CN113631227B - Anti-aging agent, antioxidant, anti-inflammatory agent, whitening agent, and cosmetic - Google Patents
Anti-aging agent, antioxidant, anti-inflammatory agent, whitening agent, and cosmetic Download PDFInfo
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- CN113631227B CN113631227B CN202080024053.0A CN202080024053A CN113631227B CN 113631227 B CN113631227 B CN 113631227B CN 202080024053 A CN202080024053 A CN 202080024053A CN 113631227 B CN113631227 B CN 113631227B
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
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Abstract
The application discloses 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 liquid, thyme fermentation liquid, herba Melissae axillaris fermentation liquid and cornflower fermentation liquid as an effective component; the antioxidant contains at least one of Artemisia plant fermentation liquor, thyme fermentation liquor, herba Melissae axillaris fermentation liquor and cornflower fermentation liquor as an active ingredient; the anti-inflammatory agent contains at least one fermentation broth of Artemisia plant fermentation broth and herba Melissae axillaris fermentation broth as an active ingredient; the whitening agent contains at least one fermentation liquor of Artemisia plant fermentation liquor, herba Melissae axillaris fermentation liquor and cornflower fermentation liquor as an effective component.
Description
Technical Field
The present invention relates to an anti-aging agent, an antioxidant, an anti-inflammatory agent, and a whitening agent each containing a fermentation broth obtained by fermenting a plant with aspergillus as an active ingredient, and a cosmetic containing at least one of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent.
Background
In recent years, active oxygen has been attracting attention as a main cause of oxidation of biological components, and adverse effects on living things have been a problem. Active oxygen is produced during energy metabolism in biological cells, and there are superoxide [ i.e., superoxide anion (O 2 -) produced by single electron reduction of oxygen molecules, hydrogen peroxide (H 2O2), singlet oxygen (1O2), hydroxyl radical (OH) ] and the like. This active oxygen is essential for the bactericidal mechanism of phagocytes and plays an important role in the clearance of viruses or cancer cells.
However, the excessive production of active oxygen attacks in vivo molecules constituting membranes and tissues in vivo, and induces various diseases. Superoxide, which is produced in the organism and becomes the starting material for other reactive oxygen species, is typically gradually cleared by the catalytic action of the superoxide dismutase (SOD, super Oxide Dismutase) contained within the cell. However, when superoxide is excessively produced 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 cases are 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 acid, cold feeling constitution, skin aging and the like.
Among these disadvantages, the skin is an organ that is prone to superoxide generation because it is directly stimulated by environmental factors such as ultraviolet rays, and thus, there are problems such as the following as the concentration of superoxide increases: the degradation, modification, or crosslinking of biological tissues such as collagen, and the oxidation of oils and fats to form peroxidized lipids which cause damage to cells, form skin wrinkles, and cause aging, inflammation, and skin pigmentation such as decrease in skin elasticity (see non-patent document 1). Therefore, it is considered that by inhibiting or suppressing the formation of active oxygen or free radicals in living bodies, it is possible to prevent, treat or improve skin aging such as wrinkle formation or reduced elasticity, tissue diseases such as rheumatoid arthritis or behcet's disease, myocardial infarction, cerebral apoplexy, cataract, diabetes, arteriosclerosis, shoulder acid, cold feeling and other diseases related to active oxygen.
Therefore, attempts to obtain active oxygen scavenging substances, radical scavenging substances, hydrogen peroxide scavenging substances, and the like from natural substances advantageous in terms of safety have been made to find that extracts of plants of the genus brassica of the family brassicaceae (see patent document 1), extracts of plants of the genus kalanchoe of the family Crassulaceae (see patent document 2), extracts of oroxylum indicum (see patent document 3), extracts of sweet potato vine (see patent document 4), and the like are effective.
In addition, the causes and pathogenesis of inflammatory diseases such as contact dermatitis (macula), psoriasis, pemphigus vulgaris, and other various skin diseases accompanied by rough skin are various. The reason for this is known to be mainly that hyaluronidase activity is increased.
Hyaluronidase is a hydrolase of hyaluronic acid. Hyaluronate, which maintains affinity with body tissues, is decomposed in an aqueous system by ultraviolet rays, oxygen, etc., and thus has a reduced molecular weight and a reduced water-retaining effect. In addition, hyaluronic acid exists in the form of intercellular tissues in living bodies and is also associated with vascular permeability. In addition, hyaluronidase is present in the obese cells, and activation thereof causes degranulation, and thus, release occurs, and functions as an inflammatory chemical mediator. Therefore, by inhibiting the activity of hyaluronidase, it is expected to enhance moisture retention, prevent inflammation, and reduce inflammation.
As a substance having such a hyaluronidase activity inhibitory action, for example, an extract of a plant of the genus golden larch (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 organisms from ultraviolet rays, but excessive generation or uneven accumulation may cause skin darkening or chloasma generation. In general, melanin is thought to be formed by the action of tyrosinase biosynthesized in pigment cells, from tyrosine to dopa, from dopa to dopaquinone, and then through intermediates such as 5, 6-dihydroxyindophenol. Therefore, it is considered that in order to prevent, treat or improve skin darkening (skin pigmentation disease), chloasma, freckle, etc., it is necessary to inhibit the activity of tyrosinase associated with melanin production or inhibit melanin production.
Conventionally, in order to prevent, treat or improve skin pigmentation, chloasma, freckle, etc., external use of a whitening agent containing a chemically synthesized product such as hydroquinone as an active ingredient has been adopted. However, there is concern that chemical synthetic products such as hydroquinone may cause side effects such as skin irritation and allergy.
Accordingly, it has been desired to develop a whitening agent containing a natural raw material with high safety as an active ingredient, and as a substance having an inhibitory effect on tyrosinase activity, for example, ampelopsis grossedentata extract (see patent document 7), polygonum hydropiper extract (see patent document 8) and the like are known. Further, as substances having a melanin production inhibiting effect, for example, an extract from the root of castor (see patent document 9), an extract from a plant belonging to the genus Saussurea (Saussurea) (see patent document 10), and the like are known.
The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and extracellular matrices such as collagen, which are located outside these cells to support the skin structure. In young skin, the interaction of these skin tissues is kept constant, whereby water retention, softness, elasticity and the like are ensured, and the skin is also firm and shiny from the external point of view, and a moist state is maintained.
However, if the skin is exposed to ultraviolet (UV-A, UV-B) light, extremely dry air, excessive skin cleansing, or the like, or the age is increased, the amount of collagen, which is a main component of the extracellular matrix, decreases, and the elastic force decreases due to crosslinking. As a result, the skin has a reduced moisturizing function and elasticity, and the keratin starts to be abnormally peeled off, so that the skin is subject to aging symptoms such as sagging, loss of luster, roughness, wrinkles, and the like.
The changes with aging of the skin, namely wrinkles, darkness, loss of texture, and reduction of elasticity, are various factors, and are related to reduction and modification of extracellular matrix components such as collagen, hyaluronic acid, and elastin. Therefore, it is considered that by promoting the production of collagen, hyaluronic acid, or the like, the aging of the skin can be prevented and improved.
Therefore, from the viewpoint of safety, the industry has attempted to obtain a substance having a collagen production promoting effect from an advantageous natural substance. As a substance having a collagen production promoting effect, for example, a carambola leaf extract (see patent document 11), a cranberry leaf extract (see patent document 12), and the like have been found.
The following are also reported: amino acids as the main component of natural moisturizing factors (Natural Moisturizing Factors) are produced by the breakdown of silk fibroin from transparent keratinous particles within the stratum corneum. The silk fibroin is expressed as silk fibrinogen in epidermal keratinocytes present in the granular layer directly below the stratum corneum. Immediately after that, the particles are phosphorylated and deposited on transparent keratin particles, and after being decomposed into silk fibroin by phosphate removal and hydrolysis, the silk fibroin is transferred to the stratum corneum, whereby the aggregation efficiency of keratin fibers is improved, and the particles participate in the internal construction of keratinocytes (see non-patent document 2).
In recent years, it has been reported that this silk fibroin is very important for moisture retention in skin, and that conditions such as drying reduce the synthesis force of silk fibroin, resulting in a decrease in the amount of amino acids in the stratum corneum (see non-patent document 3).
Therefore, it is expected that the synthesis of silk fibroin can be promoted by promoting the expression of silk fibroin mRNA in epidermal keratinocytes, thereby increasing the amount of amino acids in the stratum corneum and fundamentally improving the moisture environment of the stratum corneum.
As a natural-substance-derived silk fibroin synthesis promoter, for example, licorice extract (see patent document 13) and glycyrrhizin known as a flavonoid glycoside contained in a natural plant (see patent document 14) have been proposed, and as at least one of a natural-substance-derived silk fibroin and a silk fibroin protein production promoter, a plant extract belonging to the genus Citrus (Citrus), yeast extract (see patent document 15) or the like has been proposed.
The epidermis continuously forms new keratinocytes through division and subsequent differentiation of keratinocytes, thereby having a defensive function of protecting the skin from various stimuli from the outside. In particular, in the differentiation process of keratinocytes, proteins such as outer membrane proteins are expressed from the spiny layer to the granular layer, and are crosslinked by glutamine transaminase-1 to form a keratinized envelope (hereinafter, abbreviated as "CE") which is an insoluble cell membrane-like structure coating keratinocytes, thereby contributing to the stabilization of the cytoskeleton and structure of keratinocytes.
However, if the production amount of glutamine transaminase-1 in the epidermis is reduced due to various factors, CE formation is not complete and keratinization is not normally performed. As a result, the barrier function of the horny layer and the moisture-retaining function of the skin are lowered, and skin symptoms such as rough skin and dry skin are exhibited.
In view of the above, it is considered that by increasing the production of glutamine transaminase-1 in the epidermis by keratinocytes, promoting CE formation to normalize keratinization, lowering of the skin barrier function due to external stimulation such as dryness or ultraviolet rays can be suppressed, and various skin symptoms such as dryness or roughness of the skin can be prevented and improved.
As a natural substance-derived glutamine transaminase-1 production promoter, noni extract (see patent document 16), royal jelly extract (see patent document 17), and the like have been proposed.
In addition, it is known that aquaporins, which are known as water channels, are expressed on cell membranes in skin cells, and play a role in absorbing low-molecular substances, typified by water in the cell gap, into the cells. It is known that 13 aquaporins (AQP 0 to AQP 12) exist in humans. In epidermal cells, aquaporin 3 (Aquaporin, aqp 3) is mainly present, which is thought to take on the following roles: in addition to water, low molecular compounds such as glycerin and urea are absorbed which are involved in the water retention.
However, AQP3 decreases with age, suggesting that this decrease in AQP3 is one of the causes of decrease in water retention function, and thus it is thought that by promoting expression of AQP3, it is possible to control water retention function, barrier function, and the like, which decrease with age (see non-patent document 4). As a substance having an AQP3 expression promoting effect, for example, an extract from the leaf of carambola (see patent document 18) and the like are 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 junctions (tight junction, hereinafter abbreviated as "TJ") existing in the epidermis particle layer is defective at the gene level, the barrier function of the skin is collapsed, so TJ has been considered to play an important role in the barrier function of the skin in recent years (see non-patent document 5). TJ is a connecting device that not only seals adjacent cells to each other, but also closes the cell-cell gap, thereby controlling permeation of substances. The dense proteins (claudins) and the tight proteins (claudins) constituting the cell membrane proteins of TJ are considered to constitute the skeletons of TJ chains, 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, TJ structure is destroyed and the function as a permeation barrier for substances is lost, and thus it becomes a cause of skin symptoms such as dry skin, rough skin, atopic dermatitis, and various infectious diseases.
Therefore, it is thought that the skin symptoms can be prevented or improved by promoting the production of claudin or claudin in the epidermis to promote the formation of TJ in epidermal keratinocytes, thereby improving the barrier function and the water retention function of the skin. Based on this idea, as a substance that promotes skin barrier function by TJ formation promoting action, coptis chinensis extract (see patent document 19), spruce extract (see patent document 20) and the like derived from natural substances have been proposed.
In addition, in recent years, it has been pointed out that the involvement of matrix metalloproteinases (MMPs, matrix metalloproteinases) is a factor that induces changes accompanying skin aging. Among these MMPs, matrix metalloproteinase-1 (MMP-1) is known to be an enzyme that breaks down collagen, which is a main constituent of the dermal extracellular matrix of 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 a factor of skin wrinkles, reduction in elasticity, and the like. Thus, inhibition of MMP-1 activity is important in preventing and ameliorating skin aging symptoms.
As substances having such an MMP-1 inhibitory effect, for example, an extract of acerola (see patent document 21), an extract of zingiberaceae cassanna, or a extract of sanguinea banyan (see patent document 22), and the like are known.
On the other hand, a fermentation broth obtained by fermenting plants of the genus Artemisia with Aspergillus (see patent document 23) or a fermentation broth obtained by fermenting thyme with Aspergillus (see patent document 24) are known, but these plant fermentation broths have no effect such as an anti-aging effect, an antioxidant effect, an anti-inflammatory effect, a whitening effect, or the like.
Prior art literature
Patent literature
Patent document 1: japanese patent laid-open publication No. 2003-81848
Patent document 2: japanese patent laid-open publication 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 publication No. 2003-55242
Patent document 6: japanese patent laid-open publication No. 2003-12532
Patent document 7: japanese patent laid-open No. 2002-370962
Patent document 8: japanese patent laid-open publication No. 2004-83488
Patent document 9: japanese patent laid-open No. 2001-213757
Patent document 10: japanese patent laid-open No. 2002-201122
Patent document 11: japanese patent laid-open No. 2002-226323
Patent document 12: japanese patent laid-open publication No. 2003-146837
Patent document 13: japanese patent laid-open No. 2002-363054
Patent document 14: japanese patent laid-open publication No. 2003-146886
Patent document 15: japanese patent laid-open No. 2001-261568
Patent document 16: japanese patent laid-open No. 2010-090093
Patent document 17: japanese patent laid-open No. 2009-184955
Patent document 18: japanese patent laid-open 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 publication No. 2003-176232
Patent document 22: japanese patent laid-open publication No. 2003-176830
Patent document 23: japanese patent laid-open publication No. 2011-140453
Patent document 24: japanese patent laid-open No. 2011-130689
Non-patent literature
Non-patent document 1: "FRAGRANCE JOURNAL" temporary journal No.14, p156, 1995
Non-patent document 2: FRAGRANCE JOURNAL temporary journal, vol.17, pp.14-19 (2000)
Non-patent document 3: arch. Dermatol. Res., vol.288, pp.442-446 (1996)
Non-patent document 4: "FRAGRANCE JOURNAL",2006, vol.34, no.10, p.19-23
Non-patent document 5: J.Cell biol., vol.156, pp.1099-1111 (2002)
Non-patent document 6: japanese cosmetic science journal of cosmetics, vol.31, pp.296-301 (2007)
Disclosure of Invention
[ Problem to be solved by the invention ]
The present invention aims to solve the problems described in the past and achieve the following objects.
That is, the present invention aims to provide an anti-aging agent having an excellent anti-aging effect and high safety, an antioxidant having an excellent anti-oxidation effect and high safety, an anti-inflammatory agent having an excellent anti-inflammatory effect and high safety, and a whitening agent having an excellent whitening effect and high safety.
Further, the present invention aims to provide a cosmetic having at least 1 action selected from the group consisting of an anti-aging action, an anti-oxidation action, an anti-inflammatory action, and a whitening action, and having high safety.
[ Means of solving the problems ]
The technical means for solving the above problems are as follows. That is to say,
< 1 > An anti-aging agent, characterized in that: comprises at least one of fermentation liquor of Artemisia plants fermented by Aspergillus, fermentation liquor of thyme fermented by Aspergillus, fermentation liquor of herba Melissae axillaris fermented by Aspergillus, and fermentation liquor of cornflower fermented by Aspergillus as effective components.
< 2 > An antioxidant, characterized in that: comprises at least one of fermentation liquor of Artemisia plants fermented by Aspergillus, fermentation liquor of thyme fermented by Aspergillus, fermentation liquor of herba Melissae axillaris fermented by Aspergillus, and fermentation liquor of cornflower fermented by Aspergillus as effective components.
< 3 > An anti-inflammatory agent characterized in that: comprises at least one of fermentation liquor of Artemisia plants fermented by Aspergillus and fermentation liquor of herba Melissae axillaris fermented by Aspergillus as effective components.
< 4 > A whitening agent, characterized in that: comprises at least one of fermentation liquor of Artemisia plant fermented by Aspergillus, fermentation liquor of herba Melissae axillaris fermented by Aspergillus, and fermentation liquor of cornflower fermented by Aspergillus as effective components.
< 5 > A cosmetic characterized in that: contains at least 1 selected from the group consisting of an anti-aging agent described in < 1 >, an antioxidant described in < 2 >, an anti-inflammatory agent described in < 3 > and a whitening agent described in < 4 >.
[ Effect of the invention ]
According to the present invention, the problems described above can be solved, and the above object can be achieved by providing an anti-aging agent having an excellent anti-aging effect and high safety, an antioxidant having an excellent anti-oxidation effect and high safety, an anti-inflammatory agent having an excellent anti-inflammatory effect and high safety, and a whitening agent having an excellent whitening effect and high safety.
In addition, the present invention can provide a cosmetic having at least 1 action selected from the group consisting of an anti-aging action, an anti-oxidation action, an anti-inflammatory action, and a whitening action with high safety.
Drawings
Fig. 1A is a photograph showing an example of a droplet when the contact angle of the mountain artemisia 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 mountain artemisia fermentation broth 2 of production example 2 was measured.
Fig. 1C is a photograph showing an example of a droplet when the contact angle of the mountain artemisia extract of comparative production example 1 was measured.
FIG. 2A is a photograph showing an example of a droplet when the contact angle of the capillary artemisia fermentation broth 1 of preparation example 3 is measured.
FIG. 2B is a photograph showing an example of a droplet when the contact angle of the capillary artemisia fermentation broth 2 of preparation example 4 is measured.
Fig. 2C is a photograph showing an example of a droplet when the contact angle of the capillary artemisia extract of comparative production example 2 is measured.
Fig. 3A is a photograph showing an example of a droplet when the contact angle of thyme broth 1 of production example 5 is measured.
Fig. 3B is a photograph showing an example of a droplet when the contact angle of thyme broth 2 of production example 6 was measured.
Fig. 3C is a photograph showing an example of a droplet when the contact angle of thyme extract of comparative production example 3 was measured.
Fig. 4A is a photograph showing an example of a droplet when the contact angle of the melissa fermentation liquid 1 of production example 7 was measured.
Fig. 4B is a photograph showing an example of a droplet when the contact angle of the melissa fermentation liquid 2 of production example 8 was measured.
Fig. 4C is a photograph showing an example of a droplet when the contact angle of the melissa extract of comparative production example 4 was measured.
FIG. 5A is a photograph showing an example of a droplet when the contact angle of the cornflower fermentation liquid 1 of production example 9 was measured.
FIG. 5B is a photograph showing an example of a droplet when the contact angle of the cornflower fermentation liquid 2 of production example 10 was measured.
Fig. 5C is a photograph showing an example of a droplet when the contact angle of the cornflower extract liquid of comparative production example 5 was measured.
Detailed Description
(Anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent)
< Anti-aging Agents >)
The aging inhibitor of the present invention contains at least one of a fermentation broth of an Artemisia plant fermented by Aspergillus (hereinafter, sometimes referred to as "Artemisia plant fermentation broth"), a fermentation broth of thyme fermented by Aspergillus (hereinafter, sometimes referred to as "thyme fermentation broth"), a fermentation broth of melissa fermented by Aspergillus (hereinafter, sometimes referred to as "melissa fermentation broth"), and a fermentation broth of cornflower fermented by Aspergillus (hereinafter, sometimes referred to as "cornflower fermentation broth") as an active ingredient, and optionally contains other ingredients.
The Artemisia plant fermentation liquid, the thyme fermentation liquid, the Melissa fermentation liquid, and the cornflower fermentation liquid have at least one action selected from the group consisting of a matrix metalloproteinase-1 (matrix metalloproteinase-1, MMP-1) activity inhibitory action, a hyaluronan synthase 3 (hyaluronan synthase, HAS 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 (transglutaminase-1, TGM-1) mRNA expression promoting action, a aquaporin 3 (Aquaporin, AQP 3) mRNA expression promoting action, and a filaggrin mRNA expression promoting action, and can be used as an active ingredient of an anti-aging agent.
Accordingly, the anti-aging agent has at least 1 action selected from the group consisting of an MMP-1 activity inhibitory action, a hyaluronan synthase 3mRNA 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, a aquaporin 3mRNA expression promoting action, and a filaggrin mRNA expression promoting action.
Although details of substances that exert at least any one of the above-mentioned Artemisia plant fermentation liquid, the above-mentioned thyme fermentation liquid, the above-mentioned Melissa officinalis fermentation liquid, and the above-mentioned cornflower fermentation liquid have not been known, the above-mentioned excellent actions of MMP-1 activity inhibition, hyaluronic acid synthase 3mRNA expression promotion, type I collagen production promotion, claudin-1 mRNA expression promotion, claudin-4 mRNA expression promotion, claudin mRNA expression promotion, glutamine transaminase-1 mRNA expression promotion, aquaporin 3mRNA expression promotion, and filaggrin mRNA expression promotion have not been known at all, and the above-mentioned excellent actions of the above-mentioned Artemisia plant fermentation liquid, the above-mentioned thyme fermentation liquid, the above-mentioned Melissa officinalis fermentation liquid, and the above-mentioned cornflower fermentation liquid have not been known heretofore, and they have been used as an anti-aging agent, which has been a novel finding by the present inventors.
< Antioxidant >
The antioxidant of the present invention comprises at least one of a fermentation liquid of an Artemisia plant fermented by Aspergillus (Artemisia plant fermentation liquid), a fermentation liquid of thyme fermented by Aspergillus (thyme fermentation liquid), a fermentation liquid of Melissa officinalis fermented by Aspergillus (Melissa officinalis fermentation liquid), and a fermentation liquid of cornflower fermented by Aspergillus (cornflower fermentation liquid) as an active ingredient, and optionally comprises other ingredients.
The artemisia plant fermentation liquid, the thyme fermentation liquid, the melissa fermentation liquid and the cornflower fermentation liquid have a diphenyl-to-picrylhydrazine (DPPH, dipheny-p-picrylhydrazyl) radical scavenging effect, and can be used as an active ingredient of an antioxidant by utilizing the effect.
Thus, the antioxidant has DPPH radical scavenging effect.
Although the details of the substance that exhibits 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 been totally unknown that the artemisia plant fermentation broth, the thyme fermentation broth, the melissa fermentation broth, and the cornflower fermentation broth have such excellent action and are useful as antioxidants, which has been newly found by the present inventors.
< Anti-inflammatory Agents >)
The anti-inflammatory agent of the present invention comprises at least one of a fermentation liquid of an Artemisia plant fermented by an Aspergillus (Artemisia plant fermentation liquid) and a fermentation liquid of a Melissa herb fermented by an Aspergillus (Melissa fermentation liquid) as an active ingredient, and optionally comprises other ingredients.
The artemisia plant fermentation liquid and the melissa fermentation liquid have hyaluronidase activity inhibition effects, and can be used as active ingredients of anti-inflammatory agents by utilizing the effects.
Thus, the anti-inflammatory agent has hyaluronidase activity inhibitory effect.
Although the details of the substances that exert the hyaluronidase activity inhibitory action in the Artemisia plant fermentation liquid and the Melissa officinalis fermentation liquid are not known, it has been found that the Artemisia plant fermentation liquid and the Melissa officinalis fermentation liquid have such excellent actions and are useful as anti-inflammatory agents, which have been a new finding by the present inventors.
< Whitening agent >)
The whitening agent of the present invention comprises at least one of a fermentation liquid of an Artemisia plant fermented by Aspergillus (Artemisia plant fermentation liquid), a fermentation liquid of a Melissa officinalis fermented by Aspergillus (Melissa officinalis fermentation liquid), and a fermentation liquid of cornflower fermented by Aspergillus (cornflower fermentation liquid) as an active ingredient, and optionally other ingredients.
The Artemisia plant fermentation liquid, the Melissa officinalis fermentation liquid, and the cornflower fermentation liquid have at least 1 of tyrosinase activity inhibition and melanin production inhibition, and can be used as an active ingredient of a whitening agent by utilizing the above effects.
Therefore, the whitening agent has at least 1 of tyrosinase activity inhibition and melanin production inhibition.
Although the details of the substance exhibiting at least one of the tyrosinase activity inhibiting action and the melanin production inhibiting action of the artemisia plant fermentation liquid, the vetiver fermentation liquid, and the cornflower fermentation liquid are not known, the inventors have found that the artemisia plant fermentation liquid, the vetiver fermentation liquid, and the cornflower fermentation liquid have excellent actions and can be used as whitening agents.
Artemisia plant fermentation liquor
The Artemisia plant fermentation liquid is a fermentation liquid obtained by fermenting a plant belonging to the Artemisia (hereinafter, sometimes referred to as an "Artemisia plant") with Aspergillus.
Artemisia plant-
The Artemisia plants used as the fermentation feedstock are perennial herbs belonging to the Compositae (Compositae) Artemisia (artemia) and have been used as food or pharmaceutical feedstock since ancient times. Is widely and naturally grown or planted in japan such as north sea, this state, four countries, and ninety, and is readily available from these areas.
The species of the Artemisia plant is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include: mountain artemisia (Artemisiamontana (Nakai) pamp.), artemisia capillaris (ARTEMISIACAPILLARIS THUNBERGII), artemisia kui (ARTEMISIAPRINCEPS pampan.), artemisia japonica (Artemisiajaponica thunb.), artemisia annua (Artemisiaabsinthium l.), artemisia annua (Artemisialactiflora wall.), ascarid artemisia maritima (ARTEMISIAMARITIMA l.), swine Mao Hao (Artemisiascoparia waldst. Et kit), and the like. These may be used singly or in combination of 1 kind or 2 or more kinds.
The method for obtaining the Artemisia plant is not particularly limited, and may be appropriately selected according to the purpose, and may be obtained from the natural world or may be commercially available.
The place of use of the Artemisia plant used as the fermentation raw material is not particularly limited, and may be appropriately selected depending on the purpose, and examples thereof include: flowers, flower buds, fruits, pericarps, seeds, seed coats, stems, leaves, stalks, branches and leaves and other overground parts; underground parts such as roots and rhizomes, etc. These may be used singly or in combination of 1 kind or 2 or more kinds. Among these, the place of use of the Artemisia plant is preferably an aerial part.
The size of the Artemisia plant used as the fermentation material is not particularly limited as long as it is a size capable of culturing the Aspergillus, and examples thereof may be given as appropriate depending on the purpose: the collected original size, the size obtained by cutting into a desired size, the size obtained by pulverizing into fine powder (powder), and the like.
The state of the Artemisia plant used as the fermentation material is not particularly limited as long as it is a state in which the Aspergillus can be cultured, and examples thereof may be appropriately selected according to the purpose: the collected original state, dry state, crushed state, juiced state, extract state, etc. Among these conditions, the collected original condition, the crushed condition, the squeezed juice condition, and the extracted condition are preferable, and the collected original condition and the crushed condition are more preferable, in view of the easiness of the action of the koji mold.
The method for bringing the Artemisia plant into a dry state is not particularly limited, and may be appropriately selected depending on the purpose, and examples thereof include: a method of sun drying, a method of drying with a common dryer, and the like.
The method for bringing the plant of the genus Artemisia into the pulverized state is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a method for pulverizing by a stirrer, a sugar press, an electric mill, a jet mill, an impact mill, and the like.
The method for bringing the Artemisia plant into the juice-extracted state is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include squeezing.
The method for bringing the Artemisia plant 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.
The Artemisia plants used as the fermentation feedstock are preferably sterilized prior to inoculation with the Aspergillus. The method for sterilizing the Artemisia plant is not particularly limited, and may be appropriately selected from known methods.
Aspergillus kawachii (S. Schwann.) C.
The aspergillus for fermenting the artemisia plant is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include: aspergillus oryzae (Aspergillus oryzae), aspergillus sojae (Aspergillussojae), etc.; aspergillus niger such as Aspergillus sojae (Aspergillus luchuensis); aspergillus candidus such as Aspergillus kawachii (Aspergilluskawauchii); variants of these, and the like. These may be used singly or in combination of 1 kind or 2 or more kinds. Among these aspergillus, aspergillus oryzae (aspergillus oryzae) is preferred in terms of excellent effect of at least any one of anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect.
The method for obtaining the koji mold is not particularly limited, and may be appropriately selected according to the purpose, and may be obtained from the natural world or may be commercially available. The koji mold may be a koji mold made of rice or the like, a koji mold made of plants of the genus Artemisia as described below, or a koji mold cultured in a medium (such as an agar medium or a liquid medium). Among these koji molds, the artemisia plant koji is preferably used in view of excellent at least any one of an anti-aging effect, an antioxidant effect, an anti-inflammatory effect, and a whitening effect.
The inoculation amount of the koji mold on the artemisia plant used as the fermentation material is not particularly limited as long as the artemisia plant can be fermented, and may be appropriately selected according to the purpose, and is preferably 1×10 3/mL to 1×10 8/mL when the fermentation material is in a liquid state, and is preferably 1×10 3/g to 1×10 8/g when the fermentation material is in a solid state.
When inoculating the Artemisia plant with the Aspergillus, water is preferably added. The amount of 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 Artemisia plant.
The fermentation (culture) temperature is not particularly limited as long as it is within a temperature range in which fermentation can be performed by the koji mold, and may be appropriately selected according to the purpose, preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the fermentation temperature is lower than 20 ℃, there are cases where at least any one of the anti-aging effect, the antioxidant effect, the anti-inflammatory effect, and the whitening effect is insufficient, which do not allow the Artemisia plant to sufficiently ferment. If the temperature exceeds 50 ℃, there is a case where the koji mold cannot be proliferated.
The fermentation (cultivation) time is not particularly limited and may be appropriately selected depending on the purpose, and is preferably 10 hours to 40 hours, more preferably 20 hours to 30 hours. If the fermentation time is less than 10 hours, there are cases where at least any one of the anti-aging effect, the antioxidant effect, the anti-inflammatory effect, and the whitening effect is insufficient, which does not allow the Artemisia plant to sufficiently ferment.
The method of 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.
The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the koji mold can not grow any more, and may be appropriately selected according to the purpose, and is preferably 50℃or higher, more preferably 70℃or higher, 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 anti-oxidation 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 it is a state in which the koji mold is not allowed to grow, and may be appropriately selected according to the purpose, and is preferably 5 minutes or more, 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 anti-oxidation effect, the anti-inflammatory effect, and the whitening effect may be insufficient.
In addition, the artemisia plant fermentation broth after stopping the fermentation is preferably cooled. The cooling method is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a method of standing at ordinary temperature, standing in a refrigerator, and the like.
The number of times the Artemisia plant 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 of carrying out the fermentation a plurality of times, the koji mold may be inoculated only for the first time, may be inoculated only for a plurality of times, may be inoculated at each time of fermentation, and is preferably inoculated only for the first time.
In the case of performing the above-described fermentation a plurality of times, the fermentation temperature and the fermentation time may be different from each other or may be the same.
Artemisia plant species starter
The Artemisia plant starter is obtained by inoculating Aspergillus with the Artemisia plant as starter raw material, and allowing sufficient amount of spores to adhere to the Artemisia plant for growth. The use of the starter culture for the fermentation is advantageous in that an artemisia plant fermentation liquid having excellent skin affinity can be obtained more efficiently and easily.
The Artemisia plant used as the starter can be the same as the Artemisia plant described in the description, and the Artemisia plant is used in the same manner as the Artemisia plant in terms of the site, size, state, and the like.
As the koji mold used for producing the Artemisia plant koji, the same ones as described in the above-mentioned koji mold can be used.
The inoculation amount of the koji mold on the artemisia plant used as the starter culture raw material is not particularly limited, and may be appropriately selected according to the purpose, and is preferably 5 to 100 parts by mass (1×10 3 to 1×10 8/mL), more preferably 10 to 50 parts by mass, and particularly preferably 20 to 30 parts by mass of the koji mold suspended in the sterilizing water is inoculated to 100 parts by mass of the artemisia plant. If the inoculum size of the koji mold is less than 5 parts by mass per 100 parts by mass of the artemisia plant, there are cases where a sufficient amount of spores cannot be attached to the artemisia plant for growth, and if it exceeds 100 parts by mass, there are cases where abnormal propagation is caused by excessive moisture.
When inoculating the aspergillus with the artemisia plant used as the starter propagation material, water is preferably added. The amount of 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 Artemisia plant. If the amount of the water to be added is less than 10 parts by mass per 100 parts by mass of the Artemisia plant, there are cases where a sufficient amount of spores cannot be attached to the Artemisia plant to grow.
The culture temperature is not particularly limited as long as it is within a temperature range in which the koji mold 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 a sufficient amount of spores cannot be attached to the Artemisia plant to grow. If the temperature exceeds 50 ℃, there is a case where the koji mold cannot be proliferated.
The culture time is not particularly limited and may be appropriately selected depending on the purpose, but is preferably 80 to 210 hours, more preferably 100 to 190 hours, and particularly preferably 120 to 170 hours. If the cultivation time is less than 80 hours, there are cases where a sufficient amount of spores cannot be attached to the Artemisia plant to grow, and if it exceeds 210 hours, there are cases where the germination rate of spores is lowered.
The artemisia plant fermentation broth 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 Artemisia plant fermentation liquid is not particularly limited and may be appropriately selected depending on the purpose, and for example, the Artemisia plant fermentation liquid itself may be a purified product of the Artemisia plant fermentation liquid, a concentrate of the Artemisia plant fermentation liquid, a dilution of the Artemisia plant fermentation liquid, or the like. The Artemisia plant fermentation liquid may be a liquid obtained by mixing or dissolving a dried product of the Artemisia plant fermentation liquid in a solvent such as water or a hydrophilic solvent again.
The purified product of the Artemisia plant fermentation liquid is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a substance from which a solid component (for example, a plant body of the Artemisia plant, a cell body of Aspergillus, a precipitate, etc.) in the Artemisia plant fermentation liquid is removed.
The method of removal 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 purposes.
The dilution of the Artemisia plant fermentation liquid and the concentrate of the Artemisia plant fermentation liquid are not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a substance 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, reduced pressure concentration and the like may be mentioned.
The dried product of the Artemisia plant fermentation liquid is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a substance obtained by drying the Artemisia plant fermentation liquid.
The drying method is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include freeze drying.
The Artemisia plant fermentation liquid is not particularly limited as long as it is a fermentation liquid obtained by fermenting Artemisia plants with Aspergillus, and may be appropriately selected according to the purpose, and is preferably an Artemisia plant fermentation liquid having a contact angle of 81 ° or less, more preferably an Artemisia plant fermentation liquid having a contact angle of 78 ° or less, in view of good skin affinity.
In the present specification, the contact angle means 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 (manufactured by FTA1000 Falcon, first Ten Angstroms) and measuring the sample by a droplet method at a temperature of 22℃and a relative humidity of 20%, and obtaining a contact angle θ (°) of 1,000ms by a θ/2 method.
The contact angle is an index indicating "wetting", and is defined as "an angle (angle in the liquid) between the liquid surface and the solid surface at a position where the free surface of the stationary liquid contacts the solid wall surface" (refer to "physical chemistry dictionary" 4 th edition, rock bookstore Co., ltd.). The contact angle depends on the magnitude of the cohesion between the liquid molecules and the adhesion between the solid walls, and is acute when the liquid wets the solid (high adhesion) and obtuse when it is not wetted. Therefore, the smaller the contact angle, the more easily wetted, i.e., the better the skin affinity, and therefore, the lower limit of the contact angle of the artemisia plant fermentation broth is not particularly limited, and may be appropriately selected according to the purpose.
Thyme fermentation liquor
The thyme fermentation broth is fermentation broth obtained by fermenting thyme with aspergillus.
Thyme-like material
Thyme (Thymusvulgaris Linne) used as the fermentation raw material is a perennial woody plant belonging to the genus of capsicum (Thymus) belonging to the family Labiatae (Labiatae), is one kind of herbal medicine, and has been used as a raw material for eating or medicine since ancient times. The aliases include common thyme, etc. The origin is coastal in the Mediterranean, but also can be 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, and may be obtained from the natural world or commercially available products may be used.
The place of use of thyme as the fermentation raw material is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include: flowers, flower buds, fruits, pericarps, seeds, seed coats, stems, leaves, branches, bark, stalks, branches and leaves and other overground parts; underground parts such as roots and rhizomes, etc. These may be used alone or in combination of 1 or 2 or more. Among these, the place of use of thyme is preferably the aerial part.
The size of thyme used as the fermentation material is not particularly limited as long as it is a size capable of culturing the aspergillus, and examples thereof may be appropriately selected according to the purpose: the collected original size, the size obtained by cutting into a desired size, the size obtained by pulverizing into fine powder (powder), and the like.
The state of thyme used as the fermentation material is not particularly limited as long as it can be cultured with aspergillus, and may be appropriately selected according to the purpose, and examples thereof include an original state, a dried state, a crushed state, a squeezed state, and an extracted state. Among these conditions, the collected original condition, the crushed condition, the squeezed juice condition, and the extracted condition are preferable, and the collected original condition and the crushed condition are more preferable, in view of the easiness of the action of the koji mold.
The method for bringing thyme into a dry state is not particularly limited, and may be appropriately selected depending on the purpose, and examples thereof include: a method of sun drying, a method of drying with a common dryer, and the like.
The method for bringing thyme into the pulverized state is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a method for pulverizing with a stirrer, a sugar press, an electric mill, a jet mill, an impact mill, and the like.
The method for bringing thyme into the juice-extracted state is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include squeezing.
The method for bringing thyme 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.
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 kawachii (S. Schwann.) C.
The aspergillus for fermenting thyme is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include aspergillus described in "<" > "of the fermented liquid of plants of the genus Artemisia. These koji mold may be used alone in an amount of 1 kind or in an amount of 2 or more kinds. Among these koji molds, koji mold (aspergillus oryzae) is preferable in that it is excellent in at least one of an anti-aging effect and an antioxidant effect.
The method for obtaining the koji mold is not particularly limited, and may be appropriately selected according to the purpose, and may be obtained from the natural world or may be commercially available. The koji mold may be a koji mold made of rice or the like, a koji mold made of thyme described below, or a koji mold cultured in a medium (e.g., agar medium, liquid medium, etc.) may be used. Among these koji molds, the thyme koji is preferably used in view of the excellent effect of at least one of the anti-aging effect and the antioxidation effect.
The inoculation amount of the koji mold on the thyme used as the fermentation material is not particularly limited as long as the thyme can be fermented, and may be appropriately selected according to the purpose, and is preferably 1×10 3 pieces/mL to 1×10 8 pieces/mL when the fermentation material is in a liquid state, and is preferably 1×10 3 pieces/g to 1×10 8 pieces/g when the fermentation material is in a solid state.
When inoculating the thyme with the aspergillus, water is preferably added. The amount of 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 thyme.
The fermentation (culture) temperature is not particularly limited as long as it is within a temperature range in which fermentation can be performed by the koji mold, and may be appropriately selected according to the purpose, preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the fermentation temperature is lower than 20 ℃, the thyme may not be sufficiently fermented, and at least one of the anti-aging effect and the antioxidant effect may be insufficient.
The fermentation (cultivation) time is not particularly limited and may be appropriately selected depending on the purpose, and is preferably 10 hours to 40 hours, more preferably 20 hours to 30 hours. If the fermentation time is less than 10 hours, the thyme may not be sufficiently fermented, and at least one of the anti-aging effect and the antioxidant effect may be insufficient.
The method of 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.
The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the koji mold can not grow any more, and may be appropriately selected according to the purpose, and is preferably 50℃or higher, more preferably 70℃or higher, and particularly preferably 100℃to 130 ℃. If the heating temperature is less than 50 ℃, the fermentation may not be stopped, and if it exceeds 130 ℃, at least one of the anti-aging effect and the antioxidation effect may be insufficient.
The heating time for stopping the fermentation is not particularly limited as long as it is a state in which the koji mold is not allowed to grow, and may be appropriately selected according to the purpose, and is preferably 5 minutes or more, 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 antioxidation effect may be insufficient.
In addition, the thyme broth after the fermentation has ceased is preferably cooled. The cooling method is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a method of standing at normal temperature, a refrigerator, and the like.
The number of times of fermentation of thyme 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 of carrying out the fermentation a plurality of times, the koji mold may be inoculated only for the first time, may be inoculated only for a plurality of times, may be inoculated at each time of fermentation, and is preferably inoculated only for the first time.
In the case of performing the above-described fermentation a plurality of times, the fermentation temperature and the fermentation time may be different from each other or may be the same.
Thyme starter
The thyme starter is obtained by inoculating aspergillus to the starter raw material by using thyme as the starter raw material, and attaching enough spores to the thyme for growth. The use of the starter culture for the fermentation is advantageous in that a thyme fermentation liquid having excellent skin affinity can be obtained more efficiently and easily.
The thyme used as the starter can be the same as the plant described in the above-mentioned-thyme-and the same as the thyme in terms of the site, size, state, etc. of use.
As the Aspergillus for producing the thyme seed koji, the same ones as those described in the above-mentioned Aspergillus can be used.
The amount of the koji mold to be inoculated onto the thyme used as the starter culture raw material is not particularly limited, and may be appropriately selected according to the purpose, and is preferably 5 to 100 parts by mass (1×10 3 to 1×10 8/mL), more preferably 10 to 50 parts by mass, and particularly preferably 20 to 30 parts by mass of the koji mold suspended in the sterilizing water is inoculated to 100 parts by mass of the thyme. If the inoculum size of the koji mold is less than 5 parts by mass per 100 parts by mass of the thyme, there are cases where a sufficient amount of spores cannot be attached to the thyme for growth, and if it exceeds 100 parts by mass, there are cases where abnormal propagation is caused by excessive moisture.
When inoculating the aspergillus with the thyme used as the starter feed, water is preferably added. The amount of 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 thyme. If the amount of the water to be added is less than 10 parts by mass per 100 parts by mass of the thyme, there are cases where a sufficient amount of spores cannot be attached to the thyme to grow.
The culture temperature is not particularly limited as long as it is within a temperature range in which the koji mold 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 are cases where a sufficient amount of spores cannot be attached to the thyme for growth. If the temperature exceeds 50 ℃, there is a case where the koji mold cannot be proliferated.
The culture time is not particularly limited and may be appropriately selected depending on the purpose, but is preferably 80 to 210 hours, more preferably 100 to 190 hours, and particularly preferably 120 to 170 hours. If the cultivation time is less than 80 hours, there are cases where a sufficient amount of spores cannot be attached to the thyme for growth, and if it exceeds 210 hours, there are cases where the germination rate of spores is lowered.
The thyme fermentation liquid may contain the cells of the aspergillus or may be a fermentation liquid from which the cells of the aspergillus are removed, and preferably a fermentation liquid from which the cells of the aspergillus are 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 thyme fermentation liquid itself, a purified product of the thyme fermentation liquid, a concentrate of the thyme fermentation liquid, a diluted product of the thyme fermentation liquid, and the like may be used. The thyme fermentation liquid may be a liquid obtained by mixing or dissolving the 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 according to the purpose, and examples thereof include a substance from which a solid component (for example, a plant body of thyme, a cell body of aspergillus, a precipitate, and the like) in the thyme fermentation liquid is removed.
The method of removal 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 purposes.
The dilution of the thyme fermentation liquid and the concentrate of the thyme fermentation liquid are not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a substance prepared to have 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, reduced pressure concentration and the like may be mentioned.
The dried product of the thyme fermentation liquid is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a substance obtained by drying 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.
The thyme fermentation liquid is not particularly limited as long as it is a fermentation liquid in which thyme is fermented by aspergillus, and can be appropriately selected according to the purpose, and is preferably a thyme fermentation liquid having a contact angle of 87 ° or less, more preferably a thyme fermentation liquid having a contact angle of 81 ° or less, in view of good skin affinity.
Melissa officinalis fermentation liquor
The melissa fermentation broth is a fermentation broth obtained by fermenting melissa with aspergillus.
Melissa officinalis
The Melissa (Melissaofficinalis Linne) used as the fermentation raw material is a perennial herb belonging to the genus Melissa (melia) of the family Labiatae. It is one of the herbal medicines and has been used as a raw material for eating or medical use since ancient times. The aliases include lemon balm (Lemon balm), lemon balm flower (garden balm), etc. The origin is south europe, but natural growth or planting is also possible in japan, and can be easily obtained from these regions.
The method for obtaining the melissa is not particularly limited, and may be appropriately selected according to the purpose, may be obtained from natural sources, or may be commercially available.
The place of use of the melissa leaf used as the fermentation raw material is not particularly limited, and may be appropriately selected according to purpose, and examples thereof include: flowers, flower buds, fruits, pericarps, seeds, seed coats, stems, leaves, stalks, branches and leaves and other overground parts; underground parts such as roots and rhizomes, etc. These may be used alone or in combination of 1 or 2 or more. Of these, the place of use of the melissa is preferably an aerial part.
The size of the melissa to be 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: the collected original size, the size obtained by cutting into a desired size, the size obtained by pulverizing into fine powder (powder), and the like.
The state of the melissa 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, dry state, crushed state, juiced state, extract state, etc. Among these conditions, the collected original condition, the crushed condition, the squeezed juice condition, and the extracted condition are preferable, and the collected original condition and the crushed condition are more preferable, in view of the easiness of the action of the koji mold.
The method for bringing the melissa into a dry state is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include: a method of sun drying, a method of drying with a common dryer, and the like.
The method for bringing the melissa into the pulverized state is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a method for pulverizing by a stirrer, a sugar press, an electric mill, a jet mill, an impact mill, and the like.
The method for bringing the melissa into the juice-pressed state is not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include pressing.
The method for bringing the melissa 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 melissa used as the fermentation raw material is preferably sterilized before inoculating the aspergillus. The method for sterilizing the melissa is not particularly limited, and may be appropriately selected from known methods.
Aspergillus kawachii (S. Schwann.) C.
The aspergillus for fermenting the melissa is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include aspergillus described in "<" > "of the fermented liquid of plants of the genus Artemisia. These koji mold may be used alone in an amount of 1 kind or in an amount of 2 or more kinds. Among these aspergillus, aspergillus oryzae (aspergillus oryzae) is preferred in terms of excellent effect of at least any one of anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect.
The method for obtaining the koji mold is not particularly limited, and may be appropriately selected according to the purpose, and may be obtained from the natural world or may be commercially available. The koji mold may be a koji mold made of rice or the like, a koji mold made of melissa as described below, or a koji mold cultured in a medium (e.g., agar medium, liquid medium, etc.) may be used. Among these koji molds, the above-mentioned melissa koji is preferably used in view of excellent at least any one of anti-aging effect, antioxidant effect, anti-inflammatory effect and whitening effect.
The inoculation amount of the Aspergillus onto the melissa as the fermentation material is not particularly limited as long as it is an amount capable of fermenting the melissa, and may be appropriately selected according to the purpose, and is preferably 1X 10 3/mL to 1X 10 8/mL when the fermentation material is in a liquid state, and is preferably 1X 10 3/g to 1X 10 8/g when the fermentation material is in a solid state.
When inoculating the aspergillus to the melissa, water is preferably added. The amount of 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 fermentation can be performed by the koji mold, and may be appropriately selected according to the purpose, preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the fermentation temperature is lower than 20 ℃, there are cases where at least any one of the anti-aging effect, the antioxidant effect, the anti-inflammatory effect, and the whitening effect is insufficient.
The fermentation (cultivation) time is not particularly limited and may be appropriately selected depending on the purpose, and is preferably 10 hours to 40 hours, more preferably 20 hours to 30 hours. If the fermentation time is less than 10 hours, there are cases where at least any one of the anti-aging effect, the antioxidant effect, the anti-inflammatory effect, and the whitening effect is insufficient, which do not allow the melissa to be sufficiently fermented.
The method of 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.
The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the koji mold can not grow any more, and may be appropriately selected according to the purpose, and is preferably 50℃or higher, more preferably 70℃or higher, and particularly preferably 100℃to 130 ℃. If the heating temperature is less than 50 ℃, the fermentation may not be stopped, and if it exceeds 130 ℃, at least one of the anti-aging effect, the anti-oxidation 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 it is a state in which the koji mold is not allowed to grow, and may be appropriately selected according to the purpose, and is preferably 5 minutes or more, 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 anti-oxidation effect, the anti-inflammatory effect, and the whitening effect may be insufficient.
In addition, the fermented liquid of melissa officinalis after stopping the fermentation is preferably cooled. The cooling method is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a method of standing at normal temperature, a refrigerator, and the like.
The number of times of fermentation of the melissa using the aspergillus is not particularly limited, and may be appropriately selected according to purpose, and may be 1 time or a plurality of times.
In the case of carrying out the fermentation a plurality of times, the koji mold may be inoculated only for the first time, may be inoculated only for a plurality of times, may be inoculated at each time of fermentation, and is preferably inoculated only for the first time.
In the case of performing the above-described fermentation a plurality of times, the fermentation temperature and the fermentation time may be different from each other or may be the same.
Melissa officinalis starter propagation
The melissa starter is obtained by inoculating aspergillus to the starter raw material by using melissa as the starter raw material, and allowing enough spores to adhere to the melissa for growth. The use of the starter in the above fermentation is advantageous in that a melissa fermentation liquid excellent in skin affinity can be obtained more efficiently and easily.
The melissa material used as the starter material may be the same as the plant described in the-melissa material, the application part, size, state and other forms of the melissa are the same.
As the koji mold used for producing the melissa seed koji, the same mold as described in the above-mentioned koji mold can be used.
The inoculation amount of the koji mold on the melissa powder used as the starter is not particularly limited and may be appropriately selected according to the purpose, and is preferably 5 to 100 parts by mass of the koji mold suspended in the sterilizing water (1×10 3 to 1×10 8/mL), more preferably 10 to 50 parts by mass, and particularly preferably 20 to 30 parts by mass, based on 100 parts by mass of the melissa powder. If the inoculum size of the koji mold is less than 5 parts by mass per 100 parts by mass of the melissa, there are cases where a sufficient amount of spores cannot be attached to the melissa for growth, and if it exceeds 100 parts by mass, there are cases where abnormal propagation is caused by excessive moisture.
When inoculating the aspergillus with the melissa powder used as the starter culture raw material, water is preferably added. The amount of 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 the water to be added is less than 10 parts by mass per 100 parts by mass of the melissa, there are cases where a sufficient amount of spores cannot be attached to the melissa for growth.
The culture temperature is not particularly limited as long as it is within a temperature range in which the koji mold 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 are cases where a sufficient amount of spores cannot be attached to the melissa to grow. If the temperature exceeds 50 ℃, there is a case where the koji mold cannot be proliferated.
The culture time is not particularly limited and may be appropriately selected depending on the purpose, but is preferably 80 to 210 hours, more preferably 100 to 190 hours, and particularly preferably 120 to 170 hours. If the cultivation time is less than 80 hours, there are cases where a sufficient amount of spores cannot be attached to the melissa and the germination rate of spores is lowered if it exceeds 210 hours.
The melissa fermentation liquid may contain the fungus body of aspergillus, or may be a fermentation liquid from which the fungus body of aspergillus is removed, and preferably a fermentation liquid from which the fungus body of aspergillus is removed.
The state of the melissa fermentation liquid is not particularly limited and may be appropriately selected according to the purpose, and may be, for example, the melissa fermentation liquid itself, a purified product of the melissa fermentation liquid, a concentrate of the melissa fermentation liquid, a diluted product of the melissa fermentation liquid, or the like. The melissa fermentation liquid may be a liquid obtained by mixing or dissolving a dried product of the melissa fermentation liquid in a solvent such as water or a hydrophilic solvent again.
The purified product of the melissa fermentation liquid is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a substance from which solid components (for example, plant bodies of melissa, bacterial cells of aspergillus, and precipitates of aspergillus) in the melissa fermentation liquid are removed.
The method of removal 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 purposes.
The dilution of the melissa fermentation liquid and the concentrate of the melissa fermentation liquid are not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a substance prepared to a desired concentration of melissa fermentation liquid.
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, reduced pressure concentration and the like may be mentioned.
The dried product of the melissa fermentation liquid is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a substance obtained by drying the melissa fermentation liquid.
The drying method is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include freeze drying.
The melissa fermentation liquid is not particularly limited as long as it is a fermentation liquid obtained by fermenting melissa with aspergillus, and may be appropriately selected according to the purpose, and is preferably a melissa fermentation liquid having a contact angle of 85 ° or less, more preferably a melissa fermentation liquid having a contact angle of 79 ° or less, in view of good skin affinity.
Cornflower fermentation liquor
The cornflower fermentation broth is fermentation broth obtained by fermenting cornflower with aspergillus.
Cornflower-
Cornflower (Centaureacyanus Linne) used as the fermentation raw material is an annual herb belonging to the genus cornflower (Centaurea) of the family Compositae (Compositae), and has been used as a raw material for eating or medicines since ancient times. The aliases include rodgersflower, hibiscus sabdariffa (Centaurea), and flos Trollii (Centaurea). The origin is European, but natural growth or planting is also possible in Japan, and can be easily obtained from these regions.
The method for obtaining cornflower is not particularly limited, and may be appropriately selected according to the purpose, and may be obtained from the natural world or commercially available products may be used.
The place 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, flower buds, fruits, pericarps, seeds, seed coats, stems, leaves, stalks, branches and leaves and other overground parts; underground parts such as roots and rhizomes, etc. These may be used alone or in combination of 1 or 2 or more. Of these, the part of the cornflower where the cornflower is used is preferably an aerial part.
The cornflower to be used as the fermentation material is not particularly limited as long as it is a size capable of culturing the koji mold, and may be appropriately selected according to the purpose, and examples thereof include: the collected original size, the size obtained by cutting into a desired size, the size obtained by pulverizing into fine powder (powder), and the like.
The state of the cornflower used as the fermentation material is not particularly limited as long as the aspergillus can be cultured, and examples thereof may be appropriately selected according to the purpose: the collected original state, dry state, crushed state, juiced state, extract state, etc. Among these conditions, the collected original condition, the crushed condition, the squeezed juice condition, and the extracted condition are preferable, and the collected original condition and the crushed condition are more preferable, in view of the easiness of the action of the koji mold.
The method for bringing the cornflower into a dry state is not particularly limited, and may be appropriately selected depending on the purpose, and examples thereof include: a method of sun drying, a method of drying with a common dryer, and the like.
The method for bringing the cornflower into the pulverized state is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a method for pulverizing by a stirrer, a sugar press, an electric mill, a jet mill, an impact mill, and the like.
The method for bringing the cornflower 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 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 feedstock is preferably sterilized prior to inoculation with the aspergillus. The method for sterilizing the cornflower is not particularly limited, and may be appropriately selected from known methods.
Aspergillus kawachii (S. Schwann.) C.
The koji mold for fermenting the cornflower is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include koji mold described in "<" > "of the plant fermentation broth of Artemisia. These koji mold may be used alone in an amount of 1 kind or in an amount of 2 or more kinds. Among these aspergillus, aspergillus oryzae (aspergillus oryzae) is preferred as the aspergillus having excellent at least one of anti-aging effect, antioxidant effect and whitening effect.
The method for obtaining the koji mold is not particularly limited, and may be appropriately selected according to the purpose, and may be obtained from the natural world or may be commercially available. The koji mold may be a koji mold prepared from rice or the like, a cornflower koji mold described below, or a koji mold cultured in a medium (e.g., agar medium or liquid medium). Among these koji molds, the cornflower koji is preferably used in view of excellent at least one of an anti-aging effect, an antioxidant effect and a whitening effect.
The inoculation amount of the koji mold on the cornflower used as the fermentation material is not particularly limited as long as the cornflower can be fermented, and may be appropriately selected according to the purpose, and is preferably 1×10 3/mL to 1×10 8/mL when the fermentation material is in a liquid state, and is preferably 1×10 3/g to 1×10 8/g when the fermentation material is in a solid state.
When inoculating the cornflower with the aspergillus, water is preferably added. The amount of 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 fermentation can be performed by the koji mold, and may be appropriately selected according to the purpose, preferably 20 to 40 ℃, more preferably 25 to 35 ℃. If the fermentation temperature is lower than 20 ℃, there are cases where at least any one of the aging-preventing effect, the antioxidant effect, and the whitening effect is insufficient.
The fermentation (cultivation) time is not particularly limited and may be appropriately selected depending on the purpose, and is preferably 10 hours to 40 hours, more preferably 20 hours to 30 hours. If the fermentation time is less than 10 hours, there are cases where at least any one of the cornflower is not sufficiently fermented, and the effect of anti-aging, anti-oxidation and whitening is insufficient.
The method of 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.
The heating temperature for stopping the fermentation is not particularly limited as long as it is a temperature at which the koji mold can not grow any more, and may be appropriately selected according to the purpose, and is preferably 50℃or higher, more preferably 70℃or higher, and particularly preferably 100℃to 130 ℃. If the heating temperature is less than 50 ℃, the fermentation may not be stopped, and if it exceeds 130 ℃, at least one of the anti-aging effect, the anti-oxidation effect, and the whitening effect may be insufficient.
The heating time for stopping the fermentation is not particularly limited as long as it is a state in which the koji mold is not allowed to grow, and may be appropriately selected according to the purpose, and is preferably 5 minutes or more, 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 antioxidation effect, and the whitening effect may be insufficient.
In addition, the cornflower broth after the fermentation has been stopped is preferably cooled. The cooling method is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include a method of standing at normal temperature, a refrigerator, and the like.
The number of times the cornflower is fermented with the koji mold 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 of carrying out the fermentation a plurality of times, the koji mold may be inoculated only for the first time, may be inoculated only for a plurality of times, may be inoculated at each time of fermentation, and is preferably inoculated only for the first time.
In the case of performing the above-described fermentation a plurality of times, the fermentation temperature and the fermentation time may be different from each other or may be the same.
Cornflower seed starter
The cornflower starter is obtained by inoculating aspergillus with the cornflower starter raw material, and attaching sufficient spores to the cornflower to grow. The use of the starter culture for the fermentation is advantageous in that a cornflower fermentation liquid excellent in skin affinity can be obtained more efficiently and easily.
The cornflower used as the starter culture material may be the same as the plant described in the above-mentioned cornflower, and the cornflower may be used in the same form as the part, size, state, etc.
As the koji mold for producing the cornflower koji, the same mold as described in the above-mentioned koji mold can be used.
The inoculation amount of the koji mold on the cornflower used as the seed koji material is not particularly limited and may be appropriately selected according to the purpose, and it is preferable to inoculate 5 to 100 parts by mass of the koji mold suspended in the sterilizing water (1×10 3 to 1×10 8/mL), more preferably 10 to 50 parts by mass, and particularly preferably 20 to 30 parts by mass, with respect to 100 parts by mass of the cornflower. If the inoculum size of the koji mold is less than 5 parts by mass per 100 parts by mass of the cornflower, there are cases where a sufficient amount of spores cannot be attached to the cornflower for growth, and if it exceeds 100 parts by mass, there are cases where abnormal reproduction is caused by excessive moisture.
When inoculating the koji mold with the cornflower used as the starter culture raw material, water is preferably added. The amount of 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 cornflower. If the amount of the water to be added is less than 10 parts by mass per 100 parts by mass of the cornflower, there are cases where a sufficient amount of spores cannot be attached to the cornflower to grow.
The culture temperature is not particularly limited as long as it is within a temperature range in which the koji mold 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 are cases where a sufficient amount of spores cannot be attached to the cornflower for growth. If the temperature exceeds 50 ℃, there is a case where the koji mold cannot be proliferated.
The culture time is not particularly limited and may be appropriately selected depending on the purpose, but is preferably 80 to 210 hours, more preferably 100 to 190 hours, and particularly preferably 120 to 170 hours. If the cultivation time is less than 80 hours, there are cases where a sufficient amount of spores cannot be attached to the cornflower for growth, and if it exceeds 210 hours, there are cases where the germination rate of spores is lowered.
The cornflower fermentation liquid may contain the cells of the koji mold, or may be a fermentation liquid from which the cells of the koji mold are removed, and preferably a fermentation liquid from which the cells of the koji mold are removed.
The state of the cornflower fermentation liquid is not particularly limited and may be appropriately selected depending on the purpose, and for example, the cornflower fermentation liquid itself, a purified product of the cornflower fermentation liquid, a concentrate of the cornflower fermentation liquid, a diluted product of the cornflower fermentation liquid, and the like may be used. The cornflower fermentation liquid may be a liquid obtained by mixing or dissolving the dried product of the cornflower fermentation liquid in a solvent such as water or a hydrophilic solvent again.
The purified product of the cornflower 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 the cornflower, bacterial cells of aspergillus, and precipitates) in the cornflower fermentation liquid.
The method of removal 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 purposes.
The dilution of the cornflower fermentation liquid and the concentrate of the cornflower fermentation liquid are not particularly limited, and may be appropriately selected according to the purpose, and examples thereof include a substance prepared to have 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, reduced pressure concentration and the like may be mentioned.
The dried product of the cornflower fermentation liquid is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include a product obtained by drying the cornflower fermentation liquid.
The drying method is not particularly limited and may be appropriately selected according to the purpose, and examples thereof include freeze drying.
The cornflower fermentation liquid is not particularly limited as long as it is a fermentation liquid obtained by fermenting cornflower with aspergillus, and may be appropriately selected according to the purpose, and is preferably a cornflower fermentation liquid having a contact angle of 85 ° or less, more preferably a cornflower fermentation liquid having a contact angle of 79 ° or less, in view of good skin affinity.
Other components
The other components in 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: excipients, moisture-proof agents, preservatives, enhancers, thickeners, emulsifiers, antioxidants, sweeteners, acidulants, seasonings, colorants, fragrances, whitening agents, moisturizers, oily components, ultraviolet absorbers, surfactants, alcohols, powder components, coloring materials, aqueous components, water, skin nutrients, and the like. These may be used singly or in combination of 1 kind or 2 or more kinds.
The content of the other components is not particularly limited and may be appropriately selected according to the purpose.
Use(s)
The anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention have at least any one of excellent anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect, and therefore, are suitable for use as, for example, medicines, quasi drugs, cosmetics, foods and drinks, etc., and the amount, use, and dosage form thereof can be appropriately selected depending on the purpose of use.
The amount of the above-mentioned components may be appropriately adjusted depending on the physiological activity of the fermentation liquid. The anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent may be the fermentation broth itself.
The usage is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include: oral administration, non-oral administration, and topical application. Among these, topical application is preferred.
The dosage form is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include: oral administration agents such as tablets, powders, capsules, granules, extracts, syrups, etc.; parenteral administration such as injection, drop, and suppository; cosmetic liquid, lotion, cream, ointment, essence, skin lotion, facial mask, jelly, lipstick, powder cake, bath agent, soap, bath lotion, astringent, hair nourishing lotion, hair cream, hair styling lotion, hair conditioner, shampoo, hair conditioner, etc.
The anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention can also be used as an agent for research on the mechanism of action of anti-aging action, anti-oxidation action, anti-inflammatory action, or whitening action.
The anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent of the present invention are applicable to humans, and can be used for animals other than humans (for example, mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as the respective effects are exhibited.
(Cosmetic)
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 optionally contains other ingredients.
Anti-aging agent, antioxidant, anti-inflammatory agent, and 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 purpose, and is preferably 5% by volume or more, more preferably 20% by volume or more, with respect 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% by volume, there is a case where at least any one of the anti-aging effect, the antioxidant effect, the anti-inflammatory effect, and the whitening effect is insufficient. 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 preferably as large as possible, and the upper limit thereof is not particularly limited and may be appropriately selected according to the purpose. The cosmetic may be at least 1 substance selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent.
< Other Components >)
The cosmetic may further contain various main ingredients, auxiliary agents and other components which are usually used for producing cosmetics, if necessary, within a range not impairing the object and effect of the present invention.
The other components are not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include: astringents, bactericides, antibacterial agents, ultraviolet absorbers, cell activators, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, perfumes, and the like. These components may be used singly or in combination of 1 kind or 2 or more 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, they may act synergistically to bring about an unexpected excellent effect.
The content of the other components 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.
< Usage >
The use of the cosmetic is not particularly limited, and may be appropriately selected from usual cosmetics, and examples thereof include: skin cosmetics such as astringent, lotion, cream, ointment, essence, skin lotion, facial mask, jelly, lipstick, foundation, bath agent, soap, and bath lotion; hair cosmetics such as astringent, hair nourishing lotion, hair cream, hair styling lotion, hair tonic, shampoo, hair conditioner, etc.
The cosmetic may be formulated into any cosmetic without inhibiting the activity of at least 1 selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent, or may be a cosmetic containing at least 1 selected from the group consisting of the anti-aging agent, the antioxidant, the anti-inflammatory agent, and the whitening agent as a main component. The cosmetic may be at least 1 substance 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 suitable for use in humans, but may be used for animals other than humans (for example, mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as the respective effects are exhibited.
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, when applied to the skin, it is useful in that it exerts at least one of excellent anti-aging effect, antioxidant effect, anti-inflammatory effect, and whitening effect.
Examples
The present invention will be specifically described below with reference to production examples and test examples, but the present invention is not limited to these test examples.
Production example 1: preparation of mountain Artemisia fermentation liquor 1
Preparation of seed koji
Aspergillus oryzae (Aspergillus oryzae) was obtained by suspending Aspergillus oryzae strain name AOK1714 (manufactured by autumn and wild Co., ltd.) in 50mL of sterilized water. The number of bacteria in the Aspergillus solution was estimated using a Thoma hemocytometer (EKDS, manufactured), and found to be 1.0X10: 10 5/mL.
Then, 10g of mountain artemisia (manufactured by ALBION Co., ltd.) cut into 0.5cm to 5cm was put into a conical flask, and the mountain artemisia was sterilized by pressure, 2mL of the Aspergillus solution was inoculated to the mountain artemisia, and the mixture was allowed to stand at 30℃for 168 hours. After the completion of the cultivation, the mixture was dried at 45℃for 24 hours to obtain "mountain artemisia seed koji".
Fermentation step-
The mountain artemisia rupestris (manufactured by ALBION Co., ltd.) was crushed using a crusher (sugar press) and passed through a 2mm mesh screen to obtain a crushed mountain artemisia rupestris. 50g of the crushed mountain artemisia are mixed with 1,000mL of water, and then 20mL of mountain artemisia seed koji (the number of bacteria: about 1.0X10 6/mL) obtained in the seed koji preparation step is inoculated. Then, the cells were pre-cultured at 25℃for 22 hours. The obtained fermentation broth was filtered using celite to obtain "mountain artemisia fermentation broth 1".
Production example 2: preparation of mountain Artemisia fermentation liquor 2
In production example 1, mountain artemisia fermentation broth 2 "was obtained in the same manner as in production example 1 except that mountain artemisia fermentation broth was changed to koji (aspergillus oryzae) produced from rice (hereinafter, sometimes referred to as" rice koji "), aspergillus candidus, and tikitiki-to-date store company, inc.).
Comparative production example 1: preparation of mountain Artemisia extract
The mountain artemisia rupestris (manufactured by ALBION Co., ltd.) was crushed using a crusher (sugar press) and passed through a 2mm mesh screen to obtain a crushed mountain artemisia rupestris. 50g of the crushed mountain artemisia are mixed with 1,000mL of water, and then stirred at 25℃for 22 hours. Then, the obtained stirred material was filtered using diatomaceous earth to obtain "mountain artemisia extract".
Test example A-1 measurement of contact angle
The contact angle was measured by the following method using the mountain artemisia fermentation liquid 1 obtained in production example 1, the mountain artemisia fermentation liquid 2 obtained in production example 2, and the mountain artemisia extract obtained in comparative production example 1 as test samples.
Specifically, 3. Mu.L of each test sample was dropped onto a sample stage (made of aluminum) of a dynamic contact angle/surface tension measuring device (manufactured by FTA1000 Falcon, first Ten Angstroms) and measured by a droplet method at a temperature of 22℃and a relative humidity of 20%. The contact angle θ (°) of 1,000ms was determined by 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 an example of a droplet when the contact angle of each test sample is measured.
TABLE 1
| Test sample | Fermentation | Aspergillus | Contact angle θ (°) | |
| Production example 1 | Artemisia rupestris fermentation liquor 1 | Has the following components | Mountain artemisia seed starter | 77.44 |
| Production example 2 | Artemisia rupestris fermentation liquor 2 | Has the following components | Rice seed koji | 80.02 |
| Comparative production example 1 | Artemisia rupestris extractive solution | Without any means for | - | 81.57 |
The contact angle of the mountain artemisia fermentation broth 1 obtained in production example 1 and the mountain artemisia fermentation broth 2 obtained in production example 2 was smaller than that of the mountain artemisia extraction broth obtained in comparative production example 1, and was 81 ° or less, and the skin affinity was excellent. Further, the contact angle of the mountain artemisia fermentation liquid 1 obtained in production example 1 was 78 ° or less, and the skin affinity was more excellent.
( Test example 1-1: inhibition assay of matrix Metalloprotease-1 (MMP-1) Activity )
Using the mountain artemisia fermentation broth 1 obtained in production example 1, the mountain artemisia fermentation broth 2 obtained in production example 2, and the mountain artemisia extract obtained in comparative production example 1 as test samples, an activity inhibition test of matrix metalloproteinase-1 (MMP-1) was performed by the following test method in which a part of the Wen Shi Hehai De-Rust (Wunsch AND HEIDRICH) method was modified.
In capped test tubes, each test sample was dissolved in 0.1mol/L Tris (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 (COLLAGENASE Type IV from Clostridium histolyticum) from Clostridium histolyticum, 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 Co.) were mixed, reacted at 3℃for 30 minutes, and then 25mmol/L of citric acid solution 1mL was added to stop the reaction. In addition, the final concentration of the test sample at this time was the concentration shown in table 2 below. Then, 5mL of ethyl acetate was added and vigorously shaken. This was centrifuged at 1,600Xg for 10 minutes, and the absorbance of the ethyl acetate layer at 320nm 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 control was changed from the solution of the test sample to 0.1mol/L Tris-HCl buffer (pH 7.1) containing 20mmol/L calcium chloride without containing the test sample.
Based on the obtained measurement value of absorbance, the MMP-1 activity inhibition rate was calculated based on the following formula 1. The results are shown in table 2 below.
< 1 >
MMP-1 Activity inhibition ratio (%) = {1- (C-D)/(A-B) } ×100
In the above formula 1, a to D are each as follows.
A: absorbance at wavelength 320nm when no test sample was added and enzyme was added
B: absorbance at wavelength 320nm without addition of test sample and without addition of enzyme
C: absorbance at wavelength 320nm when the test sample and enzyme were added
D: absorbance at wavelength 320nm when the test sample was added and no enzyme was added
TABLE 2
( Test examples 1-2: test for promoting expression of hyaluronic acid synthase 3 (HAS 3) mRNA )
Using the mountain artemisia fermentation broth 1 obtained in production example 1, the mountain artemisia fermentation broth 2 obtained in production example 2, and the mountain artemisia extract obtained in comparative production example 1 as test samples, a test for an effect of promoting expression of hyaluronic acid synthase 3 (HAS 3) mRNA was performed by the following test methods.
The samples were dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB 2, manufactured by Cangfu textile Co., ltd.) so that the final concentrations became the concentrations shown in Table 3 below, and the medium to which the samples were added was prepared.
Normal human neonatal epidermal keratinocytes (Normal Human Epidermal Keratinocytes, NHEK, manufactured by Cangfu textile company limited) were cultured until confluent using a medium for normal human epidermal keratinocyte proliferation (HuMedia-KG 2, manufactured by Cangfu textile company limited) at 37 ℃ under 5% co 2, and thereafter, the cells were recovered by trypsin treatment. The cells thus recovered were adjusted to 1.5X10- 5 cells/mL using a medium (HuMedia-KG 2) for normal human epidermal keratinocyte proliferation.
Then, 2mL of the NHEK (1.5X10 5 cells/mL) was inoculated into 35mm dishes and cultured overnight at 37℃under 5% CO 2. After the completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB 2), and the culture was further carried out for 24 hours. After the completion of the culture, the medium was replaced with 2mL of the medium to which the test sample was added, and the culture was performed at 37℃under 5% CO 2 for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted with 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 200 ng/. Mu.L of total RNA was prepared using purified water.
The absorbance was measured in the same manner as described above except that 2mL of the culture medium to which the test sample was added was changed to 2mL of the normal human epidermal keratinocyte basal medium (HuMedia-KB 2) containing no test sample, and total RNA was prepared so as to be 200 ng/. Mu.L by the same method as described above.
The total RNAs were used as templates to measure the expression levels of hyaluronate synthase 3 (HAS 3) mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as internal standards. mRNA was detected 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 a SYBR (registered trademark) PRIMESCRIPT (registered trademark) RT-PCR kit (PERFECT REAL TIME (catalog number: RR063A, manufactured by Takara Bio Inc.).
The expression level of HAS3 mRNA to which no test sample was added was corrected from that of GAPDH MRNA. Based on the correction value, the HAS3 mRNA expression promotion rate was calculated based on the following expression 2. The results are shown in Table 3 below.
< 2 >
HAS3 mRNA expression promotion rate (%) =a/b×100
In the above formula 2, a and B each represent the following.
A: correction value when test sample is added
B: correction value without addition of test sample
TABLE 3
Test examples 1-3 DPPH radical scavenging test
Using the mountain artemisia fermentation broth 1 obtained in production example 1, the mountain artemisia fermentation broth 2 obtained in production example 2, and the mountain artemisia extract obtained in comparative production example 1 as test samples, a test for DPPH radical scavenging action was performed by the following test methods.
Each test sample was dissolved in an ethanol solution (manufactured by fuji film and photoplethysmography corporation) to prepare a test sample solution.
To 3mL of 150. Mu. Mol/L DPPH (diphenyl-p-picrylhydrazyl, diphenyl-p-picrylhydrazine) ethanol solution was added 3mL of the sample solution, the vessel was immediately stoppered and mixed with shaking, and after standing for 30 minutes, absorbance at a wavelength of 520nm was measured. In this case, the final concentration of the sample to be tested was as 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 test sample solution was changed to an ethanol solution (Fuji photo-alignment film and Wako pure chemical industries, ltd.) containing no test sample as a control.
Based on the obtained absorbance measurement value, the DPPH radical scavenging rate was calculated based on the following formula 3. The results are shown in table 4 below.
< 3 >
DPPH radical clearance (%) = { Α - (B-C) }/a×100
In the above formula 3, a to C are each as follows.
A: absorbance at 520nm when no test sample was added and DPPH was added
B: absorbance at 520nm when the test sample was added and DPPH was added
C: absorbance at 520nm without addition of test sample and without addition of DPPH
TABLE 4
Test examples 1 to 4 hyaluronidase Activity inhibition test
Using the mountain artemisia fermentation broth 1 obtained in production example 1, the mountain artemisia fermentation broth 2 obtained in production example 2, and the mountain artemisia extract obtained in comparative production example 1 as test samples, a test for hyaluronidase activity inhibition was performed by the following test methods.
Each test sample was dissolved in 0.1mol/L acetic acid buffer (pH 3.5) to prepare a test sample solution.
To 0.2mL of the sample solution to be tested, 0.1mL of hyaluronidase solution (type IV-S (from bovine testis), 400NF units/mL, manufactured by SIGMA Co.) was added, and the mixture was reacted at 37℃for 20 minutes. Then, 0.2mL of 2.5mmol/L calcium chloride as an activator was added, and the mixture was further reacted at 37℃for 20 minutes. To this was added 0.5mL of a sodium hyaluronate solution (from Celosis) (Fuji film and Wako pure chemical industries, ltd.) at 0.8mg/mL, and the mixture was reacted at 37℃for 40 minutes. In this case, the final concentration of the sample to be tested was as 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 boric acid solution was added to each reaction solution, and the mixture was 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 10-fold with acetic acid) was added, and the mixture was reacted at 37℃for 20 minutes. Then, 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 same procedure as described above was performed except that the test sample solution was changed to 0.1mol/L acetic acid buffer (pH 3.5) containing no test sample as a control, and the absorbance was measured.
From the obtained measured value of absorbance, the hyaluronidase activity inhibition rate was calculated based on the following formula 4. The results are shown in table 5 below.
< 4 >
Hyaluronidase activity inhibition ratio (%) = {1- (C-D)/(a-B) } ×100
In the above formula 4, a to D are each as follows.
A: absorbance at wavelength 585nm when no test sample was added and enzyme was added
B: absorbance at wavelength 585nm without addition of test sample and without addition of enzyme
C: absorbance at wavelength 585nm when the test sample and enzyme were added
D: absorbance at wavelength 585nm when the test sample was added and no enzyme was added
TABLE 5
Test examples 1 to 5 tyrosinase activity inhibition test
Using the mountain artemisia fermentation broth 1 obtained in production example 1, the mountain artemisia fermentation broth 2 obtained in production example 2, and the mountain artemisia extract obtained in comparative production example 1 as test samples, a tyrosinase activity inhibition test was performed by the following test methods.
Test sample solutions were prepared by dissolving each test sample in 25% dmso (Dimethyl sulfoxide ) solution. To a 48-well plate, 0.2mL of MCLLVAINE buffer (pH 6.8), 0.06mL of 0.3mg/mL tyrosine solution, and 0.18mL of the test sample solution were added, and the mixture was allowed to stand at 37℃for 10 minutes. To this, 0.02mL of a tyrosinase solution (manufactured by SIGMA Co.) was added at 800unit/mL, and the reaction was further carried out at 37℃for 15 minutes. After the completion of the reaction, absorbance at a wavelength of 475nm was measured. In this case, the final concentration of the sample to be tested was as 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 same procedure as described above was performed except that the test sample solution was changed to a 25% dmso solution containing no test sample as a control, and the absorbance was measured.
Based on the obtained measured value of absorbance, the tyrosinase activity inhibition rate was calculated based on the following formula 5. The results are shown in Table 6 below.
< 5 >
Tyrosinase activity inhibition ratio (%) = {1- (C-D)/(a-B) } ×100
In the above formula 5, a to D are each as follows.
A: absorbance at wavelength 475nm when no test sample was added and enzyme was added
B: absorbance at wavelength 475nm without addition of test sample and without addition of enzyme
C: absorbance at wavelength 475nm when the test sample and enzyme were added
D: absorbance at wavelength 475nm with and without enzyme added to the test sample
TABLE 6
Production example 3: preparation of herba Artemisiae Scopariae fermentation liquor 1
In the starter preparation step of production example 1, a "artemisia capillaris starter" was prepared in the same manner as in the starter preparation step of production example 1 except that artemisia capillaris was changed to artemisia capillaris (ARTEMISIA CAPILLARIS Thunbergii) (manufactured by ALBION corporation).
In addition, in the fermentation step of production example 1, a "herba Artemisiae Scopariae fermentation broth 1" was obtained in the same manner as in the fermentation step of production example 1 except that the mountain artemisia was changed to herba Artemisiae Scopariae (ARTEMISIA CAPILLARIS Thunbergii) (manufactured by ALBION corporation).
Production example 4: preparation of herba Artemisiae Scopariae fermentation liquor 2
In production example 3, a "herba Artemisiae Scopariae fermentation broth 2" was obtained in the same manner as in production example 3, except that the seed koji of herba Artemisiae Scopariae was changed to rice koji (Aspergillus oryzae, manufactured by Qingshen Liang white Aspergillus, manufactured by the company GmbH, autumn and Jinye).
Comparative production example 2: preparation of herba Artemisiae Scopariae extractive solution
In comparative production example 1, a "herba Artemisiae Scopariae extract" was obtained in the same manner as in comparative production example 1 except that the mountain artemisia was changed to herba Artemisiae Scopariae (ARTEMISIACAPILLARIS THUNBERGII) (manufactured by ALBION Co., ltd.).
(Test example A-2: measurement of contact angle)
In the test example A-1, contact angles were measured in the same manner as in the test example A-1 except that the test sample was changed to the herba Artemisiae Scopariae fermentation broth 1 obtained in production example 3, the herba Artemisiae Scopariae fermentation broth 2 obtained in production example 4, and the herba Artemisiae Scopariae extract obtained in comparative production example 2. The results are shown in Table 7 below. Fig. 2A to 2C show an example of a droplet when the contact angle of each test sample is measured.
TABLE 7
| Test sample | Fermentation | Aspergillus | Contact angle θ (°) | |
| Production example 3 | Herba Artemisiae Scopariae fermentation liquor 1 | Has the following components | Herba Artemisiae Scopariae seed koji | 77.50 |
| Production example 4 | Herba Artemisiae Scopariae fermentation liquor 2 | Has the following components | Rice seed koji | 79.58 |
| Comparative production example 2 | Herba Artemisiae Scopariae extractive solution | Without any means for | - | 87.75 |
The contact angle of each of the capillary artemisia fermentation broth 1 obtained in production example 3 and the capillary artemisia fermentation broth 2 obtained in production example 4 was smaller than that of the capillary artemisia extract obtained in comparative production example 2, and was 81 ° or less, and the skin affinity was excellent. In addition, the contact angle of the capillary artemisia fermentation broth 1 obtained in preparation example 3 was 78 ° or less, and the skin affinity was more excellent.
( Test example 2-1: test for promoting expression of hyaluronic acid synthase 3 (HAS 3) mRNA )
In test examples 1 to 2, a test was conducted by the same method as in test examples 1 to 2 except that the final concentrations of the test samples were changed to the concentrations shown in Table 8 below, except that the test samples were changed to the herba Artemisiae Scopariae fermentation broth 1 obtained in production example 3, the herba Artemisiae Scopariae fermentation broth 2 obtained in production example 4, and the herba Artemisiae Scopariae extract obtained in comparative production example 2, and the hyaluronic acid synthase 3 (HAS 3) mRNA expression promoting effect was tested. The results are shown in Table 8 below.
TABLE 8
( Test example 2-2: test for type I collagen production-promoting action )
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 test samples, a type I collagen production promoting effect was tested by the following test methods.
The samples were dissolved in dulbeck MEM (Modified Eagle's Medium, modified Eagle Medium) (manufactured by daily water pharmaceutical Co., ltd.) containing 0.25% fetal bovine serum (Fetal bovine serum, FBS, manufactured by biosera) so that the final concentration became the concentration shown in Table 9 below, and the Medium to which the samples were added was prepared.
Normal human fibroblasts (NB 1RGB, purchased from RIKEN BRC) were cultured until confluence using DMEM with 10% fbs at 37 ℃ under 5% co 2, after which the cells were recovered by trypsin treatment. The recovered cells were adjusted to 1.6X10 5 cells/mL with DMEM containing 10% FBS.
Then, the NB1RGB (1.6X10 5 cells/mL) was inoculated at 100. Mu.L per well into a 96-well microplate, and cultured overnight at 37℃under 5% CO 2. After the completion of the culture, 100. Mu.L of the medium to which the test sample was added was replaced with the medium, and the culture was carried out at 37℃for 3 days under 5% CO 2. After the completion of the culture, the amount of type I collagen in the medium of each well was measured by ELISA (Enzyme Linked Immunosorbent Assay, enzyme-linked immunosorbent assay).
Specifically, 90mL of the culture supernatant was transferred to an ELISA plate, and after adsorption on the plate was continued overnight at 4℃the solution was discarded and washed with a phosphate buffered saline (PBS-T) containing 0.05% Tween-20. Thereafter, blocking was performed using phosphate buffer containing 1% fbs. The solution was discarded, washed with a phosphate-buffered saline (PBS-T) containing 0.05% Tween-20, and reacted with an anti-human collagen I antibody (rabbit IgG, manufactured by Chemicon Co.). The solution was discarded, washed with a phosphate buffered saline (PBS-T) containing 0.05% Tween-20, reacted with an HRP-labeled anti-rabbit IgG antibody, and then subjected to a color 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.
The same procedure was performed and measurement was performed by ELISA method except that the test sample solution was changed to DMEM containing 0.25% fbs without the test sample as a control.
Based on the obtained measurement value, the promotion rate of type I collagen production was calculated based on the following formula 6. The results are shown in Table 9 below.
< 6 >
Type I collagen production acceleration rate (%) =a/b×100
In the above formula 6, a and B are each as follows.
A: type I collagen amount when test sample was added
B: type I collagen amount without addition of test sample
TABLE 9
( Test examples 2-3: test for promoting expression of claudin-1 mRNA )
Using the capillary artemisia fermentation broth 1 obtained in production example 3, the capillary artemisia fermentation broth 2 obtained in production example 4, and the capillary artemisia extract obtained in comparative production example 2 as test samples, a test for promoting the expression of claudin-1 mRNA was performed by the following test methods.
The samples were dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB 2, manufactured by Cangfu textile Co., ltd.) so that the final concentrations became the concentrations shown in Table 10 below, and the medium to which the samples were added was prepared.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Cangfu textile inc.) were cultured at 37 ℃ under 5% co 2 using a medium for normal human epidermal keratinocyte proliferation (HuMedia-KG 2, manufactured by Cangfu textile inc.) until fused, and thereafter the cells were recovered by trypsin treatment. The cells thus recovered were adjusted to 1.5X10- 5 cells/mL using a medium (HuMedia-KG 2) for normal human epidermal keratinocyte proliferation.
Then, 2mL of the NHEK (1.5X10 5 cells/mL) was inoculated into 35mm dishes and cultured overnight at 37℃under 5% CO 2. After the completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB 2), and the culture was further carried out for 24 hours. After the completion of the culture, the medium was replaced with 2mL of the medium to which the test sample was added, and the culture was performed at 37℃under 5% CO 2 for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted with 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 200 ng/. Mu.L of total RNA was prepared using purified water.
The absorbance was measured in the same manner as described above except that 2mL of the culture medium to which the test sample was added was changed to 2mL of the normal human epidermal keratinocyte basal medium (HuMedia-KB 2) containing no test sample, and total RNA was prepared so as to be 200 ng/. Mu.L by the same method as described above.
The amount of expression of the claudin-1 mRNA was measured using the total RNAs as templates, and GAPDH MRNA was used as an internal standard. mRNA was detected by a two-step real-time PCR using a real-time PCR device (THERMAL CYCLER DICE (registered trademark) manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PRIMESCRIPT (registered trademark) RT-PCR kit (PERFECT REAL TIME (catalog number: RR063A, manufactured by Takara Bio Inc.).
The expression level of the claudin-1 mRNA to which the test sample was not added was corrected from the expression level of GAPDH MRNA. Based on the correction value, the expression promotion rate of claudin-1 mRNA was calculated based on the following formula 7. The results are shown in table 10 below.
< 7 >
Claudin-1 mRNA expression promotion rate (%) =a/b×100
In the above formula 7, a and B are each as follows.
A: correction value when test sample is added
B: correction value without addition of test sample
TABLE 10
( Test examples 2 to 4: claudin-4 mRNA expression promotion assay )
The test for the promoting effect of the expression of claudin-4 mRNA was performed by the following test method using the herba Artemisiae Scopariae fermentation broth 1 obtained in production example 3, the herba Artemisiae Scopariae fermentation broth 2 obtained in production example 4, and the herba Artemisiae Scopariae extract obtained in comparative production example 2 as test samples.
The samples were dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB 2, manufactured by Cangfu textile Co., ltd.) so that the final concentrations became the concentrations shown in Table 11 below, and the medium to which the samples were added was prepared.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Cangfu textile inc.) were cultured at 37 ℃ under 5% co 2 using a medium for normal human epidermal keratinocyte proliferation (HuMedia-KG 2, manufactured by Cangfu textile inc.) until fused, and thereafter the cells were recovered by trypsin treatment. The cells thus recovered were adjusted to 1.5X10- 5 cells/mL using a medium (HuMedia-KG 2) for normal human epidermal keratinocyte proliferation.
Then, 2mL of the NHEK (1.5X10 5 cells/mL) was inoculated into 35mm dishes and cultured overnight at 37℃under 5% CO 2. After the completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB 2), and the culture was further carried out for 24 hours. After the completion of the culture, the medium was replaced with 2mL of the medium to which the test sample was added, and the culture was performed at 37℃under 5% CO 2 for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted with 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 200 ng/. Mu.L of total RNA was prepared using purified water.
In addition, as a control, total RNA was prepared in the same manner as described above to 200 ng/. Mu.L by measuring absorbance in the same manner as described above except that 2mL of the culture medium to which the test sample was added was changed to 2mL of the normal human epidermal keratinocyte basal medium (HuMedia-KB 2) containing no test sample.
The amount of expression of the claudin-4 mRNA was measured using the total RNAs as templates, and GAPDH MRNA was used as an internal standard. mRNA was detected by a two-step real-time PCR using a real-time PCR device (THERMAL CYCLER DICE (registered trademark) manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PRIMESCRIPT (registered trademark) RT-PCR kit (PERFECT REAL TIME (catalog number: RR063A, manufactured by Takara Bio Inc.).
The expression level of the claudin-4 mRNA to which the test sample was not added was corrected from the expression level of GAPDH MRNA. Based on the correction value, the expression promotion rate of claudin-4 mRNA was calculated based on the following formula 8. The results are shown in Table 11 below.
< 8 >
Claudin-4 mRNA expression promotion rate (%) =a/b×100
In the above formula 8, a and B are each as follows.
A: correction value when test sample is added
B: correction value without addition of test sample
TABLE 11
Test examples 2 to 5 test for the expression promoting effect of the protein mRNA
The test for the effect of promoting the expression of claudin mRNA was performed by using the herba Artemisiae Scopariae fermentation broth 1 obtained in production example 3, the herba Artemisiae Scopariae fermentation broth 2 obtained in production example 4, and the herba Artemisiae Scopariae extract obtained in comparative production example 2 as test samples.
The samples were dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB 2, manufactured by Cangfu textile Co., ltd.) so that the final concentration became the concentration shown in Table 12 below, and the medium to which the samples were added was prepared.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Cangfu textile inc.) were cultured at 37 ℃ under 5% co 2 using a medium for normal human epidermal keratinocyte proliferation (HuMedia-KG 2, manufactured by Cangfu textile inc.) until fused, and thereafter the cells were recovered by trypsin treatment. The cells thus recovered were adjusted to 1.5X10- 5 cells/mL using a medium (HuMedia-KG 2) for normal human epidermal keratinocyte proliferation.
Then, 2mL of the NHEK (1.5X10 5 cells/mL) was inoculated into 35mm dishes and cultured overnight at 37℃under 5% CO 2. After the completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB 2), and the culture was further carried out for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted with 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 200 ng/. Mu.L of total RNA was prepared using purified water.
In addition, as a control, total RNA was prepared in the same manner as described above to 200 ng/. Mu.L by measuring absorbance in the same manner as described above except that 2mL of the culture medium to which the test sample was added was changed to 2mL of the normal human epidermal keratinocyte basal medium (HuMedia-KB 2) containing no test sample.
The amount of expression of GAPDH MRNA was measured using each of the total RNAs as a template, and the amount of the protein mRNA was used as an internal standard. mRNA was detected by a two-step real-time PCR using a real-time PCR device (THERMAL CYCLER DICE (registered trademark) manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PRIMESCRIPT (registered trademark) RT-PCR kit (PERFECT REAL TIME (catalog number: RR063A, manufactured by Takara Bio Inc.).
The expression level of the protein mRNA to which the test sample was not added was corrected from the GAPDH MRNA expression level. Based on the correction value, the expression acceleration rate of the protein mRNA was calculated based on the following expression 9. The results are shown in table 12 below.
< 9 >
Protein mRNA expression promotion rate (%) =a/b×100
In the above formula 9, a and B are each as follows.
A: correction value when test sample is added
B: correction value without addition of test sample
TABLE 12
Production example 5: preparation of thyme fermentation liquor 1
"Thyme starter" was prepared in the same manner as in the starter preparation step of preparation example 1, except that the mountain artemisia was changed to thyme (Thymus vulgarisLinne) (manufactured by ALBION, inc.) in the starter preparation step of preparation example 1.
In addition, in the fermentation step of production example 1, thyme fermentation broth 1 was obtained in the same manner as in the fermentation step of production example 1 except that mountain artemisia was changed to thyme (Thymus vulgarisLinne) (manufactured by ALBION corporation).
Production example 6: preparation of thyme fermentation liquor 2
"Thyme fermentation broth 2" was obtained in the same manner as in production example 5, except that in production example 5, thyme koji was changed to rice koji (produced by Aspergillus oryzae, rhizoctonia solani, and Hibiscus makino, inc.).
Comparative manufacturing example 3: preparation of thyme extract
In comparative production example 1, a "thyme extract" was obtained in the same manner as in comparative production example 1 except that the mountain artemisia was changed to thyme (Thymusvulgaris Linne) (manufactured by ALBION corporation).
(Test example A-3 measurement of contact angle)
In the test example A-1, contact angles were measured in the same manner as in the test example A-1 except that the test samples were changed to 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. The results are shown in Table 13 below. Fig. 3A to 3C show an example of a droplet when the contact angle of each test sample is measured.
TABLE 13
| Test sample | Fermentation | Aspergillus | Contact angle θ (°) | |
| Production example 5 | Thyme fermentation liquor 1 | Has the following components | Thyme starter | 78.32 |
| Production example 6 | Thyme broth 2 | Has the following components | Rice seed koji | 82.77 |
| Comparative production example 3 | Thyme extract | Without any means for | - | 88.59 |
The contact angle of thyme fermentation liquid 1 obtained in production example 5 and thyme fermentation liquid 2 obtained in production example 6 was small and 87 ° or less, compared with the thyme extract obtained in comparative production example 3, and the skin affinity was excellent. In addition, the thyme broth 1 obtained in production example 5 had a contact angle of 81 ° or less, and was more excellent in skin affinity.
( Test example 3-1: glutamine transaminase-1 (TGM-1) mRNA expression promotion test )
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, a test for glutamine transaminase-1 (TGM-1) mRNA expression promotion was performed by the following test methods.
The samples were dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB 2, manufactured by Cangfu textile Co., ltd.) so that the final concentration became the concentration shown in Table 14 below, and the medium to which the samples were added was prepared.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Cangfu textile inc.) were cultured at 37 ℃ under 5% co 2 using a medium for normal human epidermal keratinocyte proliferation (HuMedia-KG 2, manufactured by Cangfu textile inc.) until fused, and thereafter the cells were recovered by trypsin treatment. The cells thus recovered were adjusted to 1.5X10- 5 cells/mL using a medium (HuMedia-KG 2) for normal human epidermal keratinocyte proliferation.
Then, 2mL of the NHEK (1.5X10 5 cells/mL) was inoculated into 35mm dishes and cultured overnight at 37℃under 5% CO 2. After the completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB 2), and the culture was further carried out for 24 hours. After the completion of the culture, the medium was replaced with 2mL of the medium to which the test sample was added, and the culture was performed at 37℃under 5% CO 2 for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted with 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 200 ng/. Mu.L of total RNA was prepared using purified water.
The absorbance was measured in the same manner as described above except that 2mL of the culture medium to which the test sample was added was changed to 2mL of the normal human epidermal keratinocyte basal medium (HuMedia-KB 2) containing no test sample, and total RNA was prepared so as to be 200 ng/. Mu.L by the same method as described above.
The total RNAs were used as templates to measure the amount of glutamine transaminase-1 (TGM-1) mRNA and GAPDH MRNA expressed as an internal standard. mRNA was detected by a two-step real-time PCR using a real-time PCR device (THERMAL CYCLER DICE (registered trademark) manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PRIMESCRIPT (registered trademark) RT-PCR kit (PERFECT REAL TIME (catalog number: RR063A, manufactured by Takara Bio Inc.).
The TGM-1mRNA expression level of the test sample without the test sample was corrected from GAPDH MRNA expression level. Based on the correction value, the TGM-1mRNA expression promotion rate was calculated based on the following formula 10. The results are shown in table 14 below.
< 10 >
TGM-1mRNA expression promotion rate (%) =A/B×100
In the above formula 10, a and B are each as follows.
A: correction value when test sample is added
B: correction value without addition of test sample
TABLE 14
( Test example 3-2: aquaporin 3 (AQP 3) mRNA expression promotion assay )
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, an experiment of aquaporin 3 (AQP 3) mRNA expression promotion was performed by the following test methods.
The samples were dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB 2, manufactured by Cangfu textile Co., ltd.) so that the final concentrations became the concentrations shown in Table 15 below, and the medium to which the samples were added was prepared.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Cangfu textile inc.) were cultured at 37 ℃ under 5% co 2 using a medium for normal human epidermal keratinocyte proliferation (HuMedia-KG 2, manufactured by Cangfu textile inc.) until fused, and thereafter the cells were recovered by trypsin treatment. The cells thus recovered were adjusted to 1.5X10- 5 cells/mL using a medium (HuMedia-KG 2) for normal human epidermal keratinocyte proliferation.
Then, 2mL of the NHEK (1.5X10 5 cells/mL) was inoculated into 35mm dishes and cultured overnight at 37℃under 5% CO 2. After the completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB 2), and the culture was further carried out for 24 hours. After the completion of the culture, the medium was replaced with 2mL of the medium to which the test sample was added, and the culture was performed at 37℃under 5% CO 2 for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted with 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 200 ng/. Mu.L of total RNA was prepared using purified water.
The absorbance was measured in the same manner as described above except that 2mL of the culture medium to which the test sample was added was changed to 2mL of the normal human epidermal keratinocyte basal medium (HuMedia-KB 2) containing no test sample, and total RNA was prepared so as to be 200 ng/. Mu.L by the same method as described above.
The expression level of aquaporin 3 (AQP 3) mRNA was measured using the total RNAs as templates, and GAPDH MRNA was used as an internal standard. mRNA was detected by a two-step real-time PCR using a real-time PCR device (THERMAL CYCLER DICE (registered trademark) manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PRIMESCRIPT (registered trademark) RT-PCR kit (PERFECT REAL TIME (catalog number: RR063A, manufactured by Takara Bio Inc.).
The AQP3 mRNA expression level without and with the test sample was corrected from GAPDH MRNA expression level. Based on the correction value, the AQP3 mRNA expression promotion rate was calculated based on the following expression 11. The results are shown in table 15 below.
< 11 >
AQP3 mRNA expression promotion rate (%) =a/b×100
In the above formula 11, a and B are each as follows.
A: correction value when test sample is added
B: correction value without addition of test sample
TABLE 15
Test example 3-3 test for the expression promoting effect of Tight mRNA
In test examples 2 to 5, a test for the effect of promoting expression of a protein mRNA was performed in the same manner as in test examples 2 to 5 except that the test samples were changed to 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, and the final concentrations of the test samples were changed to the concentrations shown in Table 16 below. The results are shown in Table 16 below.
TABLE 16
Test examples 3-4 DPPH radical scavenging test
In test examples 1 to 3, a test was conducted by the same method as in test examples 1 to 3 except that the test samples were changed to 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, and the final concentrations of the test samples were changed to the concentrations shown in table 17 below. The results are shown in Table 17 below.
TABLE 17
Production example 7: preparation of Melissa officinalis fermentation liquor 1 >
"Apis cerana seed koji" was prepared in the same manner as in the seed koji preparation step of preparation example 1, except that the mountain artemisia was changed to Apis cerana (Melissa officinalis Linne) (manufactured by ALBION Co., ltd.).
In addition, in the fermentation step of production example 1, a "melissa fermentation broth 1" was obtained in the same manner as in the fermentation step of production example 1, except that the mountain artemisia was changed to melissa (Melissa officinalis Linne) (manufactured by ALBION corporation).
Production example 8: preparation of Melissa officinalis fermentation liquor 2
A "lemon balm fermentation broth 2" was obtained in the same manner as in preparation example 7, except that the seed koji of lemon balm was changed to rice koji (Aspergillus oryzae, manufactured by Katsujopsis, inc. of GmbH).
Comparative production example 4: preparation of Melissa officinalis extract
The "lemon balm extract" was obtained in the same manner as in comparative production example 1, except that the mountain artemisia was changed to lemon balm (Melissaofficinalis Linne) (manufactured by ALBION gmbh).
(Test example A-4: measurement of contact angle)
In the test example A-1, contact angles were measured in the same manner as in the test example A-1 except that the test sample was changed to the melissa fermentation broth 1 obtained in production example 7, the melissa fermentation broth 2 obtained in production example 8, and the melissa extraction liquid obtained in comparative production example 4. The results are shown in Table 18 below. Fig. 4A to 4C show an example of a droplet when the contact angle of each test sample is measured.
TABLE 18
| Test sample | Fermentation | Aspergillus | Contact angle θ (°) | |
| PREPARATION EXAMPLE 7 | Melissa officinalis fermentation liquor 1 | Has the following components | Bee grass starter | 77.09 |
| Production example 8 | Melissa officinalis fermentation liquor 2 | Has the following components | Rice seed koji | 80.16 |
| Comparative production example 4 | Melissa officinalis extract | Without any means for | - | 86.53 |
The contact angle of the melissa fermentation liquid 1 obtained in production example 7 and the melissa fermentation liquid 2 obtained in production example 8 was smaller than that of the melissa extraction liquid obtained in comparative production example 4, and was 85 ° or less, and skin affinity was excellent. Further, the contact angle of the melissa fermented liquid 1 obtained in production example 7 was 79 ° or less, and the skin affinity was more excellent.
( Test example 4-1: test for type I collagen production-promoting action )
In test example 2-2, a test for the type I collagen production promoting effect was performed in the same manner as in test example 2-2 except that the test sample was changed to the melissa fermentation liquid 1 obtained in production example 7, the melissa fermentation liquid 2 obtained in production example 8, and the melissa extraction liquid obtained in comparative production example 4, and the final concentration of the test sample was changed to the concentration shown in table 19 below. The results are shown in Table 19 below.
TABLE 19
( Test example 4-2: aquaporin 3 (AQP 3) mRNA expression promotion assay )
In test example 3-2, a test for the aquaporin 3 (AQP 3) mRNA expression promoting effect was performed in the same manner as in test example 3-2 except that the test sample was changed to the melissa fermentation broth 1 obtained in production example 7, the melissa fermentation broth 2 obtained in production example 8, and the melissa extraction broth obtained in comparative production example 4, and the final concentration of the test sample was changed to the concentration shown in table 20 below. The results are shown in table 20 below.
TABLE 20
Test example 4-3 DPPH radical scavenging test
In test examples 1 to 3, a test was conducted in the same manner as in test examples 1 to 3 except that the test sample was changed to the melissa fermentation broth 1 obtained in production example 7, the melissa fermentation broth 2 obtained in production example 8, and the melissa extraction broth obtained in comparative production example 4, and the final concentration of the test sample was changed to the concentration shown in table 21 below. The results are shown in table 21 below.
TABLE 21
Test example 4-4 hyaluronidase Activity inhibition test
In test examples 1 to 4, a test for the hyaluronidase activity inhibitory effect was performed in the same manner as in test examples 1 to 4 except that the test samples were changed to the melissa fermentation broth 1 obtained in production example 7, the melissa fermentation broth 2 obtained in production example 8, and the melissa extraction broth 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 to 5 tyrosinase activity inhibition test
In test examples 1 to 5, a tyrosinase activity inhibition test was performed in the same manner as in test examples 1 to 5 except that the test samples were changed to the melissa fermentation broth 1 obtained in production example 7, the melissa fermentation broth 2 obtained in production example 8, and the melissa extraction broth 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 liquor 1 >
"Cornflower starter" was prepared in the same manner as in the starter preparation step of preparation example 1 except that the mountain artemisia was changed to cornflower (Centaurea cyanusLinne) (manufactured by ALBION Co., ltd.) in the starter preparation step of preparation example 1.
Further, in the fermentation step of production example 1, a "cornflower fermentation broth 1" was obtained in the same manner as in the fermentation step of production example 1 except that the mountain artemisia was changed to cornflower (Centaurea cyanusLinne) (manufactured by ALBION corporation).
Production example 10: preparation of cornflower fermentation liquor 2
In preparation example 9, a "cornflower fermentation broth 2" was obtained in the same manner as in preparation example 9, except that cornflower koji was changed to Aspergillus oryzae (manufactured by Aspergillus candidus, inc. of Qiutian and Jinye shops).
Comparative production example 5: preparation of cornflower extract
The comparative production example 1 was conducted in the same manner as in the comparative production example 1 except that the mountain artemisia was changed to cornflower (Centaureacyanus Linne) (manufactured by ALBION Co., ltd.) to obtain a "cornflower extract".
(Test example A-5 measurement of contact angle)
Contact angles were measured in the same manner as in test example A-1 except that in test example A-1, the test samples were 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. The results are shown in table 24 below. Fig. 5A to 5C show an example of a droplet when the contact angle of each test sample is measured.
TABLE 24
| Test sample | Fermentation | Aspergillus | Contact angle θ (°) | |
| Production example 9 | Cornflower fermentation liquor 1 | Has the following components | Cornflower starter | 78.52 |
| Production example 10 | Cornflower fermentation liquor 2 | Has the following components | Rice seed koji | 80.83 |
| Comparative production example 5 | Cornflower extract | Without any means for | - | 86.96 |
The contact angle of the cornflower fermentation liquid 1 obtained in production example 9 and the contact angle of the cornflower fermentation liquid 2 obtained in production example 10 were both small and 85 ° or less, compared with the cornflower extract liquid obtained in comparative production example 5, and the skin affinity was excellent. Further, the contact angle of cornflower fermentation liquid 1 obtained in production example 9 was 79 ° or less, and the skin affinity was more excellent.
( Test example 5-1: test for type I collagen production-promoting action )
In test example 2-2, a test for the type I collagen production promoting effect was performed in the same manner as in test example 2-2 except that the test sample 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 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 promotion test )
In test example 3-1, a test for the effect of promoting the expression of glutamine transaminase-1 mRNA was performed in the same manner as in test example 3-1 except that the test sample 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 test sample was changed to the concentration shown in the following table 26. The results are shown in table 26 below.
TABLE 26
( Test example 5-3: filaggrin mRNA expression promotion assay )
The procyanidin fermentation liquid 1 obtained in production example 9, the procyanidin fermentation liquid 2 obtained in production example 10, and the procyanidin extract obtained in comparative production example 5 were used as test samples, and the effect of promoting the expression of silk fibroin mRNA was tested by the following test methods.
The samples were dissolved in a normal human epidermal keratinocyte basal medium (HuMedia-KB 2, manufactured by Cangfu textile Co., ltd.) so that the final concentrations became the concentrations shown in Table 27 below, and the culture medium to which the samples were added was prepared.
Normal human neonatal epidermal keratinocytes (NHEK, manufactured by Cangfu textile inc.) were cultured at 37 ℃ under 5% co 2 using a medium for normal human epidermal keratinocyte proliferation (HuMedia-KG 2, manufactured by Cangfu textile inc.) until fused, and thereafter the cells were recovered by trypsin treatment. The cells thus recovered were adjusted to 1.5X10- 5 cells/mL using a medium (HuMedia-KG 2) for normal human epidermal keratinocyte proliferation.
Then, 2mL of the NHEK (1.5X10 5 cells/mL) was inoculated into 35mm dishes and cultured overnight at 37℃under 5% CO 2. After the completion of the culture, the medium was replaced with a normal human epidermal keratinocyte basal medium (HuMedia-KB 2), and the culture was further carried out for 24 hours. After the completion of the culture, the medium was replaced with 2mL of the medium to which the test sample was added, and the culture was performed at 37℃under 5% CO 2 for 24 hours. After completion of the culture, the culture medium was removed, total RNA was extracted with 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 200 ng/. Mu.L of total RNA was prepared using purified water.
The absorbance was measured in the same manner as described above except that 2mL of the culture medium to which the test sample was added was changed to 2mL of the normal human epidermal keratinocyte basal medium (HuMedia-KB 2) containing no test sample, and total RNA was prepared so as to be 200 ng/. Mu.L by the same method as described above.
The total RNAs were used as templates to measure the expression level of the silk fibroin mRNA and GAPDH MRNA as an internal standard. mRNA was detected by a two-step real-time PCR using a real-time PCR device (THERMAL CYCLER DICE (registered trademark) manufactured by Takara Bio Inc.) and a SYBR (registered trademark) PRIMESCRIPT (registered trademark) RT-PCR kit (PERFECT REAL TIME (catalog number: RR063A, manufactured by Takara Bio Inc.).
The expression level of the silk fibroin mRNA to which the test sample was not added was corrected from the expression level GAPDH MRNA. Based on the correction value, the expression promotion rate of the silk fibroin mRNA was calculated based on the following expression 12. The results are shown in Table 27 below.
< 12 >
Filaggrin mRNA expression promotion rate (%) =a/b×100
In the above formula 12, a and B are each as follows.
A: correction value when test sample is added
B: correction value without addition of test sample
TABLE 27
( Test examples 5 to 4: aquaporin 3 (AQP 3) mRNA expression promotion assay )
In test example 3-2, a test for aquaporin 3 (AQP 3) mRNA expression promoting effect was performed in the same manner as in test example 3-2 except that the test sample 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 test sample was changed to the concentration shown in table 28 below. The results are shown in table 28 below.
TABLE 28
( Test examples 5 to 5: test for promoting expression of hyaluronic acid synthase 3 (HAS 3) mRNA )
In test examples 1 to 2, a test was conducted in the same manner as in test examples 1 to 2 except that the final concentrations of the test samples were changed to the concentrations shown in Table 29 below, with the exception that the test samples were 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 hyaluronic acid synthase 3 (HAS 3) mRNA expression promotion effect was tested. The results are shown in table 29 below.
TABLE 29
( Test examples 5 to 6: test for promoting expression of claudin-1 mRNA )
In test examples 2 to 3, a test for promoting the expression of claudin-1 mRNA was performed in the same manner as in test examples 2 to 3 except that the test sample 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 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 to 7: claudin-4 mRNA expression promotion assay )
In test examples 2 to 4, a test for the effect of promoting the expression of claudin-4 mRNA was performed in the same manner as in test examples 2 to 4 except that the test sample 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 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 to 8 test for the expression promoting effect of the protein mRNA
In test examples 2 to 5, a test for the effect of promoting the expression of the protein mRNA 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 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 test sample was changed to the concentration shown in Table 32 below. The results are shown in table 32 below.
TABLE 32
Test example 5-9 DPPH radical scavenging test
In test examples 1 to 3, the DPPH radical scavenging test was performed in the same manner as in test examples 1 to 3 except that the test samples were 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 concentrations of the test samples were changed to the concentrations shown in table 33 below. The results are shown in table 33 below.
TABLE 33
( Test examples 5 to 10: test for inhibition of melanin production in B16 melanoma cells )
The test for inhibiting melanin production by B16 melanoma cells was performed by the following test method using the cornflower fermentation broth 1 obtained in production example 9, the cornflower fermentation broth 2 obtained in production example 10, and the cornflower extract obtained in comparative production example 5 as test samples.
Each test sample was dissolved in DMEM (manufactured by Niday Water pharmaceutical Co., ltd.) containing 10% FBS (manufactured by biosera Co.) and 1mmol/L theophylline (manufactured by Fuji film and Wako pure chemical industries, ltd.) to prepare a medium to which the test sample was added.
B16 melanoma cells were cultured using DMEM containing 10% fbs at 37 ℃ under 5% co 2 until confluent, after which the cells were recovered by trypsin treatment. The recovered cells were adjusted to 2.4X10 5 cells/mL with DMEM containing 10% FBS and 1mmol/L theophylline.
Then, the B16 melanoma cells (2.4X10 5 cells/mL) were inoculated at 300. Mu.L per well into a 48-well plate, and cultured at 37℃under 5% CO 2 for 6 hours. After the completion of the culture, 100. Mu.L of the medium to which the test sample was added was replaced with the medium, and the culture was carried out at 37℃for 3 days under 5% CO 2. After the completion of the culture, 300. Mu.L of the culture medium to which the test sample was added to each well, and the mixture was cultured at 37℃for 4 days under 5% CO 2. In this case, the final concentration of the sample to be tested was as shown in table 34 below.
After completion of the culture, the medium was removed from each well, 200. Mu.L of 2mol/L sodium hydroxide solution was added thereto, and the cells were disrupted by an ultrasonic breaker, and absorbance at 475nm was measured.
Based on the measured absorbance value, 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, the B16 melanoma cells were cultured using the medium to which the test sample was added by the same method as described above, and then the medium was removed and washed with 400 μl of PBS buffer. Then, neutral red was dissolved in DMEM containing 10% FBS at a final concentration of 0.05mg/mL, and 200. Mu.L of the resulting solution was added to each well one by one, and cultured 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 the extraction, absorbance at 540nm was measured.
The absorbance was measured in the same manner as described above, except that the test sample solution was changed to DMEM containing 10% fbs and 1mmol/L theophylline without the test sample as a control.
From the obtained measurement value, the cell survival rate was calculated based on the following formula 13, and the melanin production inhibition rate (%) corrected for the cell survival rate was calculated based on the following formula 14. The results are shown in table 34 below.
< 13 >
Cell viability (%) = (D/C) ×100
In the above formula 13, C and D are respectively represented as follows.
C: absorbance at wavelength 540nm without addition of test sample
D: absorbance at 540nm when the test sample was added
< 14 >
Melanin production inhibition ratio (%) = {1- (B/D)/(a/C) } ×100
In the above formula 14, a to D are each as follows.
A: amount of melanin in the absence of added test sample
B: amount of melanin in the case of the addition of the test sample
C: absorbance at wavelength 540nm without addition of test sample
D: absorbance at 540nm when the test sample was added
TABLE 34
Industrial applicability
The anti-aging agent, antioxidant, anti-inflammatory agent, and whitening agent of the present invention are natural substance-based products having excellent anti-aging, anti-oxidizing, anti-inflammatory, and whitening actions and high safety, and therefore can be used in any fields such as cosmetics, foods, and pharmaceuticals.
The cosmetic of the present invention is suitable for use in skin cosmetics such as lotions, emulsions, creams, ointments, essences, skin lotions, masks, jellies, lipsticks, foundation, bathing agents, soaps, body washes, and the like, because it 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; hair cosmetics such as astringent, hair nourishing lotion, hair cream, hair styling lotion, hair tonic, shampoo, hair conditioner, etc.
Claims (5)
1. An anti-aging agent characterized by: is prepared from at least one of the fermentation liquid of mountain artemisia or artemisia capillaris through fermenting with Aspergillus oryzae (Aspergillus oryzae), the fermentation liquid of melissa through fermenting with Aspergillus oryzae, and the fermentation liquid of cornflower through fermenting with Aspergillus oryzae.
2. An antioxidant, characterized in that: is prepared from at least one of the fermentation liquid of mountain artemisia or artemisia capillaris through fermenting with Aspergillus oryzae (Aspergillus oryzae), the fermentation liquid of melissa through fermenting with Aspergillus oryzae, and the fermentation liquid of cornflower through fermenting with Aspergillus oryzae.
3. An anti-inflammatory agent characterized by: is prepared from at least one of the fermentation liquid of mountain artemisia or artemisia capillaris through fermenting with Aspergillus oryzae (Aspergillus oryzae) and the fermentation liquid of melissa through fermenting with Aspergillus oryzae.
4. A whitening agent, characterized in that: is prepared from at least one of the fermentation liquid of mountain artemisia or artemisia capillaris through fermenting with Aspergillus oryzae (Aspergillus oryzae), the fermentation liquid of melissa through fermenting with Aspergillus oryzae, and the fermentation liquid of cornflower through fermenting with Aspergillus oryzae.
5. A cosmetic product characterized by: comprises 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.
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Also Published As
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|---|---|
| CN113631227A (en) | 2021-11-09 |
| JP2023171950A (en) | 2023-12-05 |
| TW202102239A (en) | 2021-01-16 |
| JP2020164486A (en) | 2020-10-08 |
| KR20210144725A (en) | 2021-11-30 |
| WO2020203933A1 (en) | 2020-10-08 |
| TW202423464A (en) | 2024-06-16 |
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