JP6986251B2 - Anti-aging agent - Google Patents

Description

The present invention relates to an anti-aging agent, and more particularly to an anti-aging agent containing an ingredient derived from a natural product as an active ingredient.

In recent years, active oxygen and free radicals (hereinafter, may be simply referred to as "radicals") have been attracting attention as factors that oxidize biological components, and their adverse effects on the living body have become a problem. Active oxygen, which occur in energy metabolism processes within living cells, as the active oxygen, superoxide (superoxide anion generated by the one-electron reduction of namely molecular oxygen: · O 2 ^_-), hydrogen peroxide (H ₂ O ₂ ) and the like can be mentioned. Normally, the active oxygen produced in the living body is sequentially eliminated by the catalytic action of various antioxidant enzymes contained in the cells, but when the production of active oxygen is excessive or the antioxidant enzyme When the action is reduced, the elimination of superoxide becomes insufficient and the superoxide concentration becomes high. Excessive superoxide causes the production of other reactive oxygen species and free radicals.

In addition to active oxygen, free radicals are generated by exposure to environmental factors such as air pollutants, radiation, ultraviolet rays, and tobacco. When these active oxygen and free radicals are excessively generated, they attack the in-vivo molecules that make up the membranes and tissues in the body, and as a result, tissue disorders such as rheumatoid arthritis and Bechet's disease, and various arteriosclerosis (ischemic heart). Diseases, myocardial infarction, cerebral ischemia, cerebral infarction, etc.), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's chorea, etc.), cancer, lung diseases caused by smoking, cataracts, diabetes, wrinkles, stiff shoulders, coldness Induces various diseases such as.

In particular, since the skin is directly stimulated by environmental factors such as ultraviolet rays, it is an organ in which active oxygen and free radicals are easily generated. Therefore, when the concentration of these chemical species increases, for example, a living body such as collagen. It is thought that it decomposes, denatures or cross-links tissues, and oxidizes fats and oils to produce lipid peroxides that damage cells, causing disorders caused by oxidative stress such as active oxygen and free radicals. , It is considered to cause aging such as skin wrinkle formation and skin elasticity decrease (see Non-Patent Document 1). Therefore, by inhibiting or suppressing the generation of radicals in the body, the above-mentioned group of diseases induced by skin aging such as wrinkle formation and decreased elasticity and oxidative stress caused by active oxygen and free radicals can be prevented. , Can be treated or improved. Extracts from the fruits of star fruit (see Patent Document 1) and the like are known to have a radical scavenging action.

The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and extracellular matrix such as collagen, elastin, and hyaluronic acid that are outside these cells and support the skin structure. In addition, a basement membrane exists at the boundary between the epidermis and the dermis that constitute the skin. The basement membrane not only connects the epidermis and the dermis, but also plays an important role in maintaining skin function (see Non-Patent Document 2). The main skeleton of the basement membrane has a network structure consisting of type IV collagen. Various glycoproteins containing laminin 5 as the main component exist at the boundary between the basement membrane and the epidermis and connect the basement membrane and the epidermis. Such laminin 5 is produced by epidermal keratinized cells existing in the epidermis. Will be done. In young skin, the interaction of skin tissues such as epidermal cells, fibroblasts, basement membranes, and extracellular matrix components maintains constancy to ensure water retention, flexibility, elasticity, etc., and the skin looks outward. It is kept taut and glossy and fresh.

However, when affected by certain external factors such as UV irradiation, significant dryness of air, and excessive skin cleaning, or as aging progresses, type I collagen, which is a major component of the extracellular matrix, and type I collagen, The production of type IV collagen, laminin 5, etc., which are the main constituents of the basement membrane, decreases, and at the same time, it causes decomposition and alteration. As a result, the moisturizing function and elasticity of the skin are lowered, and abnormal exfoliation of the keratin occurs, so that the skin loses tension and luster, and exhibits aging symptoms such as rough skin and wrinkles. Therefore, promoting the production of type I collagen, type IV collagen, laminin 5, etc. is important for preventing, treating or ameliorating skin aging.

In addition, collagen is also abundant in bones, tendons, ligaments, etc., and it is known that a decrease in collagen production due to aging or the like causes osteoporosis or the like. Furthermore, it is known that in the process of wound healing, the amount of collagen produced is increased and becomes a scaffold for fibroblasts and the like, thereby promoting wound healing. Therefore, promoting collagen production is also important from the viewpoint of prevention or treatment of osteoporosis and the like, and promotion of wound healing. As a collagen production promoting action, for example, an extract from Mallotus philippans (Patent Document 2) and the like are known.

Laminin consists of various combinations of α-chain, β-chain and γ-chain, and 15 types (laminin 1 to laminin 15) are currently known. Of these, laminin 5 (α3β3γ2) is abundantly present in the basement membrane of epithelial tissues such as skin, digestive organs, kidneys, and lungs. In genetic disorders caused by congenital abnormalities of the genes encoding each chain of laminin 5 (fatal congenital epidermolysis bullosa), the epidermis of the whole body may show fatal symptoms of exfoliation. Are known. Laminin 5 adheres cells more strongly (high cell adhesion activity) and strongly promotes cell motility (high cell motility activity) than other extracellular matrix molecules, and therefore cells in damaged skin. It is known to promote migration and promote injury healing (see Patent Document 3). That is, promoting the production of laminin 5 is important in promoting the healing of skin damage that destroys the structure of the basement membrane.

The epidermis has a function of alleviating external stimuli and controlling the loss of internal components such as water. It is composed of. Most of the cells present in each layer are keratinocytes differentiated from the basal layer. The keratinized cells that divide and proliferate in the basal layer differentiate while passing through the stratum spinosum and the stratum granulosum to become stratum corneum, which constitutes the stratum corneum composed of keratin protein fibers having strong cross-linking. Eventually it falls off the stratum corneum as dirt.

The stratum corneum is located in the outermost shell of the skin and serves as a physical barrier to external stimuli. In order to give this barrier function to the skin, the cycle (keratinization) from the production of keratinocytes in the basal layer to the exfoliation as dirt is usually repeated in a cycle of 4 weeks to metabolize the epidermis. .. However, the metabolic function of this stratum corneum also deteriorates with aging, and skin troubles such as wrinkles, dullness, pigmentation, and rough skin occur. Therefore, it is considered that the aging of the skin such as wrinkles, dullness, and pigmentation can be improved by promoting the proliferation of keratinocytes and restoring the metabolic function of the skin. Conventionally, a soil shell mother extract (see Patent Document 4) or the like is known as having an epidermal keratinocyte proliferation promoting action.

Glutathione is a tripeptide consisting of three amino acids, glutamic acid, cysteine and glycine, and is a compound having a major intracellular cysteine residue. Glutathione in cells functions as a scavenger of radicals, regulation of cell function by redox, metabolism of foreign substances, SH donor of various enzymes, and is also known as an antioxidant component against active oxygen and the like. Its onset of action is believed to be derived from cysteine residues. However, it has been reported that the amount of glutathione in cells is deficient or decreased due to excessive oxidative stress, addition of foreign substances, aging, etc., which reduces the defense ability of cells against oxidative stress and DNA of cells. And it is considered to be one of the causes of damaging constituents such as proteins.

Such diseases in which a decrease or deficiency of intracellular glutathione level is known to be related to the pathological condition include liver disorders (high alcohol content) in addition to the above-mentioned disease groups induced by oxidative stress. It is known to be caused by drinking or ingesting foreign substances such as heavy metals and chemical substances). That is, it is considered that promoting the production of glutathione can enhance the protective ability of cells against oxidative stress and prevent / treat the above-mentioned disease group caused by a decrease or deficiency of intracellular glutathione amount. .. Isoliquiritigenin (see Patent Document 5) and the like are known to have a glutathione production promoting action.

Of the basal layer, stratum spinosum, stratum granulosum, and stratum corneum that make up the epidermis, especially in the stratum granulosum, the cell membrane thickens to form a thickened cell membrane, and the action of transglutaminase-1 causes interprotein molecules. Is glutamil-lysine crosslinked to form tough keratin protein fibers. Furthermore, ceramide and the like covalently bond to a part thereof to form a hydrophobic structure, thereby supplying the base of the lamellar structure of the intercellular lipid and forming the basis of the keratin barrier function.

However, when the amount of transglutaminase-1 produced in the epidermis decreases with aging, the keratin barrier function and the moisturizing function of the skin decrease, resulting in skin aging symptoms such as rough skin and dry skin, and dry skin diseases (dry skin diseases). For example, atopic dermatitis, psoriasis, ichthyosis, etc.) will develop. Therefore, by promoting the production of transglutaminase-1 in the epidermis, it is considered that skin aging symptoms, dry skin diseases and the like can be prevented, treated or ameliorated. An extract from Hunan sweet tea (see Patent Document 6) and the like are known to have a transglutaminase-1 production promoting action.

Filaggrin is a component of the skin, is involved in the barrier function in the skin, and is thought to have the function of preventing the invasion of allergens, toxins, and infectious organisms. Decreased function due to mutation of filaggrin gene is associated with the risk of developing atopic dermatitis (eczema, skin inflammation, itchy skin, etc.), allergies, asthma, etc., and is more serious. In some cases, it is known to lead to skin diseases such as ichthyosis vulgaris (see Non-Patent Document 3).

On the other hand, amino acids, which are the main components of Natural Moisturizing Factors (NMF), are produced by degrading filaggrin derived from keratohyalin granules in the stratum corneum. This filaggrin is expressed as profilaggrin in the epidermal keratinocytes present in the stratum granulosum just below the stratum corneum. After that, it is immediately phosphorylated, accumulated in keratohyalin granules, decomposed into filaggrin via dephosphorylation and hydrolysis, and transferred to the stratum corneum to increase the aggregation efficiency of keratin filaments and participate in the internal construction of corneocytes. It is known (see Non-Patent Document 4). In recent years, it has been known that this filaggrin is very important and indispensable for water retention of the skin, and that the synthetic ability of filaggrin decreases due to conditions such as dryness, and the amount of amino acids in the stratum corneum decreases (). See Non-Patent Document 5).

Therefore, by promoting the expression of profilaggrin in epidermal keratinocytes, atopic dermatitis (eczema, skin inflammation, skin itching, etc.), allergies, asthma, and other atopic diseases can be prevented, treated, or ameliorated. it is conceivable that. In addition, it is expected that the water environment of the stratum corneum can be essentially improved by promoting the expression of profilaggrin and thereby increasing the amount of amino acids in the stratum corneum.

Conventionally, a gaiyo extract (see Patent Document 7) and the like are known as having an action of promoting the expression of profilaggrin mRNA.

In skin cells, it is known that aquaporins known as water channels are expressed on the cell membrane and play a role in taking up low molecular weight substances such as water in the intercellular spaces into the cells. In humans, the existence of 13 types of aquaporins (AQP0 to AQP12) is known. In epidermal cells, AQP3 is mainly present, and it is considered that it plays a role of taking up low molecular weight compounds such as glycerol and urea, which are involved in water retention action, in addition to water.

However, AQP3 decreases with aging, and it is suggested that this is one of the causes of the decrease in water retention function. Therefore, by promoting the expression of AQP3, the water retention function and barrier function due to aging, etc. It is considered possible to control (see Non-Patent Document 6). As a substance having an AQP3 expression promoting action, for example, an extract from the leaf portion of star fruit (see Patent Document 8) and the like are known.

In the past, it was thought that the barrier function of the skin was carried out only by the stratum corneum, but in recent years, the constituent proteins of tight junctions (hereinafter sometimes referred to as "TJ") existing in the stratum granulosum of the epidermis are genes. It has been found that the barrier function of the skin is disrupted when the skin is deficient at the level, and TJs are also considered to play an important role in the barrier function of the skin (see Non-Patent Document 7). TJ is an intercellular adhesion structure that not only brings adjacent cells into close contact with each other but also controls the permeation of a substance by sealing the gap between cells. TJ is composed of cell membrane proteins such as claudin, and these proteins are considered to constitute the skeleton of TJ strands and control the barrier function of TJ (see Non-Patent Document 8). Here, if the expression of claudin decreases for some reason, structural destruction of TJ occurs and it does not function as a permeation barrier for substances, resulting in dry skin, rough skin, atopic dermatitis, various infectious diseases, etc. It is thought to contribute to skin symptoms.

Therefore, by promoting the production of claudin in the epidermis and promoting the formation of TJ in the epidermal keratinocytes, the barrier function and water retention function of the skin are enhanced, and dry skin, rough skin, atopic dermatitis, various infectious diseases, etc. It is thought that the skin symptoms of the skin can be prevented or ameliorated. Asparagus linearis extract (Patent Document 9) and the like are known to have a claudin production promoting action.

SIRT1 is a protein included in a family called sirtuin, which is known to be present in the cell nucleus and cytoplasm and to function as a NAD-dependent protein deacetylase. In recent years, it has been clarified that this SIRT1 plays an important role in suppressing cell aging, and is particularly expressed in skin tissue and is involved in the suppression of skin aging (see Non-Patent Document 9). ). In addition to cell aging, SIRT1 has been shown to have various functions such as diabetes improving action, cardiovascular protective action, renal disease improving action, inflammatory cytokine production inhibitory action, and neuroprotective action. Therefore, if SIRT1 can be activated, it is considered to be useful for prevention, treatment or improvement of various diseases such as aging such as skin aging, diabetes, cardiovascular disease, nervous system disease and inflammatory disease.

Elastic fibers present in the dermis are essential for the elasticity of the skin. These elastic fibers are formed by depositing and cross-linking elastin proteins along microfibrils, which are extracellular fibers, and lysyl oxidase (LOX) plays an important role in this cross-linking reaction. LOX is an enzyme that converts an amino group of a lysine residue in a peptide chain or a hydroxyl group of a hydroxylysine residue into a highly reactive aldehyde group, and the aldehyde group contributes to the cross-linking reaction.

In collagen, three polypeptides are associated to form a collagen molecule (tropocollagen) having a spiral structure, which self-associates extracellularly to form collagen fibrils and further collagen fibrils (collagen fibrils). Form. Intramolecular or extramolecular cross-linked structures of collagen contribute to the strength of collagen fibers, and LOX is also involved in the formation of such cross-linked structures.

Therefore, increasing the expression level of such LOX leads to the formation of firm elastic fibers, increases the strength of collagen fibers, and prevents, treats or improves skin aging such as loss of tension and formation of wrinkles in the skin. It is considered to be connected.

In recent years, research on stratum corneum carbonylated proteins has been actively conducted in relation to skin aging and photoaging. _{Generally, carbonylated proteins are produced as a result of direct oxidation of two} NH groups of amino acid residues such as Lys, Arg, and Pro in the protein to form a carbonyl group, and lipid peroxides are oxidized to lipid peroxides. Is decomposed into a highly reactive aldehyde, which is produced by binding to a protein (see Patent Document 10). In the skin, it is considered that the protein may be directly oxidized, or the sebum on the surface of the skin may be oxidized by free radicals to generate lipid peroxide to initiate the oxidation of the protein. Once the lipid peroxide is produced, the oxidation proceeds in a chain reaction, not only stimulating the skin surface but also penetrating deep into the stratum corneum and damaging the cells.

The outermost layer of the skin, the stratum corneum, is composed of corneocytes, 85% of which is composed of the protein keratin. In recent years, it is known that this keratin is carbonylated by oxidative stress (ultraviolet rays, cigarette smoke) and the like that the skin receives on a daily basis (see Non-Patent Document 10). Normally, keratin takes up a lot of water molecules, but carbonylation eliminates water molecules, and after carbonylation, keratin cannot take in water molecules, and this also means that the skin It is known that the skin is dried and the appearance of the skin is impaired (see Patent Document 11).

It is known that such an increase in the carbonylated protein in the stratum corneum causes the skin to lose water retention (see Patent Document 12) and also loses the flexibility and elasticity of the skin (see Patent Document 12). See Patent Document 13). Therefore, by preventing the carbonylation of the stratum corneum protein of the epidermis and reducing the degree of carbonylation, it is useful for improving the water retention of the corneocytes and improving the flexibility and elasticity of the skin. Conceivable.

When the skin is exposed to ultraviolet rays, the cells of the skin are damaged or cell death is caused, the skin loses tension and elasticity, and exhibits aging symptoms such as rough skin and wrinkles, a condition called photoaging. It becomes. Therefore, prevention or improvement of skin aging can be expected by suppressing / recovering damage caused by irradiation with ultraviolet rays (for example, cell damage, cell death, etc., as well as fluctuations in the expression of genes that cause these). As a plant extract having a damage recovery effect by ultraviolet irradiation, an oil-soluble licorice extract (see Patent Document 14) and the like are known.

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An object of the present invention is to find a highly safe natural product having an excellent anti-aging effect and to provide an anti-aging agent containing the same as an active ingredient.

In order to solve the above problems, the anti-aging agent of the present invention is characterized by containing an extract from cupuas and / or an extract from acai as an active ingredient.

The extract from cupuas has a radical scavenging effect, a type I collagen production promoting effect, an IV type collagen production promoting effect, a laminin 5 production promoting effect, an epidermal keratinocyte proliferation promoting effect, a glutathione production promoting effect, and a transglutaminase-1 production. A group consisting of promoting action, profilaggrin mRNA expression promoting action, aquaporin 3 mRNA expression promoting action, claudin-1 production promoting action, sirtuin 1 mRNA expression promoting action, lysyloxidase mRNA expression promoting action, and stratum corneum protein carbonylation inhibitory action. It is preferable to have one or more kinds of actions selected from the above, and it is preferable that the extract from the acai has an ultraviolet damage suppressing action.

According to the present invention, by incorporating an extract from cupuas derived from a natural product and / or an extract from acai as an active ingredient, an antiaging agent having excellent action and effect and high safety is provided. Can be done.

Hereinafter, embodiments of the present invention will be described.
The anti-aging agent of the present embodiment contains an extract from cupuas and / or an extract from acai as an active ingredient.

Here, in the present embodiment, the "extract" is an extract obtained by using the above plant as an extraction raw material, a diluted solution or a concentrated solution of the extract, a dried product obtained by drying the extract, or these. Both crude and refined products are included.

The extraction raw materials used in this embodiment are cupuas (scientific name: Theobroma grandiflorum) and acai (scientific name: Euterpe oleracea, Euterpe precatoria).

Theobroma grandiflorum is an evergreen shrub belonging to the genus Theobroma of the Malvaceae family, which grows naturally in the Amazon region of South America and is cultivated by agroforestry in these regions and is easily available. Examples of the constituent parts of cupuas that can be used as an extraction raw material include a fruit part, a flower part, a leaf part, a trunk part, a bark part, a root part, or a mixture thereof, and the fruit part is preferable.

Acai (Euterpe oleracea, Euterpe precatoria, also known as Wakaba cabbage palm) is an evergreen tree belonging to the genus Euterpe of the palm family, which grows naturally in the Amazon region of South America and is cultivated by agroforestry in these regions. , Can be easily obtained. Examples of the constituent parts of acai that can be used as an extraction raw material include a fruit part, a leaf part, a trunk part, a bark part, a root part, or a mixture thereof, and the fruit part is preferable.

The above-mentioned extracts from cupuas and acai can be obtained by drying the extraction raw materials, crushing them as they are or using a coarse crusher, and subjecting them to extraction with an extraction solvent. Drying may be carried out in the sun or by using a commonly used dryer. Further, it may be used as an extraction raw material after being subjected to pretreatment such as degreasing with a non-polar solvent such as hexane. By performing pretreatment such as degreasing, the extraction treatment using the polar solvent of the extraction raw material can be efficiently performed.

As the extraction solvent, it is preferable to use a polar solvent, for example, water, a hydrophilic organic solvent and the like, and these are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. Is preferable.

Water that can be used as an extraction solvent includes pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmoregulation, buffering and the like. Therefore, the water that can be used as the extraction solvent in the present embodiment includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.

Hydrophilic organic solvents that can be used as the extraction solvent include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.

When a mixed solution of two or more polar solvents is used as an extraction solvent, the mixing ratio can be appropriately adjusted. For example, when a mixed solution of water and a lower aliphatic alcohol is used as an extraction solvent, the mixing ratio of water and the lower aliphatic alcohol is preferably 9: 1 to 1: 9 (volume ratio). It is more preferably 7: 3 to 2: 8 (volume ratio). When a mixed solution of water and a lower aliphatic ketone is used, the mixing ratio of water and the lower aliphatic ketone is preferably 9: 1 to 2: 8 (volume ratio), and water and a large amount are used. When a mixed solution with a valent alcohol is used, the mixing ratio of water and the polyhydric alcohol is preferably 8: 2 to 1: 9 (volume ratio).

The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be carried out according to a conventional method. For example, the extraction material is immersed in an extraction solvent 5 to 15 times the amount (mass ratio) of the extraction material, the soluble component is extracted at room temperature or under reflux heating, and then the extract is filtered to remove the extraction residue. You can get the liquid. Distilling off the solvent from the obtained extract gives a paste-like concentrate, and further drying the concentrate gives a dried product.

Since the extract from cupuas and / or the extract from acai obtained as described above has an excellent anti-aging effect, it can be used as an active ingredient of an anti-aging agent. The anti-aging agent of the present embodiment can be used in a wide range of food and drink applications such as pharmaceuticals, quasi-drugs, and cosmetics.

Here, the anti-aging action of the extract from cupuas is, for example, radical scavenging action, type I collagen production promoting action, type IV collagen production promoting action, laminin 5 production promoting action, epidermal keratinization cell proliferation promoting action, glutathione. Production promoting action, transcollagenase-1 (TG-1) production promoting action, profilaggrin mRNA expression promoting action, aquaporin 3 (AQP3) mRNA expression promoting action, claudin-1 production promoting action, sirtuin 1 (SIRT1) mRNA expression promoting action It is exerted on the basis of one or more actions selected from the group consisting of action, lysyloxidase (LOX) mRNA expression promoting action, and stratum corneum protein carbonylation inhibitory action. However, the anti-aging effect of the extract from cupuas is not limited to the anti-aging effect exerted based on the above-mentioned effect.

In addition, the extract from cupuas has its radical scavenging action, type I collagen production promoting action, type IV collagen production promoting action, laminin 5 production promoting action, epidermal keratinocyte proliferation promoting action, glutathione production promoting action, TG-1. Utilizing the production promoting action, profilaggrin mRNA expression promoting action, AQP3 mRNA expression promoting action, claudin-1 production promoting action, SIRT1 mRNA expression promoting action, LOX mRNA expression promoting action, or stratum corneum protein carbonylation inhibitory action, Radical scavenger, type I collagen production promoter, type IV collagen production promoter, laminin 5 production promoter, epidermal keratinocyte proliferation promoter, glutathione production promoter, transglutaminase-1 (TG-1) production promoter, respectively. , Profilaggrin mRNA expression promoter, aquaporin 3 (AQP3) mRNA expression promoter, claudin-1 production promoter, sirtuin 1 (SIRT1) mRNA expression promoter, ricyloxidase (LOX) mRNA expression promoter, or stratum corneum protein It may be used as an active ingredient of a carbonylation inhibitor.

In addition, in this specification, a "radical" means a molecule or an atom having one or more unpaired electrons, and includes superoxide, hydroxyl radical, DPPH and the like.

The anti-aging effect of the extract from acai is exerted, for example, based on the effect of suppressing UV damage, and above all, the effect of suppressing gene expression fluctuation due to ultraviolet (UVA) irradiation, particularly the MMP-1 (Matrix Metalloprotease-1) gene and / Or it is exerted based on the inhibitory effect of the IL-1α (Interleukin-1α) gene on the increase in mRNA expression by UVA irradiation, or the inhibitory effect on the COL1A1 (Collagen, Type I, α1) gene on the decrease in mRNA expression by UVA irradiation. However, the anti-aging effect of the extract from acai is not limited to the anti-aging effect exerted based on the above-mentioned effect. Further, the extract from acai may be used as an active ingredient of the ultraviolet damage suppressing agent by utilizing its ultraviolet damage suppressing action.

The anti-aging agent of the present embodiment may consist only of an extract from cupuas and / or an extract from acai, or may be a formulation of an extract from cupuas and / or an extract from acai. ..

The antiaging agent of the present embodiment is an arbitrary agent such as powder, granule, tablet, or liquid according to a conventional method, using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent. It can be formulated into a form. At this time, as the auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring / odorizing agent and the like can be used. The anti-aging agent can be used by blending with other compositions (for example, skin cosmetics, foods and drinks, etc.), and can also be used as an ointment, an external solution, a patch, and the like.

When the anti-aging agent of the present embodiment is formulated, the content of the extract from cupuas and / or the extract from acai is not particularly limited and can be appropriately set depending on the intended purpose.

The anti-aging agent of the present embodiment is used as an active ingredient by blending other natural extracts having an anti-aging effect and the like together with an extract from cupuas and / or an extract from acai, if necessary. be able to.

Examples of the administration method for the patient of the anti-aging agent of the present embodiment include transdermal administration, oral administration and the like, and a method suitable for prevention / treatment thereof may be appropriately selected depending on the type of the disease. In addition, the dose of the anti-aging agent of the present embodiment may be appropriately increased or decreased depending on the type of disease, severity, individual difference of patients, administration method, administration period and the like.

The anti-aging agent of the present embodiment has a radical scavenging action, a type I collagen production promoting action, an IV type collagen production promoting action, a laminin 5 production promoting action, which the extract from cupuas or the extract from acai which is an active ingredient has. Epidermal keratinocyte proliferation promoting action, collagen production promoting action, TG-1 production promoting action, profilaggrin mRNA expression promoting action, AQP3 mRNA expression promoting action, claudin-1 production promoting action, SIRT1 mRNA expression promoting action, LOX mRNA expression It is possible to prevent, treat or improve skin aging such as wrinkle formation and decreased elasticity through a promoting action, a stratum corneum protein carbonylation suppressing action, or an ultraviolet damage suppressing action. However, the anti-aging agent of the present embodiment can be used for all uses other than these uses, which are significant in exerting the above-mentioned action.

For example, the anti-aging agent of the present embodiment or the above-mentioned radical scavenging agent has tissue disorders such as rheumatoid arthritis and Bechet's disease and various arteriosclerosis (ischemic heart disease, myocardium) through the radical scavenging action of the extract from cupuas. Infarction, cerebral ischemia, cerebral infarction, etc.), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's chorea, etc.), lung diseases caused by cancer, smoking, etc., cataracts, diabetes, stiff shoulders, coldness, etc. Various diseases involving free radicals; etc. can be prevented, treated or ameliorated.

In addition to the above-mentioned uses, the anti-aging agent of the present embodiment or the above-mentioned type I collagen production promoter or type IV collagen production promoter has the type I collagen production promoting action or type IV collagen production promoting action of the extract from cupuas. Through its action, it can be used for prevention, treatment or amelioration of diseases caused by decreased collagen production such as osteoporosis; promotion of regeneration of damaged tendons and ligaments; promotion of healing of wounds or burns; and the like.

In addition to the above-mentioned uses, the anti-aging agent of the present embodiment or the above-mentioned laminin 5 production-promoting agent induces the reconstruction of the basement membrane structure through the laminin 5 production-promoting action of the extract from cupuas, and causes wounds in the skin. Can be treated and improved. In addition, the anti-aging agent of the present embodiment or the above-mentioned laminin 5 production promoter can be used as a preventive or therapeutic agent for diseases (epidermolysis bullosa, etc.) caused by deficiency (deficiency) of laminin 5.

In addition to the above-mentioned uses, the anti-aging agent of the present embodiment or the above-mentioned epidermal keratinocyte proliferation-promoting agent restores the skin metabolism through the epidermal keratinocyte proliferation-promoting action of the extract from Keratinocyte, and is agitated. It can be used for the prevention, treatment or improvement of dullness, pigmentation, etc .; regenerative medicine; etc.

In addition to the above-mentioned uses, the anti-aging agent of the present embodiment or the above-mentioned glutathione production-promoting agent can be used for liver damage (drinking of alcohol, heavy metals, chemical substances, etc.) through the glutathione production-promoting action of the extract from cupuas. It is possible to prevent, treat or ameliorate diseases in which a decrease or deficiency of intracellular glutathione amount (caused by ingestion of a foreign substance) is known to be associated with a pathological condition.

In addition to the above-mentioned uses, the anti-aging agent of the present embodiment or the above-mentioned TG-1 production-promoting agent or profilaggrin mRNA expression-promoting agent has a TG-1 production-promoting action or profilaggrin mRNA expression-promoting action of an extract from ichthyosis. Through the action, the barrier function of the skin can be strengthened, and in addition to rough skin, dry skin and the like, dry skin diseases (for example, atopic dermatitis, psoriasis, ichthyosis, etc.) can be prevented, treated or ameliorated. In addition, the anti-aging agent of the present embodiment or the above-mentioned profillagulin mRNA expression promoter or AQP3 mRNA expression promoter has a water retention ability and a barrier function due to aging through its profillagulin mRNA expression promoting action or AQP3 mRNA expression promoting action. Etc. can be improved.

In addition to the above-mentioned uses, the anti-aging agent of the present embodiment or the above-mentioned claudin-1 production-promoting agent forms tight junctions in epidermal keratinized cells through the claudin-1 production-promoting action of the extract from Kupuas. This can enhance the barrier function and water retention function of the skin, and can prevent or improve skin symptoms such as dry skin, rough skin, atopic dermatitis and various infectious diseases.

In addition to the above-mentioned uses, the anti-aging agent of the present embodiment or the above-mentioned SIRT1 mRNA expression-promoting agent has diabetes, cardiovascular disease, nervous system disease, and inflammatory disease through the SIRT1 mRNA expression-promoting action of the extract from cupuas. Various diseases such as these can be prevented / treated or ameliorated, and the life span of cells can be extended.

In addition to the above-mentioned uses, the anti-aging agent of the present embodiment or the above-mentioned LOX mRNA expression-promoting agent provides appropriate cross-linking structures of elastic fibers and collagen fibers in the skin through the LOX mRNA expression-promoting action of the extract from cupuas. It can form, lead to the formation of firm elastic fibers and increase the strength of collagen fibers, which can prevent, treat or ameliorate loss of tension and wrinkle formation.

In addition to the above-mentioned uses, the anti-aging agent of the present embodiment or the above-mentioned keratin protein carbonylation inhibitor suppresses the carbonylation of the stratum corneum protein through the stratum corneum protein carbonylation inhibitory action of the extract from cupuas. This makes it possible to suppress dullness of the skin and obtain a skin-beautifying effect.

The antiaging agent of the present embodiment or the above-mentioned ultraviolet damage inhibitor has an ultraviolet damage inhibitory effect of the extract from acai, more specifically, an inhibitory effect on gene expression fluctuation due to UVA irradiation, particularly the MMP-1 gene and / Or the IL-1α gene's UVA irradiation suppresses the increase in mRNA expression, or the COL1A1 gene's UVA irradiation suppresses the decrease in mRNA expression, thereby suppressing the damage to cells caused by UV irradiation, thereby photoaging the skin. Can be effectively prevented, treated or ameliorated.

Further, since the anti-aging agent of the present embodiment has an excellent anti-aging effect, it is suitable for blending with, for example, an external skin preparation or a food or drink. In this case, the extract from cupuas and / or the extract from acai may be blended as it is, or the antiaging agent formulated from the extract from cupuas and / or the extract from acai may be blended. May be good.

Here, the external skin preparation is not limited in its classification, and includes a wide range of skin lotions, non-pharmaceutical products, pharmaceuticals, etc. that are used transdermally, and specifically, for example, an ointment. Examples include creams, milky lotions, lotions, beauty liquids, lotions, gels, beauty oils, packs, foundations, lip creams, bathing agents, hair tonics, hair lotions, shampoos, rinses, soaps, body shampoos and the like.

Foods and drinks are those that are less likely to harm human health and are ingested by oral or gastrointestinal administration in normal social life, and are classified as foods, pharmaceuticals, quasi-drugs, etc. under administrative divisions. It is not limited to. Therefore, the "food and drink" in the present embodiment includes general foods, health foods (functional foods and drinks), health functional foods (specified health foods, nutritional functional foods), non-pharmaceutical products, and pharmaceuticals that are orally ingested. Etc. are included in a wide range.

Further, since the anti-aging agent of the present embodiment has an excellent anti-aging effect, it can be suitably used as a reagent for research on these action mechanisms.

Although the anti-aging agent of the present embodiment is suitably applied to humans, animals other than humans (for example, mice, rats, hamsters, dogs, etc.) as long as their respective actions and effects are exhibited. It can also be applied to cats, cows, pigs, monkeys, etc.).

Hereinafter, the present invention will be specifically described with reference to test examples, but the present invention is not limited to the following examples. In this test example, a cupuas fruit extract (Maruzen Pharmaceuticals Co., Ltd., sample 1) and an acai fruit extract (Maruzen Pharmaceuticals Co., Ltd., sample 2) were used as test samples.

[Test Example 1] Radical scavenging action test (DPPH method)
The radical scavenging action of the cupuas extract (sample 1) was tested as follows.

To 3 mL of a 150 μmol / L DPPH (diphenyl-p-picrylhydrazyl) ethanol solution, 3 mL of a test sample solution (sample 1, sample concentration see Table 1 below) was added, sealed, and then shaken and left for 30 minutes. After standing, the absorbance at a wavelength of 520 nm was measured. As a blank, 3 mL of the test sample solution was added to ethanol, and then the absorbance at a wavelength of 520 nm was measured immediately. Further, as a control, the same operation was performed by adding only the solvent used for dissolving the sample instead of the test sample solution, and the absorbance at a wavelength of 520 nm was measured. From the obtained results, the radical scavenging rate (%) was calculated by the following formula.

Radical scavenging rate (%) = {A- (BC)} / A × 100
Each term in the formula represents the following.
A: Absorbance at wavelength 520 nm without sample addition B: Absorbance at wavelength 520 nm with test sample addition C: Absorbance at wavelength 520 nm immediately after test sample addition (blank) Table 1 shows the results.

As shown in Table 1, the cupuas extract (sample 1) was found to have an excellent radical scavenging effect.

[Test Example 2] Type I collagen production promoting action test The cupuas extract (Sample 1) was tested for the type I collagen production promoting action as follows.

Normal human skin fibroblasts (NB1RGB) were cultured in Dulbecco MEM medium containing 10% FBS, and then the cells were recovered by trypsin treatment. After dilution with the medium to recovered cells to a cell density of 1.6 × 10 ⁵ cells / mL, seeded by 100μL per well in 96-well microplates and cultured overnight.

After completion of the culture, the medium was removed, and 100 μL of a test sample (Sample 1, sample concentration see Table 2 below) dissolved in a 0.25% FBS-containing Dalveco MEM medium was added to each well and cultured for 3 days. As a control, Dulbecco MEM medium containing 0.25% FBS without sample was used for the same culture. After culturing, the amount of type I collagen in the medium of each well was measured by the ELISA method. From the measurement results, the type I collagen production promotion rate (%) was calculated by the following formula.

Type I collagen production promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Amount of type I collagen with the addition of the test sample B: Amount of type I collagen without the addition of the sample The results are shown in Table 2.

As shown in Table 2, it was confirmed that the cupuas extract (sample 1) had an excellent type I collagen production promoting action.

[Test Example 3] Type IV collagen production promoting action test The IV type collagen production promoting action was tested on the cupuas extract (Sample 1) as follows.

Normal human skin fibroblasts (NB1RGB) were cultured in Dulbecco MEM medium containing 10% FBS, and then the cells were recovered by trypsin treatment. The collected cells were ^{diluted with the above medium to a cell density of 1.6 × 10 5} cells / mL, and then 100 μL per well was seeded on a 96-well microplate and cultured overnight.

After completion of the culture, the medium was removed, and 50 μL of a test sample (Sample 1, sample concentration see Table 3 below) dissolved in a 0.25% FBS-containing Dalveco MEM medium was added to each well and cultured for 3 days. As a control, Dulbecco MEM medium containing 0.25% FBS without sample was used for the same culture. After culturing, the amount of type IV collagen in the medium of each well was measured by the ELISA method. From the measurement results, the type IV collagen production promotion rate (%) was calculated by the following formula.

Type IV collagen production promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Amount of type IV collagen with the addition of the test sample B: Amount of type IV collagen without the addition of the sample The results are shown in Table 3.

As shown in Table 3, it was confirmed that the cupuas extract (sample 1) had an excellent type IV collagen production promoting action.

[Test Example 4] Laminin 5 production promoting action test The laminin 5 production promoting action was tested on the cupuas extract (Sample 1) as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were ^{precultured in 80 cm 2} flasks using growth medium for normal human epidermal keratinocytes (KGM), and the cells were recovered by tripsin treatment. The collected cells were diluted with a medium (KGM-BPE) obtained by removing BPE from KGM so as to have a cell density ^{of 1.0 × 10 5 cell / mL, and then seeded in a 24-well plate at a rate of 500 μL per well.} Cultured for days.

After completion of the culture, the medium was removed, and 500 μL of a test sample (sample 1, sample concentration, see Table 4 below) dissolved in the KGM-BPE medium was added to each well and cultured for 48 hours. As a control, the cells were similarly cultured using KGM-BPE medium to which no sample was added. After completion of the culture, 100 μL of the supernatant was transferred to an ELISA plate and adsorbed on the plate at 37 ° C. for 2 hours, and then the amount of adsorbed laminin 5 was measured by the ELISA method. From the obtained measurement results, the laminin 5 production promotion rate (%) was calculated by the following formula.

Laminin 5 production promotion rate (%) = A / B x 100
Each term in the formula represents the following.
A: 5 amounts of laminin with the addition of the test sample B: 5 amounts of laminin without the addition of the sample The results are shown in Table 4.

As shown in Table 4, it was confirmed that the cupuas extract (sample 1) had an excellent laminin 5 production promoting action.

[Test Example 5] Epidermal keratinization cell proliferation promoting action test The epidermal keratinocyte proliferation promoting action was tested on the cupuas extract (Sample 1) as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured in a growth medium for normal human epidermal keratinocytes (KGM), and then the cells were recovered by tripsin treatment. The collected cells were ^{diluted with KGM medium to a cell density of 3.0 × 10 4} cells / mL, and then 100 μL per well was seeded on a collagen-coated 96-well plate and cultured overnight. After completion of the culture, 100 μL of a test sample dissolved in KGM medium (Sample 1, see Table 5 below for sample concentration) was added to each well, and the cells were cultured for 3 days. As a control, the cells were similarly cultured using KGM medium to which no sample was added.

The epidermal keratinocyte proliferation promoting effect was measured using the MTT assay method. That is, after culturing for 3 days, the medium was removed, and 100 μL of MTT dissolved in PBS (−) buffer at a final concentration of 0.4 mg / mL was added to each well. After culturing for 2 hours, the intracellularly produced blue formazan was extracted with 100 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. At the same time, the absorbance at a wavelength of 650 nm was measured as the turbidity, and the difference between the two was used as the amount of blue formazan produced. From the obtained results, the epidermal keratinocyte proliferation promotion rate (%) was calculated by the following formula.

Epidermal keratinocyte proliferation promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Amount of blue formazan produced with the addition of the test sample B: Amount of blue formazan produced without the addition of the sample The results are shown in Table 5.

As shown in Table 5, it was confirmed that the cupuas extract (Sample 1) has an excellent epidermal keratinization cell proliferation promoting action.

[Test Example 6] Glutathione production promoting action test (fibroblasts)
The cupuas extract (Sample 1) was tested for glutathione production promoting action in fibroblasts as follows.

Normal human skin fibroblasts (NB1RGB) were cultured in α-MEM medium containing 10% FBS, and then the cells were recovered by trypsin treatment. The collected cells were ^{diluted with α-MEM medium containing 10% FBS so as to have a cell density of 2.0 × 10 5} cells / mL, and then 200 μL per well was seeded on a 48-well plate and cultured overnight.

After culturing, 200 μL of a test sample (Sample 1, see Table 6 below for sample concentration) dissolved in Dulbecco MEM medium containing 1% FBS was added to each well, and the cells were cultured for 24 hours. As a control, Dulbecco MEM medium containing 1% FBS without the addition of the test sample was used for the same culture. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS (−) buffer, and then the cells were lysed using 150 μL of M-PER (manufactured by PIERCE).

Total glutathione was quantified using 100 μL of this. That is, 100 μL of lysed cell extract, 50 μL of 0.1 mol / L phosphate buffer, 25 μL of 2 mmol / L NADPH, and 25 μL of glutathione reductase (final concentration 17.5 units / mL) were added to a 96-well plate, and 10 at 37 ° C. After heating for 1 minute, 25 μL of 10 mmol / L 5,5'-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm up to 5 minutes was measured to determine ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione (manufactured by Wako Pure Chemical Industries, Ltd.). After correcting the obtained value to the amount of glutathione per total protein amount, the glutathione production promotion rate (%) was calculated by the following formula.

Glutathione production promotion rate (%) = B / A x 100
Each term in the formula represents the following.
A: Glutathione amount per total protein amount without sample addition B: Glutathione amount per total protein amount with test sample addition Table 6 shows the results.

As shown in Table 6, the cupuas extract (Sample 1) was found to have an excellent glutathione production promoting action in fibroblasts.

[Test Example 7] Glutathione production promoting action test (epidermal keratinized cells)
The cupuas extract (Sample 1) was tested for glutathione production promoting action in epidermal keratinized cells as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured in a growth medium for normal human epidermal keratinocytes (KGM), and then the cells were recovered by tripsin treatment. The collected cells were ^{diluted with KGM medium to a cell density of 1.0 × 10 5} cells / mL, and then 500 μL per well was seeded on a collagen-coated 24-well plate and cultured overnight.

After completion of the culture, 400 μL of a test sample dissolved in KGM medium (Sample 1, see Table 7 below for sample concentration) was added to each well, and the cells were cultured for 24 hours. As a control, the cells were similarly cultured using KGM medium to which no sample was added. After completion of the culture, the medium was removed from each well, washed with 1 mL of PBS (-) buffer, and then the cells were lysed using 150 μL of M-PER (manufactured by PIERCE).

Total glutathione was quantified using 100 μL of this. That is, 100 μL of lysed cell extract, 50 μL of 0.1 mol / L phosphate buffer, 25 μL of 2 mmol / L NADPH, and 25 μL of glutathione reductase (final concentration 17.5 units / mL) were added to a 96-well plate, and 10 at 37 ° C. After heating for 1 minute, 25 μL of 10 mmol / L 5,5'-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm up to 5 minutes was measured to determine ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione (manufactured by Wako Pure Chemical Industries, Ltd.). After correcting the obtained value to the amount of glutathione per total protein amount, the glutathione production promotion rate (%) was calculated by the following formula.

Glutathione production promotion rate (%) = B / A x 100
Each term in the formula represents the following.
A: Glutathione amount per total protein amount without sample addition B: Glutathione amount per total protein amount with test sample addition Table 7 shows the results.

As shown in Table 7, the cupuas extract (Sample 1) was found to have an excellent glutathione production promoting action.

[Test Example 8] Transglutaminase-1 production promoting action test The transglutaminase-1 production promoting action was tested on the cupuas extract (Sample 1) as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured in a growth medium for normal human epidermal keratinocytes (KGM), and then the cells were recovered by tripsin treatment. The collected cells were ^{diluted with the above medium to a cell density of 1 × 10 5} cells / mL, and then 100 μL per well was seeded on a 96-well plate and cultured for 2 days.

After completion of the culture, 100 μL of a test sample dissolved in KGM medium (Sample 1, see Table 8 below for sample concentration) was added to each well, and the cells were cultured for 24 hours. As a control, the cells were similarly cultured using KGM medium to which no sample was added. After completion of the culture, the medium was removed, the cells were immobilized on a plate, and the amount of transglutaminase-1 expressed on the cell surface was measured by the ELISA method using a monoclonal anti-human transglutaminase-1 antibody. From the obtained measurement results, the transglutaminase-1 production promotion rate (%) was calculated by the following formula.

Transglutaminase-1 production promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Amount of transglutaminase-1 with the addition of the test sample B: Amount of transglutaminase-1 without the addition of the sample The results are shown in Table 8.

As shown in Table 8, the cupuas extract (Sample 1) was found to have an excellent transglutaminase-1 production promoting action.

[Test Example 9] Profilaggrin mRNA expression promoting action test The profilaggrin mRNA expression promoting action was tested on the cupuas extract (Sample 1) as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were precultured using growth medium for normal human epidermal keratinocytes (KGM), and the cells were recovered by tripsin treatment. The collected cells were ^{diluted with the above medium to a cell density of 20 × 10 4} cells / mL, then seeded in 2 mL each in a 35 mm petri dish (40 × 10 ⁴ cells / mL) and cultured for 24 hours.

After culturing, the medium was removed, 2 mL of a test sample dissolved in KGM medium (see Table 9 below for sample 1 and sample concentration) was added to each petri dish, and the cells were cultured for 24 hours. As a control, the cells were similarly cultured using KGM medium to which no sample was added. After culturing, the medium is removed, total RNA is extracted with ISOGEN II (Cat. No. 311-07361, manufactured by Nippon Gene), and the amount of each RNA is measured with a spectrophotometer so that the total RNA is 200 ng / μL. Total RNA was prepared in.

Using this total RNA as a template, the expression level of mRNA was measured for profilaggrin and GAPDH, which is an internal standard. Detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio Inc., code No. RR063A) using a real-time PCR device Smart Cycler (manufactured by Cepheid). .. The expression level of profilaggrin mRNA was corrected by the GAPDH value based on the total RNA standard prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the profilaggrin mRNA expression promotion rate (%) was calculated by the following formula.

Profilaggrin mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Correction value with the addition of the test sample B: Correction value without the addition of the sample The results are shown in Table 9.

As shown in Table 9, the cupuas extract (Sample 1) had an excellent effect of promoting the expression of profilaggrin mRNA.

[Test Example 10] Aquaporin 3 (AQP3) mRNA expression promoting action test The cupuas extract (Sample 1) was tested for AQP3 mRNA expression promoting action as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were precultured using growth medium for normal human epidermal keratinocytes (KGM), and the cells were recovered by tripsin treatment. The collected cells were ^{diluted with KGM medium to a cell density of 20 × 10 4} cells / mL, then seeded in 2 mL each in a 35 mm petri dish (40 × 10 ⁴ cells / mL) and cultured for 24 hours.

After culturing, the medium was removed, 2 mL of a test sample dissolved in KGM medium (Sample 1, see Table 10 below for sample concentration) was added to each petri dish, and the cells were cultured for 24 hours. As a control, the cells were similarly cultured using KGM medium to which no sample was added. After culturing, the medium is removed, total RNA is extracted with ISOGEN II (Cat. No. 311-07361, manufactured by Nippon Gene), and the amount of each RNA is measured with a spectrophotometer so that the total RNA is 200 ng / μL. Total RNA was prepared in.

Using this total RNA as a template, the expression level of mRNA was measured for AQP3 and GAPDH, which is an internal standard. Detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio Inc., code No. RR063A) using a real-time PCR device Smart Cycler (manufactured by Cepheid). .. The expression level of AQP3 mRNA was corrected by the GAPDH value based on the total RNA preparation prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the AQP3 mRNA expression promotion rate (%) was calculated by the following formula.

AQP3 mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Correction value with the addition of the test sample B: Correction value without the addition of the sample The results are shown in Table 10.

As shown in Table 10, it was confirmed that the cupuas extract (sample 1) had an excellent AQP3 mRNA expression promoting action.

[Test Example 11] Claudin-1 production promoting action test The claudin-1 production promoting action was tested on the cupuas extract (Sample 1) as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured in a growth medium for normal human epidermal keratinocytes (KGM), and then the cells were recovered by tripsin treatment. The collected cells were ^{diluted with the above medium to a cell density of 2 × 10 5} cells / mL, and then 100 μL per well was seeded on a 96-well plate and cultured overnight.

After completion of the culture, 100 μL of a test sample dissolved in KGM medium (Sample 1, see Table 11 below for sample concentration) was added to each well, and the cells were cultured for 24 hours. As a control, the cells were similarly cultured using KGM medium to which no sample was added. After completion of the culture, the medium was removed, the cells were immobilized on a plate, and the amount of claudin-1 expressed on the cell surface was measured by the ELISA method using a polyclonal anti-human claudin-1 antibody. From the obtained measurement results, the claudin-1 production promotion rate (%) was calculated by the following formula.

Claudin-1 production promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Claudin-1 amount with the addition of the test sample B: Claudin-1 amount without the sample addition The results are shown in Table 11.

As shown in Table 11, the cupuas extract (Sample 1) was found to have an excellent claudin-1 production promoting action.

[Test Example 12] Sirtuin 1 (SIRT1) mRNA expression promoting action test The SIRT1 mRNA expression promoting action was tested on the cupuas extract (Sample 1) as follows.

Normal human skin fibroblasts (NB1RGB) were cultured in α-MEM medium containing 10% FBS, and then the cells were recovered by trypsin treatment. The collected cells were ^{diluted with the above medium to a cell density of 2.0 × 10 5} cells / mL, 5 mL each was seeded in a 60 mm petri dish (10 × 10 ⁵ cells / mL), and the cells were cultured overnight. After completion of the culture, the medium was removed, replaced with 5 mL of FBS-free α-MEM medium, and further cultured for 24 hours.

After completion of the culture, the medium was removed, 5 mL of a test sample (sample 1, sample concentration, see Table 12 below) dissolved in an FBS-free α-MEM medium was added to each petri dish, and the cells were cultured for 24 hours. As a control, the cells were similarly cultured using an FBS-free α-MEM medium containing no sample. After culturing, the medium is removed, total RNA is extracted with ISOGEN II (Cat. No. 311-07361, manufactured by Nippon Gene), and the amount of each RNA is measured with a spectrophotometer so that the total RNA is 200 ng / μL. Total RNA was prepared in.

Using this total RNA as a template, the expression level of mRNA was measured for SIRT1 and GAPDH, which is an internal standard. Detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio Inc., code No. RR063A) using a real-time PCR device Smart Cycler (manufactured by Cepheid). .. The expression level of SIRT1 mRNA was corrected by the GAPDH value based on the total RNA preparation prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the SIRT1 mRNA expression promotion rate (%) was calculated by the following formula.

SIRT1 mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Correction value with the addition of the test sample B: Correction value without the addition of the sample The results are shown in Table 12.

As shown in Table 12, it was confirmed that the cupuas extract (sample 1) had an excellent SIRT1 mRNA expression promoting action.

[Test Example 13] Test of lysyl oxidase (LOX) mRNA expression promoting action The cupuas extract (Sample 1) was tested for the LOX mRNA expression promoting action as follows.

Normal human skin fibroblasts (NB1RGB) were cultured in α-MEM medium containing 10% FBS, and then the cells were recovered by trypsin treatment. The collected cells were ^{diluted with the above medium to a cell density of 2.0 × 10 5} cells / mL, 5 mL each was seeded in a 60 mm petri dish (10 × 10 ⁵ cells / mL), and the cells were cultured overnight. After completion of the culture, the medium was removed, replaced with 5 mL of FBS-free α-MEM medium, and further cultured for 24 hours.

After completion of the culture, the medium was removed, 5 mL of a test sample (sample 1, sample concentration, see Table 13 below) dissolved in an FBS-free α-MEM medium was added to each petri dish, and the cells were cultured for 24 hours. As a control, the cells were similarly cultured using an FBS-free α-MEM medium containing no sample. After culturing, the medium is removed, total RNA is extracted with ISOGEN II (Cat. No. 311-07361, manufactured by Nippon Gene), and the amount of each RNA is measured with a spectrophotometer so that the total RNA is 200 ng / μL. Total RNA was prepared in.

Using this total RNA as a template, the expression level of mRNA was measured for LOX and GAPDH, which is an internal standard. Detection was performed by real-time 2 Step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio Inc., code No. RR063A) using a real-time PCR device Smart Cycler (manufactured by Cepheid). .. The expression level of LOX mRNA was corrected by the GAPDH value based on the total RNA standard prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the LOX mRNA expression promotion rate (%) was calculated by the following formula.

LOX mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Correction value with the addition of the test sample B: Correction value without the addition of the sample The results are shown in Table 13.

As shown in Table 13, it was confirmed that the cupuas extract (sample 1) had an excellent LOX mRNA expression promoting action.

[Test Example 14] Test of inhibitory effect on carbonylation of stratum corneum protein by exhaust gas The cupuas extract (Sample 1) was tested on the inhibitory effect on carbonylation of stratum corneum protein by exhaust gas as follows.

The stratum corneum on the inner side of the upper arm, which is an unexposed portion, was peeled off and recovered by a tape stripping method using a stratum corneum checker (manufactured by Asahi Biomed). The stratum corneum checker (sample) to which the stratum corneum was attached was immersed in 1 mL of an aqueous solution of a test sample (sample 1, sample concentration is shown in Table 14 below) and treated at 37 ° C. for 16 hours. As a control, water without adding a sample was used for the same treatment. After the treatment, the sample was taken out and air-dried. Then, it was exposed to the exhaust gas of the gasoline engine collected in a plastic bag with a chuck and reacted at room temperature for 24 hours. As a blank, a sample exposed to indoor air was also prepared. The level of carbonylation of the stratum corneum was evaluated for the obtained sample by the following method.

The sample was washed twice with 1 mL of 0.1 mol / L MES (2-morpholinoethane sulfonic acid-Na) buffer (pH 5.5), then 20 μmol / L fluorescent hydrazide (fluorescein-5-thiosemicarbazide, manufactured by AnaSpec). The sample was immersed in a 0.1 mol / L MES buffer (pH 5.5) containing fluorescein and reacted at room temperature under light-shielded conditions for 1 hour to label the carbonyl group of the stratum corneum protein. After completion of the reaction, the cells were washed twice with 1 mL of PBS (-) buffer, and images were taken with a fluorescence microscope (Olympus IX71, manufactured by Olympus). The obtained image was analyzed using image analysis software (ImageJ, manufactured by National Institute of Health), and the fluorescence brightness per stratum corneum area was taken as the carbonylation level. From the obtained results, the stratum corneum protein carbonylation inhibition rate (%) was calculated by the following formula.

Weapon protein carbonylation inhibition rate (%) = {1- (AC) / (BC)} × 100
Each term in the formula represents the following.
A: Carbonylation level with exhaust gas treatment / addition of test sample B: Carbonylation level with exhaust gas treatment / no sample addition (control) C: Carbonylation level with no exhaust gas treatment / no sample addition (blank) Result Is shown in Table 14.

As shown in Table 14, the cupuas extract (Sample 1) effectively suppressed the carbonylation of the stratum corneum due to the exhaust gas.

[Test Example 15] Test of inhibitory effect on gene expression fluctuation by ultraviolet irradiation The acai extract (Sample 2) was tested for the inhibitory effect on gene expression fluctuation by ultraviolet (UVA) as follows.

Normal human skin fibroblasts (NB1RGB) were cultured in Dulbecco MEM medium containing 10% FBS, and then the cells were recovered by trypsin treatment. The collected cells were ^{diluted with Dulbecco MEM medium containing 2% FBS so as to have a cell density of 1.0 × 10 5} cells / mL, and then 1.0 mL each was seeded on a 12-well plate and cultured overnight.

After completion of the culture, the medium was removed, 1.0 mL of a test sample dissolved in FBS-free Dalveco MEM medium (Sample 2, see Table 15 below for sample concentration) was added to each well, and the cells were cultured for 24 hours. As a control, a sample-free FBS-free Dulbecco MEM medium was used for the same culture. After culturing, the medium was removed, replaced with 1.0 mL of HBSS (+) solution, ^{and irradiated with 10 J / cm 2} ultraviolet (UVA).

Immediately after UVA irradiation, the HBSS (+) solution was removed, 1.0 mL of FBS-free Dulbecco MEM medium was added to each well, and the cells were cultured for 24 hours. After culturing, the medium is removed, total RNA is extracted with ISOGEN II (Cat. No. 311-07361, manufactured by Nippon Gene), and the amount of each RNA is measured with a spectrophotometer so that the total RNA is 50 ng / μL. Total RNA was prepared in.

Using this total RNA as a template, the expression levels of mRNA were measured for MMP-1 (Matrix Metalloprotease-1), IL-1α (Interleukin-1α), COL1A1 (Collagen, TypeI, α1), and the internal standard GAPDH. .. MMP-1 and IL-1α are genes whose mRNA expression is increased by UVA irradiation, while COL1A1 is a gene whose mRNA expression is decreased by UVA irradiation. The mRNA expression level is detected by using the real-time PCR device Smart Cycler (manufactured by Cepheid) and the TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (manufactured by Takara Bio Inc., code No. RR063A) in real time 2 Step RT-. This was done by PCR reaction. The expression level of each gene mRNA was corrected by the GAPDH value based on the total RNA standard prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the mRNA expression fluctuation suppression rate (%) of each gene was calculated by the following formula.

Suppression rate of mRNA expression fluctuation (%) = {(AB)-(AC)} / (AB) × 100
Each term in the formula represents the following.
A: Correction value without UV irradiation / sample addition B: Correction value with UV irradiation / sample addition (control) C: Correction value with UV irradiation / sample addition The results are shown in Table 15.

As shown in Table 15, the acai extract (Sample 2) suppressed the increase in mRNA expression by UVA irradiation in each gene of MMP-1 and IL-1α. In addition, in the COL1A1 gene, the acai extract (Sample 2) suppressed the decrease in mRNA expression due to UVA irradiation. Therefore, it was confirmed that the acai extract is effective in preventing or ameliorating photoaging caused by UVA irradiation.

The anti-aging agent of the present invention can greatly contribute to the prevention, treatment or amelioration of skin aging symptoms; promotion of healing of wounds or burns; prevention, treatment or amelioration of dry skin diseases.

Claims (1)

The extract obtained by extracting the fruit Kupuasu a polar solvent an anti-aging agent as an active ingredient,
Type I collagen production promotion use, IV type collagen production promotion use, laminin 5 production promotion use, epidermal keratinocyte proliferation promotion use, transglutaminase-1 production promotion use, profilaggrin mRNA expression promotion use, aquaporin 3 mRNA expression promotion use, It is used for one or more uses selected from the group consisting of claudin-1 production promoting use, sirtuin 1 mRNA expression promoting use, lysyloxidase mRNA expression promoting use, and stratum corneum protein carbonylation suppressing use.
The polar solvent is one solvent selected from the group consisting of water, a lower aliphatic alcohol having 1 to 5 carbon atoms, a lower aliphatic ketone having 3 to 4 carbon atoms and a polyhydric alcohol having 2 to 5 carbon atoms. An anti-aging agent characterized by being a mixed solvent of two or more kinds.