JP2022166079A - Composition for controlling production of various factors containing mitol production-promoting component as active ingredient, mitol production promoter, and method for screening agent for controlling production of various factors with mitol production-promoting action as index - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide: a composition for promoting the production of various factors containing MITOL production-promoting components or a composition for controlling the production of the various factors; a MITOL production promoter; and an agent for promoting the production of various factors with MITOL production-promoting action as an index or a method for screening an agent for controlling the production of the various factors.

SOLUTION: Provided is an agent for suppressing the degrease of MITOL or promoting the production thereof, the MITOL containing, as an active ingredient, at least one selected from the group consisting of prunus persica extract, hamamelis extract, niacinamide, phellodendron bark extract, coptis rhizome extract, Japanese green gentian extract, β-nicotinamide mononucleotide, and D-panthenol. An SOD2 or COL17A1 production promoter or an MME production inhibitor comprising a prunus persica extract as an active ingredient is preferable.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2023,JPO&INPIT

Description

The present invention provides a composition for promoting the production of the factors listed in Table 1, a composition for suppressing the production of the factors listed in Table 2, a MITOL production promoting agent, and an MITOL production-promoting effect as an active ingredient, which contain a component that promotes MITOL production as an active ingredient. It relates to a screening method for a production regulator of various factors.

Mitochondria are one of the intracellular organelles present in cells, and their main function is to produce ATP, which is energy. Therefore, maintaining mitochondrial function leads to maintaining cell and body functions. Mitochondrial function has been shown to be regulated by mitochondrial fusion and fission, also called mitochondrial dynamics, and interactions with other intracellular organelles. MITOL, a ubiquitin ligase localized in the mitochondrial outer membrane, has been reported to be involved in mitochondrial dynamics and regulation of adhesion between mitochondria and the endoplasmic reticulum (Non-Patent Document 1). Cardiac-specific MITOL-deficient mice show decreased cardiac function, and lipofuscin deposition and increased expression of SA-β-gal, which are signs of cardiac aging, have been observed (non-patent literature). 2). In addition, aging-like findings such as white hair and hair loss have been observed in mice in which MITOL is specifically deficient in the skin epidermis (Non-Patent Document 3). From these facts, it is considered that the decrease in MITOL led to the promotion of aging symptoms. Therefore, approaches such as increasing or activating MITOL expression would be useful in anti-aging. However, no plant extract or the like that increases MITOL expression has been found.
With aging, skin aging symptoms such as dry skin, appearance of wrinkles, loss of firmness/elasticity, increase of spots and dullness, appearance of gray hair, and loss of muscle appear. Furthermore, as the muscle strength and muscle endurance of skeletal muscles decrease with age, various disorders and aging symptoms such as sluggish movement, low back pain, wrinkles and sagging, and susceptibility to gaining weight appear. Since these changes appear visually, they are a big problem.
The epidermis, the outermost layer of the skin, plays an important role in retaining moisture in the skin (Non-Patent Document 4). Most of the epidermis is composed of keratinocytes, which divide in the lowest layer of the epidermis, mature through a process called keratinization, and finally form a stratum corneum that peels off as dirt. drop down. The structure called the cornified envelope of the stratum corneum gives the skin physical strength, and its main constituents are involucrin and loricrin, which are produced during the maturation process of keratinocytes. Completed by cross-linking. The completed cornified envelope not only prevents external invasion, but also prevents water loss from the inside of the body. In addition, filaggrin is finally decomposed into amino acids during the maturation process of keratinocytes and acts as a natural moisturizing factor. Therefore, promoting the expression of involucrin and loricrin, which are components of the cornified envelope, and promoting the production of filaggrin, which is a natural moisturizing factor, are useful for retaining moisture in the skin.
The dermis layer of the skin plays an important role in wrinkle-free, firm and elastic skin. One of the causes of the appearance of wrinkles and the loss of firmness and elasticity is thought to be the decrease or alteration of collagen and elastin fibers present in the dermis layer, but the central role of synthesis and degradation of these fibers is fibroblasts. carried by cells. A collagen fiber is formed by assembling three procollagen fibers (Non-Patent Document 5). A degradative enzyme called matrix metalloprotease is involved in the degradation, and its activity is inhibited by tissue inhibitor of metalloprotease (Non-Patent Document 6). Elastin fibers are composed of fibrillin and elastin. When fibrillin aggregates, fibrils called microfibrils are formed, in which tropoelastin, a precursor protein of elastin, is deposited. An enzyme called lysyl oxidase then cross-links tropoelastins to complete elastin fibers (Non-Patent Document 7). Degradation involves elastinolytic enzymes such as fibroblast elastase and neutrophil elastase (Non-Patent Document 8, Non-Patent Document 9). An approach that targets such molecules and increases the amount of collagen fibers and elastin fibers is thought to be useful in preventing and improving wrinkles and loss of firmness and elasticity.
Dark spots are largely caused by melanin synthesized by pigment cells present in the basal portion of the epidermis. Synthesized melanin is taken up by epidermal keratinocytes, and it is believed that taking up and accumulating a large amount of melanin is one of the causes of spots and color unevenness. Activation of protease-activated receptor 2 present on epidermal keratinocytes promotes uptake of melanin (Non-Patent Document 10). Therefore, suppression of excessive uptake by epidermal keratinocytes is thought to be useful for prevention of blemish formation.
Pigment in body hair is produced by pigment cells present in the hair bulb of the hair follicle, which is the tissue of the hair. Body hair with it occurs. When the body hair regrows, the region existing below the bulge region of the hair follicle such as the old hair bulb disappears, and the pigment cells in the hair bulb disappear accordingly. When a new hair cycle begins and new body hair is produced, pigment cells are supplied from the melanocyte stem cells present in the bulge region of the hair follicle to produce pigmented body hair again. Due to aging and various stresses, melanocyte stem cells are reduced or depleted, and problems occur in the supply process of melanocytes. If melanocytes are not properly supplied, gray hair without pigment occurs.
In the bulge region, hair follicle epithelial stem cells (hereinafter referred to as hair follicle stem cells) are present adjacent to melanocyte stem cells. Hair follicle stem cells supply hair follicle epithelial cells, such as hair matrix cells, when body hair regrows, and these cells form new hair follicle tissue. Furthermore, hair follicle stem cells may have a function of forming a microenvironment for maintaining melanocyte stem cells existing adjacently (hereinafter referred to as a niche function) in addition to the function of reconstructing the epithelial cells of the hair follicle. (Non-Patent Document 11, Patent Document 1). Hair follicle stem cells maintain melanocyte stem cells by controlling the maintenance and activation of melanocyte stem cells by TGFβ and WNT signals. From this, it is considered that the decrease in melanocyte stem cells is caused by the loss of the niche function of hair follicle stem cells as a result of accumulation of damage in hair follicle stem cells due to aging and various stresses (Non-Patent Document 12). .
Furthermore, it has been elucidated that type 17 collagen plays an important role in maintaining hair follicle stem cells. Type 17 collagen is a bell-shaped collagen that forms hemidesmosomes (hemidesmosomes) that bind epithelial cells to the basement membrane, and hair follicle stem cells are also anchored to the basement membrane by this hemidesmosome structure. When type 17 collagen is deficient, hair loss is observed due to failure to maintain hair follicle stem cells. As a result, melanocyte stem cells are also lost, and graying of body hair also occurs at the same time. Furthermore, in recent years, it has been clarified that type 17 collagen is degraded with aging, hair follicle stem cells are lost, and hair loss occurs (Non-Patent Document 13). From the above, increasing the expression of type 17 collagen is useful for preventing and improving hair loss and gray hair.
It has also been found that mice in which SCF is forcibly expressed in epithelial cells, including hair follicle stem cells, show resistance to gray hair development stress. Therefore, enhancing SCF expression in epithelial cells leads to maintenance of melanocyte stem cells and is useful for prevention and improvement of gray hair (Non-Patent Document 14).
Furthermore, WNT7A, a WNT ligand, is strongly expressed in hair follicles during the hair cycle. Hair regenerated after wound healing is usually white hair that has lost its pigment, but colored hair is regenerated when the wound occurs during the growth period when epithelial cells strongly express WNT7A. This is due to the activation of melanocyte stem cells by WNT7A. In other words, increasing the expression of WNT7A is useful for preventing and improving gray hair (Non-Patent Document 15).
Skeletal muscles are roughly divided into slow-twitch fibers with slow contraction speed and fast-twitch fibers with fast contraction speed. It is known that the muscle fiber composition of marathon runners, etc., who require endurance, has a high proportion of slow-twitch fibers, while track and field sprinters, etc., who require explosive power, have a high proportion of fast-twitch fibers (Patent Document 2). .
Slow-twitch fibers contain many mitochondria. Slow twitch fibers consist of type I muscle fibers capable of sustained contraction and work during aerobic exercise. Troponin I (TNNI1) and myoglobin (MB) are proteins characteristic of type I muscle fibers (Non-Patent Document 16). There are slow-twitch and fast-twitch types of TNNI1, and it can serve as an indicator of whether a muscle fiber is slow-twitch or fast-twitch (Non-Patent Document 17). An increase in slow-twitch fibers means an increase in cells with high mitochondrial function, and continuous energy supply and ATP production are performed by β-oxidation of fatty acids. Increasing the amount of ATP improves the energy efficiency of skeletal muscle, which is the largest ATP-consuming organ in the body, and is useful for suppressing fatigue and improving endurance. Based on this, increasing the gene expression of TNNI1 or MB in skeletal muscle cells increases the number of slow-twitch fibers, and is therefore useful for making muscles slow-twitch and improving muscle endurance (Non-Patent Document 16).
Muscle endurance depends on a mechanism by which mitochondria in skeletal muscle cells produce sufficient amounts of ATP through oxidative phosphorylation. Cytochrome C oxidase (COX) is also a multi-subunit enzyme (complex IV) that catalyzes electron transfer from reduced cytochrome C to oxygen in mitochondrial respiration. The nuclear-encoded subunit COX4 is involved in the regulation and assembly of mitochondrial-encoded subunits on the mitochondrial inner membrane. In human skeletal muscle, COX4 mRNA levels have been shown to be associated with mitochondrial volume, mitochondrial elongation and VO2max (18). In other words, promotion of COX4I1 expression suggests an improvement in mitochondrial function and an increase in the amount of ATP. Therefore, enhancing COX4I1 gene expression in skeletal muscle cells is useful for improving muscle endurance.
It is known that cells undergo senescence and cell function declines with aging. Active oxygen is one of the factors that promote cell senescence. Active oxygen is generated by ultraviolet rays, mitochondrial activity, and the like, and cells have antioxidant molecules to counter active oxygen (Non-Patent Document 16). For example, glutathione itself has the ability to react with and eliminate active oxygen. Furthermore, it also has the ability to decompose hydrogen peroxide in cooperation with glutathione peroxidase. In addition, superoxide dismutase catalyzes a reaction that converts superoxide anion radicals, which are highly reactive active oxygen, into hydrogen peroxide and oxygen. Therefore, increasing the number of antioxidant molecules possessed by cells is useful for preventing deterioration of cell functions due to active oxygen. Also, the use of cell activators is useful for maintaining cell functions. As indicators of cell activation, ki67, which is a cell cycle-associated nuclear protein, which is a cell proliferation marker, and PCNA and CDC25C, which are proteins that control the cell cycle, can be used.

JP 2009-035514 A Japanese Patent Application Laid-Open No. 2015-97507

Nagashima, S. et al. (2014) Biochemistry., 86(1):63-67 Nagashima, S. et al. (2017) Journal of Japanese Pharmacology., 149(6):254-259 Yanagi, Shigeru (2016) Cosmetology research report., 24:149-152 Shimizu, H. et al. (2011) New Dermatology 2nd Edition., 7-10 Shimizu, H. et al. (2011) New Dermatology 2nd Edition., 13-16 Taihao Quan et al (2013) J Invest Dermatol., 133(5): 1362-1366 Xiaoqing Liu et al (2004) Nat Genet., 36(2):178-82. Imokawa G et al (2016) Exp Dermatol., 25:2-13. Takeuchi H et al (2010) J Dermatol Sci., 60(3):151-8 M. Seiberg et al (2000) Exp Cell Res., 254(1):25-32. Tanimura S et al (2011) Cell Stem Cell., 6(2):130-40. Aoki H et al (2013) J Invest Dermatol., 133(9):2143-51. Matsumura H et al (2016) Science., 351(6273):aad4395. Aoki H et al (2011) J Invest Dermatol. 131(9):1906-15. Yuriguchi M et al (2016) J Dermatol Sci., 84(1):80-87. Reyes NL et al (2015) PNAS. Jan 13;112(2):424-9. Wade R et al (1990) Genomics. Jul;7(3):346-57. Fukuda et al (2007) Cell. Apr 6;129(1):111-22. Nakamura, N. (2013) Nippon Medical School Journal.,9(3):164-169

An object of the present invention is to provide a composition for promoting the production of the factors listed in Table 1 or a composition for suppressing the production of the factors listed in Table 2, which contains a component that promotes MITOL production, to provide a MITOL production promoter, and It is an object of the present invention to provide a screening method for a production-enhancing agent for the factors listed in Table 1 or a production-suppressing agent for the factors listed in Table 2, using the MITOL production-promoting action as an indicator.

Therefore, as a result of intensive studies by the inventors, experiments using epidermal keratinocytes, hair follicle keratinocytes or dermal fibroblasts showed that MITOL knockdown reduces the factors listed in Table 1 and increases the factors listed in Table 2. found to do. Decrease in factors involved in cell proliferation (mki67, PCNA, and CDC25C) was confirmed, suggesting that MITOL is involved in suppressing the decrease, promoting proliferation, or activating hair follicle keratinocytes, epidermal keratinocytes, and dermal fibroblasts. I found out that In addition, since a decrease in a factor (COL17A1) involved in the maintenance of hair follicle stem cells was confirmed, it was found that MITOL is involved in suppressing the decrease, promoting proliferation, or activating hair follicle stem cells. Furthermore, the reduction of factors (WNT7A, TGFB2, and SCF) involved in the maintenance of melanocyte stem cells was confirmed, indicating that MITOL is involved in suppressing the decrease, promoting growth, or activating melanocyte stem cells. It has been reported that the decrease in hair follicle stem cells and melanocyte stem cells is involved in the development of hair loss and gray hair. Found it.

As a result of material screening, fermented rice bran extract, Phellodendron bark extract, Coptis extract, Assembly extract, Hamamelis extract, Botanpi extract, Peach extract, Eucalyptus extract, Tamogitake extract, β-estradiol, 5-aminolevulinic acid, It was found that β-nicotinamide mononucleotide, riboflavin, thiotaurine, MA5, metformin, urolithin A, niacinamide, and D-panthenol have MITOL production promoting action. Furthermore, witch hazel extract and peach extract have COL17A1 gene expression promoting action, assembly extract, hamamelis witch hazel extract, botanpi extract, peach extract and eucalyptus extract have SOD2 gene expression promoting action, Pleurotus cornucopia extract and β-nicotinamide nucleotide have a LOXL-1 gene expression promoting effect, peach extract and riboflavin have an MME gene expression suppressing effect, and hamamelis extract and eucalyptus extract have Cox4i1, Tnni1 and Mb gene expression promoting effects. As a result, it was found that the component that promotes MITOL production has an action to promote the production of the factors listed in Table 1 and an action to suppress the production of the factors listed in Table 2. Based on the above, the inventors have found that MITOL production can be used as an indicator in screening for a factor production promoter listed in Table 1 or a factor production inhibitor listed in Table 2, and have completed the present invention. .

That is, the present invention
(1) A composition for promoting the production of the factors listed in Table 1 or suppressing the production of the factors listed in Table 2, containing as an active ingredient a component that promotes MITOL production;
(2) The ingredients that promote MITOL production are rice bran fermented extract, Phellodendron bark extract, Coptis japonica extract, assembly extract, hamamelis extract, botanpi extract, peach extract, eucalyptus extract, Tamogitake extract, β-estradiol, The composition according to (1), which is at least one selected from the group consisting of 5-aminolevulinic acid, β-nicotinamide mononucleotide, riboflavin, thiotaurine, MA5, metformin, urolithin A, niacinamide, and D-panthenol,
(3) The composition according to (1), which is for suppressing the decrease, promoting growth or activating hair follicle stem cells, melanocyte stem cells, hair follicles or epidermal keratinocytes, or dermal fibroblasts,
(4) The composition according to (1), wherein the factors listed in Table 1 are COL17A1, SOD2, and LOXL-1;
(5) The composition according to (1), wherein the factor described in Table 1 is COL17A1;
(6) The composition according to (5), which is for preventing or improving gray hair,
(7) The composition according to (1), wherein the factors listed in Table 1 are COX4I1, TNNI1, and MB;
(8) A composition for promoting slow muscle growth or improving muscle endurance, characterized by containing a hamamelis extract or a eucalyptus extract,
(9) The composition according to (1), wherein the factor described in Table 2 is MME;
(10) fermented rice bran extract, Phellodendron bark extract, Coptis japonica extract, assembly extract, hamamelis extract, botanpi extract, peach extract, eucalyptus extract, oyster mushroom extract, β-estradiol, 5-aminolevulinic acid, β-nicotine MITOL reduction inhibitor or production promoter, characterized by containing at least one selected from the group consisting of amide mononucleotide, riboflavin, thiotaurine, MA5, metformin, urolithin A, niacinamide, and D-panthenol;
(11) A screening method for an agent for suppressing the decrease, promoting growth, or activating hair follicle stem cells, melanocyte stem cells, hair follicles or epidermal keratinocytes, or dermal fibroblasts, using the MITOL production promoting effect as an index,
(12) A method for screening an agent for suppressing the decrease or promoting the production of COL17A1, SOD2, LOXL-1, COX4I1, TNNI1 or MB, using the MITOL production promoting effect as an index,
(13) A method for screening an agent for promoting reduction or suppressing production of MME, using MITOL production-promoting action as an index,
(14) A screening method for the factor production promoter described in Table 1 or the factor production inhibitor described in Table 2, using the MITOL production promoting effect as an indicator;
is.

The ingredient that promotes MITOL production of the present invention has the effect of preventing or improving aging symptoms such as dryness of the skin, appearance of wrinkles, loss of firmness/elasticity, increase of spots and dullness, appearance of gray hair, and muscle loss. I can expect it. In addition, the component of the present invention that promotes MITOL production can be used as a positive control in material screening and the like. Furthermore, the present invention can be used for screening of agents for promoting the production of the factors listed in Table 1 or inhibitors for the production of the factors listed in Table 2.

FIG. 1 is a graph showing the effects of MITOL knockdown on various gene expressions in epidermal keratinocytes in test examples. FIG. 2 is a graph showing the effects of MITOL knockdown on various gene expressions in dermal fibroblasts in test examples. FIG. 3 is a graph showing the effects of MITOL knockdown on various gene expressions in follicle keratinocytes in test examples. FIG. 4 is a graph showing that test substances promote MITOL gene expression in epidermal keratinocytes in test examples. FIG. 5 is a graph showing that test substances promote MITOL gene expression in dermal fibroblasts in Test Examples. FIG. 6 is a graph showing that test substances promote MITOL gene expression in skeletal muscle cells in Test Examples. FIG. 7 is a graph showing that a component that promotes MITOL production promotes SOD2 and COL17A1 gene expression in epidermal keratinocytes in Test Examples. FIG. 8 is a graph showing that a component that promotes MITOL production promotes LOXL-1 gene expression in dermal fibroblasts and suppresses MME gene expression in test examples. FIG. 9 is a graph showing that a component that promotes MITOL production promotes Cox4i1, Tnni and Mb gene expression in skeletal muscle cells in Test Examples.

A component that promotes MITOL production in the present invention is a component that has a MITOL mRNA expression-enhancing effect or a MITOL protein expression-enhancing effect. The MITOL production promoting action can be evaluated by gene expression analysis by real-time RT-PCR, protein expression analysis by Western blotting, and the like, but the evaluation method is not particularly limited.

The factors listed in Table 1 in the present invention are factors whose expression levels are decreased by MITOL deficiency. In addition, the factors listed in Table 2 in the present invention are factors whose expression levels are increased by depletion of MITOL.

Methods for depleting MITOL are not particularly limited, and include, for example, RNA interference using siRNA. Cells deficient in MITOL are not particularly limited, but examples thereof include epidermal keratinocytes, hair follicle keratinocytes and dermal fibroblasts.

The epidermal keratinocytes in the present invention are cells that exist in the epidermal layer of the skin, divide in the basal layer, and push up to the upper layer to form the epidermal layer. Cells may be of any animal species, but human-derived cells are preferred. Whether normal cells, established cell lines or immortalized cells, normal cells are preferred.
Dermal fibroblasts in the present invention are cells that exist in the dermal layer of the skin and produce connective tissue components of the dermal dermis such as collagen and elastin. Cells may be of any animal species, but human-derived cells are preferred. Whether normal cells, established cell lines or immortalized cells, normal cells are preferred.
Melanocyte stem cells in the present invention are stem cells that exist in the hair follicle bulge to sub-bulge region and have the function of reconstructing the melanocyte lineage identified by markers such as DCT or PAX3.
The hair follicle stem cell in the present invention is a stem cell that exists in the hair follicle bulge to sub-bulge region, is identified by markers such as CD34, CD200, K15 or α6 integrin, and has the function of reconstructing the epithelial cell lineage of the hair follicle. . Note that since markers for melanocyte stem cells and hair follicle stem cells differ depending on animal species, these markers do not always need to be expressed.
Hair follicle keratinocytes in the present invention include epithelial cells reconstituted by the hair follicle stem cells described above, and epithelial cells existing throughout the hair cycle in the constant region above the bulge region of the hair follicle. Refers to epithelial cells in general.
The muscle cells in the present invention are contractile cells that form the muscle tissue of the animal body, and are also called muscle fibers because they exhibit an elongated spindle or fibrous shape. Cells may be of any animal species, but human-derived cells are preferred. Whether normal cells, established cell lines or immortalized cells, normal cells are preferred.

Collagen type 17 in the present invention refers to bell-shaped collagen that contributes to the formation of a hemidesmosome structure that binds hair follicle stem cells and keratinocytes of hair follicles and skin to the basement membrane. COL17A1 is a factor that constitutes the α chain of type 17 collagen.

In the present invention, suppression of cell decrease, promotion of proliferation, or activation of cells refers to maintenance or enhancement of cell proliferation ability. Maintenance or enhancement of cell proliferation ability can be evaluated by cell proliferation assay using MTT reagent, gene expression analysis by real-time RT-PCR method of cell proliferation marker, or protein expression analysis by Western blotting method. Not limited.

The superoxide dismutase in the present invention is an enzyme that catalyzes the reaction of generating hydrogen peroxide and oxygen from superoxide anion radicals and hydrogen ions. SOD2 is SOD containing manganese in the active center.

Lysyl oxidase in the present invention is an enzyme that cross-links lysine residues of tropoelastin. LOXL-1 is a member of the lysyl oxidase family and is a molecule known to bind to fibrin-5, one of the constituents of elastin fibers.

Elastase in the present invention is an enzyme that degrades elastin. MME is an enzyme derived from fibroblasts among enzymes that degrade elastin.

The cytochrome C oxidase in the present invention is an enzyme in the electron transport system in the mitochondrial membrane, and is an enzyme that reduces oxygen using the reduced form of cytochrome C as an electron donor. COX4I1 is one of the subunits of cytochrome C oxidase, and is a subunit encoded by genes in the nucleus.

Troponin in the present invention is a molecule that regulates calcium-dependent muscle contraction via striated muscle actin and myosin. Troponin I is an inhibitory subunit of troponin that inhibits the interaction between troponin and actin. TNNI1, a gene encoding slow-twitch troponin I, is expressed specifically in cardiac muscle and skeletal muscle during development, and is expressed only in slow-twitch fibers in adults.

Myoglobin in the present invention is a molecule that retains oxygen molecules in cardiac muscle and skeletal muscle. Myoglobin is expressed more in slow-twitch fibers with high oxygen consumption than in fast-twitch fibers with low oxygen consumption. MB is the gene encoding myoglobin.

The fermented rice bran extract used in the present invention is an extract obtained by adding yeast to a rice bran saccharified solution obtained by adding α-amylase and β-amylase to rice bran for fermentation, adding sodium chloride, and squeezing and filtering. . Rice bran is a mixture of pericarp, seed coat, germ and aleurone layer obtained when brown rice obtained from seeds of rice (scientific name Rryza sativa L.) is milled. Phellodendron bark extract is an extract obtained from the depericulated bark of yellowfin (Phellodendron amurense Ruprecht) or other congeneric plants. Coptis extract is an extract obtained from the rhizome of Coptis japonica Makino (scientific name: Coptis japonica Makino) or other plants of the same genus. Japonica japonica extract is an extract obtained from the whole plant of japonica japonica (scientific name: Swertia japonica Makino). Hamamelis extract is an extract obtained from leaves of Hamamelis (scientific name Hamamelis virginiana L.). Botanpi extract is an extract obtained from the root bark of Paeonia suffruticosa (scientific name: Paeonia suffruticosa). Peach extract is an extract obtained from the leaves of the peach (Prunus persica). Eucalyptus extract is an extract obtained from the leaves of Eucalyptus (Eucalyptus globulus Labillardiere). Pleurotus cornucopiae extract is an extract obtained from Pleurotus cornucopiae (Pleurotus cornucopiae). A method for extracting the above extract is not particularly limited, and can be carried out according to a conventional method. Either a polar solvent or a non-polar solvent can be used as the extraction solvent used for extraction. The above extract may be used as an extracted solution, or if necessary, may be used after being subjected to concentration, dilution, filtration, decolorization with activated charcoal or the like, deodorization, ethanol precipitation, or the like. Furthermore, the extracted solution may be subjected to a treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.
Commercially available fermented rice bran extract used in the present invention is fermented rice bran extract BG30 (manufactured by Maruzen Pharmaceutical Co., Ltd.). Coptis extract liquid BG30 (manufactured by Maruzen Pharmaceutical Co., Ltd.), a commercial product of japonicum extract, Japonica Extract Liquid SS (manufactured by Maruzen Pharmaceutical Co., Ltd.), a commercially available hamamelis extract, hamamelis extract liquid BG-J (manufactured by Maruzen Pharmaceutical Co., Ltd.), botanpi extract Botanpi Extract (manufactured by Maruzen Pharmaceutical Co., Ltd.) is a commercially available product, Peach Extract Liquid BG (manufactured by Maruzen Pharmaceutical Co., Ltd.) is a commercially available peach extract, and Eucalyptus Extract Liquid BG (manufactured by Maruzen Pharmaceutical Co., Ltd.) is a commercially available eucalyptus extract. , Phytothionein (manufactured by Koei Kogyo Co., Ltd.) and the like can be mentioned as commercial products of Tamogitake extract.

Phellodendron bark extract, hamamelis extract and peach extract used in the present invention are preferably extracted with a solvent containing a polyhydric alcohol (1,3-butylene glycol, propylene glycol, dipropylene glycol, glycerin, etc.). In addition to polyhydric alcohols, it may contain water, lower aliphatic alcohols (methanol, ethanol, isopropyl alcohol, etc.), lower aliphatic ketones (acetone, etc.), etc., among which mixtures of polyhydric alcohols and water It is most preferable to extract with The polyhydric alcohol preferably includes 1,3-butylene glycol or propylene glycol, with 1,3-butylene glycol being most preferred. When extracting with a solvent consisting of water and 1,3-butylene glycol, the content of 1,3-butylene glycol in the solvent is preferably 30 to 70% by volume.

Coptis extract, japonica extract, botanpi extract and eucalyptus extract used in the present invention are preferably extracted with a solvent containing a lower aliphatic alcohol (methanol, ethanol, isopropyl alcohol, etc.). In addition to lower aliphatic alcohols, water, polyhydric alcohols (1,3-butylene glycol, propylene glycol, dipropylene glycol, glycerin, etc.), lower aliphatic ketones (acetone, etc.), etc. may be contained, and this Extraction with a mixture of a lower aliphatic alcohol and water is most preferred. Ethanol is preferred as the lower aliphatic alcohol.

The extract of Pleurotus cornucopia in the present invention is preferably extracted with water. In addition to water, polyhydric alcohols (1,3-butylene glycol, propylene glycol, dipropylene glycol, glycerin, etc.), lower aliphatic alcohols (methanol, ethanol, isopropyl alcohol, etc.), lower aliphatic ketones (acetone, etc.), etc. may be contained, but those extracted with water are preferable.

β-estradiol in the present invention is a compound defined by Cas No. 50-28-2. 5-aminolevulinic acid is a compound defined by Cas No.5451-09-2. β-nicotinamide mononucleotide is a compound defined by Cas No. 1094-61-7. Riboflavin is a compound defined by CasNo.83-88-5. Thiotaurine is a compound defined by CasNo.2937-54-4. MA-5 is 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid. Metformin is a compound defined by Cas No. 1115-70-4. Urolithin A is a compound defined by CasNo.1143-70-0. Niacinamide is a compound defined by CasNo.98-92-0. D-panthenol is a compound defined by CasNo.81-13-0.

β-estradiol and metformin from Tokyo Chemical Industry Co., Ltd., β-nicotinamide mononucleotide from Oriental Yeast Co., Ltd., etc., 5-aminolevulinic acid from Cosmo Bio Co., Ltd., riboflavin, thiotaurine, niacinamide and D - Panthenol can be purchased from Wako Pure Chemical Industries, Ltd., etc., and Urolithin A can be purchased from Sigma-Aldrich, etc. MA-5 can be produced using a general synthetic method, for example, it can be produced by the method described in International Publication WO2014/080640.

The dosage form is not particularly limited, but includes external use and internal use, preferably external use applied to the skin including the scalp. Dosage forms for external application of the present invention include, for example, lotions, liquids, creams, ointments, gels, sprays, shampoos, conditioners, soaps, etc., and agents for internal application. Forms include tablets, powders, powders, granules, liquids, capsules, dry syrups, jellies, liquid foods, semi-solid foods, solid foods and the like.
These can be produced by known methods. During production, various additives that can be contained in cosmetics, quasi-drugs, pharmaceuticals, food and drink, or reagents can be blended within limits that do not impair the effects of the present invention.

The composition containing the MITOL production-promoting ingredient of the present invention as an active ingredient or the MITOL production-enhancing agent may cause, for example, dry skin, appearance of wrinkles, loss of firmness/elasticity, increase in spots and dullness, and appearance of gray hair. It can also be used as a reagent for preventing or ameliorating aging symptoms, or promoting MITOL production, and can be preferably used as a positive control drug in material screening or the like.

The amount of the component that promotes MITOL production of the present invention is 0.000001 to 10% by mass, preferably 0.000001 to 10% by mass, based on the total composition when provided as cosmetics, quasi-drugs, pharmaceuticals, food and drink, or reagents. 0.0001 to 5% by weight, more preferably 0.001 to 1% by weight.

EXAMPLES The present invention will be described in more detail with reference to examples and test examples below, but the present invention is not limited to the examples.

(Test Example 1) Evaluation of the effect of MITOL knockdown on gene expression in epidermal keratinocytes
A mixture of Opti-MEM and Lipofectamine RNAiMAX (both from Thermo Fisher Scientific) at a volume ratio of 100:1 was added to a 6-well plate at 500 μL/well. Further, 6 μL/well of 10 μM MITOL siRNA was added and allowed to stand for 20 minutes. After that, 2.5 mL of cell suspension of epidermal keratinocytes (manufactured by Kurashiki Boseki) adjusted to a density of 9 × 10 ³ cells/mL in the medium Humedia-KG2 (Kurashiki Boseki, with attached supplements other than antibacterial agents added). /well and mixed by shaking the plate. The plate was placed in an incubator set at 37°C and 5% CO2 and cultured for 1 day. The medium was replaced with fresh Humedia-KG2 and cultured for another day. After the culture was completed, the medium was removed, a lysate buffer was added to lyse the cells, and the cell lysate was collected. RNA was recovered from the cell lysate using RNeasy Mini Kit (Qiagen) according to the attached protocol, and using this as a template, cDNA was synthesized by reverse transcription using Prime Script RT Master mix (Takara Bio). Expression levels of mRNA for each of GAPDH, mki67, PCNA, TGM1, KRT1, KRT10, INV, FLG, LOR, SOD2, PAR2, and COL17A1 were determined from the synthesized cDNA using a real-time PCR system (Step One Plus, Thermo Fisher Scientific). was measured (SYBR Green method), and the expression levels of mki67, PCNA, TGM1, KRT1, KRT10, INV, FLG, LOR, SOD2, PAR2 and COL17A1 were corrected by GAPDH expression levels. The following primers were used. GAPDH: HA067812, mki67: HA177741, PCNA: HA157702, TGM1: HA171645, KRT1: HA237191, KRT10: HA230464, INV: HA226067, FLG: HA182433, LOR: HA177882, SOD2: HA169126, PAR2: HA1301 and HA13112 (also Takara Bio). The measured mRNA expression level was compared with the control group (group to which Control siRNA was added instead of MITOL siRNA) to evaluate the effect of MITOL knockdown.
<Test results>
The results are shown in FIG. MITOL knockdown was confirmed to decrease the mRNA expression levels of mki67, PCNA, TGM1, KRT1, KRT10, INV, FLG, LOR and SOD2, and to increase the mRNA expression level of PAR2. Decreases in cell proliferation markers mki67 and PCNA revealed that MITOL is important for the proliferation of epidermal keratinocytes. Based on this, the component that promotes MITOL production can be used to suppress the decrease, promote proliferation and activation of epidermal keratinocytes. MITOL production-promoting action can be used as an indicator.

(Test Example 2) Evaluation of the effect of MITOL knockdown on gene expression in dermal fibroblasts
A mixture of Opti-MEM and Lipofectamine RNAiMAX (both from Thermo Fisher Scientific) at a volume ratio of 100:0.45 was added to a 12-well plate at 250 μL/well. Furthermore, 3 μL/well of 10 μM MITOL siRNA was added and allowed to stand for 20 minutes. After that, a cell suspension of dermal fibroblasts (manufactured by Kurashiki Boseki) prepared to a density of 6 × 10 ⁴ cells/mL in the medium FibroLife BM (manufactured by Kurashiki Boseki, supplements other than antibacterial agents) was added at 1.25 mL/ml. wells and mixed by shaking the plate. The plate was placed in an incubator set at 37°C, 5% _CO2 . As for COL1A1 and ELN, the medium was replaced with the medium FibroLife BM (manufactured by Kurashiki Boseki Co., Ltd., supplements other than FBS, FGF and antibacterial agents were added) the day after the addition of siRNA, and the culture was terminated two days later. Except for COL1A1 and ELN, medium exchange was not performed, and culture was terminated 2 days after addition of siRNA. After the culture was completed, the medium was removed, a lysate buffer was added to lyse the cells, and the cell lysate was collected. RNA was recovered from the cell lysate using RNeasy Mini Kit (Qiagen) according to the attached protocol, and using this as a template, cDNA was synthesized by reverse transcription using Prime Script RT Master mix (Takara Bio). RPLP0, PCNA, CDC25C, GSS, COL1A1, ELN, MMP1, MME, FBN1, LOXL-1, TIMP-1 and SOD2 were isolated from the synthesized cDNA using a real-time PCR system (Step One Plus, Thermo Fisher Scientific). The expression level of mRNA was measured (SYBR Green method), and the expression levels of PCNA, CDC25C, GSS, COL1A1, ELN, MMP1, MME, FBN1, LOXL-1, TIMP-1 and SOD2 were corrected by the expression level of RPLP0. As the primer, the following model number manufactured by Takara Bio was used. RPLP0: HA234224, PCNA: HA157702, CDC25C: HA266272, GSS: HA285317, COL1A1: HA181838, MMP1: HA205024, MME: HA211733, FBN1: HA151534, LOXL-1: HA230268, TIMP-1: HA138168, SOD92 Takara Bio). As for the ELN primers, we asked Takara Bio to create primers specific to Accession ID: NM_000501. The level of mRNA expression was compared with a control group (a group to which Control siRNA was added instead of MITOL siRNA) to evaluate the effect of MITOL knockdown.
<Test results>
The results are shown in FIG. MITOL knockdown was confirmed to decrease the mRNA expression levels of PCNA, CDC25C, GSS, COL1A1, ELN, MMP1, LOXL-1 and TIMP-1, and to increase the mRNA expression levels of FBN1 and MME. Decreases in CDC25C and PCNA, which are cell proliferation markers, revealed that MITOL is important for the proliferation of dermal fibroblasts. From this, the component that promotes MITOL production can be used to suppress the decrease, promote proliferation and activation of dermal fibroblasts. MITOL production-promoting action can be used as an indicator.

(Test Example 3) Evaluation of the effect of MITOL knockdown on gene expression in hair follicle keratinocytes <Test method>
A mixture of Opti-MEM and Lipofectamine RNAiMAX (both from Thermo Fisher Scientific) at a volume ratio of 100:3 was added to a 6-well plate at 500 μL/well. Furthermore, 6 μL/well of 10 μM MITOL siRNA was added and allowed to stand for 20 minutes. After that, 2 mL/well of a cell suspension of hair follicle keratinocytes (Cosmo Bio) adjusted to a density of 5 × 10 ⁴ cells/mL in medium Humedia-KG2 (Kurashiki Spinning Co., Ltd., with attached supplements other than antibacterial agents added). added and mixed by shaking the plate. The plate was placed in an incubator set at 37° C. and 5% CO ₂ and cultured for 1 day. After culturing, the medium was replaced with new Humedia-KG2. SCF was further cultured for 2 days, and those other than SCF were further cultured for 3 days. After the culture was completed, the medium was removed, a lysate buffer was added to lyse the cells, and the cell lysate was collected. RNA was recovered from the cell lysate using RNeasy Mini Kit (Qiagen) according to the attached protocol, and using this as a template, cDNA was synthesized by reverse transcription using Prime Script RT Master mix (Takara Bio). Using a real-time PCR system (Step One Plus, Thermo Fisher Scientific), the expression levels of GAPDH, mki67, PCNA, COL17A1, WNT7A, TGFβ2, GSS and SCF were measured from the synthesized cDNA (SYBR Green method). , mki67, PCNA, COL17A1, WNT7A, TGFβ2, GSS and SCF expression levels were corrected by GAPDH expression levels. The following primers were used. GAPDH: HA067812, mki67: HA177741, PCNA: HA157702, COL17A1: HA132006, WNT7A: HA164443, TGFβ2: HA172380, GSS: HA285317, SCF: HA122146 (all from Takara Bio). The measured mRNA expression level was compared with the control group (group to which Control siRNA was added instead of MITOL siRNA) to evaluate the effect of MITOL knockdown.
<Test results>
The results are shown in FIG. MITOL knockdown was confirmed to decrease the mRNA expression levels of mki67, PCNA, COL17A1, WNT7A, TGFβ2, GSS and SCF. Decreases in cell proliferation markers mki67 and PCNA revealed that MITOL is important for the proliferation of hair follicle stem cells and hair follicle keratinocytes. In addition, since MITOL plays an important role in the expression of type 17 collagen, it is involved in the maintenance of hair follicle stem cells (Patent Document 13), and in addition, it is involved in the niche function of maintaining melanocyte stem cells and melanocytes. Since it is also important for the production of factors such as WNT7A, TGFβ2 and SCF, it has been found to be involved in the maintenance of melanocyte stem cells. Based on the above results, the component that promotes MITOL production can be used to suppress the decrease, promote proliferation and activation of hair follicle stem cells, melanocyte stem cells and hair follicle keratinocytes. It can be used for improvement. In addition, the production-promoting action of MITOL can be used as an index when screening components for suppressing the decrease, promoting proliferation, and activating hair follicle stem cells, melanocyte stem cells, and hair follicle keratinocytes.

(Test Example 4) Evaluation of MITOL gene expression promotion effect on epidermal keratinocytes Epidermal keratinocytes (manufactured by Kurashiki Spinning Co., Ltd.) were adjusted to 2500 cells/cm2 in a medium Humedia-KG2 (Kurashiki Spinning Co., Ltd., supplements attached). The cells were seeded in a 12-well plate and cultured for 24 hours in an incubator set at 37°C and 5% CO2. The medium was removed by aspiration, replaced with a medium containing the test substance, and cultured for an additional 24 hours. After the culture was completed, the medium was removed, a lysate buffer was added to lyse the cells, and the cell lysate was collected. RNA was recovered from the cell lysate using RNeasy Mini Kit (Qiagen) according to the attached protocol, and using this as a template, cDNA was synthesized by reverse transcription using Prime Script RT Master mix (Takara Bio). Using a real-time PCR system (Step One Plus, Thermo Fisher Scientific), the expression levels of GAPDH and MITOL mRNA were measured from the synthesized cDNA (SYBR Green method), and the expression level of MITOL was corrected by the expression level of GAPDH. . The following primers were used. GAPDH: HA067812, MITOL: HA192576 (both Takara Bio). The measured mRNA expression level was compared with that of a control group (medium containing no test substance) to evaluate the effect of the test substance on promoting MITOL gene expression.
<Test results>
The results are shown in FIG. Fermented rice bran extract, Phellodendron bark extract, Coptis japonica extract, Assembly extract, Hamamelis extract, Botanpi extract, Peach extract, Eucalyptus extract, β-estradiol, 5-aminolevulinic acid, β-nicotinamide mononucleotide and riboflavin It was confirmed that the mRNA expression level of MITOL was increased. These substances can be used as positive controls when screening for components that promote MITOL production.

(Test Example 5) Evaluation of MITOL gene expression promotion effect on dermal fibroblasts Dermal fibroblasts (manufactured by Kurashiki Boseki) were added to the medium FibroLife BM (manufactured by Kurashiki Boseki, with the attached supplement added) at a concentration of 1.0 × 10 ⁵ cells/mL. The cell density was adjusted, 0.5 mL each was seeded in a 12-well plate, and cultured in an incubator set at 37° C. and 5% CO2. On the following day, the medium was removed by aspiration, replaced with medium FibroLife BM (manufactured by Kurashiki Spinning Co., Ltd., supplements other than FBS and FGF added), and further cultured for 24 hours. The medium was removed by aspiration, replaced with a medium containing the test substance, and cultured for 24 hours. After the culture was completed, the medium was removed, a lysate buffer was added to lyse the cells, and the cell lysate was collected. RNA was recovered from the cell lysate using RNeasy Mini Kit (Qiagen) according to the attached protocol, and using this as a template, cDNA was synthesized by reverse transcription using Prime Script RT Master mix (Takara Bio). Using a real-time PCR system (Step One Plus, Thermo Fisher Scientific), the expression levels of GAPDH and MITOL mRNA were measured from the synthesized cDNA (SYBR Green method), and the expression level of MITOL was corrected by the expression level of GAPDH. . The following primers were used. GAPDH: HA067812, MITOL: HA192576 (both Takara Bio). The measured mRNA expression level was compared with that of a control group (medium containing no test substance) to evaluate the effect of the test substance on promoting MITOL gene expression.
<Test results>
The results are shown in FIG. Fermented rice bran extract, Phellodendron bark extract, coptis extract, peach extract, Tamogitake extract, β-estradiol, 5-aminolevulinic acid, β-nicotinamide mononucleotide, riboflavin, thiotaurine, MA-5, metformin, urolithin A, niacin It was confirmed that amide and D-panthenol increased the expression level of MITOL mRNA. These substances can be used as positive controls when screening for components that promote MITOL production.

(Test Example 6) Evaluation of MITOL gene expression promoting effect on mouse myoblast cell line C2C12 cells Mouse myoblast cell line C2C12 cells (DS Pharma Biomedical) were grown in DMEM containing 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific), the cells were seeded in a 6-well plate at 12,500 cells/cm 2 and cultured in an incubator set at 37° C. and 5% CO 2 . After removing the medium by aspiration and adding DMEM containing 2% Horse Serum (HS) in the differentiation medium, the culture was continued. The medium was removed by aspiration, and serum starvation medium DMEM was added and conditioned 3 hours before exposure to the test substance. The medium was removed by aspiration, replaced with a medium containing the test substance, and cultured for an additional 24 hours. After the culture was completed, the medium was removed, a lysate buffer was added to lyse the cells, and the cell lysate was recovered. RNA is recovered from the cell lysate using the RNeasy Mini Kit (Qiagen) according to the attached protocol, and using this as a template, cDNA is synthesized by reverse transcription reaction using ReverTra Ace (R) qPCR RT Master Mix (Toyobo). did. From the synthesized cDNA, the expression levels of Cyclophilin and MITOL mRNA were measured using a real-time PCR system (Step One Plus, Thermo Fisher Scientific) (SYBR Green method), and the expression level of MITOL was corrected by the expression level of Cyclophilin. . The following primers were used. MITOL: MA138812 (Takara Bio). As for Cyclophilin primers, a primer specific to Accession ID: M60456 was requested from invitrogen. The measured mRNA expression level was compared with that of a control group (medium containing no test substance) to evaluate the effect of the test substance on promoting MITOL gene expression.
<Test results>
The results are shown in FIG. It was confirmed that assembly extract, hamamelis extract, eucalyptus extract, and niacinamide increased the expression of MITOL mRNA. These substances can be used as positive controls when screening for components that promote MITOL production.

(Test Example 7) Evaluation of the effect of materials on gene expression in epidermal keratinocytes Epidermal keratinocytes (manufactured by Kurashiki Spinning Co., Ltd.) were adjusted to 2500 cells/cm2 in the medium Humedia-KG2 (Kurashiki Spinning Co., Ltd., with supplements attached). , and cultured for 24 hours in an incubator set at 37°C and 5% CO2. The medium was removed by aspiration, replaced with a medium containing the test substance, and cultured for 24 hours. After the culture was completed, the medium was removed, a lysate buffer was added to lyse the cells, and the cell lysate was recovered. RNA was recovered from the cell lysate using RNeasy Mini Kit (Qiagen) according to the attached protocol, and using this as a template, cDNA was synthesized by reverse transcription using Prime Script RT Master mix (Takara Bio). From the synthesized cDNA, the expression level of each mRNA of GAPDH, SOD2 and COL17A1 was measured using a real-time PCR system (Step One Plus, Thermo Fisher Scientific) (SYBR Green method). corrected for volume. The following primers were used. GAPDH: HA067812, SOD2: HA169126, COL17A1: HA132006 (both from Takara Bio). The measured mRNA expression level was compared with that of a control group (medium containing no test substance) to evaluate various gene expression promoting effects of the test substance.
<Test results>
The results are shown in FIG. It was confirmed that the SOD2 mRNA expression level was increased by the assembly extract, witch hazel extract, botanical extract, peach extract, and eucalyptus extract. In addition, it was confirmed that the expression level of COL17A1 mRNA was increased by the hamamelis extract and the peach extract. Based on the above, components that promote MITOL production can be used to promote the production of the factors listed in Table 1. In addition, when screening agents for promoting the production of the factors listed in Table 1, the MITOL production promoting action can be used as an indicator.

(Test Example 8) Evaluation of the influence of materials on gene expression in dermal fibroblasts Dermal fibroblasts (manufactured by Kurashiki Spinning Co., Ltd.) were added to the medium FibroLife BM (manufactured by Kurashiki Spinning Co., Ltd., with the attached supplement added) at 1.0 × 10 ⁵ cells/mL. 0.5 mL each was seeded in a 12-well plate and cultured for 24 hours in an incubator set at 37° C. and 5% CO 2 . The medium was removed by aspiration, replaced with medium FibroLife BM (manufactured by Kurashiki Boseki Co., Ltd., supplements other than FBS and FGF added), and further cultured for 24 hours. The medium was removed by aspiration, replaced with a medium containing the test substance, and cultured for 24 hours. After the culture was completed, the medium was removed, a lysate buffer was added to lyse the cells, and the cell lysate was collected. RNA was recovered from the cell lysate using RNeasy Mini Kit (Qiagen) according to the attached protocol, and using this as a template, cDNA was synthesized by reverse transcription using Prime Script RT Master mix (Takara Bio). From the synthesized cDNA, the expression levels of GAPDH, LOXL-1 and MME mRNA were measured using a real-time PCR system (Step One Plus, Thermo Fisher Scientific) (SYBR Green method), and the expression of LOXL-1 and MME was determined. The amount was corrected by GAPDH expression level. The following primers were used. GAPDH: HA067812, LOXL-1: HA230268 and MME: HA211733 (all from Takara Bio). The measured mRNA expression level was compared with that of a control group (medium containing no test substance) to evaluate various gene expression promoting effects of the test substance.
<Test results>
The results are shown in FIG. It was confirmed that the expression level of LOXL-1 mRNA was increased by the extract of Pleurotus cornucopia and β-nicotinamide mononucleotide. In addition, it was confirmed that peach extract and riboflavin decreased the expression level of MME mRNA. Based on the above, the component that promotes MITOL production can be used as an agent for promoting the production of the factors listed in Table 1 and as an agent for suppressing the production of the factors listed in Table 2. In addition, when screening the factor production-enhancing agents listed in Table 1 and the factor production-inhibiting agents listed in Table 2, the MITOL production-enhancing activity can be used as an index.

(Test Example 9) Evaluation of the influence of materials on gene expression in mouse myoblast C2C12 cells Mouse myoblast C2C12 cells (DS Pharma Biomedical) were grown in DMEM containing 10% Fetal Bovine Serum (FBS) in the growth medium. Fisher Scientific), the cells were seeded in a 6-well plate at 12,500 cells/cm 2 and cultured in an incubator set at 37° C. and 5% CO 2 . After removing the medium by aspiration and adding DMEM containing 2% Horse Serum (HS) in the differentiation medium, the culture was continued. The medium was removed by aspiration, and serum starvation medium DMEM was added and conditioned 3 hours before exposure to the test substance. The medium was removed by aspiration, replaced with a medium containing the test substance, and cultured for an additional 24 hours. After the culture was completed, the medium was removed, a lysate buffer was added to lyse the cells, and the cell lysate was collected. RNA is recovered from the cell lysate using the RNeasy Mini Kit (Qiagen) according to the attached protocol, and using this as a template, cDNA is synthesized by reverse transcription reaction using ReverTra Ace (R) qPCR RT Master Mix (Toyobo). did. From the synthesized cDNA, the expression level of each mRNA of Cyclophilin, Cox4i1, TINN1 and Mb was measured by real-time PCR system (Step One Plus, Thermo Fisher Scientific) (SYBR Green method), and the expression of Cox4i1, TINN1 and Mb was determined. Amounts were corrected for Cyclophilin expression levels. The following primers were used. Cox4i1: MA112053, TINN1: MA110779, Mb: MA117187 (all from Takara Bio). As for Cyclophilin primers, a primer specific to Accession ID: M60456 was requested from invitrogen. The measured mRNA expression level was compared with that of a control group (medium containing no test substance) to evaluate various gene expression promoting effects of the test substance.
<Test results>
The results are shown in FIG. It was confirmed that the hamamelis extract and the eucalyptus extract increased the mRNA expression levels of Cox4i1, TINN1 and Mb. Based on the above, components that promote MITOL production can be used to promote the production of the factors listed in Table 1. In addition, when screening agents for promoting the production of the factors listed in Table 1, the MITOL production promoting action can be used as an index.

The ingredient that promotes MITOL production of the present invention prevents or improves aging symptoms such as dryness of the skin, appearance of wrinkles, loss of firmness/elasticity, increase in spots and dullness, appearance of gray hair, and improvement of muscle endurance. It can be used in the fields of cosmetics, quasi-drugs, pharmaceuticals, or food and beverages. It can also be used as a positive control when screening for components that promote MITOL production. Furthermore, in the present invention, the MITOL production-promoting action can be used as an indicator in screening for the production-enhancing agents for the factors listed in Table 1 and the inhibitors for the production of the factors listed in Table 2.

Claims (6)

containing as an active ingredient at least one selected from the group consisting of peach extract, hamamelis extract, niacinamide, bark extract, coptis extract, jersey extract, β-nicotinamide mononucleotide, and D-panthenol A MITOL reduction inhibitor or production promoter characterized by the above. An SOD2 or COL17A1 production promoter or MME production inhibitor containing a peach extract as an active ingredient. An SOD2 or COL17A1 production promoter containing a hamamelis extract as an active ingredient. An SOD2 production promoter containing a japonica japonica extract as an active ingredient. A LOXL1 production promoter containing β-nicotinamide mononucleotide as an active ingredient. A composition for preventing or improving gray hair, containing peach extract or hamamelis extract as an active ingredient.

Images