JP2015221759A - Collagen synthetic promoter, hyaluronic acid synthetic promoter, and ceramide synthetic promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective collagen synthetic promoter, hyaluronic acid synthetic promoter, and ceramide synthetic promoter.SOLUTION: A collagen (hyaluronic acid, ceramide) synthetic promoter of the invention contains pine bark processed products. The collagen synthetic promoter of the invention promotes expression of COL1A1 and COL3A1 in the skin. The hyaluronic acid synthetic promoter of the invention enhances gene expression of Has2 or Has3 which is hyaluronan synthase in the skin. The ceramide synthetic promoter of the invention enhances gene expression of SPT which is ceramide synthase.

Description

  The present invention relates to an agent for promoting the synthesis of collagen, hyaluronic acid and ceramide, and an agent for promoting the expression of genes involved in the synthesis of these components.

  Collagen is the main structural protein of the biological skeleton that occupies about 30% of the biological protein. Collagen plays an important role in maintaining the structure as a supporting tissue in the body, and is present in most tissues in the body such as skin, blood vessels, tendons, bones, and teeth to protect cells and bind cells as intercellular factors. It plays an important physiological role. Among the collagens present in the body, especially the collagen present in the skin has been extensively studied so far in relation to beauty and treatment of skin diseases and skin injuries. The skin is composed of the epidermis and the dermis existing below the epidermis, and the dermis is the widest part of the skin. Collagen is a major component of the extracellular matrix, where more than 90% of the dermis is occupied by the extracellular matrix. Examples of collagen present in the skin include type I collagen and type III collagen, and type I collagen is said to account for 80% of the total collagen present in the adult dermis. Type I collagen consists of two α1 chains and one α2 chain, and is said to have a role of forming bone and skin and having elasticity. COL1A1 is known as a gene encoding this α1 chain. Type III collagen is homotrimeric collagen containing three identical α1 (III) chains. Type III collagen is a relatively thin fiber that forms a thin network structure called a reticulated fiber, creating a scaffold for cells and the like, as well as giving tissue flexibility. COL3A1 is known as a gene encoding this α1 (III) chain.

  Hyaluronic acid is a high molecular weight polysaccharide present in the epidermis and dermis in the skin, cartilage, joint fluid, etc., retention of moisture in the cell gap, retention of cells based on forming a jelly-like matrix in the tissue, It has many functions such as maintaining the lubricity and flexibility of tissues, resistance to external forces such as mechanical failure, and prevention of bacterial infection. Hyaluronic acid is present in a large amount in the extracellular matrix in the skin, particularly in the dermis, together with collagen, has a very high ability to retain moisture, and is deeply involved in the freshness, firmness and elasticity of the skin. Hyaluronic acid is produced by hyaluronic acid synthase. As mammalian hyaluronic acid synthase, Has1, Has2 and Has3 are known to exist. Has2 is a major hyaluronic acid synthase in human dermis and is said to be involved in the synthesis of high-molecular hyaluronic acid with high moisturizing power. Has3 is considered to be the main hyaluronic acid synthase in the human epidermis. Further, Has3 synthesizes low-molecular hyaluronic acid, and this low-molecular hyaluronic acid is expected to function in the immune mechanism, and its relationship with lung injury and the like due to artificial ventilation is known.

  Ceramide is a major component of interstitial cell lipids in the outermost stratum corneum in the epidermis and plays a role in suppressing the evaporation of water in the body. Specifically, ceramide exists specifically as the main component of stratum corneum intercellular lipid that covers the outermost layer of the skin, and plays an important role in maintaining the function of the skin as a barrier film between the living body and the outside world. Yes. The stratum corneum structure is compared to brick and mortar, and a strong barrier membrane is formed in such a way that intercellular lipids connect the stratum corneum cells stacked in about 15 layers. The stratum corneum retains moisture by containing a natural moisturizing factor mainly composed of amino acids in the cell, while stratum corneum lipids are mainly composed of about 50% ceramide, such as cholesterol and fatty acids. It is composed of amphipathic lipids and is characterized by a so-called lamellar structure in which hydrophobic and hydrophilic portions are alternately repeated. Ceramide is produced by the action of enzymes such as serine palmitoyltransferase, which is known as a rate-limiting enzyme for ceramide synthesis, based on serine and palmitoyl-CoA in the process of keratinization of epidermal cells.

  Collagen, hyaluronic acid, and ceramide are known to decrease due to various causes such as aging, exposure to ultraviolet rays, drying, and oxidation. For example, reduction of type I collagen in the skin is said to reduce the strength of skin, bones, etc., and reduction of type III collagen reduces skin moisture. It is known that the reduction of type I collagen and type III collagen in the skin causes skin wrinkles and talmi. In addition, it is known that the decrease in hyaluronic acid in the skin is related to dryness of the skin, weakness of the skin, decreased elasticity, wrinkles, and abnormal joint function. In addition, for ceramide, when the secretion of ceramide is impaired as a lipid (ie, interstitial cell lipid) between the stratum corneum, the skin cannot retain moisture, and a variety of factors such as atopic dermatitis are caused by lack of moisture. It is said to develop skin diseases.

  In recent years, it has been reported that the intake of a high-fat diet reduces the amount of type I collagen and hyaluronic acid in the skin as well as the gene expression level of its constituent genes or synthases (Non-patent Document 1). It has been pointed out that it is related to the delay of wound healing, worsening of bedsores, etc.

  Various compositions have been proposed as compositions having an action of promoting the production of collagen, hyaluronic acid and ceramide. For example, Patent Document 1 describes cystatin and / or a cystatin degradation product, and Patent Document 2 describes TGF-β and / or a TGF-β degradation product as having collagen production promoting action. Patent Document 3 describes a plant of Lingonberry (Vaccinium vitis-idaea) and Amla (Phyllanthus emblica or Emblica officinale) or an extract thereof, and Patent Document 4 describes glycerophospholipid. Yes. Patent Document 5 describes hyaluronic acid or a pharmaceutically acceptable salt thereof as having a hyaluronic acid production promoting action, and Patent Document 6 describes a milk basic protein fraction and / or milk. Basic protein fraction degradation products are described, and Patent Document 7 describes eggshell membranes. Moreover, sphingoids are described in patent document 8 as what has a ceramide production promotion effect | action.

JP 2012-0707019 A JP 2012-167021 A JP 2013-014561 A JP 2013-060476 A JP 2013-180989 A JP2013-234129A JP 2014-040402 A JP 2012-092032 A

Mol. Nutr. Food. Res. 2010, 54, S53-S61

  An object of the present invention is to provide an agent effective for promoting the synthesis of collagen in the skin or the like. Another object of the present invention is to provide an agent effective for promoting the synthesis of hyaluronic acid in skin and the like. Another object of the present invention is to provide an agent effective for promoting the synthesis of ceramide in the skin or the like.

The present invention provides a collagen synthesis promoter characterized by containing a treated pine bark.
The present invention provides a collagen gene COL1A1 or COL3A1 expression promoter characterized by containing a treated pine bark.
The present invention provides a hyaluronic acid synthesis promoter characterized by containing a treated pine bark.
The present invention provides a gene expression promoter for Has2 or Has3, which is a hyaluronic acid synthase, containing a treated pine bark.
The present invention provides a ceramide synthesis promoter characterized by containing a treated pine bark.
The present invention provides a gene expression promoter for SPT, which is a ceramide synthase, characterized by containing a treated pine bark.

  ADVANTAGE OF THE INVENTION According to this invention, the agent effective in promotion of the synthesis | combination of collagen in skin etc. is provided. According to the present invention, an agent effective for promoting the synthesis of hyaluronic acid in the skin or the like is provided. According to the present invention, an agent effective in promoting the synthesis of ceramide in the skin or the like is provided.

FIG. 1 is a graph showing the expression level of collagen gene COL1A1 in Examples and Reference Examples. FIG. 2 is a graph showing the expression level of collagen gene COL3A1 in Examples and Reference Examples. FIG. 3 is a graph showing the gene expression level of hyaluronic acid synthase Has2 in Examples and Reference Examples. FIG. 4 is a graph showing the gene expression level of hyaluronic acid synthase Has3 in Examples and Reference Examples. FIG. 5 is a graph showing the gene expression level of ceramide synthase SPT in Examples and Reference Examples.

  Hereinafter, the present invention will be described based on preferred embodiments thereof. The present invention includes a collagen synthesis promoter containing a processed pine bark, a collagen gene expression promoter, a hyaluronic acid synthesis promoter, a hyaluronic acid synthase gene expression promoter, a ceramide synthesis promoter, and a ceramide synthase gene expression It relates to accelerators. Preferably, a pine bark processed product is contained as an active ingredient, and more preferably, a pine bark extract is contained as an active ingredient. Hereinafter, these agents are collectively referred to as agents of the present invention. The following description applies to any of the agents of the present invention unless otherwise indicated.

  The raw material pine of the processed pine bark used in the agent of the present invention includes French pine (Pinus Martina), New Zealand pine, Finnish pine, Ekinata pine, larch, black pine, red pine, Japanese pine, Japanese pine, Korean pine, high pine, strobe pine, sugar pine , Ryukyu pine, slash pine, caribbean pine, rigid pine, tea pine, daishasho, taiwan red pine, banks pine, tsukushima pine, daimatsu, white pine, Canadian quebec aneda. Among them, the processed products of bark from French coastal pine, New Zealand pine, and Finnish pine are preferable, and French coastal pine is particularly preferable.

  French coastal pine is a marine pine that grows on the Atlantic coast of southern France. The bark of this French coastal pine contains organic acids and other physiologically active components in addition to containing proanthocyanidins, which are flavonoids, as main components. This main component, proanthocyanidins, is known to have a strong antioxidant action to remove active oxygen.

  Examples of the processed pine bark include a pine bark pulverized product obtained by pulverizing pine bark, and a pine bark extract obtained by extraction with water and / or an organic solvent. These can be used alone or in combination. Among them, it is preferable to use a pine bark extract from the viewpoint of the remarkable effect at the time of use by the above-mentioned proanthocyanidins and other physiologically active ingredients and the stability of the quality. When water is used for obtaining the pine bark extract, warm water or hot water is used. As an organic solvent used for extraction, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, butane, acetone, hexane, cyclohexane, propylene glycol, hydrous ethanol, hydrous propylene glycol, ethyl methyl ketone, Organic solvents that are acceptable for the production of food or drugs such as glycerin, methyl acetate, ethyl acetate, diethyl ether, dichloromethane, edible oils and fats, 1,1,1,2-tetrafluoroethane, 1,1,2-trichloroethene Preferably used. These water and organic solvents may be used alone or in combination. In particular, hot water, hydrous ethanol, hydrous propylene glycol and the like are preferably used.

  The extraction method from pine bark for obtaining the pine bark extract is not particularly limited, and for example, a warm extraction method, a supercritical fluid extraction method, or the like is used.

  The supercritical fluid extraction method is a method of performing extraction using a supercritical fluid that is a fluid that exceeds the critical point (critical temperature, critical pressure) of a gas-liquid substance. As the supercritical fluid, carbon dioxide, ethylene, propane, nitrous oxide (laughing gas) or the like is used, and carbon dioxide is preferably used.

  In the supercritical fluid extraction method, an extraction process for extracting a target component with a supercritical fluid and a separation process for separating the target component and the supercritical fluid are performed. In the separation step, any one of extraction separation by pressure change, extraction separation by temperature change, and extraction separation using an adsorbent / absorbent may be performed.

  Moreover, you may perform supercritical fluid extraction by the entrainer addition method. In this method, for example, ethanol, propanol, n-hexane, acetone, toluene, other aliphatic lower alcohols, aliphatic hydrocarbons, aromatic hydrocarbons, and ketones are added to the extraction fluid in an amount of 2 to 20 W / V. It is a method that drastically increases the solubility of the target extract in the extraction solvent or enhances the separation selectivity by performing supercritical fluid extraction using this fluid. It is a method of obtaining a pine bark extract.

  Since the supercritical fluid extraction method can be operated at a relatively low temperature, it can be applied to substances that are altered or decomposed at high temperatures, the advantage that the extraction fluid does not remain, and the recycling of the solvent is possible. There is an advantage that the process and the like can be omitted, and the process becomes simple.

  Further, the extraction method from pine bark for obtaining a pine bark extract may be performed by a method such as a liquid carbon dioxide batch method, a liquid carbon dioxide reflux method, a supercritical carbon dioxide reflux method, etc., in addition to the above extraction method. Good.

  The extraction method from the pine bark for obtaining the pine bark extract may be a combination of a plurality of extraction methods. By combining a plurality of extraction methods, it becomes possible to obtain pine bark extracts having various compositions.

  The extract from pine bark can be purified by ultrafiltration or column method or batch method using an adsorbent carrier (Diaion HP-20, Sephadex-LH20, chitin, etc.). To preferred. Further, if necessary, it may be concentrated or dried by a method such as vacuum concentration or freeze-drying to obtain a liquid, paste, powder (extract powder), fine granule, or granule. The processed pine bark such as pine bark extract is preferably in the form of powder, fine particles, or granules.

  The treated pine bark used in the present invention preferably contains proanthocyanidins as one of main components. Proanthocyanidins refer to a group of compounds consisting of a condensation polymer having a degree of polymerization of 2 or more, with flavan-3-ol and / or flavan-3,4-diol as a structural unit. Proanthocyanidins are powerful antioxidants produced by plants and are intensively contained in plant leaves, bark, fruit peels and seeds. This proanthocyanidin is a substance that cannot be produced in the human body.

  The proanthocyanidins contained in the treated pine bark are particularly preferably proanthocyanidins containing a large amount of a condensation polymer having a low degree of polymerization. The condensation polymer having a low polymerization degree is preferably a condensation polymer having a polymerization degree of 2 to 30 (2 to 30 mer), more preferably a condensation polymer having a polymerization degree of 2 to 10 (2 to 10 mer). Further, a condensation polymer (2 to 4 mer) having a polymerization degree of 2 to 4 is more preferable. Proanthocyanidins that are condensation polymers having a degree of polymerization of 2 to 4 (2 to 4 mer) are particularly easily absorbed into the body. In the present specification, a polymer having a degree of polymerization of 2 to 4 is referred to as an oligomeric proanthocyanidin (hereinafter referred to as “OPC”).

  The processed pine bark used in the present invention is desirably an extract containing OPC in an amount of 10% by mass or more, preferably 15% by mass or more, more preferably 20% by mass or more.

  Pine bark is rich in OPC among proanthocyanidins, and is therefore preferably used as a raw material for proanthocyanidins. However, pine bark contains not only proanthocyanidins but also components other than proanthocyanidins. For example, when the processed pine bark is an extract, various organic acids and flavonoids are also extracted by the process of extracting proanthocyanidins from the plant body. Such organic acids and flavonoids are components other than proanthocyanidins. Examples of such organic acids and flavonoids include caffeic acid, fumaric acid, quinic acid, gallic acid, vanillic acid, ferulic acid, and catechins.

  As a method for extracting from pine bark to obtain a pine bark extract, specific preferred embodiments include the following methods. However, it is not limited to this method.

  1 kg of French maritime pine bark is placed in 3 L of a saturated solution of sodium chloride and extracted at 100 ° C. for 30 minutes to obtain an extract (extraction process). Thereafter, the extract is filtered, and the resulting insoluble matter is washed with 500 mL of a saturated solution of sodium chloride to obtain a washing solution (washing step). The extract and washing solution are combined to obtain a crude extract of pine bark.

  Next, 250 mL of ethyl acetate is added to the crude extract and the phases are separated to recover the ethyl acetate layer 5 times. The collected ethyl acetate solutions are combined and added directly to 200 g of anhydrous sodium sulfate for dehydration. Thereafter, the ethyl acetate solution is filtered, and the filtrate is concentrated under reduced pressure until the original volume is reduced to one fifth. The concentrated ethyl acetate solution is poured into 2 L of chloroform, and the resulting precipitate is collected by filtration. The precipitate is dissolved in 100 mL of ethyl acetate, and then added to 1 L of chloroform again for precipitation to perform a washing step of repeating twice. By this method, for example, a pine bark extract containing 20% by mass or more of OPC can be obtained.

  The agent of the present invention contains the processed pine bark as described above.

  In the agent of the present invention, it may be composed only of the pine bark treated product, but there is no particular limitation on blending other materials other than the pine bark treated product, and if necessary, pharmaceutical Addition of an acceptable base material and carrier, and by a known formulation method, for example, powder, granule, granule, tablet, rod, plate, block, solid, round, liquid, bowl, Paste, cream, capsule like hard capsule and soft capsule, caplet, tablet, gel, jelly, gummy, wafer, biscuit, cookie, chewable, syrup, stick, etc. Thus, it can be used as a food or drink or oral preparation (including pharmaceuticals and quasi drugs).

  In addition, the agent of the present invention may be a drink obtained after wrapping a processed pine bark in a tea bag shape and leaching the components into hot water. In addition, the dosage form may be dissolved in water, hot water, milk or the like, or added to food or drink.

  In the agent of the present invention, in addition to the above components, as other materials, for example, vitamins, proteins, water-soluble dietary fibers, oligosaccharides, minerals, mucopolysaccharides, dairy products, soy milk products, plants or processed plant products In addition, microorganisms such as lactic acid bacteria, natto bacteria, butyric acid bacteria, koji molds, and yeasts can be blended. In addition, vitamins such as ascorbic acid, tocopherol, riboflavin, β-carotene, folic acid, biotin, etc., which are usually used in the food field as needed; minerals such as calcium, magnesium, selenium, iron; included in taurine, garlic, etc. Sulfur-containing compounds; Flavanoids and flavonoids such as hesperidin and quercetin; Dietary fibers such as indigestible dextrin, alginic acid, chitin, chitosan and guar gum; Proteins such as soy protein and collagen; Peptides; Amino acids; Milk fat, lard and beef tallow Animal oils such as fish oil; vegetable oils such as soybean oil and rapeseed oil; fruits such as orange, lemon, grapefruit, strawberry and their juices F royal jelly, propolis, honey, reduced maltose, lactose, sugar alcohol, liquid sugar; Excipients other than Binders, lubricants, stabilizers, diluents, bulking agents, thickeners, emulsifiers, colorants, flavors, food additives, and the like seasoning. The content of other components can be appropriately selected according to the dosage form of the agent of the present invention, the level and content of the required effect, and the like.

  As described above, the agent of the present invention may be used as a food or drink or an oral agent (including pharmaceuticals and quasi drugs). For example, when the agent of the present invention is used as a form of food or drink in which a lipid is contained in addition to the processed pine bark, the decrease in collagen, hyaluronic acid, and ceramide in the skin associated with the intake of lipids, This is preferable because it can be effectively prevented. In this case, in the agent of the present invention in the form of food and drink, the processed pine bark is preferably 0.5 parts by mass or more, particularly 1 part by mass or more with respect to 100 parts by mass of the lipid from the above viewpoint.

  In the agent of the present invention, the content of the pine bark processed product described above varies depending on the processing method of the pine bark processed product to be used, the use and dosage form of the agent, the difference in intake method, the level of required effect, and the like. However, generally, in the agent of the present invention, the treated pine bark is preferably 0.001% by mass to 50% by mass, and more preferably 0.005% by mass to 20% by mass.

  In the present invention, the content of the treated pine bark is not particularly limited. Preferably, the daily intake for adults is preferably 1 to 5000 mg, more preferably 2 to 3000 mg as a processed pine bark.

  The agent of the present invention encodes COL1A1 and / or type III collagen constituents, which are genes encoding type I collagen constituents in the skin, when taken orally, as shown in the examples described later. It promotes the synthesis of collagen through promoting the expression of the gene COL3A1. Therefore, the agent of the present invention is not only a collagen synthesis promoter, but also a collagen gene expression promoter, particularly a type I collagen gene and / or type III collagen gene expression promoter, specifically, COL1A1 and / or It can also be used as an expression promoter for COL3A1. Such an agent of the present invention can prevent or ameliorate functional deterioration such as various disorders, diseases, and illnesses associated with collagen decrease in the body, particularly skin and bone. For example, skin turnover can be promoted to improve skin firmness and elasticity, and can be moisturized to prevent or improve skin troubles such as sagging skin, roughness, rough skin, and wrinkles.

  In addition, as shown in the examples described later, the agent of the present invention, when orally ingested, promotes the expression of the genes encoding the hyaluronic acid synthases Has2 and Has3, respectively, and thereby hyaluronic acid It promotes synthesis. For this reason, the agent of the present invention is used not only as a hyaluronic acid synthesis promoter, but also as a hyaluronic acid synthase gene expression promoter, in particular, as an expression promoter for the Has2 gene and / or Has3 gene. Can do. In this way, by promoting hyaluronic acid synthesis through gene expression of hyaluronic acid synthase, the agent of the present invention prevents the deterioration of the function of various disorders, diseases, illnesses, etc. associated with the reduction of hyaluronic acid in the body, particularly the skin. Can do. For example, moisturizing the skin to improve skin firmness and elasticity and moisturize to prevent or ameliorate skin troubles such as rough skin, wrinkles, and roughness, prevent joint damage, joint ointment damage, etc. Or the effect which improves can be show | played. In particular, the agent of the present invention is a major enzyme that synthesizes hyaluronic acid in the dermis, and promotes the expression of Has2, which is said to produce high-humidity polymeric hyaluronic acid among hyaluronic acid synthases. It can have a beauty effect. In addition, the agent of the present invention can enhance the immune function in addition to promoting the synthesis of hyaluronic acid in the epidermis by promoting the expression of Has3.

  In addition, as shown in the examples described later, the agent of the present invention, when orally ingested, promotes the expression of a gene encoding serine palmitoyltransferase (SPT), which is a ceramide synthase, and thereby, It promotes synthesis. For this reason, the ceramide synthesis promoter of the present invention can also be used as a ceramide synthase gene expression promoter, in particular, as an SPT gene expression promoter. Thus, by promoting ceramide synthesis through gene expression of SPT, the agent of the present invention can prevent functional deterioration such as various disorders, diseases, and diseases associated with ceramide reduction in skin, particularly epidermis. For example, it is expected to promote moisturizing the skin and effectively prevent and improve various skin symptoms such as dry skin, rough skin, atopic dermatitis, and psoriasis.

  Furthermore, the agent of the present invention has an effective skin improvement agent for oral intake, especially a wrinkle or talmi prevention or improvement agent, a rough skin, by synergistic effect by promoting the synthesis of each component of collagen, hyaluronic acid and ceramide. It can be used as a preventive or ameliorating agent. The agent of the present invention may be used for humans and animals other than humans (such as mammals).

Furthermore, as shown in the Examples described later, the agent of the present invention can promote the expression of COL1A1 and / or COL3A1 when ingesting a high-fat diet and reduce the amount of collagen due to ingestion of a high-fat diet. It is possible to suppress. As an example of the high-fat food here, a food or drink with an energy ratio of lipid of 20% or more can be mentioned. Therefore, the agent of the present invention can also be used as an inhibitor of a decrease in collagen amount due to a high fat diet.
Similarly, the agent of the present invention can promote the expression of the Has2 gene and / or the Has3 gene when ingesting a high fat diet, and can suppress a decrease in the amount of hyaluronic acid due to ingestion of the high fat diet. Is possible. Therefore, the agent of the present invention can also be used as an inhibitor of the decrease in hyaluronic acid caused by a high fat diet.
Furthermore, the agent of the present invention can prevent the amount of ceramide and the expression level of the SPT gene that are decreased by ingesting a high fat diet. Therefore, the agent of the present invention can also be used as an inhibitor of a decrease in ceramide amount and ceramide synthase gene expression level due to a high fat diet.
Such an agent of the present invention may be particularly effective in treating, for example, skin atrophy under a high fat diet, delay in wound healing, worsening of bedsores, and the like.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the scope of the present invention is not limited to such examples. Hereinafter, unless otherwise specified, “%” represents mass%, and “part” represents mass part.

[Reference Example 1]
The standard meal based on AIN-76G was used as the sample meal of Reference Example 1. The energy ratio of the lipid of the reference meal 1 was 12%.
The composition of the sample food of Reference Example 1 is shown in Table 1 below.

[Reference Example 2]
As a model for a high fat diet, in the sample diet of Reference Example 1, the amount of sucrose was reduced from 50.0 parts to 30.0 parts, and 25.0 parts of lard was added instead of 5.0 parts of corn oil. Thus, the sample food of Reference Example 2 was obtained. The energy ratio of lipid in the reference meal 2 was 46%.

[Example 1]
As a processed pine bark, 5 parts of pine bark extract (trade name “Flavandenol” (registered trademark, manufactured by Toyo Shinyaku Co., Ltd.) is added to 100 parts of the sample food of Reference Example 2 and mixed to carry out. The sample food of Example 1 was used.

  The samples of Reference Examples 1 and 2 and Example 1 were subjected to the following <Test for promoting the synthesis of collagen, hyaluronic acid and ceramide>.

<Collagen, hyaluronic acid and ceramide synthesis promotion test>
18 rats of 4 weeks old SD strain were acclimated for 1 week in the sample diet of Reference Example 1 and then 6 rats were ingested in the sample diet of Reference Example 1 and in the sample diet of Reference Example 2, respectively. The sample food intake group of Example 1 was divided into three groups and reared for 4 weeks. Food was provided from 4:00 pm to 10:00 am the following day, and water was ad libitum.
After rearing for 4 weeks, food was removed, and after anesthesia with somnopentyl, the back hair was shaved. A slide glass coated with an instantaneous adhesive was attached to the portion along the back hair, and the lipid was collected. Next, the back skin was collected, and four discoid skins with a diameter of 18 mm were extracted from the skin near the buttocks. Among the ingested portions, RNA was extracted from the remaining skin, and the expression levels (mRNA amounts) of COL1A1, COL3A1, Has2 gene, Has3 gene, and SPT gene were measured by the following method. Further, the amount of collagen, the amount of hyaluronic acid and the amount of ceramide were measured from the disc-shaped skin by the following method.

<Measurement of gene expression level>
After the skin section was crushed under liquid nitrogen, total RNA was extracted using SV Total RNA Isolation System (Promega). After measuring the concentration of the obtained RNA, it was confirmed by agarose electrophoresis that the RNA was not degraded. This total RNA was converted to cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and then COL1A1, COL3A1, Has2 gene, Has3 using the following primers and probes with real time PCR System (Applied Biosystems). The expression level of each of the gene and the SPT gene was measured.
The expression level of each gene was calculated using β-actin as an internal standard.
The average values and standard error results of the expression levels of COL1A1 and COL3A1 are shown in Table 2 and FIGS. 1 and 2, and the average values and standard error results of the expression levels of Has2 gene and Has3 gene are shown in Table 3 and FIGS. Table 4 and FIG. 5 show the average expression level and standard error of the SPT gene, respectively. In these tables and figures, the expression level of each gene is shown as a relative value when the expression level in the intake group of Reference Example 1 is 1. About the obtained gene expression level, after testing by Kruskal-Wallis-H-test, the significant difference test was performed by confirming by multiple comparison by Student-Newman-Keuls test. When the lowercase alphabets in FIGS. 1 to 5 are different, it indicates that there is a significant difference.

Primer and probe sequences
Rat COL1A1 (Genbank accession number: Z78279)
Primers and probes described in Table 2 of Non-Patent Document 1 were used.
Rat COL3A1
Forward primer: 5'-TTGGGATGCAACTACCTTGGT-3 '/ SEQ ID NO: 1
Reverse primer: 5'-TCCCCACTCCCACACACAT-3 '/ SEQ ID NO: 2
Probe: FAM-TGACATGGTTCTGGCTTCCAGACATCTCT-TAMRA / SEQ ID NO: 3 with 5 'end modified with 6-FAM and 3' end modified with TAMRA
Rat Has2 gene (Genbank accession number: AF008201)
Primers and probes described in Table 2 of Non-Patent Document 1 were used.
Rat Has3 gene (Genbank accession number: AF543196)
Primers and probes described in Table 2 of Non-Patent Document 1 were used.
Rat SPT gene
Forward primer: 5'-CAGTGCAGCCTGCTTTGCTA-3 '/ SEQ ID NO: 4
Reverse primer: 5'-GCCTTTCGAGGATTCTTTTGATC-3 '/ SEQ ID NO: 5
Probe: FAM-CCAGAAAGGACTACAGGCATCACGCAG-TAMRA / SEQ ID NO: 6 with 5 'end modified with 6-FAM and 3' end modified with TAMRA
Rat β-actin gene (Genbank accession number: V01217)
Primers and probes described in Table 2 of Non-Patent Document 1 were used.

<Measurement of type I collagen content>
The weight of one disk-shaped skin was measured. Thereafter, the skin was shredded with dissection scissors, 2.5 ml of cold physiological saline was added per 100 mg of skin weight, and homogenized. The skin suspension was shaken at 4 ° C. for 20 hours and then centrifuged to obtain a supernatant. The tropocollagen contained in the supernatant was used as the collagen to be measured.
The amount of collagen was measured using a Western blotting method. The supernatant obtained above was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 6% polyacrylamide gel. The protein after electrophoresis is transferred to a nitrocellulose membrane, anti-human type I collagen antibody (manufactured by Lockland) is used as the primary antibody, and HRP-anti-rabbit antibody (manufactured by GE) is used as the secondary antibody. The amount of collagen was measured. Quantification was performed using a fluorescence / luminescence image analyzer LAS-3000 (manufactured by Fuji Film). The results are shown in Table 2. In the table, the amount of type I collagen is shown as a relative value when the amount of type I collagen in Reference Example 1 is 1.

<Measurement of hyaluronic acid content>
After measuring the weight of one disk-shaped skin, it was degreased with acetone and weighed again. After the skin was chopped, 5 ml of 50 mM Tris / HCl buffer (pH 7.8) per 100 mg of skin weight was added after defatting and boiled for 20 minutes. An equal amount of 2 mg / ml actinase E solution (Kaken Pharmaceutical Co., Ltd.) was added to the resulting boiled product and allowed to react at 40 ° C. for 1 week. The reaction was stopped by adding a cold trichloroacetic acid aqueous solution to a final concentration of 10%. After centrifugation, the supernatant was collected and concentrated to 1 ml with Amicon Ultra 30K (Millipore). 500 μl of this concentrated solution was treated with hyaluronidase (manufactured by Seikagaku Corporation) for 18 hours at 60 ° C., and then treated with Microcon 30 (Millipore). The amount of uronic acid in the permeated liquid of Microcon was measured to obtain the amount of hyaluronic acid. The uronic acid was measured by the method described in “Bitter, T, et al, A modeified uronic acid carbazole reaction. Anal Biochem. 1962, 4,330-334”. The results are shown in Table 3. In the table, the amount of hyaluronic acid is shown as a relative value when the amount of hyaluronic acid in Reference Example 1 is 1.

<Measurement of ceramide amount>
The rat back stratum corneum collected by applying instant adhesive to a slide glass was immersed in a hexane: ethanol (volume ratio 95: 5) solution, and lipids were extracted by ultrasonication. The solvent was evaporated from the extract with nitrogen gas to obtain a stratum corneum lipid. After making lipid concentration constant with chloroform, it was spotted on a silica gel plate (Merck, HPTLC Silica gel 60F) and developed with chloroform: methanol: acetic acid (volume ratio 192: 7: 1). After development, spray with a copper sulfate / phosphoric acid solution (8 g of phosphoric acid and copper (II) sulfate / pentahydrate 15.63 g dissolved in water to make 100 ml), and after drying, at 150 ° C. for 10 minutes Heated to develop lipid color. Ceramide spots were digitized using a fluorescence / luminescence image analyzer LAS-3000 (Fuji Film). The results are shown in Table 4. In the table, the amount of ceramide is shown as a relative value when the amount of ceramide in Reference Example 1 is 1.

  From the results of Table 2 and FIGS. 1 and 2, Reference Example 1 and Reference Example 2 that do not contain a pine bark treated product contain a pine bark treated product, whereas the expression levels of COL1A1 and COL3A1 in the skin are low. In Example 1, it can be seen that the expression levels of COL1A1 and COL3A1 are significantly high. Also, from Table 2, it can be seen that in Reference Example 2 ingesting a high fat diet, the amount of type I collagen synthesis in the skin tends to be lower than in Reference Example 1 ingesting a normal diet. Although it is ingested, it can be seen that the amount of type I collagen in the skin is as high as in Reference Example 1, which is a normal diet.

  From the results of Table 3 and FIG. 3 and FIG. 4, Reference Example 1 and Reference Example 2 that do not contain a processed pine bark have a low expression level of Has2 and Has3 genes in the skin, whereas In the contained Example 1, it turns out that the expression level of Has2 and Has3 gene is significantly high. Also, from Table 3, Reference Example 2 ingesting a high fat diet shows a tendency for the amount of hyaluronic acid in the skin to decrease compared to Reference Example 1 ingesting a normal diet. In spite of this, it can be seen that the amount of hyaluronic acid is higher than in Reference Example 1 which is a normal food.

  From the results of Table 4 and FIG. 5, Reference Example 2 ingesting a high-fat diet that does not contain a processed pine bark has a low expression level of the SPT gene in the skin, whereas a processed pine bark along with the high-fat diet It can be seen that in Example 1 ingesting SPT, the expression level of the SPT gene is significantly high. Also, from Table 4, in Reference Example 2 ingesting a high-fat diet, ceramide in the epidermis tends to be reduced compared to Reference Example 1 ingesting a normal diet. In Example 1, a high-fat diet is ingested. Nevertheless, it can be seen that the amount of ceramide in the epidermis is higher than in Reference Example 1 which is a normal diet.

Claims (6)

  A collagen synthesis promoter characterized by containing a processed pine bark.   An agent for promoting expression of COL1A1 or COL3A1, which is a collagen gene, comprising a treated pine bark.   A hyaluronic acid synthesis promoter characterized by containing a processed pine bark.   A gene expression promoter for Has2 or Has3, which is a hyaluronic acid synthase, comprising a processed pine bark.   A ceramide synthesis accelerator characterized by containing a processed pine bark.   An agent for promoting gene expression of SPT, which is a ceramide synthase, comprising a treated pine bark.

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