation broth of the artemisia plant, and the like may be used. The fermentation solution of Artemisia may be obtained by mixing or dissolving a dried product of the fermentation solution of Artemisia in a solvent such as water or a hydrophilic solvent again.

The purified product of the fermentation broth of Artemisia is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a product obtained by removing solid components (for example, plant bodies of Artemisia, cells of Aspergillus, precipitates, and the like) in the fermentation broth of Artemisia.

The removal method is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include filtration.

The filtration method is not particularly limited, and may be appropriately selected from known methods according to the purpose.

The diluted artemisia fermentation liquid and the concentrate of the artemisia fermentation liquid are not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a substance prepared by preparing the artemisia fermentation liquid to a desired concentration.

The dilution method is not particularly limited, and may be appropriately selected from known methods according to the purpose.

The concentration method is not particularly limited and may be appropriately selected according to the purpose, and for example, concentration under reduced pressure and the like are exemplified.

The dried product of the fermentation broth of Artemisia is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a product obtained by drying a fermentation broth of Artemisia.

The drying method is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include freeze drying and the like.

The fermentation liquid of the artemisia plant is not particularly limited as long as the artemisia plant is fermented by aspergillus, and may be appropriately selected according to the purpose, and from the viewpoint of good skin compatibility, the fermentation liquid of the artemisia plant with a contact angle of 81 ° or less is preferable, and the fermentation liquid of the artemisia plant with a contact angle of 78 ° or less is more preferable.

In the present specification, the contact angle is a value obtained by dropping 3. mu.L of a measurement sample onto a sample stage of a dynamic contact angle/surface tension measuring apparatus (FTA1000 Falcon, First Ten Angstroms Co., Ltd.), measuring the value by a liquid drop method under conditions of a temperature of 22 ℃ and a relative humidity of 20%, and determining a contact angle θ (. degree.) of 1,000ms by the θ/2 method.

The contact angle is used as an index indicating "wetting", and is defined as "an angle (an angle located inside a liquid) formed between a liquid surface and a solid surface at a position where a free surface of a stationary liquid and a wall surface of the solid" is in contact (refer to "physiochemical dictionary" 4 th edition, rock book store, ltd.). The contact angle depends on the magnitude relationship between the cohesive force between liquid molecules and the adhesive force between solid wall surfaces, and is an acute angle when the liquid wets the solid (the adhesive force is large) and an obtuse angle when the liquid is not wetted. Therefore, the contact angle is smaller, the wettability is higher, that is, the skin affinity is better, and therefore, the lower limit of the contact angle of the artemisia fermentation liquid is not particularly limited and may be appropriately selected according to the purpose.

Thyme fermentation liquor

The thyme fermentation liquid is fermentation liquid of thyme fermented by aspergillus.

-thyme-

Thyme(s) used as the fermentation raw material(s) ((s))Thymus vulgarisLinne belongs to LabiataeLabiatae) Sweet pepper (sweet pepper)Thymus) The perennial woody plant belongs to a kind of herbal medicine, and has been used as a raw material for food or medicine since ancient times. Also named as common thyme. The origin is mediterranean coastline, but is also naturally grown or planted in japan and can be easily obtained from these areas.

The method for obtaining thyme is not particularly limited, and may be appropriately selected according to the purpose, collected from the natural world, or commercially available products may be used.

The site of use of the thyme used as the fermentation raw material is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include: flowers, buds, fruits, pericarps, seeds, seed coats, stems, leaves, branches, barks, stalks, branches and leaves and other overground parts; root, rhizome, etc. in the lower part. These sites may be used alone in 1 kind, or may be used in combination of 2 or more kinds. Of these parts, the part of thyme to be used is preferably the aerial part.

The size of the thyme used as the fermentation raw material is not particularly limited as long as it is a size capable of culturing the aspergillus, and may be appropriately selected according to the purpose, and examples thereof include: collected original size, cut into desired size, size converted into fine powder (powder), and the like.

The state of the thyme used as the fermentation raw material is not particularly limited as long as the aspergillus is cultured, and may be appropriately selected according to the purpose, and examples thereof include a raw state, a dried state, a crushed state, a squeezed state, and an extract state. Among these conditions, the collected original condition, crushed condition, squeezed condition, and extract condition are preferable, and the collected original condition and crushed condition are more preferable, from the viewpoint that the aspergillus easily acts.

The method for drying the thyme is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include: a method of drying by sun, a method of drying by a conventional dryer, and the like.

The method for pulverizing the thyme is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of pulverizing the thyme by a blender, a sugar mill, an electric mill, a jet mill, an impact mill, or the like.

The method for bringing the thyme into the squeezed state is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include squeezing.

The method for making the thyme in the extract state is not particularly limited, and a method commonly used in plant extraction may be appropriately selected according to the purpose.

The thyme used as the fermentation feedstock is preferably sterilized prior to inoculation with the aspergillus. The method for sterilizing thyme is not particularly limited, and may be appropriately selected from known methods.

Aspergillus-

The aspergillus used for fermenting thyme is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include the aspergillus described in "artemisia plant fermentation broth". These Aspergillus may be used alone in 1 kind, or may be used in combination of 2 or more kinds. Among these aspergillus, aspergillus oryzae (aspergillus oryzae) is preferable because it has excellent at least one of anti-aging effect and anti-oxidation effectAspergillus oryzae)。

The method for obtaining the aspergillus is not particularly limited, and may be appropriately selected according to the purpose, collected from the nature, or a commercially available product may be used. As the aspergillus, a koji using rice or the like as a raw material, a thyme koji described below, or an aspergillus cultured in a medium (agar medium, liquid medium, or the like) may be used. Among these aspergillus, the above-mentioned thyme koji is preferably used in that at least one of the anti-aging effect and the anti-oxidation effect is excellent.

With respect to the amount of inoculation of the Aspergillus on the thyme used as the fermentation feedstockThe amount of the thyme that can be fermented is not particularly limited and may be appropriately selected according to the purpose, and when the fermentation raw material is in a liquid state, it is preferably 1 × 10³1X 10 to one/mL⁸The amount per mL is preferably 1X 10 in the case where the fermentation raw material is in a solid state³Per g to 1X 10⁸Per gram.

When the thyme is inoculated with the aspergillus, water is preferably added. The amount of the water to be added is not particularly limited and may be appropriately selected according to the purpose, and is preferably 500 to 5,000 parts by mass, more preferably 1,000 to 4,000 parts by mass, and particularly preferably 1,500 to 3,000 parts by mass, based on 100 parts by mass of the thyme.

The fermentation (culture) temperature is not particularly limited as long as it is within a temperature range in which the fermentation by the aspergillus is possible, and may be appropriately selected according to the purpose, and is preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the fermentation temperature is lower than 20 ℃, the thyme cannot be sufficiently fermented, and at least one of the anti-aging effect and the anti-oxidation effect may be insufficient.

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

The method for stopping the fermentation (culture) is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of heating and the like.

The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the aspergillus cannot grow any more, and may be appropriately selected according to the purpose, and is preferably 50 ℃ or more, more preferably 70 ℃ or more, and particularly preferably 100 to 130 ℃. If the heating temperature is less than 50 ℃, the fermentation may not be stopped, and if the heating temperature exceeds 130 ℃, at least one of the anti-aging effect and the anti-oxidation effect may be insufficient.

The heating time for stopping the fermentation is not particularly limited as long as the aspergillus can be prevented from growing, and may be appropriately selected according to the purpose, and is preferably 5 minutes or more, and more preferably 10 minutes to 20 minutes. If the heating time is less than 5 minutes, the fermentation may not be stopped, and if it exceeds 20 minutes, at least one of the anti-aging effect and the antioxidant effect may be insufficient.

Further, the thyme fermentation broth after the fermentation is stopped is preferably cooled. The cooling method is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of standing at room temperature, a refrigerator, and the like.

The number of times the thyme is fermented by the aspergillus is not particularly limited, and may be appropriately selected according to the purpose, and may be 1 time or a plurality of times.

In the case where the fermentation is carried out a plurality of times, the Aspergillus may be inoculated only at the first inoculation, may be inoculated only a plurality of times, may be inoculated at each fermentation, and is preferably inoculated only at the first inoculation.

In the case where the fermentation is performed a plurality of times, the fermentation temperature and the fermentation time may be different from each other or may be the same.

- - -thyme seed-koji-

The thyme starter is obtained by using the thyme as a starter raw material, inoculating aspergillus to the starter raw material and attaching sufficient spores to the thyme for growth. The use of the koji in the fermentation is advantageous in that a thyme fermentation liquid excellent in skin affinity can be obtained more efficiently and easily.

The thyme used as the starting material for the koji may be the same as the thyme described in the above-thyme, and the thyme may be used in the same manner in terms of the parts, size, state and the like.

As the above-mentioned Aspergillus used for producing the thyme koji, the same bacteria as described in the above-mentioned Aspergillus can be used.

The amount of the koji mold to be inoculated onto the thyme used as the koji material is not particularly limited and may be appropriately selected depending on the purpose, and it is preferable to inoculate 5 to 100 parts by mass of the koji mold (1 × 10) suspended in sterilizing water to 100 parts by mass of the thyme³1X 10 to one/mL⁸one/mL), more preferably from 10 to 50 parts by mass, and particularly preferably from 20 to 30 parts by mass. If the amount of the aspergillus oryzae to be inoculated is less than 5 parts by mass with respect to 100 parts by mass of the thyme, spores may not be attached to the thyme in a sufficient amount to grow, and if the amount exceeds 100 parts by mass, abnormal growth may be caused by excessive moisture.

When the thyme used as the koji material is inoculated with the aspergillus, water is preferably added. The amount of the water to be added is not particularly limited and may be appropriately selected according to the purpose, and is preferably 10 to 250 parts by mass, more preferably 20 to 200 parts by mass, and particularly preferably 30 to 150 parts by mass, based on 100 parts by mass of the thyme. If the amount of water added is less than 10 parts by mass per 100 parts by mass of the thyme, spores may not be attached to the thyme in a sufficient amount to grow.

The culture temperature is not particularly limited as long as it is within a temperature range in which the aspergillus can grow, and may be appropriately selected according to the purpose, and is preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the culture temperature is lower than 20 ℃, there is a case where sufficient spores cannot be attached to the thyme to grow. Further, if it exceeds 50 ℃, the Aspergillus cannot be proliferated in some cases.

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

The thyme fermentation broth may contain the fungus of aspergillus, may be a fermentation broth from which the fungus of aspergillus has been removed, and is preferably a fermentation broth from which the fungus of aspergillus has been removed.

The state of the thyme fermentation liquid is not particularly limited and may be appropriately selected depending on the purpose, and for example, the state may be the thyme fermentation liquid itself, or may be a purified thyme fermentation liquid, a concentrate of thyme fermentation liquid, a diluted thyme fermentation liquid, or the like. The thyme fermentation liquid may be a liquid obtained by mixing or dissolving a dried thyme fermentation liquid in a solvent such as water or a hydrophilic solvent again.

The purified product of the thyme fermentation liquid is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a product obtained by removing solid components (for example, plant bodies of thyme, cells of aspergillus, precipitates, and the like) in the thyme fermentation liquid.

The removal method is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include filtration.

The filtration method is not particularly limited, and may be appropriately selected from known methods according to the purpose.

The diluted thyme fermentation liquid and the concentrate of thyme fermentation liquid are not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a thyme fermentation liquid prepared to a desired concentration.

The dilution method is not particularly limited, and may be appropriately selected from known methods according to the purpose.

The concentration method is not particularly limited and may be appropriately selected according to the purpose, and for example, concentration under reduced pressure and the like are exemplified.

The dried product of the thyme fermentation liquid is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a product obtained by drying a thyme fermentation liquid.

The drying method is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include freeze drying and the like.

The thyme fermentation liquid is not particularly limited as long as thyme is fermented by aspergillus, and may be appropriately selected according to the purpose, and from the viewpoint of good skin affinity, the thyme fermentation liquid having a contact angle of 87 ° or less is preferable, and the thyme fermentation liquid having a contact angle of 81 ° or less is more preferable.

Melissa officinalis fermentation liquor

The melissa officinalis fermentation liquid is a fermentation liquid of melissa officinalis fermented by aspergillus.

Melissa officinalis

Melissa officinalis used as the fermentation raw material (A)Melissa officinalisLinne belongs to LabiataeLabiatae) Bee pollen (bee pollen), (b)Melissa) Belonging to perennial herbaceous plants. It is one of the herbs and has been used as a raw material for food or medicine since ancient times. Also called Lemon balm (Lemon balm), melissa (garden balm), etc. The origin is south europe, but is also naturally grown or cultivated in japan, and can be easily obtained from these areas.

The method for obtaining the melissa officinalis is not particularly limited, and may be appropriately selected according to the purpose, collected from the natural world, or a commercially available product may be used.

The site of use of the melissa officinalis used as the fermentation raw material is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include: flowers, buds, fruits, pericarps, seeds, seed coats, stems, leaves, stalks, branches and leaves and other overground parts; root, rhizome, etc. in the lower part. These sites may be used alone in 1 kind, or may be used in combination of 2 or more kinds. Among these parts, the use part of the melissa officinalis is preferably an aerial part.

The size of the melissa officinalis used as the fermentation raw material is not particularly limited as long as it is a size capable of culturing the aspergillus, and may be appropriately selected according to the purpose, and for example, there may be mentioned: collected original size, cut into desired size, size converted into fine powder (powder), and the like.

The state of the melissa officinalis used as the fermentation raw material is not particularly limited as long as it is a state capable of culturing the aspergillus, and may be appropriately selected according to the purpose, and for example, there may be mentioned: the collected original state, dried state, pulverized state, squeezed state, and extract state. Among these conditions, the collected original condition, crushed condition, squeezed condition, and extract condition are preferable, and the collected original condition and crushed condition are more preferable, from the viewpoint that the aspergillus easily acts.

The method for drying the melissa officinalis is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include: a method of drying by sun, a method of drying by a conventional dryer, and the like.

The method for pulverizing the aforementioned balm grass is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of pulverizing the balm grass by a blender, a sugar mill, an electric mill, a jet mill, an impact mill, or the like.

The method for bringing the lemon balm into the squeezed state is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include squeezing.

The method for bringing the melissa officinalis into the state of the extract is not particularly limited, and a method commonly used in plant extraction may be appropriately selected according to the purpose.

It is preferable that the melissa officinalis used as the fermentation raw material is sterilized before the aspergillus is inoculated. The method for sterilizing the melissa officinalis is not particularly limited, and may be appropriately selected from known methods.

Aspergillus-

The Aspergillus to be fermented with the melissa is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include the Aspergillus described in the "Artemisia plant fermentation broth". These Aspergillus may be used alone in 1 kind, or may be used in combination of 2 or more kinds. Of these aspergillus, aspergillus oryzae (aspergillus oryzae) is preferable as the aspergillus having excellent at least one of anti-aging effect, anti-oxidation effect, anti-inflammatory effect and whitening effectAspergillus oryzae)。

The method for obtaining the aspergillus is not particularly limited, and may be appropriately selected according to the purpose, collected from the nature, or a commercially available product may be used. The aspergillus may be a koji using rice or the like as a raw material, a melissa koji described below, or an aspergillus cultured in a medium (e.g., an agar medium, a liquid medium, etc.). Among these aspergillus, the aforementioned melissa officinalis koji is preferably used in that at least one of an anti-aging effect, an antioxidant effect, an anti-inflammatory effect, and a whitening effect is excellent.

The amount of the Aspergillus to be inoculated to the Melissa officinalis used as the fermentation material is not particularly limited as long as the Melissa officinalis can be fermented, and may be appropriately selected according to the purpose, and is preferably 1X 10 when the fermentation material is in a liquid state³1X 10 to one/mL⁸The amount per mL is preferably 1X 10 in the case where the fermentation raw material is in a solid state³Per g to 1X 10⁸Per gram.

When the melissa officinalis is inoculated with the aspergillus, water is preferably added. The amount of the water to be added is not particularly limited and may be appropriately selected according to the purpose, and is preferably 500 to 5,000 parts by mass, more preferably 1,000 to 4,000 parts by mass, and particularly preferably 1,500 to 3,000 parts by mass, based on 100 parts by mass of the melissa officinalis.

The fermentation (culture) temperature is not particularly limited as long as it is within a temperature range in which the fermentation by the aspergillus is possible, and may be appropriately selected according to the purpose, and is preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the fermentation temperature is lower than 20 ℃, the melissa officinalis cannot be sufficiently fermented, and at least any one of the anti-aging effect, the antioxidant effect, the anti-inflammatory effect, and the whitening effect may be insufficient.

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

The method for stopping the fermentation (culture) is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of heating and the like.

The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the aspergillus cannot grow any more, and may be appropriately selected according to the purpose, and is preferably 50 ℃ or more, more preferably 70 ℃ or more, and particularly preferably 100 to 130 ℃. If the heating temperature is less than 50 ℃, the fermentation may not be stopped, and if the heating temperature exceeds 130 ℃, at least one of the anti-aging effect, the antioxidant effect, the anti-inflammatory effect, and the whitening effect may be insufficient.

The heating time for stopping the fermentation is not particularly limited as long as the aspergillus can be prevented from growing, and may be appropriately selected according to the purpose, and is preferably 5 minutes or more, and more preferably 10 minutes to 20 minutes. If the heating time is less than 5 minutes, the fermentation may not be stopped, and if it exceeds 20 minutes, at least one of the anti-aging effect, the antioxidant effect, the anti-inflammatory effect, and the whitening effect may be insufficient.

Further, the melissa officinalis fermented liquid after the fermentation is stopped is preferably cooled. The cooling method is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of standing at room temperature, a refrigerator, and the like.

The number of times the melissa officinalis is fermented by the aspergillus is not particularly limited, and may be appropriately selected according to the purpose, and may be 1 time or more.

In the case where the fermentation is carried out a plurality of times, the Aspergillus may be inoculated only at the first inoculation, may be inoculated only a plurality of times, may be inoculated at each fermentation, and is preferably inoculated only at the first inoculation.

In the case where the fermentation is performed a plurality of times, the fermentation temperature and the fermentation time may be different from each other or may be the same.

- - -Melissa seed-

The melissa officinalis mother culture is obtained by using the melissa officinalis as a mother culture raw material, inoculating aspergillus to the mother culture raw material and enabling sufficient spores to be attached to the melissa officinalis to grow. The use of the koji for the fermentation is advantageous in that a melissa officinalis fermentation liquid excellent in skin compatibility can be obtained more efficiently and easily.

The plant described in the above-described Melissa officinalis can be used as the Melissa officinalis used as the starting material for the koji, and the use part, size, state and other forms of the Melissa officinalis are also the same.

As the Aspergillus used for producing the melissa serissoide, the same may be used as the above-mentioned Aspergillus.

The amount of the koji mold to be inoculated to the melissa officinalis used as the koji material is not particularly limited and may be appropriately selected depending on the purpose, and it is preferable to inoculate 5 to 100 parts by mass of the koji mold (1 × 10) suspended in sterilizing water to 100 parts by mass of the melissa officinalis³1X 10 to one/mL⁸one/mL), more preferably from 10 to 50 parts by mass, and particularly preferably from 20 to 30 parts by mass. If the inoculation amount of the aspergillus is less than 5 parts by mass with respect to 100 parts by mass of the melissa officinalis, a sufficient amount of spores may not be attached to the melissa officinalis and grow, and if it exceeds 100 parts by mass, abnormal propagation may be caused by excessive moisture.

When the melissa officinalis used as the koji material is inoculated with the aspergillus, water is preferably added. The amount of the water to be added is not particularly limited and may be appropriately selected according to the purpose, and is preferably 10 to 250 parts by mass, more preferably 20 to 200 parts by mass, and particularly preferably 30 to 150 parts by mass, based on 100 parts by mass of the melissa officinalis. If the amount of water added is less than 10 parts by mass per 100 parts by mass of the melissa officinalis, a sufficient amount of spores may not be attached to the melissa officinalis and grow.

The culture temperature is not particularly limited as long as it is within a temperature range in which the aspergillus can grow, and may be appropriately selected according to the purpose, and is preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the cultivation temperature is lower than 20 ℃, there is a case where sufficient spores cannot be attached to the melissa folium to grow. Further, if it exceeds 50 ℃, the Aspergillus cannot be proliferated in some cases.

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

The melissa officinalis fermentation broth may contain the fungus of the aspergillus, or may be a fermentation broth from which the fungus of the aspergillus is removed, preferably a fermentation broth from which the fungus of the aspergillus is removed.

The state of the melissa officinalis fermentation liquid is not particularly limited and may be appropriately selected depending on the purpose, and for example, the melissa officinalis fermentation liquid itself, a purified product of the melissa officinalis fermentation liquid, a concentrate of the melissa officinalis fermentation liquid, a diluted product of the melissa officinalis fermentation liquid, or the like may be used. The melissa officinalis fermented liquid may be a liquid obtained by mixing or dissolving a dried product of the melissa officinalis fermented liquid again in water or a solvent such as a hydrophilic solvent.

The purified product of the melissa officinalis fermentation solution is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a product obtained by removing solid components (for example, plant bodies of the melissa officinalis, cells of aspergillus oryzae, precipitates, and the like) in the melissa officinalis fermentation solution.

The removal method is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include filtration.

The filtration method is not particularly limited, and may be appropriately selected from known methods according to the purpose.

The diluted matter of the melissa officinalis fermentation liquid and the concentrated matter of the melissa officinalis fermentation liquid are not particularly limited and may be appropriately selected according to the purpose, and for example, a matter prepared by preparing the melissa officinalis fermentation liquid to a desired concentration and the like may be mentioned.

The dilution method is not particularly limited, and may be appropriately selected from known methods according to the purpose.

The concentration method is not particularly limited and may be appropriately selected according to the purpose, and for example, concentration under reduced pressure and the like are exemplified.

The dried matter of the melissa officinalis fermented liquid is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a matter obtained by drying a melissa officinalis fermented liquid.

The drying method is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include freeze drying and the like.

The melissa officinalis fermentation liquid is not particularly limited as long as it is a fermentation liquid of melissa officinalis fermented by aspergillus, and may be appropriately selected according to the purpose, and from the viewpoint of good skin affinity, it is preferably a melissa officinalis fermentation liquid having a contact angle of 85 ° or less, more preferably a melissa officinalis fermentation liquid having a contact angle of 79 ° or less.

Cornflower fermentation liquor

The cornflower fermentation liquor is fermentation liquor of cornflower fermented by aspergillus.

Cornflower-

Cornflower (A) for use as the fermentation feedstockCentaurea cyanusLinne belongs to the family of CompositaeCompositae) Cornflower genus (Centaurea) The annual herbaceous plant of (1) has been used as a raw material for food or medicine since ancient times. Also known as Rodgersia podophylla, Rodgersia rosea (Centaurea), Cartesian flower (Centaurea), etc. The origin is europe, but it is also natural growth or planting in japan, and can be easily obtained from these areas.

The method for obtaining the cornflower is not particularly limited, and may be appropriately selected depending on the purpose, collected from the natural world, or a commercially available product may be used.

The site of use of the cornflower used as the fermentation raw material is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include: flowers, buds, fruits, pericarps, seeds, seed coats, stems, leaves, stalks, branches and leaves and other overground parts; root, rhizome, etc. in the lower part. These sites may be used alone in 1 kind, or may be used in combination of 2 or more kinds. Of these sites, the use site of the cornflower is preferably the aerial part.

The size of the cornflower used as the fermentation raw material is not particularly limited as long as it is a size capable of culturing the aspergillus, and may be appropriately selected according to the purpose, and examples thereof include: collected original size, cut into desired size, size converted into fine powder (powder), and the like.

The state of the cornflower used as the fermentation raw material is not particularly limited as long as it is a state in which the aspergillus can be cultured, and may be appropriately selected according to the purpose, and examples thereof include: the collected original state, dried state, pulverized state, squeezed state, and extract state. Among these conditions, the collected original condition, crushed condition, squeezed condition, and extract condition are preferable, and the collected original condition and crushed condition are more preferable, from the viewpoint that the aspergillus easily acts.

The method for bringing the cornflower into a dry state is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include: a method of drying by sun, a method of drying by a conventional dryer, and the like.

The method for pulverizing the cornflower into the pulverized state is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of pulverizing the cornflower by a blender, a sugar mill, an electric mill, a jet mill, an impact mill, or the like.

The method for bringing the cornflower into the juiced state is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include squeezing.

The method for bringing the cornflower into the extract state is not particularly limited, and a method commonly used in plant extraction may be appropriately selected according to the purpose.

The cornflower used as the fermentation raw material is preferably sterilized before the aspergillus is inoculated. The method for sterilizing the cornflower is not particularly limited, and may be appropriately selected from known methods.

Aspergillus-

The Aspergillus to be used for the cornflower fermentation is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include the Aspergillus described in the "Artemisia fermentation broth". These Aspergillus may be used alone in 1 kind, or may be used in combination of 2 or more kinds. Among these aspergillus, aspergillus oryzae (aspergillus oryzae) is preferable because it has excellent at least one of anti-aging effect, anti-oxidation effect and whitening effectAspergillus oryzae)。

The method for obtaining the aspergillus is not particularly limited, and may be appropriately selected according to the purpose, collected from the nature, or a commercially available product may be used. As the Aspergillus, a koji using rice or the like as a raw material, a cornflower koji described below, or an Aspergillus cultured in a medium (such as an agar medium or a liquid medium) may be used. Among these aspergillus, the above-mentioned cornflower koji is preferably used in terms of being excellent in at least any one of an anti-aging effect, an anti-oxidation effect, and a whitening effect.

The inoculation amount of the Aspergillus on the cornflower used as the fermentation raw material is not particularly limited as long as it is an amount capable of fermenting the cornflower, and may be appropriately selected according to the purpose, and in the case where the fermentation raw material is in a liquid state, it is preferably 1X 10³1X 10 to one/mL⁸The amount per mL is preferably 1X 10 in the case where the fermentation raw material is in a solid state³Per g to 1X 10⁸Per gram.

When the cornflower is inoculated with the aspergillus, water is preferably added. The amount of the water to be added is not particularly limited and may be appropriately selected according to the purpose, and is preferably 500 to 5,000 parts by mass, more preferably 1,000 to 4,000 parts by mass, and particularly preferably 1,500 to 3,000 parts by mass, based on 100 parts by mass of the cornflower.

The fermentation (culture) temperature is not particularly limited as long as it is within a temperature range in which the fermentation by the aspergillus is possible, and may be appropriately selected according to the purpose, and is preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the fermentation temperature is less than 20 ℃, the cornflower cannot be sufficiently fermented, and at least one of the anti-aging effect, the anti-oxidation effect, and the whitening effect may be insufficient.

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

The method for stopping the fermentation (culture) is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of heating and the like.

The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the aspergillus cannot grow any more, and may be appropriately selected according to the purpose, and is preferably 50 ℃ or more, more preferably 70 ℃ or more, and particularly preferably 100 to 130 ℃. If the heating temperature is less than 50 ℃, the fermentation may not be stopped, and if the heating temperature exceeds 130 ℃, at least one of the anti-aging effect, the antioxidant effect, and the whitening effect may be insufficient.

The heating time for stopping the fermentation is not particularly limited as long as the aspergillus can be prevented from growing, and may be appropriately selected according to the purpose, and is preferably 5 minutes or more, and more preferably 10 minutes to 20 minutes. If the heating time is less than 5 minutes, the fermentation may not be stopped, and if it exceeds 20 minutes, at least one of the anti-aging effect, the antioxidant effect, and the whitening effect may be insufficient.

Further, the cornflower broth after the fermentation is stopped is preferably cooled. The cooling method is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a method of standing at room temperature, a refrigerator, and the like.

The number of times the cornflower is fermented with the aspergillus is not particularly limited, and may be appropriately selected according to the purpose, and may be 1 time or a plurality of times.

In the case where the fermentation is carried out a plurality of times, the Aspergillus may be inoculated only at the first inoculation, may be inoculated only a plurality of times, may be inoculated at each fermentation, and is preferably inoculated only at the first inoculation.

In the case where the fermentation is performed a plurality of times, the fermentation temperature and the fermentation time may be different from each other or may be the same.

- -cornflower seed koji-

The cornflower seed koji is obtained by using the cornflower as a seed koji raw material, inoculating aspergillus to the seed koji raw material and attaching sufficient spores to the cornflower to grow. The use of the koji in the fermentation is advantageous in that a cornflower fermentation solution having excellent skin affinity can be obtained more efficiently and easily.

The plant described in the above-cornflower can be used as the cornflower used as the koji material, and the forms such as the site, size, state and the like of the cornflower can be also used.

As the Aspergillus used for producing the cornflower koji, the same may be used as the fungus described in the above-Aspergillus.

The amount of the Aspergillus on the cornflower used as the koji material is not particularly limited and may be appropriately selected depending on the purpose, and it is preferable to inoculate 5 to 100 parts by mass of Aspergillus (1X 10) suspended in sterilizing water to 100 parts by mass of the cornflower³1X 10 to one/mL⁸one/mL), more preferably from 10 to 50 parts by mass, and particularly preferably from 20 to 30 parts by mass. If the amount of the Aspergillus is less than 5 parts by mass relative to 100 parts by mass of the cornflower, spores may not be attached to the cornflower in a sufficient amount to grow, and if the amount exceeds 100 parts by mass, abnormal propagation may occur due to excessive moisture.

When the cornflower used as the koji material is inoculated with the aspergillus, water is preferably added. The amount of the water to be added is not particularly limited and may be appropriately selected according to the purpose, and is preferably 10 to 250 parts by mass, more preferably 20 to 200 parts by mass, and particularly preferably 30 to 150 parts by mass, based on 100 parts by mass of the cornflower. If the amount of water added is less than 10 parts by mass relative to 100 parts by mass of the cornflower, there is a case where a sufficient amount of spores cannot be attached to the cornflower for growth.

The culture temperature is not particularly limited as long as it is within a temperature range in which the aspergillus can grow, and may be appropriately selected according to the purpose, and is preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the culture temperature is less than 20 ℃, there are cases where sufficient spores cannot be attached to the cornflower for growth. Further, if it exceeds 50 ℃, the Aspergillus cannot be proliferated in some cases.

The culture time is not particularly limited and may be appropriately selected according to the purpose, and is preferably 80 to 210 hours, more preferably 100 to 190 hours, and particularly preferably 120 to 170 hours. If the cultivation time is less than 80 hours, there is a case where sufficient spores are not allowed to attach to the cornflower for growth, and if it exceeds 210 hours, there is a case where the germination rate of spores is decreased.

The cornflower fermentation broth may contain the cells of the Aspergillus, may be a fermentation broth from which the cells of the Aspergillus have been removed, and is preferably a fermentation broth from which the cells of the Aspergillus have been removed.

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

The purified product of the cornflower fermentation broth is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a product obtained by removing solid components (for example, plant bodies of the cornflower, cells of aspergillus, precipitates, and the like) in the cornflower fermentation broth.

The removal method is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include filtration.

The filtration method is not particularly limited, and may be appropriately selected from known methods according to the purpose.

The dilution of the cornflower fermentation broth and the concentrate of the cornflower fermentation broth are not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include those in which the cornflower fermentation broth is prepared to a desired concentration.

The dilution method is not particularly limited, and may be appropriately selected from known methods according to the purpose.

The concentration method is not particularly limited and may be appropriately selected according to the purpose, and for example, concentration under reduced pressure and the like are exemplified.

The dried product of the cornflower fermentation broth is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a product obtained by drying a cornflower fermentation broth.

The drying method is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include freeze drying and the like.

The cornflower fermentation liquid is not particularly limited as long as it is a cornflower fermentation liquid fermented by aspergillus, and may be appropriately selected according to the purpose, and from the viewpoint of good skin affinity, the cornflower fermentation liquid having a contact angle of 85 ° or less is preferable, and the cornflower fermentation liquid having a contact angle of 79 ° or less is more preferable.

Other components

The other components of the anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent are not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include: excipient, moisture-proof agent, antiseptic, enhancer, thickener, emulsifier, antioxidant, sweetener, sour agent, flavoring agent, colorant, perfume, whitening agent, humectant, oily component, ultraviolet absorbent, surfactant, thickener, alcohol, powder component, coloring material, aqueous component, water, skin nourishing agent, etc. These may be used alone in 1 kind, or may be used in combination of 2 or more kinds.

The content of the other components is not particularly limited and may be appropriately selected according to the purpose.

Application-

The anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention have at least one of excellent anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect, and therefore are suitable for use as, for example, pharmaceuticals, quasi drugs, cosmetics, foods, drinks, and the like, and the amount, usage, and formulation thereof can be appropriately selected according to the purpose of use.

The amount of the above-mentioned fermentation solution may be appropriately adjusted according to the physiological activity of the fermentation solution. In addition, the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent may be the fermentation liquid itself.

The use is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include: oral, parenteral, topical, etc. Of these, external use is preferable.

The dosage form is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include: oral preparations such as tablet, powder, capsule, granule, extract, and syrup; non-oral administration preparations such as injection, drop, and suppository; lotions, milky lotions, creams, ointments, essences, skin lotions, masks, jellies, lipsticks, pressed powders, bath agents, soaps, bath lotions, astringent lotions, hair tonics, pomades, shampoos and the like.

In addition, the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent of the present invention can also be used as an agent for research relating to the action mechanism of anti-aging action, anti-oxidation action, anti-inflammatory action, or whitening action.

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

(cosmetics)

The cosmetic of the present invention contains at least 1 selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent of the present invention, and further contains other components as necessary.

Anti-aging agent, antioxidant, anti-inflammatory agent, whitening agent

The content of at least 1 selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent in the cosmetic is not particularly limited, and may be appropriately selected according to the purpose, and is preferably 5% by volume or more, and more preferably 20% by volume or more, relative to the total amount of the cosmetic. If the content of at least 1 selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent is less than 5 vol%, at least one of the anti-aging effect, the anti-oxidative effect, the anti-inflammatory effect, and the whitening effect may be insufficient. In addition, the more the content of at least 1 selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent, the better, the upper limit is not particularly limited and may be appropriately selected according to the purpose. In addition, the cosmetic may be at least 1 substance itself selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent.

< other ingredients >

The cosmetic may further contain various main agents, auxiliary agents and other components which are generally used for the production of cosmetics, as necessary, within a range not impairing the object and the effect of the present invention.

The other components are not particularly limited and may be appropriately selected according to the purpose, and examples thereof include: astringent, bactericide, antibacterial agent, ultraviolet absorbent, cell activator, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactant, perfume, etc. These components can be used alone in 1 kind, also can be combined with more than 2 kinds. When these components are used in combination with at least 1 selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent, the components may act synergistically to provide an excellent effect beyond expectation.

The content of the other ingredients in the cosmetic is not particularly limited as long as the effect of the present invention is not impaired, and may be appropriately selected according to the purpose.

< use >)

The use of the cosmetic is not particularly limited, and may be appropriately selected from general cosmetics, and examples thereof include: skin cosmetics such as astringent, lotion, cream, ointment, essence, skin lotion, facial mask, jelly, lipstick, pack, bathing agent, soap, and bath lotion; scalp and hair cosmetics such as astringent, hair tonic, hair cream, hair conditioner, hair oil, shampoo, and hair conditioner.

The cosmetic may be prepared by blending at least 1 selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent in any cosmetic without inhibiting the activity thereof, or may be a cosmetic mainly composed of at least 1 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 1 substance itself selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent.

The cosmetic of the present invention is suitably used for humans, but may be used for animals other than humans (for example, mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as they exhibit their respective effects.

The cosmetic of the present invention contains at least 1 selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent, and therefore exhibits at least one of excellent anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect when applied to the skin, and is useful in this respect.

Examples

The present invention will be specifically described below by way of production examples and test examples, but the present invention is not limited to these test examples at all.

Production example 1: preparation of Artemisia tridentate fermentation broth 1

A seed preparation step

Aspergillus (Aspergillus oryzae, strain name: AOK1714, manufactured by Okamura japonica Koidz Co., Ltd.) was suspended in 50mL of sterilizing water using platinum loop (Aspergillus loop) to prepare an Aspergillus solution. The number of the cells of the Aspergillus solution was estimated using a Thoma blood cell count plate (manufactured by EKDS) and found to be 1.0X 10⁵one/mL.

Then, 10g of sagebrush (manufactured by ALBION GmbH.) cut into 0.5-5 cm was put into a conical flask, sterilized under pressure, inoculated with 2mL of the above-mentioned Aspergillus solution, and subjected to static culture at 30 ℃ for 168 hours. After the culture is finished, drying for 24 hours at the temperature of 45 ℃ to obtain the "sagebrush mother liquor".

A fermentation step-

The artemisia tridentate (manufactured by ALBION corporation) was pulverized using a pulverizer (sugar press), and passed through a 2mm mesh screen to obtain a pulverized artemisia tridentate. The pulverized material of Artemisia tridentate is mixed with 1,000mL of water (50 g), and inoculated with the seed koji obtained in the seed koji preparation step (number of bacteria: about 1.0X 10) ⁶pieces/mL) 20 mL. Then, it was pre-cultured at 25 ℃ for 22 hours. The obtained fermentation liquor is filtered by using diatomite to obtain the artemisia tridentate fermentation liquor 1.

Production example 2: preparation of Artemisia tridentate fermentation broth 2

In the above production example 1, the sagebrush seed koji was changed to a seed koji made from rice (Aspergillus oryzae) ((Aspergillus oryzae))Aspergillus oryzae)"Artemisia tridentate fermentation broth 2" was obtained in the same manner as in production example 1, except that Aspergillus leucissus, manufactured by autumn filed store Ltd. (hereinafter, sometimes referred to as "Miqu").

Comparative production example 1: preparation of artemisia tridentate extract

The artemisia tridentate (manufactured by ALBION corporation) was pulverized using a pulverizer (sugar press), and passed through a 2mm mesh screen to obtain a pulverized artemisia tridentate. This pulverized material of Artemisia tridentate was mixed with 1,000mL of water (50 g), followed by stirring at 25 ℃ for 22 hours. Then, the obtained stirred material was filtered with celite to obtain "artemisia tridentate extract".

(test example A-1: measurement of contact Angle)

The artemisia tridentate fermentation liquid 1 obtained in production example 1, the artemisia tridentate fermentation liquid 2 obtained in production example 2, and the artemisia tridentate extract liquid obtained in comparative production example 1 were used as test samples, and the contact angle was measured by the following method.

Specifically, 3. mu.L of each test sample was dropped onto a sample stage (aluminum) of a dynamic contact angle/surface tension measuring apparatus (FTA1000 Falcon, manufactured by First Ten anchors Co., Ltd.) and measured by a liquid drop method under conditions of a temperature of 22 ℃ and a relative humidity of 20%. The contact angle θ (°) of 1,000ms was obtained by the θ/2 method. The contact angle was measured 3 times, and the average value was obtained. The results are shown in table 1 below. Fig. 1A to 1C show examples of the liquid droplets when the contact angle of each test sample is measured.

[ Table 1]

	Test sample	Fermentation of	Aspergillus sp	Contact angle theta (°)
					Production example 1	Artemisia tridentate fermentation liquor 1	Is provided with	Three-tooth sagebrush seed koji	77.44
Production example 2	Artemisia tridentate fermentation liquor 2	Is provided with	Rice koji	80.02
					Comparative production example 1	Herba Artemisiae Scopariae extractive solution	Is free of	-	81.57

The contact angles of the artemisia tridentate fermentation liquid 1 obtained in production example 1 and the artemisia tridentate fermentation liquid 2 obtained in production example 2 were both small, 81 ° or less, and excellent in skin compatibility, compared to the artemisia tridentate extraction liquid obtained in comparative production example 1. Furthermore, the contact angle of the artemisia tridentate fermentation liquid 1 obtained in production example 1 was 78 ° or less, and the skin compatibility was further excellent.

(test example 1-1: inhibition of matrix metalloproteinase-1 (MMP-1) Activity test)

Using the artemisia tridentate fermentation broth 1 obtained in production example 1, the artemisia tridentate fermentation broth 2 obtained in production example 2, and the artemisia tridentate extract obtained in comparative production example 1 as samples to be tested, a test for the inhibitory activity of matrix metalloproteinase-1 (MMP-1) was performed by the following test method, in which a part of the saururus chinensis and Heidrich (Wunsch and Heidrich) method was changed.

In a test tube with a cap, each sample to be tested was dissolved in 0.1mol/L Tris (hydroxymethyl) aminomethane, Tris-HCl buffer (pH 7.1) containing 20mmol/L calcium chloride. Then, 50. mu.L of a solution of 50. mu. L, MMP-1 (collagenase IV from Clostridium histolyticum IV, manufactured by Sigma Co.) and 400. mu.L of a solution of Pz-peptide (Pz-Pro-Leu-Gly-Pro-D-Arg-OH, manufactured by BACHEM Feinchemikalien AG) were mixed, and reacted at 3 ℃ for 30 minutes, and then 1mL of 25mmol/L citric acid solution was added to stop the reaction. In addition, the final concentration of the sample to be tested at this time was the concentration shown in table 2 below. Then, ethyl acetate 5mL was added, and vigorously shaken. This was centrifuged at 1,600 Xg for 10 minutes, and the absorbance at 320nm of the ethyl acetate layer was measured.

The absorbance was measured in the same manner as described above except that the MMP-1 solution (enzyme solution) was changed to 0.1mol/L Tris-HCl buffer (pH 7.1) as a blank sample.

The absorbance was measured in the same manner as described above except that the solution of the sample to be tested was changed to 0.1mol/L Tris-HCl buffer (pH 7.1) containing 20mmol/L calcium chloride and not containing the sample to be tested as a control.

From the obtained measured values of absorbance, the MMP-1 activity inhibitory rate was calculated based on the following formula 1. The results are shown in table 2 below.

< formula 1 >

MMP-1 activity inhibition ratio (%) { 1- (C-D)/(a-B) } × 100

In the formula 1, A to D each represent the following.

A: absorbance at 320nm of a wavelength when the sample to be tested is not added and the enzyme is added

B: absorbance at 320nm without sample and enzyme

C: absorbance at 320nm of a sample to be tested added and an enzyme added

D: absorbance at 320nm of a sample without enzyme

[ Table 2]

(test examples 1-2: hyaluronic acid synthetase 3(HAS3) mRNA expression promoting action test)

Using the artemisia tridentate fermentation broth 1 obtained in production example 1, the artemisia tridentate fermentation broth 2 obtained in production example 2, and the artemisia tridentate extract obtained in comparative production example 1 as test samples, a test for promoting the expression of hyaluronan synthase 3(HAS3) mRNA was performed by the following test method.

Each sample to be tested was dissolved in a basal medium for normal human epidermal keratinocytes (HuMedia-KB2, manufactured by sank textile gmbh) so that the final concentration thereof became the concentration shown in table 3 below, and a medium to which the sample to be tested was added was prepared.

A medium for proliferating normal human epidermal keratinocytes (HuMedia-KG2, manufactured by Korea textile Co., Ltd.) were used at 37 ℃ with 5% CO₂Normal Human neonatal Epidermal Keratinocytes (Normal Human Epidermal Keratinocytes, NHEK, manufactured by sank textile gmbh) were cultured until confluent, and thereafter, the cells were recovered by trypsin treatment. The collected cells were adjusted to 1.5X 10 using a medium for normal human epidermal keratinocyte growth (HuMedia-KG2)⁵cells/mL.

Then, the NHEK (1.5X 10)⁵cells/mL) 2mL were inoculated into 35mm dishes at 37 ℃ with 5% CO₂Was cultured overnight under the conditions of (1). After completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB2), and the cells were further cultured for 24 hours. After completion of the culture, the medium was replaced with 2mL of the sample-added medium, and the mixture was incubated at 37 ℃ with 5% CO₂Cultured under the conditions of (1) for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted using an RNA extraction reagent (ISOGEN II (catalog No. 311-07361), manufactured by NIPPONGENE Co., Ltd.), the amount of each RNA was measured by a spectrophotometer, and total RNA was prepared so as to be 200 ng/. mu.L using purified water.

In addition, as a control, total RNA was prepared in the same manner as described above so as to be 200 ng/. mu.l, except that 2mL of the medium to which the sample to be tested was added was changed to 2mL of a normal human epidermal keratinocyte basal medium (HuMedia-KB2) containing no sample to be tested, and absorbance was measured.

The total RNAs were used as templates to measure the expression levels of hyaluronic acid synthase 3(HAS3) mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal standard. The detection of mRNA was carried out by a two-step RT-PCR reaction using a Real-Time PCR (polymerase chain reaction) apparatus (Thermal Cycler Dice (registered trademark) Real-Time System III (Real Time System III), manufactured by Takara Bio Inc.) and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR kit (Perfect Real Time (catalog No.: RR063A, manufactured by Takara Bio Inc.).

The expression levels of HAS3 mRNA in the samples without and with the samples were corrected for the expression level of GAPDH mRNA. From this corrected value, the HAS3 mRNA expression promotion rate was calculated based on the following formula 2. The results are shown in table 3 below.

< formula 2 >

HAS3 mRNA expression promotion Rate (%) ═ A/B X100

In the formula 2, a and B each represent the following.

A: correction value when sample under test is added

B: correction value without sample under test

[ Table 3]

(test examples 1-3: DPPH radical scavenging action test)

The DPPH radical scavenging action was tested by the following test method using the artemisia tridentate fermentation broth 1 obtained in production example 1, the artemisia tridentate fermentation broth 2 obtained in production example 2, and the artemisia tridentate extract obtained in comparative production example 1 as test samples.

Each sample to be tested was dissolved in an ethanol solution (manufactured by fuji film and wako pure chemical industries, ltd.) to prepare a sample solution to be tested.

To 3mL of a 150. mu. mol/L DPPH (diphenyl-p-propylhydrazyl) ethanol solution, 3mL of the sample solution was added, the sample solution was immediately mixed by shaking with a stopple, and after standing for 30 minutes, the absorbance at 520nm was measured. In addition, the final concentration of the sample to be tested at this time was the concentration shown in table 4 below.

The absorbance was measured in the same manner as described above except that the DPPH ethanol solution was changed to an ethanol solution containing no DPPH as a blank sample.

The absorbance was measured in the same manner as described above except that the sample solution to be tested was changed to an ethanol solution (manufactured by fuji film and wako pure chemical industries, ltd.) containing no sample to be tested as a control.

From the obtained measured value of absorbance, the DPPH radical clearance was calculated based on the following formula 3. The results are shown in table 4 below.

< formula 3 >

DPPH radical clearance (%) { A- (B-C) }/A × 100

In the above formula 3, A to C each represent the following.

A: absorbance at 520nm of a wavelength when DPPH is added without a sample to be tested

B: absorbance at 520nm of a wavelength when a sample to be tested is added and DPPH is added

C: absorbance at 520nm without addition of sample to be tested and without addition of DPPH

[ Table 4]

(test examples 1-4: Hyaluronidase Activity inhibition test)

The hyaluronidase activity inhibition test was performed by the following test method using the artemisia tridentate fermentation broth 1 obtained in production example 1, the artemisia tridentate fermentation broth 2 obtained in production example 2, and the artemisia tridentate extract obtained in comparative production example 1 as test samples.

Each sample to be tested was dissolved in 0.1mol/L acetate buffer (pH 3.5) to prepare a sample solution to be tested.

0.1mL of hyaluronidase solution (IV-S type (from bovine testis), 400NF units/mL, manufactured by SIGMA corporation) was added to 0.2mL of the sample solution to be tested, and the mixture was reacted at 37 ℃ for 20 minutes. Then, 0.2mL of 2.5mmol/L calcium chloride was added as an activating agent, and the reaction was further carried out at 37 ℃ for 20 minutes. To this, 0.5mL of a 0.8mg/mL sodium hyaluronate solution (derived from rooster comb) (manufactured by Fuji film and Wako pure chemical industries, Ltd.) was added, and the mixture was reacted at 37 ℃ for 40 minutes. In addition, the final concentration of the sample to be tested at this time was the concentration shown in table 5 below. Then, 0.2mL of 0.4mol/L sodium hydroxide was added to stop the reaction, and after cooling, 0.2mL of a boric acid solution was added to each reaction solution and boiled for 3 minutes. After water cooling, 6mL of p-DABA reagent (a reagent prepared by dissolving 10g of p-dimethylaminobenzaldehyde in a mixture of 12.5mL of 10N hydrochloric acid and 87.5mL of acetic acid and diluting the solution with acetic acid by a factor of 10) was added, and the reaction was carried out at 37 ℃ for 20 minutes. Then, the absorbance at a wavelength of 585nm was measured.

The absorbance was measured in the same manner as described above except that the hyaluronidase solution (enzyme solution) was changed to 0.1mol/L acetate buffer (pH 3.5) as a blank sample.

The absorbance was measured in the same manner as described above except that the sample solution to be tested was changed to 0.1mol/L acetate buffer (pH 3.5) containing no sample to be tested as a control.

From the obtained measurement values of absorbance, the hyaluronidase activity inhibition ratio was calculated based on the following formula 4. The results are shown in table 5 below.

< formula 4 >

Hyaluronidase activity inhibition rate (%) { 1- (C-D)/(a-B) } × 100

In the above formula 4, A to D each represent the following.

A: absorbance at a wavelength of 585nm when the test sample is not added and the enzyme is added

B: absorbance at wavelength 585nm without addition of sample to be tested and without addition of enzyme

C: absorbance at a wavelength of 585nm when the sample to be tested is added and the enzyme is added

D: absorbance at a wavelength of 585nm when the sample is added and the enzyme is not added

[ Table 5]

(test examples 1-5: tyrosinase Activity inhibition test)

The tyrosinase activity inhibitory activity was tested by the following test method using the artemisia tridentate fermentation liquid 1 obtained in production example 1, the artemisia tridentate fermentation liquid 2 obtained in production example 2, and the artemisia tridentate extract obtained in comparative production example 1 as test samples.

Each sample to be tested was dissolved in a 25% DMSO (Dimethyl sulfoxide) solution to prepare a sample solution to be tested. To a 48-well plate, 0.2mL of Mclvaine buffer (pH 6.8), 0.06mL of 0.3 mg/mL of the tyrosine solution, and 0.18mL of the sample solution to be tested were added, and the mixture was allowed to stand at 37 ℃ for 10 minutes. To this, 0.02mL of 800unit/mL tyrosinase solution (manufactured by SIGMA corporation) was added, and the reaction was further carried out at 37 ℃ for 15 minutes. After the reaction, the absorbance at a wavelength of 475nm was measured. In addition, the final concentration of the sample to be tested at this time was the concentration shown in table 6 below.

The absorbance was measured in the same manner as described above except that the tyrosinase solution (enzyme solution) was changed to Mcllvaine buffer (pH 6.8) as a blank sample.

The absorbance was measured in the same manner as described above except that the test sample solution was changed to a 25% DMSO solution containing no test sample as a control.

From the obtained measurement values of 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 above formula 5, A to D each represent the following.

A: absorbance at wavelength 475nm when no sample to be tested was added and enzyme was added

B: absorbance at wavelength 475nm without sample and enzyme

C: absorbance at wavelength of 475nm when the sample to be tested is added and the enzyme is added

D: absorbance at wavelength of 475nm when sample to be tested was added and no enzyme was added

[ Table 6]

Production example 3: preparation of Artemisia capillaris fermentation liquor 1

In the koji preparation step of production example 1, Artemisia tridentate was changed to Artemisia capillaris, (A)Artemisia capillaris"seed koji of Artemisia capillaris" was prepared by the same method as the seed koji preparation procedure of the preparation example 1 except for Thunbergii (manufactured by ALBION GmbH).

In the fermentation step of production example 1, artemisia capillaris thunb was changed to artemisia capillaris thunb (Artemisia capillaris"Artemisia capillaris Thunbergii fermentation broth 1" was obtained by the same method as the fermentation step of production example 1, except that Thunbergii (manufactured by ALBION GmbH.).

Production example 4: preparation of Artemisia capillaris fermentation liquor 2

In the above production example 3, the Artemisia capillaris Thunb seed koji was changed to Mi koji ((Aspergillus oryzae"artemisia capillaris fermentation liquid 2" was obtained in the same manner as in production example 3 except that aspergillus leuciscus for white spirit, produced by oka japan store gmbh).

Comparative production example 2: preparation of artemisia capillaris extracting solution

In comparative production example 1, Artemisia tridentate was replaced with Artemisia capillaris (A)Artemisia capillarisThunbergii) (manufactured by ALBION corporation), in addition to,"an extract solution of artemisia capillaris was obtained by the same method as in comparative production example 1.

(test example A-2: measurement of contact Angle)

In the test example a-1, the contact angle was measured by the same method as in the test example a-1 except that the test sample was changed to artemisia capillaris fermentation liquid 1 obtained in production example 3, artemisia capillaris fermentation liquid 2 obtained in production example 4, and artemisia capillaris extract obtained in comparative production example 2. The results are shown in table 7 below. Fig. 2A to 2C show examples of the liquid droplets when the contact angle of each test sample is measured.

[ Table 7]

	Test sample	Fermentation of	Aspergillus sp	Contact angle theta (°)
					Production example 3	Artemisia capillaris fermenting liquid 1	Is provided with	Seed koji of Artemisia capillaris	77.50
Production example 4	Artemisia capillaris fermenting liquid 2	Is provided with	Rice koji	79.58
					Comparative production example 2	Herba Artemisiae Scopariae extractive solution	Is free of	-	87.75

The contact angles of the artemisia capillaris fermentation liquid 1 obtained in the production example 3 and the artemisia capillaris fermentation liquid 2 obtained in the production example 4 were both small, 81 ° or less, and the skin compatibility was excellent, compared to the artemisia capillaris extraction liquid obtained in the comparative production example 2. Further, the contact angle of the artemisia capillaris fermentation liquid 1 obtained in production example 3 was 78 ° or less, and the skin compatibility was further excellent.

(test examples 2-1: hyaluronic acid synthetase 3(HAS3) mRNA expression promoting action test)

In test examples 1-2, the effect of promoting expression of hyaluronic acid synthase 3(HAS3) mRNA was tested in the same manner as in test example 1-2 except that the sample to be tested was changed to artemisia capillaris fermentation liquid 1 obtained in production example 3, artemisia capillaris fermentation liquid 2 obtained in production example 4, and artemisia capillaris extract obtained in comparative production example 2, and the final concentration of the sample to be tested was changed to the concentration shown in table 8 below. The results are shown in table 8 below.

[ Table 8]

(test example 2-2 type I collagen production promoting Effect test)

A test for the collagen type I production promoting effect was performed by the following test method using the artemisia capillaris fermentation liquid 1 obtained in production example 3, the artemisia capillaris fermentation liquid 2 obtained in production example 4, and the artemisia capillaris extract obtained in comparative production example 2 as samples to be tested.

Each sample to be tested was dissolved in dulbecco MEM (Modified Eagle's Medium) (manufactured by japan pharmaceuticals limited) containing 0.25% Fetal bovine serum (FBS, biosera) so that the final concentration thereof was as shown in table 9 below, to prepare a Medium to which the sample to be tested was added.

DMEM with 10% FBS at 37 deg.C and 5% CO₂Normal human fibroblasts (NB1RGB, purchased from RIKEN BRC) were cultured until confluent, after which the cells were recovered by trypsin treatment. The recovered cells were adjusted to 1.6X 10 with DMEM containing 10% FBS⁵cells/mL.

Then, NB1RGB (1.6 × 10)⁵cells/mL) was seeded at 100. mu.L per well in 96-well microplates at 37 ℃ with 5% CO₂Was cultured overnight under the conditions of (1). After completion of the culture, the medium was replaced with 100. mu.L of the medium containing the sample to be tested, and the medium was incubated at 37 ℃ with 5% CO₂Cultured for 3 days. After the completion of the culture, the amount of type I collagen in the culture medium of each well was measured by ELISA (Enzyme Linked Immunosorbent Assay).

Specifically, 90mL of the culture supernatant was transferred to an ELISA plate, and after adsorbing the culture plate overnight at 4 ℃, the solution was discarded and washed with a phosphate physiological saline buffer (PBS-T) containing 0.05% Tween-20. Thereafter, the blocking operation was performed using a phosphate physiological saline buffer containing 1% FBS. The solution was discarded, washed with 0.05% Tween-20 in phosphate physiological saline buffer (PBS-T), and reacted with an anti-human collagen type I antibody (rabbit IgG, manufactured by Chemicon). The solution was discarded, washed with a 0.05% Tween-20-containing phosphate physiological saline buffer (PBS-T), reacted with an HRP-labeled anti-rabbit IgG antibody, and then subjected to a color development reaction by the same washing procedure.

The amount of type I collagen was calculated by performing the ELISA using a standard and preparing a calibration curve.

In addition, measurement was performed by ELISA in the same manner as described above except that the sample solution to be tested was changed to DMEM containing 0.25% FBS without the sample to be tested as a control.

From the obtained measurement values, the type I collagen production promoting rate was calculated based on the following formula 6. The results are shown in table 9 below.

< formula 6 >

Type I collagen production promoting rate (%) ═ a/B × 100

In the above formula 6, a and B each represent the following.

A: amount of type I collagen when a sample to be tested is added

B: amount of type I collagen without addition of subject sample

[ Table 9]

(test examples 2-3: St. Ex. 1 mRNA expression promoting action)

An experiment was conducted to determine the effect of promoting the expression of claudin-1 mRNA by the following test method using artemisia capillaris fermentation liquid 1 obtained in production example 3, artemisia capillaris fermentation liquid 2 obtained in production example 4, and artemisia capillaris extract obtained in comparative production example 2 as samples to be tested.

Each sample to be tested was dissolved in a basal medium for normal human epidermal keratinocytes (HuMedia-KB2, manufactured by sank textile gmbh) so that the final concentration thereof became the concentration shown in table 10 below, and a medium to which the sample to be tested was added was prepared.

A medium for proliferating normal human epidermal keratinocytes (HuMedia-KG2, manufactured by Korea textile Co., Ltd.) were used at 37 ℃ with 5% CO₂Normal human neonatal epidermal keratinocytes (NHEK, manufactured by canker textile gmbh) were cultured under the conditions of (1) until confluency, and thereafter the cells were recovered by trypsin treatment. The collected cells were adjusted to 1.5X 10 using a medium for normal human epidermal keratinocyte growth (HuMedia-KG2)⁵cells/mL.

Then, the NHEK (1.5X 10)⁵cells/mL) 2mL were inoculated into 35mm dishes at 37 ℃ with 5% CO₂Incubated overnight under conditions. After the culture is finished, the culture medium is changed to normalHuman epidermal keratinocytes basal medium (HuMedia-KB2) were further cultured for 24 hours. After completion of the culture, the medium was replaced with 2mL of the sample-added medium, and the mixture was incubated at 37 ℃ with 5% CO₂Cultured under the conditions of (1) for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted using an RNA extraction reagent (ISOGEN II (catalog No. 311-07361), manufactured by NIPPONGENE Co., Ltd.), the amount of each RNA was measured by a spectrophotometer, and total RNA was prepared so as to be 200 ng/. mu.L using purified water.

In addition, as a control, total RNA was prepared in the same manner as described above so as to be 200 ng/. mu.l, except that 2mL of the medium to which the sample to be tested was added was changed to 2mL of a normal human epidermal keratinocyte basal medium (HuMedia-KB2) containing no sample to be tested, and absorbance was measured.

The total RNAs were used as templates to determine the expression levels of claudin-1 mRNA and GAPDH mRNA as an internal standard. The detection of mRNA was carried out by a two-step Real-Time PCR reaction using a Real-Time PCR apparatus (Thermal Cycler Dice (registered trademark) Real-Time system III, manufactured by Takara Bio Inc.) and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR kit (Perfect Real Time (catalog No.: RR063A, manufactured by Takara Bio Inc.).

The expression level of claudin-1 mRNA in the samples not added and added was corrected by the expression level of GAPDH mRNA. From the corrected value, the rate of promoting expression of claudin-1 mRNA was calculated based on the following formula 7. The results are shown in table 10 below.

< formula 7 >

Expression promotion rate (%) of claudin-1 mRNA

In the above formula 7, a and B each represent the following.

A: correction value when sample under test is added

B: correction value without sample under test

[ Table 10]

(test examples 2-4: St. Ex. 4mRNA expression promoting action)

The artemisia capillaris fermentation liquid 1 obtained in production example 3, the artemisia capillaris fermentation liquid 2 obtained in production example 4, and the artemisia capillaris extract obtained in comparative production example 2 were used as samples to be tested, and the effect of promoting the expression of claudin-4 mRNA was tested by the following test method.

Each sample to be tested was dissolved in a basal medium for normal human epidermal keratinocytes (HuMedia-KB2, manufactured by sank textile gmbh) so that the final concentration thereof became the concentration shown in table 11 below, and a medium to which the sample to be tested was added was prepared.

A medium for proliferating normal human epidermal keratinocytes (HuMedia-KG2, manufactured by Korea textile Co., Ltd.) were used at 37 ℃ with 5% CO₂Normal human neonatal epidermal keratinocytes (NHEK, manufactured by canker textile gmbh) were cultured under the conditions of (1) until confluency, and thereafter the cells were recovered by trypsin treatment. The collected cells were adjusted to 1.5X 10 using a medium for normal human epidermal keratinocyte growth (HuMedia-KG2)⁵cells/mL.

Then, the NHEK (1.5X 10)⁵cells/mL) 2mL were inoculated into 35mm dishes at 37 ℃ with 5% CO₂Incubated overnight under conditions. After completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB2), and the cells were further cultured for 24 hours. After completion of the culture, the medium was replaced with 2mL of the sample-added medium, and the mixture was incubated at 37 ℃ with 5% CO₂Cultured under the conditions of (1) for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted using an RNA extraction reagent (ISOGEN II (catalog No. 311-07361), manufactured by NIPPONGENE Co., Ltd.), the amount of each RNA was measured by a spectrophotometer, and total RNA was prepared so as to be 200 ng/. mu.L using purified water.

In addition, as a control, total RNA was prepared in the same manner as described above so as to be 200 ng/. mu.l, except that 2mL of the medium to which the sample to be tested was added was changed to 2mL of a normal human epidermal keratinocyte basal medium (HuMedia-KB2) containing no sample to be tested, and absorbance was measured.

The total RNAs were used as templates to determine the expression levels of claudin-4 mRNA and GAPDH mRNA as an internal standard. The detection of mRNA was carried out by a two-step Real-Time PCR reaction using a Real-Time PCR apparatus (Thermal Cycler Dice (registered trademark) Real-Time system III, manufactured by Takara Bio Inc.) and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR kit (Perfect Real Time (catalog No.: RR063A, manufactured by Takara Bio Inc.).

The expression levels of claudin-4 mRNA in the samples not added and added were corrected by the expression level of GAPDH mRNA. From the corrected value, the rate of promoting expression of claudin-4 mRNA was calculated based on the following formula 8. The results are shown in table 11 below.

< formula 8 >

Expression promotion rate (%) of claudin-4 mRNA

In the above formula 8, a and B each represent the following.

A: correction value when sample under test is added

B: correction value without sample under test

[ Table 11]

(test examples 2-5: Tissurin mRNA expression promoting action test)

The artemisia capillaris fermentation liquid 1 obtained in production example 3, the artemisia capillaris fermentation liquid 2 obtained in production example 4, and the artemisia capillaris extract obtained in comparative production example 2 were used as samples to be tested, and the effect of promoting expression of claudin mRNA was tested by the following test method.

Each sample to be tested was dissolved in a basal medium for normal human epidermal keratinocytes (HuMedia-KB2, manufactured by sank textile gmbh) so that the final concentration thereof became the concentration shown in table 12 below, and a medium to which the sample to be tested was added was prepared.

A medium for proliferating normal human epidermal keratinocytes (HuMedia-KG2, manufactured by Korea textile Co., Ltd.) were used at 37 ℃ with 5% CO₂Normal human neonatal epidermal keratinocytes (NHEK, manufactured by canker textile gmbh) were cultured under the conditions of (1) until confluency, and thereafter the cells were recovered by trypsin treatment. The collected cells were adjusted to 1.5X 10 using a medium for normal human epidermal keratinocyte growth (HuMedia-KG2)⁵cells/mL.

Then, the NHEK (1.5X 10) ⁵cells/mL) 2mL were inoculated into 35mm dishes at 37 ℃ with 5% CO₂Incubated overnight under conditions. After completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB2), and the cells were further cultured for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted using an RNA extraction reagent (ISOGEN II (catalog No. 311-07361), manufactured by NIPPONGENE Co., Ltd.), the amount of each RNA was measured by a spectrophotometer, and total RNA was prepared so as to be 200 ng/. mu.L using purified water.

In addition, as a control, total RNA was prepared in the same manner as described above so as to be 200 ng/. mu.l, except that 2mL of the medium to which the sample to be tested was added was changed to 2mL of a normal human epidermal keratinocyte basal medium (HuMedia-KB2) containing no sample to be tested, and absorbance was measured.

The expression levels of the Tight protein mRNA and the GAPDH mRNA as an internal standard were determined using each of the total RNAs as a template. The detection of mRNA was carried out by a two-step Real-Time PCR reaction using a Real-Time PCR apparatus (Thermal Cycler Dice (registered trademark) Real-Time system III, manufactured by Takara Bio Inc.) and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR kit (Perfect Real Time (catalog No.: RR063A, manufactured by Takara Bio Inc.).

The expression levels of the claudin mRNA in the samples not added and added were corrected by the expression level of GAPDH mRNA. From this corrected value, the expression promotion rate of claudin mRNA was calculated based on the following formula 9. The results are shown in table 12 below.

< formula 9 >

Tight protein mRNA expression promotion rate (%) ═ A/B × 100

In the above formula 9, a and B each represent the following.

A: correction value when sample under test is added

B: correction value without sample under test

[ Table 12]

Production example 5: preparation of thyme fermentation broth 1

In the koji preparation step of production example 1, Artemisia tridentata was changed to Thymus vulgaris: (Thymus vulgarisLinne) (manufactured by ALBION GmbH), except that the same procedure as described for the preparation of the mother starter in production example 1 was used, a "thyme mother starter" was prepared.

In the fermentation step of production example 1, the artemisia trirata was changed to thyme(s) ((s))Thymus vulgarisLinne) (manufactured by ALBION GmbH), except that the same method as the fermentation step of production example 1 was used, a "thyme fermentation broth 1" was obtained.

< production example 6: preparation of thyme fermentation broth 2 >

In the above production example 5, the thyme seed koji was changed to the koji (Aspergillus oryzae"thyme fermentation liquid 2" was obtained in the same manner as in production example 5, except that "white spirit aspergillus leucatus" was produced by oka japan store gmbh).

< comparative production example 3: preparation of thyme extract >

In the comparative production example 1, the herb of three-tooth wormwood was changed to thyme (thyme: (a))Thymus vulgarisLinne) (manufactured by ALBION GmbH) was used to obtain a "thyme extract" in the same manner as in comparative production example 1.

(test example A-3: measurement of contact Angle)

In the test example a-1, the contact angle was measured by the same method as in the test example a-1 except that the test sample was changed to the thyme fermentation liquid 1 obtained in production example 5, the thyme fermentation liquid 2 obtained in production example 6, and the thyme extract obtained in comparative production example 3. The results are shown in table 13 below. Fig. 3A to 3C show examples of the liquid droplets when the contact angle of each test sample is measured.

[ Table 13]

	Test sample	Fermentation of	Aspergillus sp	Contact angle theta (°)
					Production example 5	Thyme fermentation broth 1	Is provided with	Thyme seed koji	78.32
Production example 6	Thyme fermentation broth 2	Is provided with	Rice koji	82.77
					Comparative production example 3	Thyme extract	Is free of	-	88.59

The contact angles of the thyme fermentation liquid 1 obtained in production example 5 and the thyme fermentation liquid 2 obtained in production example 6 were small, 87 ° or less, and the thyme extract obtained in comparative production example 3 was excellent in skin compatibility. Further, the thyme fermentation liquid 1 obtained in production example 5 had a contact angle of 81 ° or less, and was excellent in skin compatibility.

Test example 3-1 Glutamine transaminase-1 (TGM-1) mRNA expression promoting action test

A test for promoting the expression of glutamine transaminase-1 (TGM-1) mRNA was performed by the following test method using thyme fermentation liquid 1 obtained in production example 5, thyme fermentation liquid 2 obtained in production example 6, and thyme extract obtained in comparative production example 3 as samples to be tested.

Each sample to be tested was dissolved in a basal medium for normal human epidermal keratinocytes (HuMedia-KB2, manufactured by sank textile gmbh) so that the final concentration thereof became the concentration shown in table 14 below, and a medium to which the sample to be tested was added was prepared.

A medium for proliferating normal human epidermal keratinocytes (HuMedia-KG2, manufactured by Korea textile Co., Ltd.) were used at 37 ℃ with 5% CO₂Normal human neonatal epidermal keratinocytes (NHEK, manufactured by canker textile gmbh) were cultured under the conditions of (1) until confluency, and thereafter the cells were recovered by trypsin treatment. The collected cells were adjusted to 1.5X 10 using a medium for normal human epidermal keratinocyte growth (HuMedia-KG2)⁵cells/mL.

Then, the NHEK (1.5X 10)⁵cells/mL) 2mL were inoculated into 35mm dishes at 37 ℃ with 5% CO ₂Incubated overnight under conditions. After completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB2), and the cells were further cultured for 24 hours. After the culture is finished, the culture medium is culturedThe medium was replaced with 2mL of the test sample-added medium, and the medium was incubated at 37 ℃ with 5% CO₂Cultured under the conditions of (1) for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted using an RNA extraction reagent (ISOGEN II (catalog No. 311-07361), manufactured by NIPPONGENE Co., Ltd.), the amount of each RNA was measured by a spectrophotometer, and total RNA was prepared so as to be 200 ng/. mu.L using purified water.

In addition, as a control, total RNA was prepared in the same manner as described above so as to be 200 ng/. mu.l, except that 2mL of the medium to which the sample to be tested was added was changed to 2mL of a normal human epidermal keratinocyte basal medium (HuMedia-KB2) containing no sample to be tested, and absorbance was measured.

The total RNAs were used as templates to determine the expression levels of transglutaminase-1 (TGM-1) mRNA and GAPDH mRNA as an internal standard. The mRNA was detected by a two-step Real-Time PCR reaction using a Real-Time PCR device (Thermal Cycler Dice (registered trademark) Real-Time system III, manufactured by Takara Bio Inc.) and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR kit (Perfect Real Time (catalog No. RR063A, manufactured by Takara Bio Inc.).

The expression levels of TGM-1 mRNA obtained without and with the addition of the test sample were corrected for the expression level of GAPDH mRNA. From the corrected 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 >

The promotion rate (%) of TGM-1 mRNA expression is A/B.times.100

In the above formula 10, a and B each represent the following.

A: correction value when sample under test is added

B: correction value without sample under test

[ Table 14]

(test examples 3-2-Purchase Activity test for aquaporin 3(AQP3) mRNA expression)

A test for promoting the expression of aquaporin 3(AQP3) mRNA was performed by the following test method using thyme fermentation broth 1 obtained in production example 5, thyme fermentation broth 2 obtained in production example 6, and thyme extract obtained in comparative production example 3 as test samples.

Each sample to be tested was dissolved in a basal medium for normal human epidermal keratinocytes (HuMedia-KB2, manufactured by sank textile gmbh) so that the final concentration thereof became the concentration shown in table 15 below, and a medium to which the sample to be tested was added was prepared.

A medium for proliferating normal human epidermal keratinocytes (HuMedia-KG2, manufactured by Korea textile Co., Ltd.) were used at 37 ℃ with 5% CO ₂Normal human neonatal epidermal keratinocytes (NHEK, manufactured by canker textile gmbh) were cultured under the conditions of (1) until confluency, and thereafter the cells were recovered by trypsin treatment. The collected cells were adjusted to 1.5X 10 using a medium for normal human epidermal keratinocyte growth (HuMedia-KG2)⁵cells/mL.

Then, the NHEK (1.5X 10)⁵cells/mL) 2mL were inoculated into 35mm dishes at 37 ℃ with 5% CO₂Incubated overnight under conditions. After completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB2), and the cells were further cultured for 24 hours. After completion of the culture, the medium was replaced with 2mL of the sample-added medium, and the mixture was incubated at 37 ℃ with 5% CO₂Cultured under the conditions of (1) for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted using an RNA extraction reagent (ISOGEN II (catalog No. 311-07361), manufactured by NIPPONGENE Co., Ltd.), the amount of each RNA was measured by a spectrophotometer, and total RNA was prepared so as to be 200 ng/. mu.L using purified water.

In addition, as a control, total RNA was prepared in the same manner as described above so as to be 200 ng/. mu.l, except that 2mL of the medium to which the sample to be tested was added was changed to 2mL of a normal human epidermal keratinocyte basal medium (HuMedia-KB2) containing no sample to be tested, and absorbance was measured.

The expression levels of aquaporin 3(AQP3) mRNA and GAPDH mRNA as an internal standard were determined using each of the total RNAs as a template. The detection of mRNA was carried out by a two-step Real-Time PCR reaction using a Real-Time PCR apparatus (Thermal Cycler Dice (registered trademark) Real-Time system III, manufactured by Takara Bio Inc.) and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR kit (Perfect Real Time (catalog No.: RR063A, manufactured by Takara Bio Inc.).

The expression levels of AQP3 mRNA were corrected for GAPDH mRNA expression levels for the samples without and with the samples. From this corrected value, the rate of promoting AQP3 mRNA expression 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 above formula 11, a and B each represent the following.

A: correction value when sample under test is added

B: correction value without sample under test

[ Table 15]

(test examples 3-3: Tissurin mRNA expression promoting action test)

In test examples 2 to 5, a test for a claudin mRNA expression promoting effect was performed in the same manner as in test examples 2 to 5 except that the sample to be tested was changed to the thyme fermentation liquid 1 obtained in production example 5, the thyme fermentation liquid 2 obtained in production example 6, and the thyme extract obtained in comparative production example 3, and the final concentration of the sample to be tested was changed to the concentration shown in table 16 below. The results are shown in Table 16 below.

[ Table 16]

(test examples 3-4: DPPH radical scavenging action test)

In test examples 1 to 3, a DPPH radical scavenging action was performed in the same manner as in test examples 1 to 3 except that the samples to be tested were changed to the thyme fermentation liquid 1 obtained in production example 5, the thyme fermentation liquid 2 obtained in production example 6, and the thyme extract obtained in comparative production example 3, and the final concentrations of the samples to be tested were changed to the concentrations shown in table 17 below. The results are shown in table 17 below.

[ Table 17]

< production example 7: preparation of Melissa fermentation broth 1

In the koji preparation step of production example 1, Artemisia tridentata is changed to Melissa officinalis (ii)Melissa officinalisLinne) (manufactured by ALBION GmbH), except for the above, a "Melissa seed koji" was prepared by the same method as the seed koji preparation procedure of the production example 1.

In addition, in the fermentation step of the above production example 1, Artemisia tridentate was changed to Melissa officinalis (S. mongolica and/or S. mongolica)Melissa officinalisLinne) (manufactured by ALBION GmbH), except for the above, a "Melissa officinalis broth 1" was obtained in the same manner as in the fermentation step of the above-mentioned production example 1.

< production example 8: preparation of Melissa fermentation broth 2

In the above production example 7, the Melissa seed koji was changed to a koji ( Aspergillus oryzae"Melissa fermentation broth 2" was obtained in the same manner as in production example 7, except that Aspergillus leuciscus for white spirit, produced by autumn filed store, Ltd.).

< comparative production example 4: preparation of Melissa extract

In the comparative production example 1, Artemisia tridentate was changed to Melissa officinalis: (Melissa officinalisLinne) (manufactured by ALBION GmbH), in addition to the above, the following compounds were usedThe same procedure as in comparative production example 1 was used to obtain a "Melissa officinalis extract".

(test example A-4: measurement of contact Angle)

In the test example a-1, the contact angle was measured in the same manner as in the test example a-1 except that the test sample was changed to the melissa officinalis fermented liquid 1 obtained in production example 7, the melissa officinalis fermented liquid 2 obtained in production example 8, and the melissa officinalis extract obtained in comparative production example 4. The results are shown in Table 18 below. Fig. 4A to 4C show examples of the liquid droplets when the contact angle of each test sample is measured.

[ Table 18]

	Test sample	Fermentation of	Aspergillus sp	Contact angle theta (°)
					Production example 7	Melissa fermentation broth 1	Is provided with	Melissa seed koji	77.09
Production example 8	Melissa fermentation liquor 2	Is provided with	Rice koji	80.16
					Comparative production example 4	Melissa extract	Is free of	-	86.53

The contact angles of both the melissa officinalis fermented liquid 1 obtained in production example 7 and the melissa officinalis fermented liquid 2 obtained in production example 8 were small, 85 ° or less, and the skin compatibility was excellent, compared to the melissa officinalis extract obtained in comparative production example 4. Further, the melissa officinalis fermented liquid 1 obtained in production example 7 had a contact angle of 79 ° or less, and was more excellent in skin compatibility.

Test example 4-1 type I collagen production promoting Effect test

In test example 2-2, a test for the collagen type I production promoting effect was performed in the same manner as in test example 2-2 except that the sample to be tested was changed to the melissa officinalis fermented liquid 1 obtained in production example 7, the melissa officinalis fermented liquid 2 obtained in production example 8, and the melissa officinalis extract obtained in comparative production example 4, and the final concentration of the sample to be tested was changed to the concentration shown in the following table 19. The results are shown in Table 19 below.

[ Table 19]

(test examples 4-2-aquaporin 3(AQP3) mRNA expression promoting action test)

In test example 3-2, a test for the aquaporin 3(AQP3) mRNA expression promoting effect was performed in the same manner as in test example 3-2, except that the test samples were changed to the melissa officinalis fermented liquid 1 obtained in production example 7, the melissa officinalis fermented liquid 2 obtained in production example 8, and the melissa officinalis extract obtained in comparative production example 4, and the final concentrations of the test samples were changed to the concentrations shown in the following table 20. The results are shown in table 20 below.

[ Table 20]

(test examples 4-3: DPPH radical scavenging action test)

In test examples 1 to 3, a DPPH radical scavenging action was performed in the same manner as in test examples 1 to 3 except that the test samples were changed to the melissa officinalis fermentation liquid 1 obtained in production example 7, the melissa officinalis fermentation liquid 2 obtained in production example 8, and the melissa officinalis extract obtained in comparative production example 4, and the final concentrations of the test samples were changed to the concentrations shown in table 21 below. The results are shown in table 21 below.

[ Table 21]

(test examples 4-4: Hyaluronidase Activity inhibition test)

In test examples 1 to 4, the hyaluronidase activity inhibitory activity was tested in the same manner as in test examples 1 to 4 except that the test samples were changed to the melissa officinalis fermented liquid 1 obtained in production example 7, the melissa officinalis fermented liquid 2 obtained in production example 8, and the melissa officinalis extract obtained in comparative production example 4, and the final concentrations of the test samples were changed to the concentrations shown in table 22 below. The results are shown in Table 22 below.

[ Table 22]

(test examples 4-5: tyrosinase Activity inhibition test)

In test examples 1 to 5, a tyrosinase activity inhibitory effect was performed in the same manner as in test examples 1 to 5 except that the test samples were changed to the melissa officinalis fermentation liquid 1 obtained in production example 7, the melissa officinalis fermentation liquid 2 obtained in production example 8, and the melissa officinalis extract obtained in comparative production example 4, and the final concentrations of the test samples were changed to the concentrations shown in table 23 below. The results are shown in Table 23 below.

[ Table 23]

Production example 9: preparation of cornflower fermentation broth 1

In the koji preparation step of production example 1, sagebrush was changed to cornflower (R) ((R))Centaurea cyanusLinne) (manufactured by ALBION GmbH), except for the above, a "cornflower koji" was prepared by the same method as the koji preparation procedure of the production example 1.

In addition, in the fermentation step of production example 1, sagebrush was changed to cornflower (S) ((S))Centaurea cyanusLinne) (manufactured by ALBION GmbH), except that the same method as the fermentation step of the manufacturing example 1 was used, a "cornflower fermentation broth 1" was obtained.

Production example 10: preparation of cornflower fermentation broth 2

In production example 9, the cornflower koji was changed to the meldonium koji (ii)Aspergillus oryzae"cornflower fermentation broth 2" was obtained in the same manner as in production example 9 except that "koji mold (aspergillus leuciscus) was used.

Comparative production example 5: preparation of cornflower extract

In comparative production example 1, sagebrush was changed to cornflower: (Centaurea cyanusLinne) (manufactured by ALBION GmbH), except that "cornflower extract" was obtained in the same manner as in comparative manufacturing example 1.

(test example A-5: measurement of contact Angle)

The contact angle was measured in the same manner as in test example A-1 except that the test sample was changed to cornflower broth 1 obtained in production example 9, cornflower broth 2 obtained in production example 10, and cornflower extract obtained in comparative production example 5 in test example A-1. The results are shown in Table 24 below. Fig. 5A to 5C show examples of the liquid droplets when the contact angle of each test sample is measured.

[ Table 24]

	Test sample	Fermentation of	Aspergillus sp	Contact angle theta (°)
					Production example 9	Cornflower fermentation liquor 1	Is provided with	Cornflower seed cake	78.52
Production example 10	Cornflower fermentation liquor 2	Is provided with	Rice koji	80.83
					Comparative production example 5	Cornflower extract	Is free of	-	86.96

The cornflower fermentation liquids 1 and 2 obtained in production examples 9 and 10 each had a smaller contact angle of 85 ° or less than that of the cornflower extract obtained in comparative production example 5, and had excellent skin compatibility. The cornflower fermentation solution 1 obtained in production example 9 had a contact angle of 79 ° or less, and was more excellent in skin compatibility.

Test example 5-1 type I collagen production promoting Effect test

In test example 2-2, a collagen type I production promoting effect was tested in the same manner as in test example 2-2 except that the test sample was changed to cornflower fermentation liquid 1 obtained in production example 9, cornflower fermentation liquid 2 obtained in production example 10, and cornflower extract obtained in comparative production example 5, and the final concentration of the test sample was changed to the concentration shown in table 25 below. The results are shown in Table 25 below.

[ Table 25]

Test example 5-2 Glutamine transaminase-1 mRNA expression-promoting Effect test

In test example 3-1, a glutamine transaminase-1 mRNA expression-promoting effect was tested in the same manner as in test example 3-1 except that the test sample was changed to the cornflower fermentation liquid 1 obtained in production example 9, the cornflower fermentation liquid 2 obtained in production example 10, and the cornflower extract liquid obtained in comparative production example 5, and the final concentration of the test sample was changed to the concentration shown in Table 26 below. The results are shown in table 26 below.

[ Table 26]

(test examples 5-3: experiment of promoting expression of filaggrin mRNA)

A test for the effect of promoting the expression of filaggrin mRNA was carried out by the following test method using the cornflower fermentation liquid 1 obtained in production example 9, the cornflower fermentation liquid 2 obtained in production example 10, and the cornflower extract liquid obtained in comparative production example 5 as samples to be tested.

Each sample to be tested was dissolved in a basal medium for normal human epidermal keratinocytes (HuMedia-KB2, manufactured by sank textile gmbh) so that the final concentration thereof became the concentration shown in table 27 below, and a medium to which the sample to be tested was added was prepared.

A medium for proliferating normal human epidermal keratinocytes (HuMedia-KG2, manufactured by Korea textile Co., Ltd.) were used at 37 ℃ with 5% CO₂Normal human neonatal epidermal keratinocytes (NHEK, manufactured by canker textile gmbh) were cultured under the conditions of (1) until confluency, and thereafter the cells were recovered by trypsin treatment. The collected cells were adjusted to 1.5X 10 using a medium for normal human epidermal keratinocyte growth (HuMedia-KG2)⁵cells/mL.

Then, the NHEK (1.5X 10)⁵cells/mL) 2mL were inoculated into 35mm dishes at 37 ℃ with 5% CO₂Incubated overnight under conditions. After completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB2), and the cells were further cultured for 24 hours. After completion of the culture, the medium was replaced with 2mL of the sample-added medium, and the mixture was incubated at 37 ℃ with 5% CO ₂Cultured under the conditions of (1) for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted using an RNA extraction reagent (ISOGEN II (catalog No. 311-07361), manufactured by NIPPONGENE Co., Ltd.), the amount of each RNA was measured by a spectrophotometer, and total RNA was prepared so as to be 200 ng/. mu.L using purified water.

In addition, as a control, total RNA was prepared in the same manner as described above so as to be 200 ng/. mu.l, except that 2mL of the medium to which the sample to be tested was added was changed to 2mL of a normal human epidermal keratinocyte basal medium (HuMedia-KB2) containing no sample to be tested, and absorbance was measured.

The total RNAs were used as templates to determine the expression levels of filaggrin mRNA and GAPDH mRNA as an internal standard. The detection of mRNA was carried out by a two-step Real-Time PCR reaction using a Real-Time PCR apparatus (Thermal Cycler Dice (registered trademark) Real-Time system III, manufactured by Takara Bio Inc.) and SYBR (registered trademark) PrimeScript (registered trademark) RT-PCR kit (Perfect Real Time (catalog No.: RR063A, manufactured by Takara Bio Inc.).

The expression levels of filaggrin mRNA without and with the subject sample were corrected for GAPDH mRNA expression. From this corrected value, the acceleration rate of filaggrin mRNA expression was calculated based on the following formula 12. The results are shown in table 27 below.

< formula 12 >

Filaggrin mRNA expression promotion rate (%) ═ a/B × 100

In formula 12, a and B each represent the following.

A: correction value when sample under test is added

B: correction value without sample under test

[ Table 27]

(test examples 5-4-aquaporin 3(AQP3) mRNA expression promoting action test)

In test example 3-2, a test for the aquaporin 3(AQP3) mRNA expression promoting effect was performed in the same manner as in test example 3-2 except that the sample to be tested was changed to 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, and the final concentration of the sample to be tested was changed to the concentration shown in Table 28 below. The results are shown in Table 28 below.

[ Table 28]

(test examples 5-5: hyaluronic acid synthetase 3(HAS3) mRNA expression promoting action test)

In test examples 1-2, the effect of promoting expression of hyaluronic acid synthase 3(HAS3) mRNA was tested in the same manner as in test example 1-2 except that the test sample was changed to cornflower fermentation liquid 1 obtained in production example 9, cornflower fermentation liquid 2 obtained in production example 10, and 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. The results are shown in table 29 below.

[ Table 29]

(test examples 5-6: St. Ex. 1 mRNA expression promoting action)

In test examples 2 to 3, a test for the claudin-1 mRNA expression promoting effect was carried out in the same manner as in test example 2 to 3 except that the test sample was changed to the cornflower fermentation liquid 1 obtained in production example 9, the cornflower fermentation liquid 2 obtained in production example 10, and the cornflower extract obtained in comparative production example 5, and the final concentration of the test sample was changed to the concentration shown in Table 30 below. The results are shown in table 30 below.

[ Table 30]

(test examples 5-7: St.4 mRNA expression-promoting action)

In test examples 2 to 4, a test for the claudin-4 mRNA expression promoting effect was carried out in the same manner as in test examples 2 to 4 except that the test sample was changed to the cornflower fermentation liquid 1 obtained in production example 9, the cornflower fermentation liquid 2 obtained in production example 10, and the cornflower extract obtained in comparative production example 5, and the final concentration of the test sample was changed to the concentration shown in Table 31 below. The results are shown in table 31 below.

[ Table 31]

(test examples 5-8: Tissurin mRNA expression promoting action test)

In test examples 2 to 5, a test for a claudin mRNA expression promoting effect was carried out in the same manner as in test examples 2 to 5 except that the test sample was changed to the cornflower fermentation liquid 1 obtained in production example 9, the cornflower fermentation liquid 2 obtained in production example 10, and the cornflower extract obtained in comparative production example 5, and the final concentration of the test sample was changed to the concentration shown in Table 32 below. The results are shown in table 32 below.

[ Table 32]

(test examples 5-9: DPPH radical scavenging action test)

The DPPH radical scavenging effect test was carried out in the same manner as in test examples 1 to 3 except that the samples to be tested were changed to the cornflower fermentation liquid 1 obtained in production example 9, the cornflower fermentation liquid 2 obtained in production example 10, and the cornflower extract obtained in comparative production example 5, and the final concentrations of the samples to be tested were changed to the concentrations shown in Table 33 below. The results are shown in table 33 below.

[ Table 33]

(test examples 5-10: test for inhibitory Effect on melanin production by B16 melanoma cells)

The test for the melanin production-inhibiting effect of B16 melanoma cells was carried out by the following test method using the cornflower fermentation liquid 1 obtained in production example 9, the cornflower fermentation liquid 2 obtained in production example 10, and the cornflower extract liquid obtained in comparative production example 5 as test samples.

Each sample was dissolved in DMEM (Nippon pharmaceutical Co., Ltd.) containing 10% FBS (biosera) and 1mmol/L theophylline (Fuji film and Wako pure chemical industries, Ltd.) to prepare a medium to which the sample was added.

DMEM with 10% FBS at 37 deg.C and 5% CO ₂B16 melanoma cells were cultured under the conditions of (a) until confluency, after which the cells were recovered by trypsin treatment. The recovered cells were adjusted to 2.4X 10 with DMEM containing 10% FBS and 1mmol/L theophylline⁵cells/mL.

Then, the B16 melanoma cells (2.4X 10)⁵cells/mL) at 300. mu.L per well in 48-well plates at 37 ℃ with 5% CO₂Cultured under the conditions of (1) for 6 hours. After completion of the culture, the medium was replaced with 100. mu.L of the medium containing the sample to be tested, and the medium was incubated at 37 ℃ with 5% CO₂Cultured for 3 days. After completion of the culture, the subject sample-added medium was added to each well in an amount of 300. mu.L, and the mixture was incubated at 37 ℃ with 5% CO₂Cultured for 4 days. The final concentration of the sample to be tested at this time was a concentration shown in table 34 below.

After completion of the culture, the medium was removed from each well, 200. mu.L of a 2mol/L sodium hydroxide solution was added thereto, the cells were disrupted by an ultrasonicator, and the absorbance at a wavelength of 475nm was measured.

From the absorbance values measured, the amount of melanin was calculated based on a calibration curve prepared using synthetic melanin (manufactured by SIGMA).

In addition, in order to measure the cell viability, B16 melanoma cells were cultured using the above-described medium to which the sample to be tested was added, and then the medium was removed and washed with 400 μ L of PBS buffer, in the same manner as described above. Then, neutral red was dissolved in DMEM containing 10% FBS at a final concentration of 0.05mg/mL, and the resulting solution was added to each well in 200. mu.L portions, followed by incubation for 2.5 hours. After the completion of the culture, the neutral red solution was removed, and 200. mu.L of an ethanol-acetic acid solution (ethanol: acetic acid: water: 50: 1: 49 (volume ratio)) was added to each well one by one to extract the pigment. After extraction, the absorbance at a wavelength of 540nm was measured.

The absorbance was measured in the same manner as described above except that the sample solution was changed to DMEM containing 10% FBS and 1mmol/L theophylline without the sample.

From the obtained measurement values, the cell survival rate was calculated based on the following formula 13, and the melanin production inhibition (%) corrected for the cell survival rate 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 above formula 13, C and D represent the following, respectively.

C: absorbance at wavelength 540nm without sample addition

D: absorbance at a wavelength of 540nm when a sample to be tested is added

< formula 14 >

Melanin production inhibition rate (%) { 1- (B/D)/(a/C) } × 100

In the above formula 14, a to D each represent the following.

A: amount of melanin when no sample is added

B: amount of melanin when a sample to be tested is added

C: absorbance at wavelength 540nm without sample addition

D: absorbance at a wavelength of 540nm when a sample to be tested is added

[ Table 34]

Industrial applicability

The anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention are natural products having excellent anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect, and high safety, and therefore can be used in any fields such as cosmetics, foods, and pharmaceuticals.

The cosmetic of the present invention contains at least 1 selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent of the present invention, and thus is suitably used for skin cosmetics such as lotions, milky lotions, creams, ointments, essences, skin lotions, facial masks, jellies, lipsticks, pressed powders, bath agents, soaps, and body washes; scalp and hair cosmetics such as astringent, hair tonic, hair cream, hair conditioner, hair oil, shampoo, and hair conditioner.