JP2018150262A - Anti-aging agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To find a natural product having an excellent anti-aging effect from among highly safe natural products, and to provide an anti-aging agent containing the same as an active ingredient. An extract from acai and / or an extract from cupuas is contained as an active ingredient of the anti-aging agent of the present invention. The extract from acai preferably has an ultraviolet damage suppressing effect, and the extract from cupuas has a radical scavenging effect, a type I collagen production promoting effect, a type IV collagen production promoting effect, a laminin 5 production promoting effect, and an epidermal keratin. Keratinocyte proliferation promoting action, glutathione 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 promoting action , And one or more selected from the group consisting of stratum corneum protein carbonylation inhibitory action. [Selection diagram] None

Description

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

In recent years, active oxygen and free radicals (hereinafter sometimes simply referred to as “radicals”) have attracted attention as factors that oxidize biological components, and their adverse effects on living organisms have become a problem. Active oxygen is generated in the process of energy metabolism in living cells. Examples of active oxygen include superoxide (that is, superoxide anion generated by one-electron reduction of oxygen molecules: .O ₂ ⁻ ), hydrogen peroxide (H ₂ O ₂ ) and the like. Normally, active oxygen produced in the body is sequentially eliminated by the catalytic action of various antioxidant enzymes contained in the cells. However, when the production of active oxygen is excessive, or When the action is lowered, superoxide is not sufficiently erased and the superoxide concentration is increased. Excess superoxide causes generation of other active 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 oxygens and free radicals are generated excessively, they attack in vivo molecules that make up membranes and tissues in the body, resulting in tissue damage such as rheumatoid arthritis and Behcet's disease, various arteriosclerosis (ischemic heart Diseases, myocardial 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, wrinkles, stiff shoulders, coldness Induce various diseases.

  In particular, since the skin is directly affected by environmental factors such as ultraviolet rays, it is an organ that easily generates active oxygen and free radicals. Therefore, when the concentration of these chemical species increases, for example, a living body such as collagen It is thought to break down tissue, denature or cross-link, oxidize fats and oils to produce lipid peroxides that damage cells, and damage 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 reduction (see Non-Patent Document 1). Therefore, by inhibiting or suppressing the generation of in vivo radicals, the above-mentioned diseases caused by skin aging such as wrinkle formation and reduced elasticity, and oxidative stress due to active oxygen, free radicals, etc. can be prevented. It can be treated or improved. As a substance having a radical scavenging action, an extract from the fruit of a star fruit (see Patent Document 1) and the like are known.

  The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and extracellular matrices such as collagen, elastin, and hyaluronic acid that are outside these cells and support the skin structure. In addition, a basement membrane is present at the boundary between the epidermis and dermis constituting the skin. The basement membrane not only connects the epidermis and 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 composed of type IV collagen. Various glycoproteins mainly composed of laminin 5 are present at the boundary between the basement membrane and the epidermis and connect the basement membrane and the epidermis. Such laminin 5 is produced from epidermal keratinocytes present in the epidermis. Is done. In young skin, the skin tissue such as epidermis cells, fibroblasts, basement membranes, extracellular matrix components, etc. maintain the homeostasis, ensuring moisture retention, flexibility, elasticity, etc. It is also kept fresh with tension and gloss.

  However, when there is an influence of certain external factors such as irradiation of ultraviolet rays, significant drying of air, excessive skin washing, etc., or when aging progresses, type I collagen, which is a major component of the extracellular matrix, The production of type IV collagen, laminin 5 and the like, which are the main constituents of the basement membrane, decreases, and causes degradation 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 its tension and gloss, and exhibits aging symptoms such as rough skin and wrinkles. Therefore, promoting the production of type I collagen, type IV collagen, laminin 5 and the like is important for preventing, treating or improving skin aging.

  Collagen is also abundant in bones, tendons, ligaments and the like, and it is known that a decrease in collagen production due to aging or the like causes osteoporosis and the like. Furthermore, it is known that in the wound healing process, the amount of collagen produced is increased and becomes a scaffold for fibroblasts to promote wound healing. Therefore, promoting the production of collagen is important from the viewpoint of preventing or treating osteoporosis and the like, and promoting wound healing. As what has a collagen production promotion effect, the extract (patent document 2) etc. from Kusunohagashi are known, for example.

  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 present in large amounts in the basement membrane of epithelial tissues such as skin, digestive organs, kidneys and lungs. In genetic diseases caused by congenital abnormalities of the genes encoding each chain of laminin 5 (lethal congenital epidermolysis bullosa, Herlitz junctional epidermolysis bullosa), the epidermis of the whole body may show fatal symptoms Are known. And laminin 5 strongly adheres cells (high cell adhesion activity) and strongly promotes cell motility (high cell motility activity) compared to other extracellular matrix molecules, so cells in damaged skin It is known to promote movement and promote wound healing (see Patent Document 3). That is, promoting the production of laminin 5 is important in promoting healing of skin damage that destroys the structure of the basement membrane.

  The epidermis functions to alleviate external stimuli and control the loss of body components such as moisture, and starts with the basal layer, the lowest layer, and continues to the spiny layer, granule layer, and stratum corneum. It is composed of Most cells present in each layer are keratinocytes differentiated from the basal layer. The keratinocytes that divide and proliferate in the basal layer differentiate into keratinocytes while passing through the spinous layer and the granule layer, forming a stratum corneum composed of keratin protein fibers with strong cross-linking, Eventually it will fall off the stratum corneum as plaque.

  The stratum corneum is present in the outermost shell of the skin and serves as a physical barrier against irritation from the outside world. In order to have this barrier function in the skin, the cycle (keratinization) from the production of keratinocytes in the basal layer to the peeling off of the keratinocytes (keratinization) is usually repeated at a cycle of 4 weeks to perform epidermal metabolism. . 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 skin aging such as wrinkles, dullness, and pigmentation can be improved by promoting the proliferation of keratinocytes and restoring the metabolic function of the skin. Conventionally, as a thing which has an epidermal keratinocyte proliferation promotion effect, a shellfish mother extract (refer to patent documents 4) etc. are known.

  Glutathione is a tripeptide consisting of three amino acids, glutamic acid, cysteine and glycine, and is a compound having a major cysteine residue in the cell. Intracellular glutathione functions as a radical donor, regulation of cell function by redox, foreign body metabolism, SH donor of various enzymes, and is also known as an antioxidant component against active oxygen and the like. Its action expression is thought 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., and this reduces the protective ability of cells against oxidative stress, and the cellular DNA In addition, it is considered to be a cause of damaging components such as proteins.

  In addition to the above-mentioned diseases that are induced by oxidative stress, diseases such as a decrease or deficiency in intracellular glutathione are known to be associated with the pathological condition. It is caused by drinking or ingestion of foreign substances such as heavy metals and chemical substances). That is, it is considered that promoting the production of glutathione can enhance the ability of cells to protect against oxidative stress, and can prevent and treat the above-mentioned disease group caused by the decrease or lack of intracellular glutathione level. . As a substance having a glutathione production promoting action, liquiritigenin (see Patent Document 5) and the like are known.

  Among the basal layer, spiny layer, granule layer, and stratum corneum constituting the epidermis, in particular, in the granule layer, the cell membrane is thickened to form a thickened cell membrane, and the action of transglutaminase-1 causes interprotein molecules. Are glutamyl-lysine cross-linked to form tough keratin protein fibers. Furthermore, ceramide or the like is covalently bonded to a part of the ceramide to form a hydrophobic structure, thereby providing a foundation for 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 skin moisturizing function decrease, so that skin aging symptoms such as rough skin and dry skin may occur, or dry skin diseases ( For example, atopic dermatitis, psoriasis, ichthyosis, etc.) may develop. Therefore, it is considered that skin aging symptoms, dry skin diseases, and the like can be prevented, treated, or improved by promoting production of transglutaminase-1 in the epidermis. Extracts from Hunan cocoon tea (see Patent Document 6) and the like are known as having transglutaminase-1 production promoting action.

  Filaggrin is a structural component of the skin, is involved in the barrier function in the skin, and is considered to have a function of preventing the entry of allergens, toxins and infectious organisms. Reduced function due to filaggrin gene mutation is associated with the risk of developing atopic diseases including atopic dermatitis (eczema, skin inflammation, skin itch, etc.), allergy, asthma, etc. 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 the degradation of filaggrin derived from keratohyalin granules in the stratum corneum. This filaggrin is expressed as profilagrin in epidermal keratinocytes present in the granule layer immediately below the stratum corneum. After that, it is immediately phosphorylated, accumulated in keratohyalin granules, decomposed to filaggrin through dephosphorylation and hydrolysis, transferred to the stratum corneum, increases the aggregation efficiency of keratin filaments and participates in the internal construction of keratinocytes It is known (see Non-Patent Document 4). In recent years, it is known that this filaggrin is very important and indispensable for moisture retention of the skin, and that the synthetic power of filaggrin is reduced by conditions such as drying and the amount of amino acids in the stratum corneum is reduced ( Non-patent document 5).

  Therefore, by promoting the expression of profilagrin in epidermal keratinocytes, it is possible to prevent, treat or improve atopic diseases including atopic dermatitis (eczema, skin inflammation, skin itch, etc.), allergy, asthma, etc. it is conceivable that. Moreover, it is expected that the water environment of the stratum corneum can be essentially improved by promoting the expression of profilagrin and thereby increasing the amount of amino acids in the stratum corneum.

  Conventionally, as a thing which has a profilaggrin mRNA expression promotion effect, a gaiyo extract (refer patent document 7) etc. are known.

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

  However, AQP3 decreases with aging, and it is suggested that this contributes to a decrease in the water retention function. Therefore, by promoting the expression of AQP3, the water retention function and barrier function due to aging, etc. Can be controlled (see Non-Patent Document 6). As what has an AQP3 expression promotion effect, the extract from the leaf part of a star fruit (refer patent document 8) etc. are known, for example.

  In the past, it was thought that only the stratum corneum was responsible for the barrier function of the skin, but in recent years, the protein constituting the tight junction (hereinafter sometimes referred to as “TJ”) present in the epidermal granule layer is genetically encoded. It has been found that the skin barrier function is disrupted when it is deficient at the level, and TJ is also considered to play an important role in the skin barrier function (see Non-Patent Document 7). TJ is an intercellular adhesion structure that controls not only the adhesion between adjacent cells but also the permeation of substances by sealing the gaps between cells. TJ is constituted by claudin or the like, which is a cell membrane protein, and these proteins constitute the skeleton of TJ strands and are thought to control the barrier function of TJ (see Non-Patent Document 8). Here, when Claudin expression is reduced 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, it promotes TJ formation of epidermal keratinocytes, thereby enhancing the skin barrier function and water retention function, such as dry skin, rough skin, atopic dermatitis and various infectious diseases It is considered that the skin symptoms can be prevented or ameliorated. Asparasine lineias extract (patent document 9) etc. are known as what has a claudin production promotion effect.

  SIRT1 is a protein included in a family called a sirtuin, is present in the cell nucleus and in the cytoplasm, and is known to function as an NAD-dependent protein deacetylase. In recent years, SIRT1 plays an important role in suppressing cell aging, and it has been revealed that it is particularly expressed in skin tissue and is involved in 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 improvement action, cardiovascular protection action, renal disease improvement action, inflammatory cytokine production suppression action, neuroprotection action and the like. Therefore, if SIRT1 can be activated, it is considered useful for the prevention / treatment or improvement of various diseases such as aging such as skin aging, diabetes, cardiovascular disease, nervous system disease, inflammatory disease and the like.

  Elastic fibers present in the dermis are essential for the property (elasticity) of the skin to expand and contract. This elastic fiber is formed by depositing and cross-linking elastin protein along the microfibril which is an extracellular fiber. 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 hydroxy lysine residue into a highly reactive aldehyde group, and the aldehyde group contributes to a crosslinking reaction.

  Collagen forms a collagen molecule (tropocollagen) having a helical structure by associating three polypeptides, and this self-associates extracellularly to produce collagen fibrils, and further collagen fibers (collagenous fibers). Form. Collagen intramolecular or extramolecular cross-linked structures 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 LOX leads to the formation of firm elastic fibers, increases the strength of collagen fibers, and prevents, treats or improves skin aging such as the disappearance of skin tension and the formation of wrinkles. It is thought to be connected.

In recent years, researches on stratum corneum carbonylated proteins have been actively conducted in relation to aging and photoaging with skin aging. Generally, carbonylated proteins include those produced as a result of direct oxidation of NH ₂ groups of amino acid residues such as Lys, Arg and Pro in proteins to carbonyl groups, lipids 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 protein oxidation may be initiated when protein is directly oxidized, or when sebum on the skin surface is oxidized by free radicals and lipid peroxide is generated. Once lipid peroxides are generated, oxidation proceeds in a chain, not only stimulating the skin surface, but also penetrates deep into the stratum corneum and damages cells.

  Further, the stratum corneum, which is the outermost layer of the skin, consists of keratinocytes, 85% of which is composed of keratin, a protein. In recent years, it has been known that this keratin is carbonylated by oxidative stress (ultraviolet rays, cigarette smoke) or the like that is routinely applied to the skin (see Non-Patent Document 10). Normally, keratin takes in a lot of water molecules, but carbonylation eliminates water molecules, and keratin after carbonylation cannot take up water molecules. It is known that the skin is dried and the appearance of the skin is impaired (see Patent Document 11).

  It is known that the increase in the carbonylated protein in the stratum corneum results in loss of water retention of the skin (see Patent Document 12) and loss of skin flexibility / elasticity ( (See Patent Document 13). Therefore, by preventing carbonylation of the horny layer protein of the epidermis or reducing its carbonylation degree, it is useful for improving the water retention of keratinocytes and improving the flexibility and elasticity of the skin. Conceivable.

  When the skin is irradiated with ultraviolet rays, the cells of the skin are damaged or cell death is caused, the skin loses its elasticity and elasticity, and it exhibits aging symptoms such as rough skin and wrinkles, so-called photoaging. It becomes. Therefore, prevention or improvement of skin aging can be expected by suppressing / recovering damage caused by ultraviolet irradiation (for example, cell damage, cell death, and the like, including gene expression fluctuations that cause these). Oil-soluble licorice extracts (see Patent Document 14) and the like are known as plant extracts having a damage recovery effect by ultraviolet irradiation.

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  An object of the present invention is to find an anti-aging agent having an excellent anti-aging action from natural products having high safety, and to provide an anti-aging agent having the active ingredient as an active ingredient.

  In order to solve the above-mentioned 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 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, transglutaminase-1 production Group consisting of promoting action, profilagrin mRNA expression promoting action, aquaporin 3 mRNA expression promoting action, claudin-1 production promoting action, sirtuin 1 mRNA expression promoting action, lysyl oxidase mRNA expression promoting action, and stratum corneum protein carbonylation inhibiting action It is preferable to have one or more selected actions, and it is preferable that the extract from the acai has a UV damage suppressing action.

  ADVANTAGE OF THE INVENTION According to this invention, the anti-aging agent which is excellent in an effect and is highly safe by containing the extract from the cupuas derived from a natural product and / or the extract from an acai as an active ingredient is provided. Can do.

Embodiments of the present invention will be described below.
The anti-aging agent of this embodiment contains the extract from a cupuas and / or the extract from an acai as an active ingredient.

  Here, in the present embodiment, the “extract” refers to an extract obtained using the above plant as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or these Either a crude product or a purified product is included.

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

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

  Acai (Euterpe oleracea, Euterpe precatoria, aka: Wakaba cabbage) is an evergreen tree belonging to the genus Euterpe, and is cultivated by agroforestry in these regions, as well as growing naturally in the Amazon region of South America. Can be easily obtained. Examples of the constituent part 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 a fruit part is preferable.

  The extract from the above cupuas and acai can be obtained by drying the extraction raw material, pulverizing it as it is or using a crusher, and subjecting it to extraction with an extraction solvent. Drying may be performed in the sun or using a commonly used dryer. Moreover, after performing pretreatment, such as degreasing, with a nonpolar solvent such as hexane, it may be used as an extraction raw material. By performing a pretreatment such as degreasing, the extraction material can be efficiently extracted with a polar solvent.

  As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water and hydrophilic organic solvents. These are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. It is preferable.

  Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, and those subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, and the like. Therefore, the water that can be used as the extraction solvent in this embodiment includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

  Examples of hydrophilic organic solvents that can be used as extraction solvents 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 using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when a mixed liquid of water and lower aliphatic alcohol is used as an extraction solvent, the mixing ratio of water and lower aliphatic alcohol is preferably 9: 1 to 1: 9 (volume ratio), More preferably, it is 7: 3 to 2: 8 (capacity ratio). In addition, when a mixed liquid 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). When using the liquid mixture with a monohydric alcohol, it is preferable that the mixing ratio of water and a polyhydric alcohol is 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 performed according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent 5 to 15 times (mass ratio) of the extraction raw material, the soluble components are extracted at room temperature or under reflux, and then filtered to remove the extraction residue. A liquid can be obtained. When the solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and when this concentrate is further dried, a dried product is obtained.

  Since the extract from Cupuas and / or the extract from Acai obtained as described above has an excellent anti-aging action, it can be used as an active ingredient of an anti-aging agent. The anti-aging agent of this embodiment can be used for a wide range of food and drink products 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 keratinocyte proliferation promoting action, glutathione, Production promotion action, transglutaminase-1 (TG-1) production promotion action, profilaggrin mRNA expression promotion action, aquaporin 3 (AQP3) mRNA expression promotion action, claudin-1 production promotion action, sirtuin 1 (SIRT1) mRNA expression promotion It is exhibited based on one or more actions selected from the group consisting of action, lysyl oxidase (LOX) mRNA expression promoting action, and stratum corneum protein carbonylation inhibiting action. However, the anti-aging action which the extract from Cupuas has is not limited to the anti-aging action exhibited based on the said action.

  In addition, the extract from cupuas has 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 production promoting action, profilagrin 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 inhibiting 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 , Aquaporin, profilagrin mRNA expression promoter (AQP3) Used as an active ingredient of mRNA expression promoter, claudin-1 production promoter, sirtuin 1 (SIRT1) mRNA expression promoter, lysyl oxidase (LOX) mRNA expression promoter, or stratum corneum protein carbonylation inhibitor May be.

  In this specification, “radical” means a molecule or atom having one or more unpaired electrons, and includes superoxide, hydroxy radical, DPPH and the like.

  The anti-aging effect of the extract from Acai is exerted on the basis of, for example, an ultraviolet damage suppression effect, and in particular, an inhibitory effect on gene expression fluctuation caused by ultraviolet (UVA) irradiation, particularly an MMP-1 (Matrix Metalloprotease-1) gene and It is exhibited based on the suppressive action of the mRNA expression increase by UVA irradiation of the IL-1α (Interleukin-1α) gene or the suppressive action of mRNA expression decrease by UVA irradiation of the COL1A1 (Collagen, TypeI, α1) gene. However, the anti-aging action which the extract from an acai has is not limited to the anti-aging action exhibited based on the said action. Moreover, you may use the extract from an acai as an active ingredient of a ultraviolet-ray damage inhibitor using the ultraviolet-ray damage inhibitory effect.

  The anti-aging agent of the present embodiment may be composed 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 anti-aging agent of the present embodiment is an arbitrary agent such as powder, granule, tablet, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier such as dextrin and cyclodextrin and any other auxiliary agent. It can be formulated into a form. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring / flavoring 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), and can also be used as an ointment, a liquid for external use, a patch and the like.

  When the anti-aging agent of this 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 according to the purpose.

  In addition, the anti-aging agent of this embodiment mix | blends the other natural extract etc. which have an anti-aging effect with the extract from a cupuas and / or the extract from an acai as an active ingredient as needed. be able to.

  Examples of the administration method of the anti-aging agent of this embodiment to a patient include transdermal administration, oral administration, and the like, and a suitable method for the prevention / treatment or the like may be appropriately selected according to the type of 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 differences among patients, administration method, administration period, and the like.

  The anti-aging agent of the present embodiment includes a radical scavenging action, an type I collagen production promoting action, a type IV collagen production promoting action, a laminin 5 production promoting action possessed by an extract from cupuas or an extract from acai as an active ingredient, Epidermal keratinocyte proliferation promoting action, glutathione 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 Through accelerating action, stratum corneum protein carbonylation inhibiting action, or UV damage inhibiting action, skin aging such as wrinkle formation and reduced elasticity can be prevented, treated or improved. However, the anti-aging agent of this embodiment can be used for all uses other than these uses that are meaningful to exhibit the above-described effects.

  For example, the anti-aging agent of the present embodiment or the radical scavenger described above can cause tissue damage such as rheumatoid arthritis and Behcet's disease, 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., active oxygen such as cataracts, diabetes, stiff shoulders, coldness, etc. Various diseases involving free radicals, etc. can be prevented, treated or ameliorated.

  In addition to the above-described uses, the anti-aging agent of the present embodiment or the above-described type I collagen production promoter or type IV collagen production promoter is a type I collagen production promoting action or type IV collagen production promotion possessed by an extract from Cupuas. Through the action, it can be used for applications such as prevention, treatment or amelioration of diseases caused by a decrease in collagen production such as osteoporosis; promotion of regeneration of damaged tendons and ligaments; promotion of healing of wounds or burns;

  In addition to the above-described uses, the anti-aging agent of the present embodiment or the laminin 5 production promoter described above induces remodeling of the basement membrane structure through the laminin 5 production promotion action of the extract from cupuas, thereby causing wounds in the skin. Can be treated and improved. Moreover, the anti-aging agent of this embodiment or the laminin 5 production promoter described above can be used as a prophylactic or therapeutic agent for diseases (such as epidermolysis bullosa) caused by laminin 5 deficiency (deficiency).

  In addition to the above-described uses, the anti-aging agent of the present embodiment or the above-described epidermal keratinocyte proliferation promoter restores skin metabolism through the action of promoting the epidermal keratinocyte proliferation possessed by the extract from Cupuas, and is It can be used for applications such as prevention, treatment or improvement of dullness, pigmentation, etc .; regenerative medicine;

  In addition to the above-described uses, the anti-aging agent of the present embodiment or the above-described glutathione production promoter is capable of liver damage (drinking 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 a disease or the like in which a decrease or deficiency in intracellular glutathione is known to be associated with a disease state, such as a cause of ingestion of a foreign substance.

  In addition to the above-described uses, the anti-aging agent of the present embodiment, the above-described TG-1 production promoter or the profilagrin mRNA expression promoter is a TG-1 production promotion action or a profilagulin mRNA expression promotion possessed by an extract from Cupuas. Through the action, the barrier function of the skin can be strengthened to prevent, treat or ameliorate dry skin diseases (eg, atopic dermatitis, psoriasis, ichthyosis, etc.) as well as rough skin and dry skin. In addition, the anti-aging agent of the present embodiment, the profilagrin mRNA expression promoter or the AQP3 mRNA expression promoter described above is capable of retaining water content and barrier function due to aging through its profilagrin mRNA expression promoting action or AQP3 mRNA expression promoting action. Etc. can be improved.

  In addition to the above-described uses, the anti-aging agent of the present embodiment or the above-mentioned claudin-1 production promoter is capable of forming tight junctions in epidermal keratinocytes through the claudin-1 production promoting action of the extract from Cupuas. Thus, the skin barrier function and moisture retention function can be enhanced, and skin symptoms such as dry skin, rough skin, atopic dermatitis and various infectious diseases can be prevented or improved.

  In addition to the above-described uses, the anti-aging agent of the present embodiment or the above-mentioned SIRT1 mRNA expression promoting agent can be used for diabetes, cardiovascular disease, nervous system disease, inflammatory disease through the SIRT1 mRNA expression promoting action of the extract from Cupuas. It is possible to prevent, treat or ameliorate various diseases such as, and to prolong the life of cells.

  In addition to the above-described uses, the anti-aging agent of the present embodiment or the above-described LOX mRNA expression promoting agent provides an appropriate cross-linked structure of elastic fibers and collagen fibers in the skin through the LOX mRNA expression promoting action of the extract from Cupuas. Forming, leading to the formation of firm elastic fibers and increasing the strength of the collagen fibers, thereby preventing, treating or ameliorating loss of tension and wrinkle formation.

  In addition to the use described above, the anti-aging agent of the present embodiment or the keratin protein carbonylation inhibitor described above suppresses the carbonylation of the horny layer protein through the inhibitory action of the horny layer protein carbonylation possessed by the extract from Cupuas. As a result, skin dullness can be suppressed and a beautiful skin effect can be obtained.

  The anti-aging agent of the present embodiment or the above-described ultraviolet damage inhibitor is an ultraviolet damage inhibitory action of an extract from Acai, more specifically, an action of suppressing gene expression fluctuations caused by UVA irradiation, particularly the MMP-1 gene and Inhibiting damage to cells caused by UV irradiation through suppression of mRNA expression increase by UVA irradiation of IL-1α gene or suppression of decrease of mRNA expression by UVA irradiation of COL1A1 gene, and thereby photoaging of skin Can be effectively prevented, treated or improved.

  Moreover, since the anti-aging agent of this embodiment has the outstanding anti-aging effect, it is suitable for mix | blending with a skin external preparation or food-drinks, for example. In this case, the extract from cupuas and / or the extract from acai may be blended as they are, or the anti-aging agent formulated from the extract from cupuas and / or the extract from acai may be blended. Also good.

  Here, the topical skin preparation is not limited in its category, and includes a wide range of skin cosmetics, quasi-drugs, pharmaceuticals, and the like used transdermally. Creams, milky lotions, lotions, cosmetics, lotions, gels, cosmetic oils, packs, foundations, lip balms, bath salts, hair nicks, hair lotions, shampoos, rinses, soaps, body shampoos and the like.

  Food and drink are those that are less likely to harm human health and are taken orally or by gastrointestinal administration in normal social life, and are classified as administrative divisions such as food, pharmaceuticals, and quasi drugs. It is not limited to. Therefore, “food and drink” in the present embodiment includes general foods that are taken orally, health foods (functional foods and drinks), health functional foods (special health foods, nutritional functional foods), quasi drugs, and pharmaceuticals. And so on.

  Moreover, since the anti-aging agent of this embodiment has the outstanding anti-aging effect | action, it can be utilized suitably also as a reagent for the research regarding these action mechanisms.

  In addition, although the anti-aging agent of this embodiment is applied suitably with respect to a human, as long as each effect is show | played, animals (for example, a mouse, a rat, a hamster, a dog, Cats, cattle, pigs, monkeys, etc.).

  Hereinafter, although a test example is shown and this invention is demonstrated concretely, this invention is not restrict | limited to each following example at all. In this test example, cupuas fruit extract (manufactured by Maruzen Pharmaceutical Co., Ltd., sample 1) and acai fruit extract (manufactured by Maruzen Pharmaceutical Co., Ltd., sample 2) were used as test samples.

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

  3 mL of a test sample solution (sample 1, sample concentration: see Table 1 below) was added to 3 mL of 150 μmol / L DPPH (diphenyl-p-picrylhydrazyl) ethanol solution, sealed, then shaken and allowed to stand 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. As a control, only the solvent used for dissolving the sample was added instead of the test sample solution and the same operation was performed, 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 a wavelength of 520 nm without addition of a sample B: Absorbance at a wavelength of 520 nm with addition of a test sample C: Absorbance at a wavelength of 520 nm immediately after addition of the test sample (blank) Table 1 shows the results.

  As shown in Table 1, it was recognized that the cupuas extract (sample 1) has an excellent radical scavenging action.

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

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

  After completion of the culture, the medium was removed, 100 μL of a test sample dissolved in Dulbecco's MEM medium containing 0.25% FBS (Sample 1, see Table 2 below for sample concentration) was added to each well and cultured for 3 days. As a control, the culture was similarly performed using Dulbecco's MEM medium containing 0.25% FBS without any sample. After culture, the amount of type I collagen in the medium of each well was measured by ELISA. 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: Type I collagen amount with test sample added B: Type I collagen amount without sample added Table 2 shows the results.

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

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

Normal human skin fibroblasts (NB1RGB) were cultured using Dulbecco's MEM medium containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with the above medium to a cell density of 1.6 × 10 ⁵ cells / mL, then seeded at 100 μL per well in 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, refer to Table 3 below for sample concentration) dissolved in 0.25% FBS-containing Dulbecco's MEM medium was added to each well and cultured for 3 days. As a control, the culture was similarly performed using Dulbecco's MEM medium containing 0.25% FBS without any sample. After culture, the amount of type IV collagen in the medium of each well was measured by ELISA. 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: Type IV collagen amount with test sample added B: Type IV collagen amount without sample added Table 3 shows the results.

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

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

Normal human newborn epidermal keratinocytes (NHEK) were pre-cultured in a 80 cm ² flask using normal human epidermal keratinocyte growth medium (KGM), and the cells were collected by trypsin treatment. The collected cells are diluted with a medium (KGM-BPE) obtained by removing BPE from KGM so that the cell density becomes 1.0 × 10 ⁵ cells / mL, and then seeded at 500 μL per well in a 24-well plate. Cultured for days.

  After completion of the culture, the medium was removed, and 500 μL of a test sample dissolved in KGM-BPE medium (Sample 1, see Table 4 below for sample concentration) was added to each well and cultured for 48 hours. In addition, it culture | cultivated similarly using the KGM-BPE culture medium without a sample as control. After completion of the culture, 100 μL of the supernatant was transferred to an ELISA plate, adsorbed on the plate at 37 ° C. for 2 hours, and then the amount of adsorbed laminin 5 was measured by ELISA. From the obtained measurement results, the laminin 5 production promotion rate (%) was calculated by the following formula.

Laminin 5 production promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Laminin 5 amount with addition of test sample B: Laminin 5 amount without addition of sample Table 4 shows the results.

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

[Test Example 5] Epidermal keratinocyte proliferation promoting action test The cupuas extract (sample 1) was tested for epidermal keratinocyte proliferation promoting action as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured using a growth medium (KGM) for normal human epidermal keratinocytes, and then cells were collected by trypsin treatment. The collected cells were diluted with KGM medium to a cell density of 3.0 × 10 ⁴ cells / mL, and then seeded at 100 μL per well in 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 cultured for 3 days. As a control, the same culture was performed using a sample-free KGM medium.

  The epidermal keratinocyte proliferation promoting action was measured using the MTT assay. 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. After culturing for 2 hours, blue formazan produced in the cells 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 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: Blue formazan production amount with addition of test sample B: Blue formazan production amount without addition of sample Table 5 shows the results.

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

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

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

  After culturing, 200 μL of a test sample (sample 1, refer to Table 6 below for sample concentration) dissolved in 1% FBS-containing Dulbecco's MEM medium was added to each well and cultured for 24 hours. As a control, the culture was similarly performed using Dulbecco's MEM medium containing 1% FBS without addition of the test sample. 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 (PIERCE).

  Of these, 100 μL was used to quantify total glutathione. Specifically, 100 μL of lysed cell extract, 50 μL of 0.1 mol / L phosphate buffer, 2 μL of NADPH and 25 μL of glutathione reductase (final concentration 17.5 unit / mL) were added to a 96-well plate, After warming for 5 minutes, 25 mmol of 10 mmol / L 5,5′-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm until 5 minutes was measured to obtain 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 × 100
Each term in the formula represents the following.
A: Glutathione amount per total protein amount without addition of sample B: Glutathione amount per total protein amount with test sample added Table 6 shows the results.

  As shown in Table 6, it was recognized that the cupuas extract (sample 1) has an excellent glutathione production promoting action in fibroblasts.

[Test Example 7] Glutathione production promoting action test (epidermal keratinocytes)
About the cupuas extract (sample 1), the glutathione production promoting effect in epidermal keratinocytes was tested as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured using a growth medium (KGM) for normal human epidermal keratinocytes, and then cells were collected by trypsin treatment. The collected cells were diluted with KGM medium to a cell density of 1.0 × 10 ⁵ cells / mL, and then seeded on a collagen-coated 24-well plate at 500 μL per well 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 cultured for 24 hours. As a control, the same culture was performed using a sample-free KGM medium. 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 (PIERCE).

  Of these, 100 μL was used to quantify total glutathione. Specifically, 100 μL of lysed cell extract, 50 μL of 0.1 mol / L phosphate buffer, 2 μL of NADPH and 25 μL of glutathione reductase (final concentration 17.5 unit / mL) were added to a 96-well plate, After warming for 5 minutes, 25 mmol of 10 mmol / L 5,5′-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm until 5 minutes was measured to obtain 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 × 100
Each term in the formula represents the following.
A: Glutathione amount per total protein amount when no sample was added B: Glutathione amount per total protein amount when the test sample was added Table 7 shows the results.

  As shown in Table 7, it was recognized that the cupuas extract (sample 1) had an excellent glutathione production promoting action.

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

Normal human neonatal epidermal keratinocytes (NHEK) were cultured using a growth medium for normal human epidermal keratinocytes (KGM), and then cells were collected by trypsin treatment. The collected cells were diluted with the above medium to a cell density of 1 × 10 ⁵ cells / mL, then seeded at 100 μL per well in 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 cultured for 24 hours. As a control, the same culture was performed using a sample-free KGM medium. After completion of the culture, the medium was removed, the cells were fixed to a plate, and the amount of transglutaminase-1 expressed on the cell surface was measured by ELISA using a monoclonal anti-human transglutaminase-1 antibody. 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: Transglutaminase-1 amount with addition of test sample B: Transglutaminase-1 amount without addition of sample Table 8 shows the results.

  As shown in Table 8, it was recognized that the cupuas extract (sample 1) has an excellent transglutaminase-1 production promoting action.

[Test Example 9] Profilaggrin mRNA expression promoting effect test The cupuas extract (sample 1) was tested for profilagrin mRNA expression promoting action as follows.

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

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

  Using this total RNA as a template, the expression level of mRNA was measured for profilaggrin and GAPDH as an internal standard. Detection was performed by real-time 2-step RT-PCR reaction using a real-time PCR device Smart Cycler (Cepheid) using a TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). . The expression level of profilagrin mRNA was determined as a GAPDH value based on the total RNA preparation prepared from cells cultured in “addition of test sample” and “no sample”, respectively. From the obtained value, the profilagrin 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 addition of test sample B: Correction value without addition of sample Table 9 shows the results.

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

[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 newborn epidermal keratinocytes (NHEK) were pre-cultured using a growth medium for normal human epidermal keratinocytes (KGM), and the cells were collected by trypsin treatment. The collected cells were diluted with KGM medium so as to have 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 / Petri dish), and cultured for 24 hours.

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

  Using this total RNA as a template, the expression level of mRNA was measured for AQP3 and GAPDH as an internal standard. Detection was performed by real-time 2-step RT-PCR reaction using a real-time PCR device Smart Cycler (Cepheid) using a TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). . The expression level of AQP3 mRNA was determined as a GAPDH value based on the total RNA preparation prepared from cells cultured in “addition of test sample” and “no sample addition”, respectively. From the obtained value, 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 addition of test sample B: Correction value without addition of sample Table 10 shows the results.

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

[Test Example 11] Claudin-1 production promoting action test With respect to the cupuas extract (sample 1), the claudin-1 production promoting action was tested as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured using a growth medium for normal human epidermal keratinocytes (KGM), and then cells were collected by trypsin treatment. The collected cells were diluted with the above medium to a cell density of 2 × 10 ⁵ cells / mL, then seeded at 100 μL per well in 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 cultured for 24 hours. As a control, the same culture was performed using a sample-free KGM medium. After completion of the culture, the medium was removed, the cells were fixed on a plate, and the amount of claudin-1 expressed on the cell surface was measured by ELISA 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 addition of test sample B: Claudin-1 amount without addition of sample Table 11 shows the results.

  As shown in Table 11, it was recognized that the cupuas extract (sample 1) has an excellent claudin-1 production promoting action.

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

Normal human skin fibroblasts (NB1RGB) were cultured using α-MEM medium containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with the above medium so as to have a cell density of 2.0 × 10 ⁵ cells / mL, then seeded with 5 mL each in a 60 mm petri dish (10 × 10 ⁵ cells / Petri dish) and 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, and 5 mL of the test sample dissolved in the FBS-free α-MEM medium (sample 1, refer to Table 12 below for sample concentration) was added to each dish and cultured for 24 hours. As a control, the same culture was performed using an FBS-free α-MEM medium containing no sample. After culturing, the medium is removed, total RNA is extracted with ISOGEN II (Nippon Gene, Cat. No. 311-07361), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 ng / μL. Total RNA was prepared.

  Using this total RNA as a template, the expression level of mRNA was measured for SIRT1 and GAPDH as an internal standard. Detection was performed by real-time 2-step RT-PCR reaction using a real-time PCR device Smart Cycler (Cepheid) using a TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). . The expression level of SIRT1 mRNA was determined by a GAPDH value based on the total RNA preparation prepared from the cells cultured in “addition of test sample” and “no sample addition”, respectively. From the obtained value, 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 addition of test sample B: Correction value without addition of sample Table 12 shows the results.

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

[Test Example 13] Lysyl oxidase (LOX) mRNA expression promoting effect test The cupuas extract (sample 1) was tested for LOX mRNA expression promoting action as follows.

Normal human skin fibroblasts (NB1RGB) were cultured using α-MEM medium containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with the above medium so as to have a cell density of 2.0 × 10 ⁵ cells / mL, then seeded with 5 mL each in a 60 mm petri dish (10 × 10 ⁵ cells / Petri dish) and 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, and 5 mL of each test sample (sample 1, refer to Table 13 below for sample concentration) dissolved in the FBS-free α-MEM medium was added and cultured for 24 hours. As a control, the same culture was performed using an FBS-free α-MEM medium containing no sample. After culturing, the medium is removed, total RNA is extracted with ISOGEN II (Nippon Gene, Cat. No. 311-07361), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 ng / μL. Total RNA was prepared.

  Using this total RNA as a template, the expression level of mRNA was measured for LOX and GAPDH as an internal standard. Detection was performed by real-time 2-step RT-PCR reaction using a real-time PCR device Smart Cycler (Cepheid) using a TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). . The expression level of LOX mRNA was determined as a GAPDH value based on the total RNA preparation prepared from cells cultured in “addition of test sample” and “no sample addition”, respectively. From the obtained value, 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 addition of test sample B: Correction value without addition of sample Table 13 shows the results.

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

[Test Example 14] Inhibition test of stratum corneum protein carbonylation by exhaust gas The cupuas extract (sample 1) was tested for its inhibitory action on stratum corneum protein carbonylation by exhaust gas as follows.

  The stratum corneum on the inner side of the upper arm, which is a non-exposed portion, was peeled and collected by a tape stripping method using a horny checker (manufactured by Asahi Biomed). The stratum corneum checker (sample) to which the stratum corneum adhered was immersed in 1 mL of an aqueous solution of a test sample (Sample 1, see Table 14 below for sample concentration), and treated at 37 ° C. for 16 hours. As a control, the same treatment was performed using water with no sample added. After treatment, the sample was removed and allowed to air dry. Subsequently, it was exposed to the exhaust gas of a 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 room air was also prepared. About the obtained sample, the level of stratum corneum carbonylation was evaluated 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), and then 20 μmol / L fluorescent hydrazide (fluorescein-5-thiosemicarbazide, manufactured by AnaSpec) The sample was immersed in a 0.1 mol / L MES buffer solution (pH 5.5) containing 1 and reacted for 1 hour at room temperature under light-shielding conditions to label the carbonyl group of the stratum corneum protein. After completion of the reaction, the plate was 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 luminance per stratum corneum area was defined as the carbonylation level. From the obtained results, the stratum corneum protein carbonylation inhibition rate (%) was calculated by the following formula.

Corneal layer protein carbonylation inhibition rate (%) = {1− (A−C) / (B−C)} × 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 added (control) C: Carbonylation level without exhaust gas treatment / sample added (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 by exhaust gas.

[Test Example 15] Inhibition of Gene Expression Variation due to Ultraviolet Irradiation The Acai extract (Sample 2) was tested for its suppression of gene expression variation due to ultraviolet (UVA) as follows.

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

After completion of the culture, the medium was removed, and 1.0 mL of a test sample dissolved in Dulbecco's MEM medium containing no FBS (Sample 2, see Table 15 below for sample concentration) was added to each well and cultured for 24 hours. In addition, it culture | cultivated similarly as control using the FBS-free Dulbecco MEM culture medium without a sample. After culturing, the medium was removed and replaced with 1.0 mL of HBSS (+) solution and irradiated with 10 J / cm ² of ultraviolet rays (UVA).

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

  Using this total RNA as a template, the expression level of mRNA was measured for MMP-1 (Matrix Metalloprotease-1), IL-1α (Interleukin-1α), COL1A1 (Collagen, TypeI, α1), and GAPDH as an internal standard. . 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 using a real-time PCR device Smart Cycler (Cepheid) and a real-time 2-Step RT-TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). Performed by PCR reaction. The expression level of each gene mRNA was determined as a GAPDH value based on the total RNA preparation prepared from the cells cultured in “addition of test sample” and “no sample addition”. From the obtained value, the mRNA expression variation suppression rate (%) of each gene was calculated by the following formula.

mRNA expression fluctuation suppression rate (%) = {(A−B) − (A−C)} / (A−B) × 100
Each term in the formula represents the following.
A: Correction value with no UV irradiation / no sample added B: Correction value with UV irradiation / no sample added (control) C: Correction value with UV irradiation / test sample added Table 15 shows the results.

  As shown in Table 15, the acai extract (sample 2) suppressed the increase in mRNA expression due to UVA irradiation in each gene of MMP-1 and IL-1α. Moreover, 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 improving photoaging caused by UVA irradiation.

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

Claims (2)

  An anti-aging agent comprising an extract from cupuas and / or an extract from acai as an active ingredient.   The extract from cupuas has 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, transglutaminase-1 production Group consisting of promoting action, profilagrin mRNA expression promoting action, aquaporin 3 mRNA expression promoting action, claudin-1 production promoting action, sirtuin 1 mRNA expression promoting action, lysyl oxidase mRNA expression promoting action, and stratum corneum protein carbonylation inhibiting action The anti-aging agent according to claim 1, wherein the anti-aging agent has one or more selected actions, and the extract from the acai has a UV damage suppressing action.