JP2016056116A - Composition for promoting collagen production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition having a significant collagen production promoting ability by searching for a component involved in collagen production promotion.SOLUTION: The invention provides a collagen production-promoting composition containing (A) one or more kinds selected from the group consisting of berberine, berberine analogs, and their salts, as well as a plant extract containing berberine, in order to promote secretion of collagen from inside to outside out of cells, and (B) a collagen synthesis promoter for promoting synthesis of collagen within cells.SELECTED DRAWING: None

Description

  The present invention relates to a composition for promoting collagen production that is suitable for external preparations for skin, foods and drinks, pharmaceuticals, and the like.

  Collagen is a major protein constituting dermis, bone, cartilage, tendon, etc., and occupies about 30% of the total protein content of the human body. Collagen is the main structural component of connective tissue, is found in the interstitial tissue of the parenchymal organ, and is involved in maintaining the morphology of the organ and tissue. In addition, it has been found that collagen plays a role in information transmission that gives proliferation and differentiation signals to cells in contact. Since collagen has such a function, it may cause a malfunction in the living body due to a decrease in collagen due to aging or the like. For example, corneal disorders, joint disorders, inflammatory diseases and the like.

  In the skin, about 70% of the dry weight of the skin is collagen, and collagen fibers are gathered and bundled in the dermis to form a collagen fiber bundle. The organization form is maintained by stretching this in a mesh shape. Moreover, it is known that collagen is excellent in moisture retention function. Collagen having these functions greatly contributes to the elasticity and strength of the skin. This is why many young people have moderate elasticity on their skin. However, the skin of the elderly has no firmness and many wrinkles and tarmi are seen. This is thought to be due to a decrease in collagen due to natural aging, photoaging, and the like.

  In order to improve the biological phenomenon due to the decrease in collagen as described above, development of collagen production promoting substances has been promoted, and various collagen production promoting substances have been found so far. For example, plant extracts such as retinoic acid (Non-Patent Document 1), glycine, proline and alanine (Patent Document 1), licorice, Sakuhakuhi, Aloe, horsetail, goldfish, grasshopper, gaiyou or gentian (Patent Document) 2) TGF-β, ascorbic acids and the like are known.

  As described above, various collagen production promoting substances have been developed, and some components that enhance the ability to promote collagen production by combining them have been found. For example, L-ascorbic acid 2-glucoside, which is a collagen production promoting substance, enhances its function of royal jelly (Patent Document 3), trimethylglycine (Patent Document 4), which enhances the function of ascorbic acids, which are collagen production promoting substances. ) Etc. are known.

R. Marks et al., British Journal of Dermatology, 122, 91-98, 1990. JP-A-7-194375 JP 2001-206835 A Japanese Patent No. 4456796 Japanese Patent No. 5241054

  As described above, since the role of collagen in the living body is important, it is desired to search for further components involved in the promotion of collagen production and to develop a composition having a remarkable ability to promote collagen production. .

  In the conventional search for collagen production promoting substances, the ability to promote collagen production has been evaluated as increasing the amount of collagen inside or outside the cell, regardless of whether the collagen is inside or outside the cell. However, collagen is cross-linked extracellularly to form a fibrous structure and has various biological functions as described above. Therefore, it promotes the secretion of collagen synthesized inside the cell and reduces the amount of extracellular collagen. It is important to increase.

  In view of the above-mentioned present situation, the present invention searches for a component capable of promoting the secretion of collagen synthesized intracellularly from cells and increasing the amount of collagen outside the cell. It aims at providing the composition which has a production promotion ability.

  As a result of intensive studies to solve the above problems, the present inventors have found that an extract of Coptis japonica has an action of promoting the secretion of collagen from cells. Clarified that it is Berberine. Collagen is synthesized inside the cell, then secreted outside the cell, aggregates and becomes fibrous, and participates in the formation of the extracellular matrix. Therefore, in the living body, the action of secreting collagen synthesized intracellularly to the outside of the cell is important. It has been reported that berberine is involved in promoting the production of collagen, but as described above, it has an action of actively secreting collagen synthesized inside the cell to the outside. This is a new finding by the present inventors that has not been known so far.

  Furthermore, the present inventors synergistically enhance the ability to promote collagen production by combining the above-described berberine and a substance having an action of promoting intracellular collagen synthesis, rather than using each of them alone. I found that I can do it. Among various substances having an action of promoting intracellular collagen synthesis, in particular, a component having a transforming growth factor-β (TGF-β) and / or a TGF-β-like action is combined with the above berberine. It has been found that a remarkable ability to promote collagen production can be seen by combining them. It has not been known so far that combining berberine with TGF-β and the like significantly enhances the ability to promote collagen production, which is a new finding by the present inventors. Based on these findings, the present invention has been completed.

  That is, the present invention is selected from the group consisting of (A) berberine, berberine analogs and salts thereof, and a plant extract containing berberine for promoting the secretion of collagen from the inside of the cell to the outside of the cell. And (B) a collagen synthesis promoter for promoting collagen synthesis in cells.

  In another aspect, the present invention relates to a skin external preparation, food or drink, or pharmaceutical product containing the composition for promoting collagen production.

  In another aspect, the present invention provides an intracellular to extracellular mixture containing one or more selected from the group consisting of berberine, berberine analogs, and salts thereof, and plant extracts containing berberine. The present invention relates to a collagen secretion promoter for promoting collagen secretion.

  According to the present invention, there can be provided a composition for promoting collagen production that can solve the above-mentioned problems and has a remarkable ability to promote collagen production, and a skin external preparation, food and drink, and pharmaceuticals using the same. Can do.

It is the graph which showed the change of the amount of extracellular (in culture supernatant) collagen when various extracts are added to the culture solution of a normal human skin fibroblast (NHDF). It is the graph which showed the change of the amount of intracellular collagen when various extracts are added to the culture solution of a normal human skin fibroblast (NHDF). When cultured with normal human skin fibroblasts (NHDF), berberine and / or collagen synthesis promoter (TGF-β, IGF-1, or curcumin) alone or in combination is added to the extracellular (in culture) It is the graph which showed the change of the amount of collagen. When the berberine and / or collagen synthesis promoter (TGF-β, IGF-1, or curcumin) is added alone or in combination to a culture solution of normal human skin fibroblasts (NHDF), It is the graph which showed the change. Extracellular (in culture supernatant) collagen when berberine and / or collagen synthesis promoter (whey protein or nettle extract) is added alone or in combination to normal human skin fibroblast (NHDF) culture It is the graph which showed the change of quantity. Changes in the amount of intracellular collagen when berberine and / or collagen synthesis promoter (whey protein or nettle extract) was added alone or in combination to the culture medium of normal human skin fibroblasts (NHDF) It is a graph.

[Composition for promoting collagen production]
The composition for promoting collagen production according to the present invention comprises (A) at least one selected from the group consisting of berberine, berberine analogs, and salts thereof, and a plant extract containing berberine (in the present specification, , Sometimes referred to as “berberine”) and (B) a collagen synthesis promoter.

  In the conventional literature, the terms “synthesis” and “production” for collagen are often used synonymously. However, in the present specification, these terms are used in principle, although depending on the context. Differentiate and use. That is, in this specification, “synthesis promotion” of collagen refers to the action of promoting collagen synthesis performed in cells producing collagen such as fibroblasts, osteoblasts, chondrocytes, etc. This can be confirmed, for example, by detecting an increase in intracellular collagen when a target substance is allowed to act on cells that produce collagen, as shown in the Examples described later. On the other hand, in this specification, “production promotion” of collagen refers to not only the synthesis of collagen in cells but also the involvement of any of a series of processes including the subsequent secretion of collagen outside the cells. This refers to the action of finally increasing the amount of collagen present outside the cell. This is, for example, when a target substance is allowed to act on cells that produce collagen, as shown in the examples described later. This can be confirmed by detecting the increased amount of collagen secreted extracellularly (collagen present in the culture supernatant). The increased amount of collagen inside or outside the cell can be detected by a conventional method, for example, at the protein level inside or outside the cell before and after acting the target substance. The amount of collagen can be measured using an antibody and detected by the difference between the measured amounts. Ultimately, it is desirable to increase the amount of collagen that functions as an extracellular matrix between cells. Therefore, in the present invention, in addition to “stimulation of synthesis” of collagen in the cell, secretion outside the cell is also performed. The main purpose is to “promote production” of the included collagen.

  In the present specification, “collagen” may be at any stage in the biosynthesis process. That is, collagen is synthesized as single-chain pro-α chains in cells that produce collagen, and these three are gathered together and secreted outside the cell as three-chain procollagen. Thereafter, some amino acids at the end are cleaved by enzymatic degradation to form tropocollagen, aggregate to form fibers, and are further crosslinked to form mature collagen fibers. In this specification, those in any of these stages are included in “collagen”.

(A) Berberine Berberine is blended in a composition for promoting collagen production for the purpose of promoting the secretion of collagen synthesized inside the cell by acting on the cell producing collagen. Berberines increase the amount of collagen outside the cell while decreasing the amount of collagen inside the cell. Therefore, by incorporating berberine into the composition for promoting collagen production, it is possible to efficiently secrete the collagen synthesized inside the cell and increase the number of collagen fibers that actually function between cells. Conceivable.

  Examples of berberines include berberine, berberine analogs, berberine or a salt of berberine analogs, and plant extracts containing berberine, which can be used in the fields of pharmaceuticals, quasi drugs, cosmetics, or foods. If it is, it can be used without particular limitation. Examples of berberine analogs include berberine chloride, berberine sulfate, berberine tannate, palmatin, coptisine, wollenin, jatrolidine, and sanguinarine. Examples of berberine or berberine analog salts include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium, ammonium salts, organic amine salts such as triethanolamine and triethylamine, and bases such as lysine and arginine. Amino acid salts and the like. Examples of plant extracts containing berberine include an extract of Coptis japonica, an extract of agron (Phrodendron amurense bark), and the like. As for the oren extract, an extract from a rhizome is particularly preferable. These can be used individually by 1 type or in combination of 2 or more types.

  The plant extract containing berberine can be obtained, for example, by extracting from the above plant according to a conventional method. For example, it can be obtained by extracting and purifying from the above plant with water, glycerin, propylene glycol, ethanol, butylene glycol, or a water-soluble solvent, or a mixture thereof, at room temperature or by heating treatment. The obtained extract may be used as it is, or may be used after removing inactive impurities. Alternatively, the concentrated / dried product may be dissolved again in water or a polar solvent and used. Furthermore, it should be used after subjecting the extract to purification of decolorization, deodorization, and desalting, and fractional extraction by column chromatography within a range that does not impair the desired level of secretion promoting action. You can also. Moreover, as a plant extract containing berberine, a commercial item can also be used, for example, Ouren extract-J (made by Maruzen Pharmaceutical Co., Ltd.) etc. are mentioned.

  The content of berberine in the composition for promoting collagen production is not particularly limited and can be appropriately selected according to the purpose. However, as the amount of berberine (including analogs and salts) as an active ingredient, It is 0.0001-50 mass%, 0.01-5 mass% is preferable, and 0.025-0.5 mass% is more preferable. When the content of berberine is within a preferable range, it is advantageous from the viewpoint of promoting collagen secretion and preventing problems caused by coloring of the composition with a large amount of berberine.

(B) Collagen synthesis promoter A collagen synthesis promoter is blended with the above-described berberine to blend in a composition for promoting collagen production for the purpose of producing a synergistic collagen production promoting effect as a whole. The reason why the combination of berberines and a collagen synthesis promoter has a remarkable collagen production promotion effect is not particularly limited to the mechanism, but the increased intracellular collagen by the action of the collagen synthesis promoter is not limited. It is considered that the amount of collagen existing outside the cell can be efficiently increased as a whole by actively secreting it outside the cell by the action of the above berberines.

  The collagen synthesis promoter is used without particular limitation as long as it has an action of promoting collagen synthesis in cells at least and can be used in the field of pharmaceuticals, quasi drugs, cosmetics, or foods. For example, swordfish, giant moss, south rare involcata, coxnohagashi, hibiscus, broken tree, dragon leaf, kirimizukuzu, anthecephalus indicas, anaphalis busua, blue grass, kiyosumi moss, gentian bark Sumner, Convertible Grass, Lotus Flower, Gossa, Yellow Skin Leaves, Oni Strawberry, Ceylon Matsuri, Chlorella, Saximakhuyo, Hosobaokinagoke, Oshiragagoke, Pyramid, Vulgaris, Lotus Germ, Sennenken, Moon Peach, Water Eggplant, Five Finger Peach, America Halibutki, Chang Bat, Dokai Mother, Amor Co, Coelospondius axillaris, Nemunoki, Nogateto, Roman Chamomile, Bush, Ozukinkaburitake, Hitorishizuka, Passionfruit, Big Flower Scenic, Sekiji Kagetenten, Daylily, Sakuhakuhi, Aloe, Sugina, Kinginga, Oubakaku , Plant extracts such as gayyo or gentian, retinoic acid, glycine, proline and alanine, three amino acids, TGF-β, IGF-1, ascorbic acid, brassinolide, epibrassinolide, menaquinone-7, phosphatidic acid Etc. These can be used individually by 1 type or in combination of 2 or more types.

  Among them, it is preferable to use TGF-β (transforming growth factor-β) as a collagen synthesis promoter. TGF-β is a naturally occurring growth factor with many functions, and is important for cell growth and differentiation, including extracellular matrix protein production and deposition, epithelial cell growth inhibition, immune cell function inhibition, etc. It is known to play a role. TGF-β induces transcriptional expression of target genes by binding to TGF-β receptors existing on the surface of target cells such as fibroblasts and phosphorylating Smad2 and Smad3, which are proteins responsible for signal transmission And leads to the synthesis of collagen, an extracellular matrix protein. As described above, it is generally known that TGF-β promotes the synthesis of collagen. However, when used in combination with the above-described berberines, it has so far been effective in promoting collagen production. This is a new finding by the present inventors.

  Considering the mechanism of action of TGF-β as described above, as with TGF-β, a component having a TGF-β-like action (for example, a substance that promotes production of TGF-β, other than TGF-β, And substances that activate the TGF-β signal transduction pathway) are also expected to have an effect of promoting collagen synthesis. That is, a component having such a TGF-β-like action can also be preferably used as a collagen synthesis promoter in the present invention. Examples of components having a TGF-β-like action include whey protein, whey protein fragment, nettle extract, winged bean extract, daifu cow extract, aloe vera extract, curcumin, fucoxanthin, fucoxanthinol, chitin, β -1,4-mannobiose and the like. Among these, as a component having a TGF-β-like action, whey protein and / or a whey protein fragment are preferable.

  Whey protein is a general term for proteins contained in whey obtained by removing casein and milk fat from raw milk such as cows, sheep and humans. Examples of main whey proteins include β-lactoglobulin, α-lactalbumin, immunoglobulin, glycomacropeptide, and lactoferrin. As the whey protein, bovine β-lactoglobulin (SEQ ID NO: 6) is preferably used. Further, as the whey protein, it is also preferable to use a whey protein prepared so as to contain β-lactoglobulin in a high concentration. Moreover, CBP (Concentrated Bovine milk active protein; Concentrated whey active protein) which concentrated the low molecular weight fraction (1-30 kDa) of whey protein can also be used.

  As the whey protein fragment, a β-lactoglobulin fragment is preferably used, and particularly preferably, Tyr-Leu-Leu-Phe (SEQ ID NO: 1), which is the amino acid sequence at positions 102 to 105 of β-lactoglobulin, Ala-Leu-Pro-Met (SEQ ID NO: 2) which is amino acid sequence 142-145 of β-lactoglobulin, His-Ile-Arg-Leu (sequence) which is amino acid sequence 146-149 of β-lactoglobulin No. 3), Asp-Ile-Gln-Lys-Val-Ala-Gly-Thr-Trp (SEQ ID NO: 4), which is the 11th to 19th amino acid sequence of β-lactoglobulin, and 150- Ser-Phe-Asn-Pro-Thr-Gln- which is the 162nd amino acid sequence It is possible to use one or more peptide fragments selected from the group consisting of eu-Glu-Glu-Gln-Cys-His-Ile (SEQ ID NO: 5).

  Whey protein can be obtained, for example, by extraction from raw bovine milk, ultrafiltration, electrodialysis, evaporation, reverse osmosis and the like. Or what was biosynthesized using microorganisms and what was artificially synthesized by organic synthetic chemistry may be used. Moreover, the whey protein fragment can be obtained by hydrolyzing the whey protein. Or what was biosynthesized using microorganisms and what was artificially synthesized by organic synthetic chemistry may be used. Moreover, the whey protein and the whey protein fragment may be derivatized by modifying the N-terminus and / or the C-terminus.

  The content of the collagen synthesis promoter in the composition for promoting collagen production is not particularly limited and can be appropriately selected according to the purpose, but is usually 0.0000001 to 50% by mass, and 0.000001 to 10%. % By mass is preferable, and 0.000001 to 1.2% by mass is more preferable. When the content of the collagen synthesis accelerator is within the preferred range, it is advantageous from the viewpoint of promoting collagen synthesis and preventing undesirable effects on the quality such as color and properties by a large amount of collagen synthesis accelerator.

  In the composition for promoting collagen production, the content ratio of the berberine to the collagen synthesis promoter is usually berberine (the active ingredient berberine (analogue, salt) relative to 100 parts by mass of the collagen synthesis promoter. Inclusive) is 0.05 parts by mass or more, preferably 0.05 to 20000000 parts by mass, and more preferably 0.125 to 50000 parts by mass. When the content ratio is within the preferred range, it is advantageous in terms of promoting collagen production.

  The composition for promoting collagen production may consist of only the above two components (berberine and collagen synthesis promoter), but if necessary, a normal pharmaceutical, quasi-drug, cosmetic or Various optional ingredients that can be used in foods, such as oils, powders, surfactants, adhesives, resins, amino acids, sugars, organic UV absorbers, natural plant extract ingredients, solvents, preservatives, metals An ion sequestering agent, an anti-inflammatory component, a medicinal component, and the like can be appropriately contained. These may be used individually by 1 type and may use 2 or more types together. Moreover, content of these arbitrary components in a composition can be suitably selected within the range which does not impair the effect of this invention.

  Since the composition for promoting collagen production according to the present invention has an excellent ability to promote collagen production, for example, a composition for preventing or treating cosmetic problems and various disorders caused by collagen reduction or denaturation. In particular, it is useful as a composition for preventing and / or improving wrinkle or tarmi, reduction of elasticity, and a composition for preventing and / or treating corneal disorders, joint disorders, inflammatory diseases, etc. Can be used for In addition, the living body to which the composition for promoting collagen production is applied is mainly human, but it can also be applied to animals other than humans. The composition for promoting collagen production can also be used to promote collagen production in in vitro collagen-producing cells such as cultured fibroblasts.

  The method of using the composition for promoting collagen production is not particularly limited, and the active ingredients (berberine and collagen synthesis promoter) can be allowed to act on cells that produce collagen in vivo or in vitro. I can do it. For example, the target living body can take the composition orally or parenterally (for example, transdermally). In addition, when the target is an ex vivo cell, the composition can be added to the cell culture medium. There is no restriction | limiting in particular also in the intake amount of the composition in a biological body, or the addition amount of the composition with respect to the cell outside a living body, and it can select suitably in the range which does not impair the effect of this invention.

[External preparation for skin, food and drink, pharmaceutical]
The present invention also relates to an external preparation for skin, a food and drink, and a pharmaceutical comprising the above-described composition for promoting collagen production. These have an excellent ability to promote collagen production based on a combination of berberine and a collagen synthesis promoter, so that for example, prevention or improvement of wrinkles and tarmi caused by reduction or degeneration of collagen, corneal disorders, joints, etc. It can be suitably used for the prevention or treatment of diseases such as disorders and inflammatory diseases.

  The topical skin preparation may be a quasi-drug or cosmetic, for example, a quasi-drug in the form of a lotion, emulsion, cream, ointment, skin care cosmetics such as lotion, emulsion, cream, cosmetic oil, foundation, etc. , Powder, lipstick, blusher, eyeshadow, nail enamel makeup cosmetics, aerosols, pumps, extrusions, and other cosmetics, tablets, capsules, granules, powders, liquids, etc. Agents and the like. The external preparation for skin contains at least berberine and a collagen synthesis promoter, and further contains optional ingredients that can be blended into quasi-drugs and cosmetics, and is manufactured in an arbitrary dosage form according to a conventional method. Can do.

  Examples of food and drink include general processed foods such as breads, confectionery, noodles, dairy products, powders, beverages, various processed products, nutritional supplements such as vitamins, nutritional supplements, and animal health foods. Specific health foods, nutritional functional foods, health functional foods, and the like. The food or drink contains at least a berberine and a collagen synthesis accelerator, and further contains any component that can be blended in the food or drink, and can be produced in any dosage form according to a conventional method.

  Examples of pharmaceuticals include oral pharmaceuticals comprising solid compositions such as tablets, granules, powders, powders, capsules, etc., oral pharmaceuticals comprising liquid compositions such as normal solutions, suspensions and emulsions, and injections. And parenteral drugs such as transdermal absorption agents and external preparations. The pharmaceutical contains at least berberine and a collagen synthesis promoter, and further contains any component that can be blended with the pharmaceutical, and can be produced in any dosage form according to a conventional method.

  The content of active ingredients (berberine and collagen synthesis promoter) in external preparations for skin, foods and beverages, and pharmaceuticals is not particularly limited, and the desired degree of collagen production promoting action and the type of active ingredient used. , External preparations for skin, foods and drinks, pharmaceutical dosage forms, etc., can be appropriately selected according to the purpose. For example, when using a plant extract containing berberine as the berberine, depending on the content of the berberine in the plant extract, 0.001 to 30 mass in the skin external preparation, food and drink, and pharmaceutical products % Can be included. For example, when using whey protein as a collagen synthesis promoter, it can be contained in the range of 0.0025-30 mass% in skin external preparation, food-drinks, and a pharmaceutical. Moreover, as content ratio of the said 2 component in skin external preparation, food-drinks, and a pharmaceutical, for example, it is set as mass ratio, it is set as the plant extract containing berberine: whey protein = 20: 1-1: 200. Can do.

[Collagen secretion promoter]
The present invention also relates to a collagen secretion promoter containing at least one selected from the group consisting of berberine, berberine analogs and salts thereof, and a plant extract containing berberine. As a collagen secretion promoter, the above berberines may be used alone or in combination with other optional components. Moreover, there is no restriction | limiting in particular in content and dosage form of berberine in a collagen secretion promoter, According to the objective, it can select suitably. The collagen secretagogue according to the present invention has an action of efficiently secreting collagen synthesized in the cell to the outside of the cell. Therefore, for example, for cells that produce collagen such as cultured fibroblasts in vitro. Thus, it can be beneficially used for the purpose of promoting the secretion of collagen and for the purpose of preventing or improving cosmetic and health problems caused by the decrease in collagen between cells in the living body.

  The present invention will be described in more detail with reference to Examples and Formulation Examples below, but the present invention is not limited to these Examples and Formulation Examples.

[Test Example 1: Screening for a component capable of promoting collagen secretion in dermal fibroblasts]
Skin fibroblasts were used to screen for components that promoted secretion of collagen from the cell into the culture supernatant. Specifically, when various components such as plant extracts are added to the culture solution, changes in the amount of collagen inside and outside the cell (in the culture supernatant) are measured to synthesize collagen in the cell. And the effect | action of the various components with respect to the secretion of collagen outside a cell was tested as follows. In addition, as various components, β-lactoglobulin-rich whey protein (Seperex Nutritional) was used as the whey protein. The β-lactoglobulin-rich whey protein was prepared by a proprietary process owned by Seperex Nutritional, and the outline of the process is as follows. Cattle-derived raw milk was degreased, pasteurized, ultrafiltered, and spray-dried. The resulting concentrate was diluted and major non-whey protein was removed by diafiltration (30 kDa membrane). Furthermore, the obtained permeate was subjected to ultrafiltration / diafiltration (1 kDa membrane) to remove low molecules. The obtained whey protein contains a protein of 1 to 30 kDa, and particularly contains β-lactoglobulin at a high concentration. As plant extracts, Asenyaku extract (Asenyaku extract, manufactured by Maruzen Pharmaceutical Co., Ltd.), soybean extract (Phytopolyamine-S, manufactured by Toyobo Co., Ltd.), Ouren extract (Ouren extract-J, manufactured by Maruzen Pharmaceutical Co., Ltd.), Alfalfa extract (vitanol, manufactured by Shirabu) was used.

Normal human skin fibroblasts (NHDF) were cultured in 12-well culture plates. More specifically, the cells were seeded at a density of 8.0 × 10 ⁴ cells / well and cultured at 37 ° C. in an environment of 5% carbon dioxide gas and 95% air for 24 hours. As a culture solution, a medium containing 2% by mass of fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) was used at 1 mL per well. Subsequently, whey protein was added at 200 μg / mL, and other extracts were added at 0.25% by mass. After completion of the culture, the amount of collagen in the culture supernatant was determined by ELISA (Human Collagen Type I ELISA kit; Manufactured). The results are shown in FIG. In addition, the amount of collagen was compared with the amount of collagen in the culture supernatant when no whey protein or other extract was added as 100.

  From the results shown in FIG. 1, the amount of collagen in the culture supernatant was most increased in the cells cultured in the culture solution to which the aurene extract was added, compared to the cells cultured in other culture solutions.

  Subsequently, in order to confirm a change in the amount of collagen in the cell, the amount of collagen in the cell was detected. First, the cells after collection of the culture supernatant were washed with PBS (−), and RIPA buffer was added. After that, SDS sample buffer was added and heat reduction treatment was performed. This was subjected to SDS-PAGE, and intracellular collagen was detected by Western blotting using an antibody (Anti-Collagen Type I Antibody; manufactured by Merck Millipore). The results are shown in FIG. The amount of collagen in the cells when no whey protein or other extract was added was taken as 100, and the amount of collagen was compared.

  From the results shown in FIG. 2, the amount of collagen in the cells was greatly reduced in the cells cultured in the culture solution to which the aurene extract was added as compared with the other culture solutions.

  From the results of FIG. 1 and FIG. 2, it was found that the auren extract decreases the amount of collagen in the cells and increases the amount of collagen in the culture supernatant. From this, it was suggested that the auren extract promotes the secretion of collagen from the cell to the culture supernatant. It is known that the main component of the auren extract is berberine, and it is considered that berberine has an action of promoting secretion of collagen from the inside of the cell to the outside of the cell.

[Example 1: Collagen production upon costimulation of berberine and a collagen synthesis promoter (compound) in dermal fibroblasts]
In Test Example 1 above, berberine, which is a major component of the auren extract that has been shown to promote collagen secretion, and a compound (TGF-β, IGF-1, or curcumin) that has a collagen synthesis promoting effect are added in combination. In this case, the effect of promoting the synthesis of collagen in cells and the secretion of collagen outside the cells was tested as follows. In addition, berberine chloride (manufactured by Wako Pure Chemical Industries, Ltd.) is used as berberine, transforming growth factor-β1 human (manufactured by Sigma Aldrich) as TGF-β, and Insulin-like Growth Factor-I human (manufactured by Sigma Aldrich). Sigma Aldrich) and curcumin were curcumin (Wako Pure Chemical Industries).

Normal human skin fibroblasts (NHDF) were cultured in 12-well culture plates. More specifically, the cells were seeded at a density of 8.0 × 10 ⁴ cells / well and cultured at 37 ° C. in an environment of 5% carbon dioxide gas and 95% air for 24 hours. As a culture solution, a medium containing 2% by mass of fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) was used at 1 mL per well. Subsequently, berberine was added at 1 μg / mL, and collagen synthesis promoter (TGF-β, IGF-1, or curcumin) was added at 10 ng / mL, either alone or both. The amount of collagen was measured by ELISA (Human Collagen Type I ELISA kit; manufactured by Acer Corporation). The measurement results are shown as relative values in FIG. 3, with the amount of collagen when no berberine or collagen synthesis promoter is added (“−” in the figure) being 100.

  From the results shown in FIG. 3 in which the amount of collagen in the culture supernatant was examined, the cells cultured in a culture solution to which both berberine and a collagen synthesis promoter were added had a higher collagen content than those cultured in a single culture solution. Increased. From this, it was confirmed that collagen secretion to the outside of the cell was remarkably enhanced by combining berberine and a collagen synthesis promoter. Further, when a certain amount of collagen synthesis promoter and berberine were combined, the combination of TGF-β and berberine most promoted the secretion of collagen to the outside of the cell. TGF-β has the largest molecular weight among the three types of collagen synthesis promoters tested, and the number of molecules added was small. Therefore, even when compared with a certain amount of substance, TGF-β is higher than other compounds. Also, it can be said that collagen secretion into the culture supernatant when combined with berberine was most prominent.

  Subsequently, a change in the amount of intracellular collagen was detected. The cells after collection of the culture supernatant were washed with PBS (−), and RIPA buffer was added. After that, SDS sample buffer was added and heat reduction treatment was performed. This was subjected to SDS-PAGE, and intracellular collagen was detected by Western blotting using an antibody (Anti-Collagen Type I Antibody; manufactured by Merck Millipore). The measurement results are shown as relative values in FIG. 4, with the amount of collagen when no berberine or collagen synthesis promoter is added being taken as 100.

  From the results shown in FIG. 4 in which the amount of collagen in the cells was examined, the amount of collagen in the cells cultured in the culture solution to which the collagen synthesis promoter was added alone increased, indicating an effective collagen synthesis promoting ability. On the other hand, when combined with berberine, the amount of collagen decreased compared to when the collagen synthesis promoter was used alone, suggesting that berberine promotes secretion of collagen out of the cell.

  From the results of FIGS. 3 and 4, the amount of intracellular collagen decreased and the amount of collagen in the culture supernatant significantly increased when co-stimulated with berberine and a collagen synthesis promoter than when stimulated alone. From these results, it was shown that by combining berberine and a collagen synthesis promoter such as TGF-β, the amount of collagen secreted to the outside of the cell can be remarkably increased and the production of collagen as a whole can be promoted. .

[Example 2: Collagen production during costimulation of berberine and collagen synthesis promoter (extract) in dermal fibroblasts]
When berberine and an extract having a collagen synthesis promoting action (whey protein or nettle extract) are added in combination, collagen synthesis in the cell and promotion of collagen secretion outside the cell are as follows. Were tested as follows. As berberine, berberine chloride (manufactured by Wako Pure Chemical Industries, Ltd.) was used, and as whey protein, the same β-lactoglobulin-rich whey protein (manufactured by Seperex Nutritional) as used in Test Example 1 was used. . As the nettle extract, nettle extract BG (manufactured by Maruzen Pharmaceutical Co., Ltd.) was used.

Normal human skin fibroblasts (NHDF) were cultured in 12-well culture plates. More specifically, the cells were seeded at a density of 8.0 × 10 ⁴ cells / well and cultured at 37 ° C. in an environment of 5% carbon dioxide gas and 95% air for 24 hours. As a culture solution, a medium containing 2% by mass of fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) was used at 1 mL per well. Subsequently, berberine is added at 1 μg / mL, and a collagen synthesis promoter (whey protein or nettle extract) is added alone or both so that the solid content is 25 μg / mL. The amount of collagen was measured by ELISA (Human Collagen Type I ELISA kit; manufactured by Acer Corporation). The measurement results are shown as relative values in FIG. 5, where the amount of collagen when no berberine or collagen synthesis promoter is added (“−” in the figure) is 100.

  From the result of FIG. 5, the amount of collagen in the culture supernatant increased in the cells cultured in the culture solution to which both berberine and the collagen synthesis promoter were added, compared to the cells cultured in the single culture solution. From this, it was recognized that the combination of berberine and a collagen synthesis promoter significantly enhances the secretion of collagen to the outside of the cell. When a certain amount of collagen synthesis promoter and berberine were combined, the combination with whey protein enhanced the secretion of collagen most extracellularly.

  Subsequently, in order to confirm a change in the amount of collagen in the cell, the amount of collagen in the cell was detected. The cells after collection of the culture supernatant were washed with PBS (−), and RIPA buffer was added. After that, SDS sample buffer was added and heat reduction treatment was performed. This was subjected to SDS-PAGE, and intracellular collagen was detected by Western blotting using an antibody (Anti-Collagen Type I Antibody; manufactured by Merck Millipore). The measurement results are shown as relative values in FIG. 6 with the amount of collagen when no berberine or collagen synthesis promoter is added being 100.

  From the result of FIG. 6, the amount of collagen in the cells of the cells cultured in the culture solution to which the collagen synthesis promoter was added alone increased. On the other hand, when combined with berberine, the amount of collagen decreased compared to the case of the collagen synthesis promoter alone. These results suggest that berberine promotes extracellular secretion of collagen.

  From the results of Example 1 and Example 2 above, by combining berberine and various collagen synthesis promoters, the amount of collagen secreted outside the cell is remarkably increased, and as a result, collagen production is effectively improved. I found that it can be promoted. In addition, among the collagen synthesis promoters, it has been found that a higher collagen production promoting effect can be achieved by combining TGF-β or whey protein with berberine.

[Formulation Example 1: Lotion]
A lotion having the composition shown below was prepared as follows. Ingredients (1) to (7) were added to ingredient (8) and mixed.
(Ingredient) (mass%)
(1) Ouren root extract 0.01
(2) Whey protein 0.10
(3) PEG-50 hydrogenated castor oil 0.20
(4) Glycerin 5.00
(5) BG 5.00
(6) pH adjuster appropriate amount (7) preservative appropriate amount
(8) Purified water balance 100

[Formulation Example 2: Emulsion]
An emulsion having the composition shown below was prepared as follows. Ingredients (3) to (7) were heated and mixed at about 80 ° C. To this, components (8), (9), (12) and (13) heated and mixed at about 80 ° C. were added and cooled to room temperature. Components (10) and (11) were added sequentially, and components (1) and (2) were added last.
(Ingredient) (mass%)
(1) Ouren root extract 0.10
(2) Whey protein 1.00
(3) Squalane 0.50
(4) Dimethicone 0.50
(5) Polysorbate 80 0.50
(6) Glyceryl stearate 1.00
(7) Behenyl alcohol 1.00
(8) Glycerin 5.00
(9) BG 8.00
(10) Carbomer 0.20
(11) Na hydroxide 0.06
(12) Preservative appropriate amount
(13) Purified water balance 100

[Formulation Example 3: Cream]
A cream having the following composition was prepared as follows. Components (3) to (9) were heated and mixed at about 80 ° C. To this, components (10), (11), (14) and (15) heated and mixed at about 80 ° C. were added and cooled to room temperature. Components (12) and (13) were added sequentially, and components (1) and (2) were added last.
(Ingredient) (mass%)
(1) Ouren root extract 0.10
(2) Whey protein 1.00
(3) Squalane 0.70
(4) Dimethicone 1.00
(5) Stearic acid 5.50
(6) PEG-40 stearate 1.20
(7) PEG-25 stearate 0.40
(8) Behenyl alcohol 3.50
(9) Cetyl ethylhexanoate 4.60
(10) Glycerin 2.00
(11) BG 3.00
(12) Carbomer 0.25
(13) Na hydroxide 0.08
(14) Preservative appropriate amount
(15) Purified water balance 100

[Formulation Example 4: Hard capsule]
Hard capsules having the composition shown below were prepared as follows. Components (1) to (6) were mixed and filled into capsules to obtain hard capsules.
(Ingredient) (mass%)
(1) Ouren root extract 10.00
(2) Whey protein 10.00
(3) Sodium ascorbate 18.00
(4) Dextrin 24.00
(5) Glycerin fatty acid ester 0.50
(6) Powdered sugar Total remaining 100

  Since the composition for promoting collagen production according to the present invention has an excellent ability to promote collagen production, for example, prevention or treatment of an anti-wrinkle composition, an anti-tarmi composition, a corneal disorder, a joint disorder, an inflammatory disease, etc. It can be suitably used as a composition for use.

Claims (8)

(A) one or more selected from the group consisting of berberine, berberine analogs and salts thereof, and a plant extract containing berberine for promoting the secretion of collagen from the inside of the cell to the outside of the cell;
(B) A composition for promoting collagen production, comprising a collagen synthesis promoter for promoting the synthesis of collagen in cells.   The composition for promoting collagen production according to claim 1, wherein the component (B) is a component having a TGF-β and / or TGF-β-like action.   The composition for promoting collagen production according to claim 1, wherein the component (B) is at least one selected from the group consisting of TGF-β, whey protein, and whey protein fragments.   The composition for promoting collagen production according to any one of claims 1 to 3, comprising 0.05 part by mass or more of the component (A) with respect to 100 parts by mass of the component (B).   A skin external preparation containing the composition for promoting collagen production according to any one of claims 1 to 4.   Food / beverage products containing the composition for a collagen production promotion in any one of Claims 1-4.   The pharmaceutical containing the composition for collagen production promotion in any one of Claims 1-4.   Collagen for promoting secretion of collagen from inside to outside of cell, comprising at least one selected from the group consisting of berberine, berberine analogs and salts thereof, and plant extract containing berberine Secretagogue.

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