JP5859200B2 - Anti-inflammatory agent, anti-aging agent, hair restorer, and cosmetics - Google Patents

Description

  The present invention relates to an anti-inflammatory agent, an anti-aging agent, a hair restorer, and a cosmetic.

Conventionally, fermentation technology using yeast has been used in the production of fermented foods such as dairy products such as yogurt and cheese, wine, and pickles, and has attracted attention in various fields and regions.
In recent years, yeast fermented liquor is highly safe, and many valuable products have been produced. Therefore, pharmaceuticals such as anti-inflammatory agents, anti-aging agents, and hair restorers that contain these products as active ingredients, In addition, demand for cosmetics is increasing.

  The anti-inflammatory agent has an action of suppressing symptoms caused by inflammation such as redness, edema, fever, pain, dysfunction. It is known that the symptoms caused by such inflammation are advanced by the production of prostaglandins through cyclooxygenase (COX) activity found in human tissues and organs. Therefore, it is considered that the inflammation can be suppressed by inhibiting the activity of the cyclooxygenase. For the purpose of suppressing the inflammation, for example, aspirin (for example, see Non-Patent Document 1), mangosteen peel extract Among them, cyclooxygenase activity inhibitors such as α-mangostin and γ-mangostin (see, for example, Patent Document 1) have been proposed. Among the cyclooxygenases, the activity of cyclooxygenase-2 (COX-2) is the main cause of the inflammation. For the purpose of suppressing the inflammation, for example, 2-phenyl-1,2-benziso A cyclooxygenase-2 (COX-2) activity inhibitor comprising selenazol-3 (2H) -one, a salt thereof, or a hydrate thereof has been proposed (for example, see Patent Document 2). From such a background, a substance having higher safety and excellent cyclooxygenase-2 (COX-2) activity inhibitory action (anti-inflammatory action) has been demanded.

The anti-aging agent has an action of suppressing an aging phenomenon that irreversibly causes a state in which cells stop dividing and cannot grow. Substances that play an important role in replenishing the energy of the cells, because it is necessary to replenish the cells with energy necessary for division in order to promote cell division by increasing the function of the cells that have been lowered by aging. As ATP (adenosine 5'-triphosphate) is known. Therefore, it is considered that the aging phenomenon can be suppressed by promoting the production of ATP. For the purpose of suppressing the aging phenomenon, for example, an ATP production promoter using vegetable oil is proposed. (For example, see Patent Document 3). From such a background, a substance having higher safety and excellent ATP production promoting action (anti-aging action) has been demanded.
The anti-aging agent also has an action of improving skin symptoms such as rough skin and dry skin caused by an aging phenomenon. The epidermis always creates new keratinocytes through the division and subsequent differentiation of keratinocytes, and has the function of protecting the skin from stimuli from the outside world. In particular, in the differentiation process of the keratinocytes, it is known that transglutaminase-1 promotes the formation of a cornified envelope (CE) and contributes to the structural stability of the keratinocytes. Therefore, it is believed that by promoting the production of the transglutaminase-1, it is considered that the skin symptoms associated with the aging phenomenon can be improved. For the purpose of improving the skin symptoms associated with the aging phenomenon, A transglutaminase-1 production promoter using calcium chloride, which is a constituent component, has been proposed (see, for example, Patent Document 4). From such a background, there is a demand for a substance having higher safety and excellent transglutaminase-1 production promoting action (anti-aging action).

The said hair restorer has the effect | action which suppresses the symptom regarding hair loss, such as a male type bun. These symptoms are the cause of testosterone, which is one of the male hormones collectively called androgen, is reduced by testosterone 5α-reductase to dihydrotestosterone, accumulates in the scalp, atrophys the hair root, and induces hair loss. It is known. Therefore, it is considered that the symptoms related to hair loss can be suppressed by inhibiting the activity of the testosterone 5α-reductase. For the purpose of preventing and treating the symptoms related to hair loss, for example, plants belonging to the genus Choerospondias ( For example, testosterone 5α-reductase activity using, for example, Japanese cedar, birdcage, hood umbrella, and perennial lotus (see, for example, Patent Document 7) Inhibitors have been proposed. From such a background, a substance having higher safety and excellent testosterone 5α-reductase activity inhibitory action (hair growth action) has been demanded.

  As described above, it has an anti-inflammatory agent having a cyclooxygenase-2 (COX-2) activity inhibitory action, an anti-aging agent having an ATP production promoting action and a transglutaminase-1 production promoting action, and a testosterone 5α-reductase activity inhibiting action The demand for hair restorers is extremely high. Moreover, among the fermented liquids by yeast as mentioned above, the fermented liquid derived from yeast that can be used for food is considered to be highly safe, and each agent using such a highly safe fermented liquid. The demand for is extremely high.

Among the yeasts, Kluyveromyces thermotolerance is isolated from wine brewing, plum jam, fruits and the like (see, for example, Non-Patent Document 2), so that the fermented liquid is safe. Although it is considered to be high, it has not been known so far that the fermented liquid from the yeast has the anti-inflammatory action, the anti-aging action, and the hair-growth action. Also, an anti-inflammatory agent having an excellent anti-inflammatory effect using such a highly safe fermentation broth, an anti-aging agent having an excellent anti-aging effect, a hair-restoring agent having an excellent hair-restoring effect, and a cosmetic Currently, prompt provision is strongly demanded.

JP 2002-047180 A JP 2000-016935 A Japanese Patent Laid-Open No. 06-219926 JP 2004-051596 A JP 2003-055162 A JP 2002-241296 A JP 2002-087976 A

Pharmacology Atlas (P184), translated by Takehiko Fukuhara, Bunkodo The Yeasts. A Taxonomic Study, 4th ed. Elsevier Science BV, Amsterdam. 240-241 (1998)

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, an object of the present invention is to provide an anti-inflammatory agent having excellent anti-inflammatory action and high safety. Another object of the present invention is to provide an anti-aging agent having an excellent anti-aging action and high safety. Another object of the present invention is to provide a hair restoring agent that has an excellent hair restoring action and is highly safe. Another object of the present invention is to provide a cosmetic that has at least one of excellent anti-inflammatory action, anti-aging action, and hair-growth action and has excellent moisture retention.

In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following findings. That is, it has been found that the fermented liquid by Kluyveromyces thermotolerance has excellent anti-inflammatory action, anti-aging action, and hair-growth action. In addition, a cosmetic using a fermentation liquid by Kluyveromyces thermotolerance has at least one of excellent anti-inflammatory action, anti-aging action, and hair-growth action, and has excellent moisture retention. Found to have.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> An anti-inflammatory agent characterized by containing a fermented liquid by Kluyveromyces thermotolerans ( Kluyveromyces thermotolerans ).
<2> The anti-inflammatory agent according to <1>, wherein the fermentation broth is a brown sugar fermentation broth.
<3> The anti-inflammatory agent according to any one of <1> to <2>, which has a cyclooxygenase-2 (COX-2) activity inhibitory action.
<4> An anti-aging agent characterized by containing a fermentation broth by Kluyveromyces thermotolerance ( Kluyveromyces thermotolerans ).
<5> The anti-aging agent according to <4>, wherein the fermentation broth is a brown sugar fermentation broth.
<6> The anti-aging agent according to any one of <4> to <5>, which has at least one of an ATP production promoting action and a transglutaminase-1 production promoting action.
<7> A hair restorer characterized by containing a fermented liquid by Kluyveromyces thermotolerance ( Kluyveromyces thermotolerans ).
<8> The hair restorer according to <7>, wherein the fermentation broth is a brown sugar fermentation broth.
<9> The hair restorer according to any one of <7> to <8>, which has a testosterone 5α-reductase activity inhibitory action.
<10> The anti-inflammatory agent according to any one of <1> to <3>, the anti-aging agent according to any one of <4> to <6>, and any one of <7> to <9> A cosmetic comprising at least one of the hair-restoring agents described above.

According to the anti-inflammatory agent of the present invention, conventional problems can be solved, and an anti-inflammatory agent having excellent anti-inflammatory action and high safety can be provided.
According to the anti-aging agent of the present invention, conventional problems can be solved, and an anti-aging agent having an excellent anti-aging action and high safety can be provided.
According to the hair restorer of the present invention, it is possible to solve various problems in the prior art, and to provide a hair restorer having an excellent hair restoring action and high safety.
According to the cosmetic of the present invention, it is possible to solve various problems in the prior art, and have a superior anti-inflammatory action, anti-aging action, and hair restoration action, and a cosmetic having excellent moisture retention. Can be provided.

FIG. 1 is a diagram showing the results of a moisture retention test of the cosmetic of the present invention.

(Anti-inflammatory agents, anti-aging agents, and hair restorers)
The anti-inflammatory agent, anti-aging agent, and hair- restoring agent of the present invention contain a fermentation broth by Kluyveromyces thermotolerans , and further contain other components as necessary.

The Kluyveromyces thermotolerance is a lactic acid-producing yeast isolated from wine brewing or fermented plum jam. In addition, the Kluyveromyces thermotolerance is described in The Yeasts. A Taxonomic Study, 4th ed. Elsevier Science BV, Amsterdam. 240-241 (1998) and has biochemical and physiological properties.

The culture method of Kluyveromyces thermotolerans is not particularly limited as long as it is a culture method usually used for yeast, and can be performed according to a conventionally known method, for example, The Yeasts. A Taxonomic Study, 4th ed. Elsevier Science BV, Amsterdam. 240-241 (1998).

The medium components such as carbon source, nitrogen source, and inorganic salts used for culturing the Kluyveromyces thermotolerans are not particularly limited. For example, yeast comprising a carbon source such as glucose or sucrose, a nitrogen source such as yeast extract, peptone, corn steep liquor or ammonium nitrate, or an inorganic salt such as potassium dihydrogen phosphate or magnesium sulfate. In general, medium components commonly used are mentioned.

There is no restriction | limiting in particular as a culture medium used for culture | cultivation of the said Kluyveromyces thermotolerance ( Kluyveromyces thermotolerans ), According to the objective, it can select suitably according to the objective. The culture conditions such as pH, dissolved oxygen, culture temperature, and culture time in the medium are not particularly limited as long as they are culture conditions normally used for yeast, and can be appropriately selected according to the purpose. it can.

There is no restriction | limiting in particular as a fermentation liquid by the said Kluyveromyces thermotolerance ( Kluyveromyces thermotolerans ), Although it can select suitably according to the objective, Brown sugar fermentation is a point rich in minerals, such as magnesium and potassium. Liquid is preferred.

The method for preparing the fermentation broth by the Kluyveromyces thermotolerance is not particularly limited and can be appropriately selected according to the purpose. However, a brown sugar aqueous solution prepared with black sugar in water is aerated and stirred. After sterilization with the fermenter, adjust to a suitable temperature, inoculate Kluyveromyces thermotolerans ( Kluyveromyces thermotolerans ) pre-cultured with the medium into the brown sugar aqueous solution, perform aeration and agitation while controlling the culture temperature, fermentation And the method of preparation is preferred. In addition, there is no restriction | limiting in particular as said aeration stirring type fermenter, According to the objective, it can select suitably, For example, the aeration stirring type fermenter for bacteria, yeast, and filamentous fungi etc. are mentioned.

The brown sugar used in the method for preparing a fermentation broth by Kluyveromyces thermotolerans is produced by concentrating sugarcane juice as a raw material.

  There is no restriction | limiting in particular as said brown sugar, According to the objective, it can select suitably, For example, Wasanbon, molasses, black honey, etc. are mentioned.

  There is no restriction | limiting in particular as content in the brown sugar aqueous solution of the said brown sugar, Although it can select suitably according to the objective, 3 mass%-15 mass% are preferable, and 5 mass%-8 mass% are more preferable.

  The water in the brown sugar aqueous solution is not particularly limited and can be appropriately selected according to the purpose.For example, pure water, tap water, well water, mineral water, mineral water, hot spring water, spring water, fresh water, purified water, Examples include ion-exchanged water, physiological saline, phosphate buffer, and phosphate buffered saline.

There is no restriction | limiting in particular as a method of collect | recovering the fermented liquor by the said Kluyveromyces thermotolerance ( Kluyveromyces thermotolerans ), According to the objective, it can select suitably, For example, it collects using normal methods, such as centrifugation. can do. The recovered product can be used as it is or in a dried state.

The fermentation solution by Kluyveromyces thermotolerans is not particularly limited and can be appropriately selected according to the purpose. For example, the diluted solution or concentrated solution of the fermentation solution, Examples thereof include a dried product, or a crude product or a purified product thereof. There is no restriction | limiting in particular as a solution used when diluting the said fermentation broth, For example, a physiological saline solution is mentioned. There is no restriction | limiting in particular as a method of obtaining the dried material of the said fermented liquid, For example, it can obtain by methods, such as freeze drying and spray drying.

The fermentation liquid by Kluyveromyces thermotolerans can be purified for the purpose of decolorization, deodorization, etc. within a range that does not cause a decrease in its physiological activity. There is no restriction | limiting in particular as said refinement | purification, According to the objective, it can select suitably, For example, it can carry out by activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, etc.

A fermentation broth by Kluyveromyces thermotolerans has cyclooxygenase-2 (COX-2) activity inhibitory action, and the present invention is based on the cyclooxygenase-2 (COX-2) activity inhibitory action. The anti-inflammatory action of the anti-inflammatory agent is exhibited.
The fermentation broth by the Kluyveromyces thermotolerans has at least one of an ATP production promoting action and a transglutaminase-1 production promoting action, the ATP production promoting action, and the transglutaminase. The anti-aging effect of the anti-aging agent of the present invention is exerted on the basis of at least one of the -1 production promoting effects.
The fermented liquor by Kluyveromyces thermotolerans has testosterone 5α-reductase activity inhibitory action, and the hair growth effect of the hair restorer of the present invention is demonstrated based on the testosterone 5α-reductase activity inhibitory action. Is done.
In addition, the fermented liquid by the said Kluyveromyces thermotolerance ( Kluyveromyces thermotolerans ) is based on each above-mentioned effect | action, a cyclooxygenase-2 activity inhibitor, an ATP production promoter, a transglutaminase-1 production promoter, and testosterone 5α- Each can also be suitably used as a reductase activity inhibitor.

There is no restriction | limiting in particular as content of the fermented liquid by the said Kluyveromyces thermotolerance ( Kluyveromyces thermotolerans ) in the said anti-inflammatory agent, an anti-aging agent, and a hair restorer, It can select suitably according to the objective. However, 0.0001% by mass to 100% by mass is preferable, and the fermentation broth may be used at a low concentration, or the fermentation broth may be used as it is.

As other components other than the fermented liquid by Kluyveromyces thermotolerans , which can be contained in the anti-inflammatory agent, anti-aging agent, and hair restorer, within the range that does not impair the effects of the present invention. If there is no particular limitation, it can be appropriately selected according to the purpose. For example, a physiological saline for diluting the fermentation broth by Kluyveromyces thermotolerans to a desired concentration is available. Can be mentioned. Moreover, there is no restriction | limiting in particular as content of the said other component of the said anti-inflammatory agent, an anti-aging agent, and a hair restorer, According to the objective, it can select suitably.

  The dosage form of the anti-inflammatory agent, anti-aging agent, and hair restorer is not particularly limited and may be appropriately selected depending on the intended purpose. For example, any dosage form such as powder, granule, tablet, etc. Is mentioned. There is no restriction | limiting in particular as usage of the said anti-inflammatory agent, an anti-aging agent, and a hair restorer, According to the objective, it can select suitably, For example, oral, parenteral, external administration forms etc. are mentioned.

  The anti-inflammatory agent, anti-aging agent, and hair restorer of the present invention have excellent anti-inflammatory action, anti-aging action, and hair-restoring action, and are excellent in safety. For example, to the cosmetic of the present invention described later. It is suitable for use.

(Cosmetics)
The cosmetic of the present invention contains at least one of the above-described anti-inflammatory agent, anti-aging agent, and hair restorer of the present invention, and further contains other components as necessary.

The cosmetic may be a blend of a fermented liquid by the Kluyveromyces thermotolerance ( Kluyveromyces thermotolerans ) in an arbitrary cosmetic so as not to interfere with its activity. It may be a cosmetic mainly composed of a fermented liquid by thermotolerance ( Kluyveromyces thermotolerans ). Further, the cosmetic may be a fermented liquid by the Kluyveromyces thermotolerance itself.

There is no restriction | limiting in particular as said cosmetics, According to the objective, it can select suitably, For example, skin cosmetics, scalp cosmetics, hair cosmetics, etc. are mentioned.
The use of the cosmetic is not particularly limited and may be appropriately selected depending on the intended purpose. For example, ointment, cream, emulsion, lotion, lotion, pack, jelly, lip balm, lipstick, bath preparation, astringent , Hair tonic, shampoo, rinse, etc.

  The cosmetic can further contain other components such as various main agents and auxiliaries usually used in the production of the cosmetic as long as the purpose and action and effects of the present invention are not impaired. The other components are not particularly limited and may be appropriately selected depending on the intended purpose. For example, astringents, bactericides, antibacterial agents, whitening agents, ultraviolet absorbers, humectants, cell activators, fats and oils Waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances and the like.

The content of at least one of the anti-inflammatory agent, anti-aging agent, and hair restorer in the cosmetic is not particularly limited, and may be appropriately adjusted depending on the type of cosmetic and the physiological activity of the fermentation broth. However, for example, the amount of the fermentation liquid by Kluyveromyces thermotolerans is preferably 0.0001% by mass to 100% by mass, and the fermentation liquid may be used at a low concentration. May be used as is.

  The cosmetic of the present invention has high safety and at least one of an excellent cyclooxygenase-2 activity inhibitory action, ATP production promoting action, transglutaminase-1 production promoting action, and testosterone 5α-reductase activity inhibiting action. Demonstrates excellent moisture retention.

(effect)
The anti-inflammatory agent, anti-aging agent, hair restorer, and cosmetics of the present invention can be used on a daily basis, and are fermented liquids by the above-mentioned Kluyveromyces thermotolerance ( Kluyveromyces thermotolerans ). By the action of the above, anti-inflammatory action based on cyclooxygenase-2 activity inhibiting action, anti-aging action based on at least one of ATP production promoting action and transglutaminase-1 production promoting action, hair growth based on testosterone 5α-reductase activity inhibiting action The function and the moisture retention can be exhibited extremely effectively.

The anti-inflammatory agent, anti-aging agent, hair restorer, and cosmetics of the present invention are suitably applied to humans, but as long as their effects are exerted, animals other than humans (for example, Mouse, rat, hamster, dog, cat, cow, pig, monkey, etc.). In addition, the anti-inflammatory agent, anti-aging agent, hair restorer, and cosmetics of the present invention comprise a fermented solution by the above Kluyveromyces thermotolerance as an active ingredient, and are excellent in safety. This is also advantageous.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Preparation Example 1)
<Preparation of brown sugar fermentation broth by Kluyveromyces thermotolerans >
-Pre-culture-
Kluyveromyces thermotolerance ( Kluyveromyces thermotolerans ) was obtained from Akita Imano store and liquid culture was performed using YDP medium (pH 6.0). One platinum loop was taken from the liquid culture solution and inoculated into a YM medium (solid medium), and precultured at 25 ° C. for 2 days.

-Preparation of brown sugar aqueous solution-
500 parts by mass of pure water was added to 25 parts by mass of brown sugar to obtain a 5% by mass brown sugar aqueous solution. The 5 mass% brown sugar aqueous solution was sterilized together with the aeration and stirring type fermenter, and then adjusted to an appropriate temperature.

-Fermentation process-
The colony obtained by the pre-culture of Kluyveromyces thermotolerans was suspended in physiological saline, and 1.0 × 10 ⁹ CFU / mL to 1.0 × 10 ¹⁰ CFU / mL. 1% by mass of the total amount of the 5% by mass brown sugar aqueous solution is added to the 5% by mass brown sugar aqueous solution, and the shaking suspension (shaking condition: 100 rpm) is performed at 30 ° C. for 2 days. went. The culture solution after the shaking culture was sterilized by autoclaving (conditions: 121 ° C., 15 minutes) to prepare a brown sugar fermentation broth of Kluyveromyces thermotolerans ( Kluyveromyces thermotolerans ).

(Preparation Example 2)
<Preparation of ethanol-treated brown sugar fermentation broth of Kluyveromyces thermotolerans >
To the brown sugar fermentation liquid prepared in Preparation Example 1, 128 mL of 99 mass% ethanol was added to prepare an ethanol-treated brown sugar fermentation liquid.

(Comparative Preparation Example 1)
<Preparation of unfermented brown sugar from Kluyveromyces thermotolerans >
In the preparation of the brown sugar aqueous solution of Preparation Example 1, the prepared 5 mass% brown sugar aqueous solution was used as the brown sugar unfermented liquid.

(Production Example 1)
<Manufacture of cosmetic 1>
150 parts by mass of a butylene glycol (BG) solution is added to 350 parts by mass of the brown sugar fermentation broth prepared in Preparation Example 1 so that the BG solution is contained in an amount of 30% by mass with respect to the total amount of the solution. : 121 ° C., 15 minutes) to obtain a sterilized BG 30% by mass solution. Thereafter, the sterilized BG 30% by mass solution was prepared with a potassium hydroxide solution so as to have a pH of 6.5, and then impurities and residues were removed by Celite filtration (W-50, C # 65). The filtrate obtained by the celite filtration was prepared to have a solid content concentration of 0.1% by mass, BG of 10% by mass, and phenoxyethanol of 10% by mass. After the preparation, Celite filtration (W-50, C # 65) was performed again to produce Cosmetic 1.

(Comparative Production Example 1)
<Manufacture of comparative cosmetic 1>
A 10% by mass BG solution obtained by adding 90 parts by mass of pure water to 10 parts by mass of butylene glycol was produced as comparative cosmetic 1.

(Comparative Production Example 2)
<Manufacture of comparative cosmetics 2>
A 0.1 mass% glucose solution (solid content concentration 0.1 mass%) obtained by adding pure water to 100 mass parts by adding 0.1 mass parts of glucose was produced as comparative cosmetic 2.

(Experimental example 1)
<COX-2 activity inhibition test>
Brown sugar fermentation broth (Example) prepared in Preparation Example 1, ethanol-treated brown sugar fermentation liquid (Example) prepared in Preparation Example 2, and brown sugar unfermented liquid prepared in Comparative Preparation Example 1 (Comparative Example) Was used as a test sample, and the COX-2 activity inhibitory action was tested by the following test method.

Mouse macrophage cells (RAW264.7) were cultured using Dulbecco's MEM containing 10% by mass FBS and then collected with a cell scraper. The collected cells were diluted with Dulbecco MEM containing 10% by mass FBS so as to have a concentration of 2.0 × 10 ⁵ cells / mL, then seeded at 100 μL per well on a 96-well plate, and cultured for 18 hours. After completion of the culture, in order to acetylate and inactivate COX-1 already present and COX-2 expressed in a small amount, the medium was changed to a medium containing 500 μmol / L aspirin and cultured for 4 hours. The cells were washed 3 times with PBS (−), and 100 μL of the test sample dissolved in Dulbecco MEM containing 10% by mass FBS containing 0.5% by mass final DMSO was added to each well, and the final concentration was 1 μg / mL. 100 μL of lipopolysaccharide (LPS, E. coli 0111; B4, manufactured by DIFCO) dissolved in 10% by mass FBS-containing Dulbecco MEM was added and cultured for 16 hours. After completion of the culture, the amount of prostaglandin E _{2 in} the culture supernatant of each well was quantified using PGE2 EIA Kit (manufactured by Cayman Chemical). The results are shown in Table 1.
The calculation method of COX-2 activity inhibition rate is as follows.
COX-2 activity inhibition rate (%) = {1− (A−C) / (B−C)} × 100
A: Prostaglandin E ₂ amount at the time of test sample addition / LPS stimulation B: Prostaglandin E ₂ amount at the time of no test sample addition / LPS stimulation C: Prostaglandin E ₂ at the time of no test sample addition / LPS stimulation amount

  From Table 1, it was confirmed that the brown sugar fermentation broth has a concentration-dependent and significant COX-2 activity inhibitory action in the entire concentration range tested. Moreover, in the ethanol-treated brown sugar fermentation broth, it was confirmed that the COX-2 activity inhibitory action becomes weaker as the concentration increases.

(Experimental example 2)
<ATP production promoting action test>
Brown sugar fermentation broth (Example) prepared in Preparation Example 1, ethanol-treated brown sugar fermentation liquid (Example) prepared in Preparation Example 2, and brown sugar unfermented liquid prepared in Comparative Preparation Example 1 (Comparative Example) Was used as a test sample, and the ATP production promoting action was tested by the following test method.

Normal human neonatal foreskin keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte long-term culture growth medium (EpiLife-KG2), and then cells were collected by trypsin treatment. The collected cells were diluted with the above medium to a concentration of 2.0 × 10 ⁵ cells / mL, then seeded at 100 μL per well on a collagen-coated 96-well plate and cultured overnight. After completion of the culture, the medium was removed, and 100 μL of the test sample dissolved in the medium was added to each well and cultured for 2 hours. For ATP production promoting action, the amount of ATP in the cells was measured using a firefly luciferase luminescence method. That is, after completion of the culture, 100 μL of ““ Cellular ”ATP measurement reagent” (manufactured by Toyo B-Net Co., Ltd.) was added to each well. After the reaction, the amount of chemiluminescence was measured. The results are shown in Table 2.
The calculation method of the ATP production promotion rate is as follows.
ATP production promotion rate (%) = A / B × 100
A: Chemiluminescence amount in cells when test sample is added B: Chemiluminescence amount in cells when test sample is not added

  From Table 2, it was confirmed that the brown sugar fermented liquid has an ATP production promotion effect compared with the brown sugar non-fermented liquid. It was also confirmed that the ethanol-treated brown sugar fermentation broth has an ATP production promoting effect.

(Experimental example 3)
<Transglutaminase-1 production promoting action test>
Brown sugar fermentation broth (Example) prepared in Preparation Example 1, ethanol-treated brown sugar fermentation liquid (Example) prepared in Preparation Example 2, and brown sugar unfermented liquid prepared in Comparative Preparation Example 1 (Comparative Example) Was used as a test sample, and the transglutaminase-1 production promoting effect was tested by the following test method.

Human normal neonatal skin epidermal keratinocytes (NHEK) were cultured using human normal neonatal epidermal keratinocyte medium (KGM), and then cells were collected by trypsin treatment. The collected cells were diluted with the medium so as to have a concentration of 1 × 10 ⁵ cells / mL, then seeded at 100 μL per well on a 96-well plate, and cultured for 2 days. After completion of the culture, 100 μL of the test sample dissolved in the medium was added to each well and cultured for 24 hours. After completion of the culture, the medium was removed, the cells were fixed to a plate, and the amount of transglutaminase-1 expressed on the cell surface was measured by ELISA using a monoclonal anti-human transglutaminase-1 antibody. The results are shown in Table 3.
The calculation method of the transglutaminase-1 production promotion rate is as follows.
Transglutaminase-1 production promotion rate (%) = A / B × 100
A: Absorbance at a wavelength of 405 nm when a test sample is added B: Absorbance at a wavelength of 405 nm when no test sample is added (control)

  From Table 3, a significant transglutaminase-1 production promoting action was confirmed in the brown sugar fermentation broth. Moreover, the transglutaminase-1 production promotion effect was confirmed also about the ethanol-treated brown sugar fermentation broth.

(Experimental example 4)
<Testosterone 5α-reductase activity inhibitory action test>
Brown sugar fermentation broth (Example) prepared in Preparation Example 1, ethanol-treated brown sugar fermentation liquid (Example) prepared in Preparation Example 2, and brown sugar unfermented liquid prepared in Comparative Preparation Example 1 (Comparative Example) Was used as a test sample, and the testosterone 5α-reductase activity inhibitory action was tested by the following test method.

  Tris-HCl buffer solution (concentration 5 mmol / mL, pH 7.13) containing 20 μL of testosterone (concentration 4.2 mg / mL) prepared with propylene glycol and NADPH (concentration 1 mg / mL) in a V-bottom test tube with a lid 825 μL was mixed. To this, 80 μL of the test sample prepared with 50% by mass of ethanol and 75 μL of rat hepatocytes (S-9) were added and mixed again. After reacting at 37 ° C. for 30 minutes, 1 mL of methylene chloride was added to react. Stopped. This was centrifuged (1,600 × g, 10 minutes) with a centrifuge, and the methylene chloride layer was subjected to gas chromatography analysis under the following conditions. Moreover, the blank test was done by the same method. The rat hepatocyte (S-9) is an SD rat male administered with an enzyme inducer (phenobarbital, 5,6-benzoflavone) intraperitoneally, and then the liver is ground to 9,000 × g. It is the centrifuged supernatant.

-Gas chromatography conditions-
Equipment used: Shimadzu GC-7A
Column: DB-1701 (φ0.53 mm × 30 m, film thickness; 1.0 μm)
Column / injection temperature: 240 ° C / 300 ° C
Detector: FID
Carrier gas: Nitrogen gas

In advance, 3α-androstanediol, dihydrotestosterone (DHT), and methylene chloride solutions of standard testosterone were similarly analyzed by gas chromatography, and the amount of compounds per peak area was determined by precise weighing and peak area of these three compounds. Has been calculated. And the density | concentration per each peak area in 3 (alpha) -androstanediol, dihydrotestosterone (DHT), and a testosterone after reaction by a rat hepatocyte (S-9) was calculated | required by following formula (1). Then, according to following formula (2), the conversion rate of the test sample was calculated | required. And based on the said conversion rate, according to following formula (3), the testosterone 5 alpha-reductase activity inhibition rate was calculated | required. The testosterone 5α-reductase activity inhibition rate is shown in Table 4. Further, Table 5 shows the results of calculating the 50% inhibitory activity concentration (IC ₅₀ : μg / mL).
Concentration (%) = peak area of test sample x standard product concentration / peak area of standard product (1)
Conversion rate (%) = (A + B) / (A + B + C) Equation (2)
A: concentration of 3α-androstanediol B: concentration of dihydrotestosterone (DHT) C: concentration of testosterone Testosterone 5α-reductase activity inhibition rate (%)
= (1-E / D) × 100 Formula (3)
D: Conversion rate in blank test E: Conversion rate in addition of test sample

  From Table 4 and Table 5, all the test samples showed concentration-dependent testosterone 5α-reductase activity inhibitory action, but the testosterone 5α-reductase activity inhibitory action by ethanol-treated brown sugar fermentation broth was the highest, and then It was confirmed that the testosterone 5α-reductase activity inhibitory action by the brown sugar fermentation broth was high.

(Experimental example 5)
<Moisture retention test of cosmetics>
Cosmetic 1 prepared in Production Example 1 (Example), Comparative Cosmetic 1 prepared in Comparative Production Example 1 (Comparative Example), and Comparative Cosmetic 2 prepared in Comparative Production Example 2 (Comparative Example), In addition, purified water (comparative example) was used as a test sample, and the moisture retention was tested by the following test method.

  20 μL of each test sample was dropped onto a paper disk having a diameter of 8 mm (manufactured by ADVANTEC, mass of about 0.017 g). This was left to stand in a test room (room temperature 25 ° C., humidity 60%), and the weight was measured every minute for 0 minutes to 10 minutes. The water residual rate of each test sample was determined with the mass for 0 minute as 100%. The results are shown in Table 6 and FIG.

  From Table 6 and FIG. 1, it was confirmed that the cosmetic 1 containing brown sugar fermented liquid has moisture retention as compared with the comparative cosmetic 1 and the comparative cosmetic 2. Moreover, it turned out that the said cosmetics 1 are cosmetics which can anticipate moisture retention.

Since the anti-inflammatory agent of the present invention has an excellent cyclooxygenase-2 (COX-2) activity inhibitory action, it can be blended in anti-inflammatory drugs and other skin external preparations, beauty cosmetics and pharmaceuticals, Furthermore, it can be suitably used as a reagent for research.
Since the anti-aging agent of the present invention has at least one of an excellent ATP production promoting action and an excellent transglutaminase-1 production promoting action, the anti-aging agent is blended in an external preparation for skin including anti-aging drugs, It can be used in cosmetic cosmetics and pharmaceuticals, and can be suitably used as a research reagent.
Since the hair restorer of the present invention has an excellent testosterone 5α-reductase activity inhibitory action, it can be added to skin external preparations such as hair cosmetics, beauty cosmetics and pharmaceuticals, and further for research. It can be suitably used as a reagent.
Since the cosmetic of the present invention has excellent moisturizing properties, it can be suitably used as a cosmetic for cosmetics or a cosmetic preparation for external use.

Claims (7)

Kluyveromyces thermotolerance ( Kluyveromyces thermo)
an anti-inflammatory agent characterized by containing a brown sugar fermentation broth by Tolerans ). The anti-inflammatory agent according to claim 1, which has an activity of inhibiting cyclooxygenase-2 (COX-2) activity. Kluyveromyces thermotolerance (Kluyveromyces thermo)
an anti-aging agent characterized by containing a brown sugar fermentation broth by Tolerans). At least one of ATP production promoting action and transglutaminase-1 production promoting action
The anti-aging agent of Claim 3 which has these. Kluyveromyces thermotolerance (Kluyveromyces thermo)
a hair growth agent characterized by containing a brown sugar fermentation liquor by Tolerans). The hair restorer according to claim 5, which has a testosterone 5α-reductase activity inhibitory action. The anti-inflammatory agent according to any one of claims 1 to 2, and the anti-aging agent according to any one of claims 3 to 4.
And at least one of the hair restorer according to any one of claims 5 to 6.
Cosmetics characterized by.

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