WO2023149408A1 - Cosmetic composition, cosmetic composition for epidermal keratinocyte proliferation, epidermal keratinocyte proliferation agent, antioxidant, cosmetic composition for dermal papilla cell proliferation, dermal papilla cell proliferation agent, hair restorer, loricrin gene expression promoter, heme oxygenase-1 gene expression promoter, and cornified envelope formation promoter - Google Patents

Abstract

The present invention provides a cosmetic composition, a cosmetic composition for epidermal keratinocyte proliferation, an epidermal keratinocyte proliferation agent, an antioxidant, a cosmetic composition for dermal papilla cell proliferation, a dermal papilla cell proliferation agent, and a hair restorer.　The problem is solved by a cosmetic composition, a cosmetic composition for epidermal keratinocyte proliferation, an epidermal keratinocyte proliferation agent, an antioxidant, a cosmetic composition for dermal papilla cell proliferation, a dermal papilla cell proliferation agent, and a hair restorer containing a Euglena-derived substance and an animal cell-derived culture supernatant. The Euglena-derived substance may be a Euglena aqueous solvent extract or a water-soluble component extracted upon performing protein enzymatic decomposition of cells of algae belonging to the genus Euglena. The culture supernatant may be a culture supernatant of an embryonic membrane derived from a fertilized bird egg, or specifically a supernatant of a chicken extraembryonic membrane cell conditioned medium.

Description

Cosmetic composition, cosmetic composition for proliferation of epidermal keratinocytes, agent for proliferation of epidermal keratinocytes, antioxidant, cosmetic composition for proliferation of dermal papilla cells, agent for proliferation of dermal papilla cells, hair restorer, promotion of loricrin gene expression agent, heme oxygenase-1 gene expression promoter and cornified envelope formation promoter

The present invention provides a cosmetic composition, a cosmetic composition for proliferating epidermal keratinocytes, an agent for proliferating epidermal keratinocytes, an antioxidant, a cosmetic composition for proliferating dermal papilla cells, an agent for proliferating dermal papilla cells, a hair restorer, The present invention relates to a loricrin gene expression promoter, a heme oxygenase-1 gene expression promoter and a cornified envelope formation promoter.

Algae belonging to the genus Euglena have traditionally been studied as supplements and feed, and are also used as raw materials for hair restorers and cosmetics. Patent Document 1 describes a cosmetic containing, as an active ingredient, a water-soluble component extracted by proteolytic decomposition of algal cells belonging to the genus Euglenoids.

In addition, in cultures using tissues and cells taken from animals, culture supernatants containing cell metabolism/secretion factors are used to promote cell growth/differentiation during culture. Patent Document 2 describes an animal cell culture growth promoter containing, as an active ingredient, culture supernatant of embryonic membranes derived from fertilized eggs of birds or reptiles.

JP 2010-90065 A WO2020/096004

There is a demand for technology that maintains the condition of the skin. An object of the present invention is to provide a cosmetic composition, a cosmetic composition for proliferation of epidermal keratinocytes, an agent for proliferation of epidermal keratinocytes, an antioxidant, a cosmetic composition for proliferation of dermal papilla cells, an agent for proliferation of dermal papilla cells, and a hair restorer. agent, a loricrin gene expression promoter, a heme oxygenase-1 gene expression promoter, and a cornified envelope formation promoter.

The above problems are solved by a cosmetic composition containing a Euglena-derived substance and an animal cell-derived culture supernatant.
The above-mentioned problems are solved by a cosmetic composition for epidermal keratinocyte proliferation containing a Euglena-derived substance and a culture supernatant of embryonic membranes derived from avian fertilized eggs.
The above problems are solved by an epidermal keratinocyte proliferating agent containing a Euglena-derived substance and a culture supernatant of embryonic membranes derived from avian fertilized eggs.
The above problems are solved by an antioxidant containing a Euglena-derived substance and a culture supernatant of embryonic membranes derived from avian fertilized eggs.
The above problems are solved by a cosmetic composition for dermal papilla cell proliferation containing a Euglena-derived substance and an animal cell-derived culture supernatant.
The above problems are solved by a dermal papilla cell proliferation agent containing a Euglena-derived substance and an animal cell-derived culture supernatant.
The above problems are solved by a hair restorer containing a Euglena-derived substance and an animal cell-derived culture supernatant.
The above problems are solved by a loricrin (LOR) gene expression promoter containing a Euglena-derived substance and a culture supernatant of embryonic membranes derived from avian fertilized eggs.
The above-mentioned problems are solved by a heme oxygenase-1 (HMOX1) gene expression promoter containing a Euglena-derived substance and a culture supernatant of an embryonic membrane derived from avian fertilized eggs.
The above problems are solved by a cornified envelope formation promoter containing a Euglena-derived substance and a culture supernatant of embryonic membranes derived from avian fertilized eggs.
At this time, the Euglena-derived substance is preferably an aqueous solvent extract of Euglena or a water-soluble component extracted by proteolytic decomposition of algal cells belonging to the genus Euglena.
At this time, the culture supernatant is preferably a culture supernatant of embryonic membranes derived from fertilized avian eggs.
At this time, the culture supernatant is preferably the supernatant of chicken extraembryonic membrane cell conditioned medium.

According to the present invention, a cosmetic composition capable of maintaining skin condition, a cosmetic composition for epidermal keratinocyte proliferation, an epidermal keratinocyte proliferation agent, an antioxidant, and a cosmetic for dermal papilla cell proliferation A composition, dermal papilla cell proliferation agent, hair restorer, loricrin gene expression promoter, heme oxygenase-1 gene expression promoter, and cornified envelope formation promoter can be provided.

Fig. 4 is a graph showing proliferation of human epidermal keratinocytes when various raw materials were added (***p<0.001, **p<0.01, t-test (Bonferroni correction) vs. control). 1 is a graph showing the results of loricrin (LOR) gene expression analysis. FIG. 10 is a graph showing the results of heme oxygenase (HMOX1) gene expression analysis. FIG. Graph showing proliferation of human dermal papilla cells upon addition of various raw materials (+p<0.1, *p<0.05, ***p<0.001, t-test (Bonferroni correction) vs. control). .

An embodiment (this embodiment) of the present invention will be described below with reference to FIGS. 1 to 4. FIG. The present embodiment provides a cosmetic composition, a cosmetic composition for epidermal keratinocyte proliferation, and an epidermal keratinocyte proliferation agent containing a Euglena-derived substance and a culture supernatant of an embryonic membrane derived from avian fertilized eggs. , antioxidant, cosmetic composition for dermal papilla cell proliferation, dermal papilla cell proliferation agent, hair restorer, loricrin (LOR) gene expression promoter, heme oxygenase-1 (HMOX1) gene expression promoter and cornified envelope formation promoter It relates to agents.

<Euglena>
In the present embodiment, the term "euglena" includes microorganisms taxonomically classified into the genus Euglena, variants thereof, variants thereof, and closely related species of the family Euglenaceae. Here, the genus Euglena is a group of eukaryotic organisms belonging to Excoverta, Euglenozoa, Euglenomycetes, Euglenoides, and Euglenaceae.

Specific examples of species included in the genus Euglena include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, and Euglena viridis. As Euglena, Euglena gracilis, particularly E. gracilis Z strain can be used, but in addition, Euglena gracilis Z strain mutant strain SM-ZK strain (chloroplast-deficient strain), variants of E. gracilis var.

The genus Euglena is widely distributed in freshwater such as ponds and marshes, and it may be used by separating it from these, or any already isolated Euglena genus may be used. The genus Euglena includes all its variants. These mutant strains also include those obtained by genetic methods such as recombination, transduction, and transformation.

(Euglenoid algae)
In the present embodiment, Euglenoid algae can be used as Euglena. Euglenoid cells separated by centrifugation, filtration, sedimentation, or the like can be used as they are as Euglenoid alga bodies. Euglena living cells can be used as they are after being harvested from the culture vessel, but they are preferably washed with water or physiological saline. Alternatively, the Euglenoid alga body may be used in the form of a dispersion in a liquid such as water. In the present embodiment, it is preferable to use dry Euglena alga bodies (Euglena powder) obtained by freeze-drying or spray-drying living Euglena cells as Euglena alga bodies.

Furthermore, a mechanically processed product of algal bodies obtained by subjecting living Euglena cells to ultrasonic irradiation treatment or mechanical treatment such as homogenization may be used as Euglena algal bodies. Alternatively, a dried mechanically processed product obtained by subjecting a mechanically processed product to a drying treatment may be used as the Euglenoid algal body.

(Euglena extract)
In the present embodiment, it is possible to use an Euglena extract (Euglena extract) as Euglena, and it is particularly preferable to use an Euglena aqueous solvent extract. In the present embodiment, "Euglena aqueous solvent extract" means an extract extracted from Euglena using an aqueous solvent. It is preferable to use a water extract, a hot water extract, an alcohol extract, or a glycol extract of Euglena extracted for several seconds to several tens of hours at ℃. The water used for extraction does not necessarily have to be distilled, pure, or ultrapure water, and may, for example, be tap water or contain impurities, but may contain components that interfere with the extraction of the active ingredient. Free water is preferred.

In the present embodiment, "water extract" means an extract with water at 0 to 50°C (excluding 0°C). Here, "water" means water at 0 to 50°C (excluding 0°C). The temperature of the water is not particularly limited as long as the active ingredient can be sufficiently extracted without affecting the active ingredient. Especially preferred is 10 to 30°C.

In the present embodiment, the "hot water extract" means an extract with water at a temperature higher than 50°C, and can also be called a "hot water extract". Here, “hot water” means water having a temperature higher than 50° C., and is a concept that includes “hot water”, including water in a boiling state. Moreover, it is not limited to hot water in a liquid state, but also includes hot water in a gaseous state and a supercritical state. The temperature of the hot water is not particularly limited as long as the active ingredient can be extracted sufficiently without affecting the active ingredient. °C and 100°C or less.

The pH of the water used for extraction is not particularly limited as long as the active ingredient can be sufficiently extracted without affecting the active ingredient, preferably pH 4 to 10, more preferably pH 5 to 9. , particularly preferably pH 6-8.

In the present embodiment, as the aqueous solvent, one or more solvents that can sufficiently extract the active ingredient without affecting the active ingredient and that can be used for extraction are usually selected. may be used. Examples include, but are not limited to, water, alcohols, glycols, and the like. Alcohols include ethanol, methanol, n-propanol, isopropanol and the like. Glycols include 1,3-butylene glycol (BG) and propylene glycol. Other aqueous solvents include acetone and the like. These solvents may be used alone or as an aqueous solution, or may be used as a mixed solvent of any two or three or more.

The temperature of the aqueous solvent used for extraction is, for example, 0°C or higher, and is not particularly limited as long as it does not affect the active ingredients. Aqueous solvents in a boiling or supercritical state can also be used, but it is preferred to use aqueous solvents at 5°C to 600°C, more preferably 10°C to 200°C. Therefore, the aqueous solvent for extraction includes an aqueous solvent in a boiling state or a supercritical state. The amount of the aqueous solvent used for extraction is preferably an amount that can sufficiently dissolve the water-soluble active ingredient contained in Euglena.

The extraction method is also not particularly limited. For example, extraction can be performed by the method shown below, but it is not limited to this, and a normal extraction method can be freely selected and used. For example, a dry powder of Euglena algae is immersed in an aqueous solvent for a predetermined period of time and then centrifuged or filtered, or a dry powder of Euglena algae is added to an aqueous solvent and shaken to uniformly disperse, followed by centrifugation or filtration. method, etc. Moreover, in order to promote extraction, it is also possible to heat the aqueous solvent after adding Euglena.

Euglena can be extracted with water by the usual method shown below, but it is not limited to this. For example, the Euglena tissue and water are placed in a container and allowed to stand for a predetermined period of time with appropriate stirring or shaking, and the obtained extract can be used as a water extract as it is. Moreover, for example, the supernatant obtained by centrifuging such an extract can also be used as an aqueous extract. Also, such an extract or supernatant can be concentrated and dried to remove water and used as an aqueous extract. The water extraction may be carried out by adding a small amount of alcohol, preferably ethanol, to water in an amount of, for example, 10% by mass or less, in order to increase the extraction efficiency and shorten the extraction time. The extraction time for water extraction is not particularly limited as long as the active ingredient is extracted, and can be appropriately set in the range of several seconds to several tens of hours depending on the extraction temperature.

The extraction with hot water can be performed by a commonly used method as shown below, but it is not limited to this. After Euglena is introduced into a commonly used extractor together with water, it is extracted by heating. When boiling water or water in a supercritical state is used for extraction, it is necessary to use an extractor that can withstand the vapor pressure of water. The pressure during extraction can be set to 1 to 5000 atmospheres, preferably 60 to 400 atmospheres.

When extracting at high temperature and high pressure, if the extraction time is too long, the active ingredients may decompose or chemical reactions may occur. Therefore, when the extraction is performed under high temperature and high pressure, the extraction time is short, for example, preferably within 3 minutes, more preferably within 1 minute, and particularly preferably within 30 seconds.

The extracted Euglena extract can be used as it is for the epidermal keratinocyte proliferation agent according to the present embodiment, the cosmetic composition for dermal papilla cell proliferation, the dermal papilla cell proliferation agent, the hair restorer, the loricrin gene expression promoter, and the heme oxygenase-1. Although it can be used as an active ingredient of a gene expression promoter and a cornified envelope formation promoter, the extract is further subjected to an appropriate separation means (e.g., partition extraction, gel filtration, silica gel chromatography, reverse phase or normal phase). It is also possible to fractionate and use a highly active fraction by high-performance liquid chromatography, etc.). Alternatively, the Euglena extract or a fraction thereof can be concentrated and dried to remove the aqueous solvent, and this can be used as an aqueous solvent extract.

Alternatively, an extract extracted using BG may be used as the Euglena extract. The Euglena extract may be in the form of a complex with other cosmetic raw materials, such as permeation type collagen, permeation type elastin, permeation type hyaluronic acid, and hexapeptide-33 in a multi-layered capsule.

(Hydrolyzed euglena extract)
As the Euglena extract, a water-soluble component extracted by enzymatically decomposing algal cells belonging to the genus Euglena, specifically, a hydrolyzed Euglena extract obtained by enzymatically hydrolyzing and extracting Euglena powder (Euglena algae body) is used. can be Hydrolyzed Euglena extract can be prepared according to the method described in JP-A-2010-90065.

The method for preparing the hydrolyzed Euglena extract will be explained below. Purified water is preferably added in an amount (weight) 100 times the amount (weight) of the dried Euglena, and pressure and heat treatment is performed. After that, a proteolytic enzyme is added to treat the algal body. After completion of the treatment, it is deactivated, for example, at 90° C., and the residue and water-soluble components are separated by centrifugation or filtration.

Specifically, the pressure and heat treatment conditions are preferably 100 to 150 ° C. using an autoclave, atmospheric pressure to 0.255 MPa, and heat and pressure treatment for 1 minute to 30 minutes. It is preferable to heat and pressurize at 0.14 MPa and 121° C. for 10 minutes. As the proteolytic enzyme, for example, pepsin, pancreatin, papain, and other commonly used enzymes having protease activity may be used alone or in combination. It is preferred to employ an endo-type peptidase to decompose into .

As commercially available proteases, Pantidase MP and Alloase AP-10 manufactured by Yakult Pharmaceutical Co., Ltd. can also be used. The concentration of the enzyme added, the pH and reaction temperature of the reaction solution, and other conditions may be selected optimally for each enzymatic agent.

The water-soluble component obtained in this way can be used as it is, but it can also be used after undergoing purification operations such as fractionation, deodorization, decolorization, and concentration within the range that does not lose the effect of the present invention.

<Paramylon>
"Paramylon" is a porous polymer (β-1,3-glucan) in which about 700 glucoses are polymerized by β-1,3-bonds, and is a storage polysaccharide contained in the genus Euglena. is. Paramylon particles are flattened spheroidal particles and are formed by helically intertwined β-1,3-glucan chains.

Paramylon exists as granules in the Euglena cells of all species and varieties, and the number, shape, and uniformity of the particles are characteristic for each species. Paramylon is composed only of glucose, and the average degree of polymerization of paramylon obtained from E. gracilis Z wild strain and chloroplast-deficient strain SM-ZK is about 700 glucose units. Paramylon is insoluble in water and hot water, but soluble in dilute alkali, strong acid, dimethyl sulfoxide, formaldehyde and formic acid. The average density of paramylon is 1.53 for E. gracilis Z and 1.63 for E. gracilis var. bacillaris SM-L1.

According to X-ray analysis using the powder pattern method, paramylon has a gentle helical structure in which three straight-chain β-1,3-glucans are twisted like a right-handed rope. Several of these glucan molecules aggregate to form paramylon granules. Paramylon granules have a very large crystal structure portion, accounting for about 90%, and are compounds with the highest crystal structure rate among polysaccharides (Euglena, Physiology and Biochemistry, edited by Shozaburo Kitaoka, Gakkai Shuppan Center). The particle size distribution of paramylon (manufactured by Euglena Co., Ltd.) has a median diameter of 1.5 to 2.5 μm when measured with a laser diffraction/scattering particle size distribution analyzer.

Paramylon particles are isolated from cultured Euglena cells by any appropriate method and purified into fine particles, and are usually provided as powder. For example, paramylon particles can be (1) cultured Euglena cells in any appropriate medium, (2) separation of Euglena cells from the medium, (3) isolation of paramylon from the separated Euglena cells, ( 4) purification of the isolated paramylon, and optionally (5) cooling and subsequent lyophilization. Isolation of paramylon is performed, for example, using non-ionic or anionic detergents of the type that are largely biodegradable. Purification of paramylon occurs substantially simultaneously with isolation.

(Processed product of paramylon)
Processed products of paramylon include water-soluble paramylon obtained by chemically or physically treating paramylon by various known methods, sulfated paramylon, and paramylon derivatives.

Examples of processed products of paramylon include amorphous paramylon and emulsion paramylon. Amorphous paramylon is a substance obtained by amorphizing crystalline paramylon derived from Euglena. Amorphous paramylon has a relative crystallinity of 1 to 20% with respect to crystalline paramylon produced from Euglena by a known method. However, this relative crystallinity is obtained by the method described in JP-A-2011-184592.

That is, amorphous paramylon and paramylon were each pulverized with a pulverizer (ball mill MM400 manufactured by Retsh) for 5 minutes at a frequency of 20 times/second, and then analyzed using an X-ray diffractometer (H'PertPRO manufactured by Spectris). At a voltage of 45 KV and a tube current of 40 mA, scanning is performed in the range of 2θ from 5° to 30° to obtain diffraction peaks Pc and Pa near 2θ=20° of paramylon and amorphous paramylon. Using the values of Pc and Pa, the relative crystallinity of amorphous paramylon is calculated by the relative crystallinity of amorphous paramylon=Pa/Pc×100 (%).

Amorphous paramylon is prepared according to the method described in JP-A-2011-184592 by treating crystalline paramylon powder with an alkali, then neutralizing it with an acid, followed by washing, removing water, and drying. Processed products of paramylon also include water-soluble paramylon obtained by chemically or physically treating paramylon by various known methods, sulfated paramylon, and paramylon derivatives.

"Emulsion paramylon" is a substance also called emulsion paramylon because its processing method and physical properties are similar to emulsions, and is obtained by adding water to paramylon according to the method described in JP-A-2016-199650. It is processed paramylon that is swollen by bonding with water four times or more, obtained by performing a collision treatment in which the fluid is ejected from a fine nozzle at an ultrahigh pressure to collide with an object to be collided.

Emulsion paramylon is a known physical property modification device that ejects a slurry obtained by adding a water-soluble solvent to a solid such as powder at an ultrahigh pressure from a fine-pore nozzle to collide with an object to be collided (for example, JP-A-2011-88108). It can be obtained by performing collision treatment one or more times at a nozzle pressure of 245 MPa at the time of jetting using a device described in Japanese Patent Application Laid-Open No. 6-47264.

Emulsion paramylon has a median diameter of 5 times or more that of paramylon and 7 μm or more when the particle size is measured with a laser diffraction/scattering particle size distribution analyzer, and the particles are separated from adjacent particles by an optical electron microscope. Adhesion was observed, and it swelled by binding with water four times or more that of paramylon.

Slurry, which is a mixture of raw material paramylon and water, is a free-flowing fluid. It has excellent stickiness and elasticity, and has a paste-like feel. The processed paramylon obtained is called emulsion paramylon in this specification because of its processing method and physical properties, but it is unknown whether or not it is emulsified. is.

<Culture supernatant of embryonic membrane derived from fertilized bird eggs>
Cosmetic composition of the present embodiment, cosmetic composition for epidermal keratinocyte proliferation, epidermal keratinocyte proliferation agent, cosmetic composition for dermal papilla cell proliferation, dermal papilla cell proliferation agent, hair restorer, loricrin (LOR) The gene expression promoter, heme oxygenase-1 (HMOX1) gene expression promoter, and cornified envelope formation promoter contain the culture supernatant of avian egg embryo membrane (animal cell-derived culture supernatant) as an active ingredient. . Examples of birds from which embryonic membranes are collected include quails, chickens, and the like, but are not limited to these, and any avian animal having egg embryonic membranes may be used.

Here, embryonic membrane means a membranous tissue that is formed outside the embryo during the development process of birds, performs functions such as protection, nutrition, and respiration of the embryo, and does not participate in the construction of the body after hatching or birth. , for example, the outermost chorion of the embryo, the amniotic membrane that envelops the embryo body, the allantois that forms the allantois, the chorioallantoic membrane (CAM) that partially adheres to the chorion and the allantoic membrane, and the yolk that envelops the yolk. Sac etc. are mentioned.

The embryonic membrane culture supernatant is the medium in which the embryonic membrane is isolated and cultured. Embryonic membranes to be cultured may be one of the above-described serous membranes, amniotic membranes, allantoic membranes, allantoic membranes, yolk sacs, or the like, or may be cultured by mixing a plurality of types.

The embryonic membrane can be collected from the egg by a known method. For example, specifically, embryonic membranes can be isolated by the following method. First, a fertilized egg is incubated under conditions suitable for artificial incubation for a predetermined period of time, and then the eggshell is cracked. (2) the amniotic membrane can be obtained by removing the transparent membranous tissue that does not have blood vessels running around the fetus; (4) the serosa can be obtained by removing the outermost membranous tissue surrounding all elements within the egg; Furthermore, with longer incubation times, the chorioallantoic membrane partially fuses with the allantoic membrane to form the chorioallantoic membrane. At this stage, the chorioallantoic membrane can be obtained by removing the membranous tissue that is in contact with the eggshell and has blood vessels running through it. The incubation period may be appropriately determined depending on each animal and the target membrane tissue. For example, in the case of chicken eggs, 4 to 21 days is preferable, 10 to 18 days is more preferable, and 13 to 13 days is preferable. 15 days is more preferred. When these embryonic membranes are cultured, they may be cultured as they are in a membranous state. Alternatively, embryonic membrane-derived cells may be isolated and cultured from the embryonic membrane.

For culturing the embryonic membrane, a medium generally used for culturing animal cells can be used, and examples thereof include, but are not limited to, DMEM and F12. Supplements that are usually blended as medium additives may be added to this medium. As for the culture conditions, the conditions used for normal animal cell culture can be used, typically using a carbonate buffer-based medium and culturing at 5% CO ₂ at 37° C. The culture conditions are as follows. is not limited to, and can be appropriately selected by those skilled in the art. The culture time is not particularly limited, but preferably 1 to 7 days, more preferably 2 to 6 days. After culturing, the supernatant may be collected and added to the cell culture medium as it is, or a medium obtained by removing solids such as cell debris by filtration or centrifugation may be added. good. The amount of the culture supernatant added to the cell culture medium is not particularly limited as long as it is an effective amount at which the growth effect of cultured cells is observed, but the final concentration in the medium is preferably 20% or more, more preferably 35% or more. , more preferably 50% or more.

<Epidermal keratinocyte proliferation agent>
The epidermal keratinocyte proliferation agent according to the present embodiment contains a Euglena-derived substance and a culture supernatant of an embryonic membrane derived from avian fertilized eggs, and is used to promote the proliferation of epidermal keratinocytes. It is.

<Dermal papilla cell proliferation agent>
The dermal papilla cell proliferation agent according to the present embodiment contains a Euglena-derived substance and a culture supernatant of embryonic membrane derived from avian fertilized eggs, and is used to promote the proliferation of dermal papilla cells. be.

<Hair restorer>
The hair restorer according to the present embodiment contains a Euglena-derived substance and a culture supernatant of an embryonic membrane derived from avian fertilized eggs, and is used to promote hair growth through its effect of promoting proliferation of dermal papilla cells. It is.

<Loricrin (LOR) gene expression promoter>
The loricrin (LOR) gene expression promoter according to the present embodiment contains a Euglena-derived substance and a culture supernatant of an embryonic membrane derived from avian fertilized eggs, and promotes loricrin (LOR) gene expression. It is used for “Promote gene expression” includes not only expression at the transcription level (expression as mRNA) but also expression at the translation level (expression as protein).

Lochlorin is a hydrophobic protein rich in glycine, serine, cysteine, etc. Lochlorin is the major constituent of the cornified envelope. After the base of the cornified envelope is formed in the keratinocyte membrane by involucrin, cystatin A, etc., a firm cornified envelope is formed while loricrin bridges.

<Cornified envelope formation accelerator>
The cornified envelope is a proteinous membranous structure that envelops the corneocytes that make up the outermost layer of the skin. The cornified envelope is an organelle that plays a major role in the stratum corneum barrier function.

The cornified envelope formation promoter according to the present embodiment contains a Euglena-derived substance and a culture supernatant of embryonic membranes derived from avian fertilized eggs, and is used to promote the formation of cornified envelopes in keratin. It is something that can be done.

<Heme oxygenase-1 (HMOX1) gene expression promoter>
The heme oxygenase-1 (HMOX1) gene expression promoter according to the present embodiment contains a Euglena-derived substance and a culture supernatant of an embryonic membrane derived from avian fertilized eggs, and the heme oxygenase-1 (HMOX1) gene is used to promote the expression of “Promote gene expression” includes not only expression at the transcription level (expression as mRNA) but also expression at the translation level (expression as protein).

Heme oxygenase-1 (HMOX1) is an enzyme responsible for degrading heme, and its expression is induced when cells are exposed to reactive oxygen species or heavy metals, and exhibits antioxidant activity and cytoprotection.

<Antioxidant>
The antioxidant according to the present embodiment contains a Euglena-derived substance and a culture supernatant of avian fertilized egg-derived embryonic membranes.

<About target cells>
Target cells are not particularly limited, but eukaryotic cells are exemplified. Examples of cells include skin-related cells such as keratinocytes (keratinocytes), epithelial cells, fibroblasts, and dermal papilla cells, but other cells may also be used.

<Application>
The epidermal keratinocyte proliferation agent, dermal papilla cell proliferation agent, hair restorer, loricrin (LOR) gene expression promoter, heme oxygenase-1 (HMOX1) gene expression promoter, and cornified envelope formation promoter according to the present embodiment are It is configured as a food composition such as a cosmetic composition, a pharmaceutical composition, and a health food, and is preventively used, administered, ingested, and administered in order to maintain the skin condition.

(Cosmetic composition)
The epidermal keratinocyte proliferation agent, dermal papilla cell proliferation agent, hair restorer, loricrin (LOR) gene expression promoter, heme oxygenase-1 (HMOX1) gene expression promoter, and cornified envelope formation promoter according to the present embodiment are Epidermal keratinocyte proliferative action, dermal papilla cell proliferative action, hair growth action, loricrin (LOR) gene expression promotion action, heme oxygenase-1 (HMOX1) gene expression promotion action and cornified envelope formation promotion action are used to produce cosmetics. It can be suitably used for the composition. The cosmetic composition can be applied to any form of cosmetics. For example, skin care cosmetics such as cleansing agents, facial cleansers, lotions, lotions, milky lotions, creams, serums, packs, and scrubs, foundations, concealers, makeup bases, lipsticks, blushers, eyeshadows, eyeliners, hair growth lotions, It can be applied to shampoos, treatments, makeup cosmetics such as eyelash serums, sunscreen cosmetics, and the like.

The cosmetic composition according to the present embodiment includes the epidermal keratinocyte proliferation agent, the dermal papilla cell proliferation agent, the hair restorer, the loricrin (LOR) gene expression promoter, and the heme oxygenase-1 (HMOX1) gene according to the present embodiment. In addition to the expression enhancer and the cornified envelope formation enhancer, it is possible to freely select and blend one or two or more components that are commonly used in cosmetic compositions. For example, base materials, preservatives, emulsifiers, coloring agents, preservatives, surfactants, ultraviolet absorbers, antioxidants, moisturizing agents, ultraviolet absorbers, fragrances, antiseptic antifungal agents, extender pigments, coloring pigments, alcohols, All additives that can normally be used in the cosmetic field, such as water, can be included.

In the cosmetic composition according to the present embodiment, an epidermal keratinocyte proliferation agent, a dermal papilla cell proliferation agent, a hair restorer, a loricrin (LOR) gene expression promoter, a heme oxygenase-1 (HMOX1) gene expression promoter, and cornified The content of the enveloping promoter is not particularly limited, and can be freely set according to the purpose.

(Pharmaceutical composition)
The epidermal keratinocyte proliferation agent, dermal papilla cell proliferation agent, hair restorer, loricrin (LOR) gene expression promoter, heme oxygenase-1 (HMOX1) gene expression promoter, and cornified envelope formation promoter of the present embodiment are pharmaceuticals. in the field of epidermal keratinocyte proliferation, dermal papilla cell proliferation, hair growth, loricrin (LOR) gene expression promotion, heme oxygenase-1 (HMOX1) gene expression promotion and cornified envelope formation promotion. A pharmaceutical composition having the effect is provided by adding a pharmaceutically acceptable carrier and additives together with a sufficient amount of Euglena-derived substance and culture supernatant of embryonic membrane derived from avian fertilized eggs. be. The pharmaceutical composition may be a drug or a quasi-drug.

The pharmaceutical composition may be applied internally or externally. Therefore, the pharmaceutical composition is used in the form of an oral preparation, an injection such as an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection and/or an intraperitoneal injection, a transmucosal application, a transdermal application, and the like. be able to. The dosage form of the pharmaceutical composition can be appropriately set depending on the application form. Semi-solid formulations such as ointments or gels are included.

The pharmaceutical composition according to the present embodiment can contain one or more pharmaceutically acceptable additives that are freely selected. For example, when the pharmaceutical composition according to this embodiment is applied to an oral formulation, for example, excipients, binders, disintegrants, surfactants, preservatives, coloring agents, flavoring agents, fragrances, stabilizers, preservatives All additives that can be commonly used in the field of pharmaceutical formulations, such as agents, antioxidants, etc., can be contained. In addition, a drug delivery system (DDS) can be used to prepare a sustained-release preparation or the like.

(Food composition)
The epidermal keratinocyte proliferation agent, dermal papilla cell proliferation agent, hair restorer, loricrin (LOR) gene expression promoter, heme oxygenase-1 (HMOX1) gene expression promoter, and cornified envelope formation promoter of the present embodiment are foods. in the field of epidermal keratinocyte proliferation, dermal papilla cell proliferation, hair growth, loricrin (LOR) gene expression promotion, heme oxygenase-1 (HMOX1) gene expression promotion and cornified envelope formation promotion. By adding an effective amount of Euglena-derived substances that can be exhibited and the culture supernatant of embryonic membranes derived from avian fertilized eggs as food materials to various foods, food compositions having such effects can be provided. . That is, the present invention can provide a food composition for a food labeled as for epidermal keratinocyte proliferation or the like in the field of foods. Examples of the food composition include general foods, foods for specified health uses, foods with nutrient function claims, foods with function claims, foods for hospital patients, supplements, and the like. It can also be used as a food additive.

Examples of the food composition include seasonings, processed meat products, processed agricultural products, beverages (lactic acid beverages, soft drinks, alcoholic beverages, carbonated beverages, milk beverages, fruit juice beverages, tea, coffee, nutritional drinks, etc.), powders Beverages (powdered juice, powdered soup, etc.), concentrated drinks, confectionery (candies, cookies, biscuits, gums, gummies, chewables, tablets, chocolate, etc.), breads, cereals and the like. In the case of food for specified health uses, food with nutrient function claims, food with function claims, etc., the shape may be capsule, troche, syrup, granule, powder, or the like.

Foods for Specified Health Uses are foods that contain functional health ingredients that affect physiological functions, etc., and can be labeled as suitable for specific health uses with the permission of the Director-General of the Consumer Affairs Agency. be. In the present invention, it is a food product that is marketed with an indication of a specific health use related to proliferation of epidermal keratinocytes.

A food with nutrient function claims is a food that is used to supplement nutritional ingredients (vitamins, minerals), and that displays the functions of the nutritional ingredients. In order to be marketed as a food with nutrient function claims, the amount of nutrients contained in the recommended daily intake must be within the specified upper and lower limits. also need to

Foods with functional claims are foods labeled with functionalities based on scientific evidence under the responsibility of the business operator. Information on the grounds for safety and functionality was submitted to the Commissioner of the Consumer Affairs Agency before sales.

In the food composition according to the present embodiment, in addition to the Euglena-derived substance and the culture supernatant of embryonic membrane derived from avian fertilized eggs, one or more components that can be used in normal food compositions are added. It is possible to freely select and blend them. For example, all additives commonly used in the food field, such as various seasonings, preservatives, emulsifiers, stabilizers, flavoring agents, coloring agents, preservatives, and pH adjusters, can be contained.

The present invention will be specifically described below based on specific examples, but the present invention is not limited to these.

<Sample 1: Hydrolyzed Euglena extract>
As Sample 1, a hydrolyzed Euglena extract, which is a water-soluble component extracted by proteolytic decomposition of Euglena gracilis cells, was used. The hydrolyzed Euglena extract is obtained by adding 100 times the amount of purified water to 1 g of Euglena gracilis powder (Euglena algae, manufactured by Euglena Co., Ltd.), heating to a pressure of 0.1 MPa and a temperature of 12 ° C., and heating and pressurizing for 10 minutes. processed. After that, 5% of the total amount of proteolytic enzyme of endo-type protease (for example, pantidase MP manufactured by Yakult Pharmaceutical Co., Ltd.) was added, and the mixture was reacted with stirring at 35° C. for 16 hours. After the reaction, the water-soluble component was fractionated by filtering using a filter paper. A sample obtained by sterilizing the water-soluble component with a 0.45 μm filter was used as 100% hydrolyzed Euglena extract undiluted solution.

<Sample 2: Euglena extract (Euglena extract EX)>
As Sample 2, an Euglena extract was used. The Euglena extract was prepared using Euglena gracilis powder (Euglena algal body, manufactured by Euglena Co., Ltd.) as a raw material and 1,3-butylene glycol (BG) as a solvent.

<Sample 3: Chick egg extraembryonic membrane cell conditioned medium (cerament)>
As sample 3, a chicken egg-derived cell culture supernatant (trade name: CELLAMENT (registered trademark), Integriculture Co., Ltd.) was used. The egg-derived cell culture supernatant contains 75% chicken egg extraembryonic membrane cell conditioned medium, 20% BG, and 5% pentylene glycol.

<Sample 4: Euglena serum formulation>
A sample 4 Euglena serum formulation was prepared by combining the hydrolyzed Euglena extract of sample 1, the Euglena extract of sample 2 (Euglena extract EX), and the chick extraembryonic membrane cell conditioned medium (cerament) of sample 3.

<Test 1: Proliferation of human epidermal keratinocytes>
In Test 1, proliferation of human epidermal keratinocytes by the Euglena-cerament formulation (Sample 4) was evaluated.

(Method of Test 1)
Human epidermal keratinocytes (7500 cells/well) were seeded in a 24-well plate using HuMedia-KG2 medium (growth medium). Next, Euglena Cerament Formulation (Sample 4), Hydrolyzed Euglena Extract Alone (Sample 1), Euglena Extract EX Alone (Sample 2), and Cerament Alone (Sample 3) were each dissolved in HuMedia-KB2 medium (non-proliferation medium). and changed the medium. After replacing the medium, the cells were cultured for 3 days, and the cell count was measured using Cell Counting Kit-8.

(Results of Test 1)
Hydrolyzed Euglena extract alone (Sample 1), Euglena extract EX alone (Sample 2), and Cerament alone (Sample 3) promote the proliferation of human epidermal keratinocytes compared to controls cultured without adding anything. However, it was revealed that human epidermal keratinocytes proliferated additively in the Euglena-cerament formulation (Sample 4) (Fig. 1). This indicates the possibility that a higher effect can be achieved by combining the raw materials, rather than by using each raw material alone.

<Test 2: Gene change analysis using next-generation sequencer>
In Test 2, human epidermal keratinocytes (2.5×10 ⁴ cells/well) were seeded in a 12-well plate using HuMedia-KG2 medium (growth medium). Next, the Euglena-cerament formulation (Sample 4) was dissolved in HuMedia-KB2 medium (non-proliferation medium), and the medium was replaced. After replacing the medium, the cells were cultured for 3 days, the culture supernatant was removed, and total RNA was isolated from the cells using RNAiso Plus, followed by purification using NucleoSpin (registered trademark) RNA. RNA library construction and sequencing was performed using the TruSeq Stranded mRNA Library Prep Kit and NovaSeq 6000 (Illumina Inc, USA). In this test (n=1), FPKM values were calculated, and genes with |logFC|>1 were defined as variable genes. A next-generation sequencer detected 13,152 genes, and the addition of the Euglena-cerament formulation increased 24 genes and decreased 87 genes compared to the control cultured without adding anything. Enrichment analysis was performed on the variable genes whose expression level was increased by the euglena serum prescription, and it was found that autocrine signaling (hormones and chemical messengers secreted by cells act on the cells themselves), nuclear receptors meta-pathway (Nuclear receptors meta-pathway; pathway related to receptors that cause activation or suppression of transcription of DNA in the nucleus), estrogen signaling pathway (Estrogen signaling pathway), hair follicle development: It was found that gene clusters such as cytodifferentiation, aging, and skin development are included.

<Test 3: Formation of cornified envelope and analysis of antioxidant-related genes>
In human epidermal keratinocytes, Euglena-cerament formulation (Sample 4), hydrolyzed Euglena extract alone (Sample 1), Euglena extract EX alone (Sample 2), and Cerament alone (Sample 3) were added and cultured. Genetic changes were comprehensively analyzed using a generational sequencer. In addition, RNA was reverse transcribed to synthesize cDNA, and the gene expression level was evaluated by real-time PCR. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal standard for real-time PCR.

Regarding the expressed genes, when confirming the genes whose expression was increased by more than 2 times or whose expression was decreased to 1/2 or less, the gene expression level of loricrin (LOR), which is the main component of the cornified envelope increased about 4-fold in the serum formulation compared to the control cultured without adding anything (Fig. 2).

In addition, the gene expression of heme oxygenase-1 (HMOX1), an antioxidant enzyme, increased approximately twice in the serum formulation compared to the control (Fig. 3). These results were similar in experiments using RT-qPCR.

From the above results, it was shown that the Euglena serum formulation may promote the proliferation of human epidermal keratinocytes, and may be effective in the formation of cornified envelopes and antioxidants. In other words, by using Euglena-derived substances as active ingredients in combination with the culture supernatant of embryonic membranes derived from avian fertilized eggs, it is possible to promote the growth of human epidermal keratinocytes, promote the formation of cornified envelopes, It was shown that the antioxidative action can be effectively exhibited.

<Test 4: Proliferation of human dermal papilla cells>
In Test 4, the proliferation of human dermal papilla cells by the Euglena serum formulation (Sample 4) was evaluated.

(Method of Test 4)
Euglena serum formulation (sample 4), hydrolyzed euglena extract alone (sample 1), euglena extract EX alone (sample 2), and serum alone (sample 3) were added to human dermal papilla cells (4,000 cells/well/100 uL). The cells were added, cultured for 3 days, and the number of cells was measured using Cell Counting Kit-8.

(Results of test 4)
Hydrolyzed Euglena extract alone (Sample 1), Euglena extract EX alone (Sample 2), and Cerament alone (Sample 3) promote the proliferation of human epidermal keratinocytes compared to controls cultured without adding anything. However, in the Euglena serum formulation (Sample 4), it was revealed that human dermal papilla cells proliferated more cooperatively (Fig. 4). This indicates the possibility that a higher effect can be achieved by combining the raw materials, rather than by using each raw material alone.

Claims (13)

1. a Euglena-derived substance;
   A cosmetic composition containing an animal cell-derived culture supernatant.
2. The cosmetic composition according to claim 1, wherein the Euglena-derived substance is an aqueous solvent extract of Euglena or a water-soluble component extracted by proteolytic decomposition of algal cells belonging to the genus Euglena.
3. The cosmetic composition according to claim 2, wherein the culture supernatant is a culture supernatant of an embryonic membrane derived from avian fertilized eggs.
4. The cosmetic composition according to claim 3, wherein the culture supernatant is the supernatant of chicken extraembryonic membrane cell conditioned culture medium.
5. a Euglena-derived substance;
   and a culture supernatant derived from animal cells.
6. a Euglena-derived substance;
   An epidermal keratinocyte proliferation agent containing an animal cell-derived culture supernatant.
7. a Euglena-derived substance;
   and a culture supernatant derived from animal cells.
8. a Euglena-derived substance;
   A dermal papilla cell proliferation agent containing a culture supernatant derived from animal cells.
9. a Euglena-derived substance;
   A hair restorer containing an animal cell-derived culture supernatant.
10. a Euglena-derived substance;
    A cornified envelope formation promoter containing an animal cell-derived culture supernatant.
11. a Euglena-derived substance;
    A loricrin (LOR) gene expression promoter containing a culture supernatant derived from animal cells.
12. a Euglena-derived substance;
    A heme oxygenase-1 (HMOX1) gene expression promoter containing an animal cell-derived culture supernatant.
13. a Euglena-derived substance;
    An antioxidant containing a culture supernatant derived from animal cells.

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