JP4939766B2 - Basement membrane stabilizer - Google Patents

Description

  The present invention relates to a novel basement membrane stabilizer. More specifically, the present invention relates to a basement membrane stabilizer comprising various biological extracts and hydrolysates thereof as active ingredients. The present invention also relates to an extracellular matrix production promoter comprising the above-mentioned biological extract or the like as an active ingredient, a method for producing the same, and a method for producing artificial skin using the extract or the like.

  Skin covering the entire body of various animals including human beings is exposed to external factors such as sunlight, dryness, oxidation, environmental stress, mental stress, and wrinkle formation due to aging, hardening, spots, dullness, elasticity It is exposed to changes such as decline. Here, the skin is roughly divided into two layers, the epidermis and the dermis. Between the epidermis and the dermis there is a thin and delicate membrane called the basement membrane. Epidermal metabolism depends on factors and blood supply produced by dermal cells through this basement membrane. Epidermal growth and differentiation in the skin is regulated by the basement membrane and dermis. Therefore, the communication between the epidermis and dermis via the basement membrane plays an important role in regulating the function of the skin epidermis.

  The skin basement membrane has a special structure called an anchoring complex, which plays a role in stabilizing the adhesion and communication between the two tissues, the epidermis and dermis. The anchoring complex proteins are linked to both keratin cytoskeleton of keratinocytes and connective tissue proteins of the dermal papillary layer. One important component of the anchoring complex is laminin 5 (Rousselle et al., J Cell Biol. 1991 Aug; 114 (3): 567-76). Previous studies have revealed that genetic variation in the gene encoding the three strands of laminin 5 is responsible for the congenital genetic disease that forms severe blistering, Herritz junctional epidermolysis bullosa. (Aberdam et al., Nat Genet. 1994 Mar; 6 (3): 299-304). This indicates that laminin 5 is an essential substance for adhesion of the epidermis. In addition, laminin 5 forms an anchoring filament and has been elucidated to bind to integrin α6β4 (a transmembrane integrin present in hemidesmosome) known as a receptor for laminin 5 (Niessen). et al., Exp Cell Res. 1994 Apr; 211 (2): 360-7).

  Laminin 5 not only binds directly to type VII collagen, which forms anchoring fibers connected to the dermal papillary layer, but also forms a complex with other laminin 6 and 7, which is responsible for naidogen. It is reported that it binds to type IV collagen, which is the skeleton of the basement membrane.

  The expression level of type IV collagen constituting the complex has been observed to decrease with aging (Vazquez F et al., Maturitas 1996, 25: 209-215), and type VII collagen to which laminin 5 binds. As for dermal fibroblasts derived from elderly people, the production ability is reduced at the protein level and mRNA level compared to dermal fibroblasts derived from young people (Chen et al., J. Invest. Dermatol., 102: 205-209, 1994). In addition, anchoring fibers composed of type VII collagen are reported to decrease in normal skin with physiological aging and photoaging (Takuo Tsuji, Nisshinkai 105: 963-975, 1995, Tidman et al., J Invest Dermatol., 83: 448-453, 1984). In addition to the characteristics of these individual components, the basement membrane itself is known to exhibit more than a structure such as multiplexing and rupture with skin aging (Lavker et al., J. Invest. Dermatol. 1979 73: 59-66), as a result of this structural change, it seems that the appearance of senile features such as wrinkles and sagging, and a decrease in skin function associated with aging. Therefore, promoting the production of laminin 5, type IV and type VII collagen constituting the basement membrane skeleton is extremely important for maintaining the basement membrane structure in a good state and preventing skin aging such as wrinkles. Conceivable. In fact, when the skin basement membrane structure is observed in detail, since the basement membrane damage is frequently observed from the late 20s to the early 30s, the structural change of the basement membrane plays an important role in the induction of skin aging. (IFSCC Magazine, 2000 4 (4): 15-23).

  So far, preparations derived from soybeans, lysophosphatidylcholine, lysophosphatidic acid for the purpose of enhancing the ability to produce laminin 5, the main component of the basement membrane, from the viewpoint of maintaining skin homeostasis Furthermore, extracts of plants belonging to the genera Bacconia, Psophocarpus, Cassia, and Erythraea have been proposed (Japanese Patent Laid-Open Nos. 11-343226 and 2000-226308). And JP-A-2003-313135). Similarly, as for the production promoters of type IV and type VII collagen, various plant-derived extracts such as beech buds, cuckoos, western ivy, beech family beech genus extracts are known (Japanese Patent Application Laid-Open (JP-A)). 2004-18471, JP-A-2004-75661, etc.). However, there are no known substances that promote the production of laminin 5, type IV, and type VII collagen, which are important constituents of the basement membrane, other than the cuckoo extract (Japanese Patent Laid-Open Nos. 2003-137767 and 2004-18471). issue).

JP 2003-267822 A JP 2002-12548 A JP 2001-10945 A JP-A-10-330222 Japanese National Patent Publication No. 9-512284 JP-A-10-330221 JP 2003-34631 A JP-A-6-24937 JP 2000-159627 A JP 2001-114634 A JP 2000-247897 A JP-A-10-330222 JP 2001-114634 A JP 2001-114636 A Japanese Patent Laid-Open No. 10-36279 JP-A-9-291023 JP 2001-181169 A JP 2003-221770 A JP 2003-277227 A JP-A-7-277939 Japanese Patent Laid-Open No. 6-211625

  The present invention relates to the provision of a novel basement membrane stabilizer.

  As a result of intensive studies on various biological extracts and the like for the purpose of obtaining a basement membrane stabilizing action, the present inventors have found that the following biological extracts and the like are laminin 5, type IV collagen, which is an important component of the basement membrane, It was revealed that the production of type VII collagen was remarkably promoted. In addition, the solvent used in extraction of the said biological extract etc. is listed for the purpose of illustration, and is not intending limiting.

  Dendrobins (scientific name Calendula officinalis Linne): Extracts of Dendrobins, a member of the family Asteraceae, are obtained by extracting from the head flowers with water, ethanol, propylene glycol, 1,3-butylene glycol or a mixture of these. . The extract is known to have an antioxidant effect (Japanese Patent Laid-Open No. 2003-267822) and a phototoxicity-inhibiting effect (Aging by products generated by UVA irradiation of air pollutants, Japanese Patent Laid-Open No. 2002-12548). Collagen production promoting action and basement membrane action are not known.

  Hazelnut (Guevina avellana Mol): An extract of hazelnut, a kind of genus Porphyra, is a fatty oil obtained from its seeds. The extract is known to have a moisturizing and rough skin action (Japanese Patent Laid-Open No. 2001-10945), but is not known to promote collagen production or to act on the basement membrane.

  Cornflower (scientific name Centaurea cyanus Linne): An extract of cornflower, which is a member of the family Asteraceae, is obtained by extraction from its head flower with water, ethanol, propylene glycol, 1,3-butylene glycol or a mixture thereof. Although the extract is known to be used as a normalizing agent for dermal collagen fiber bundles (Japanese Patent Laid-Open No. 10-330222), its action on the basement membrane is not known.

  Barley (scientific name: Hordeum vulgare Linne): An extract of barley, a member of the gramineae family, is obtained by extracting water from its seeds with water, propylene glycol or a mixture thereof. Although the extract is known to have an anti-wrinkle action and the like (Tokuhei 9-512284), it is not known about the basement membrane stabilizing action.

  Olysam (scientific name Lamium album Linne): An extract of Olysium, a member of the family Lamiaceae, is obtained by extraction from its flowers, stems and leaves with water, propylene glycol, 1,3-butylene glycol or a mixture thereof. The extract is known to have a dermal collagen fiber bundle normalizing action and the like (Japanese Patent Laid-Open No. 10-330221), but is not known about a basement membrane stabilizing action.

  Kyonin: Kyonin is a seed of honanzu (scientific name: Prunus armeniaca Linne), apricot (scientific name: Prunus armeniaca Linne var. Ansu Maximowicz), or other related plant, and its extract contains 30 seeds. Obtained by extraction with a% ethanol solution. The extract is known to have a fibroblast proliferation promoter or the like (Japanese Patent Application Laid-Open No. 2003-34631), but is not known about the basement membrane stabilizing action.

  Generasho (Geranium thunbergii Siebold et Zuccarini): An extract of Genokosho, a member of the family Octeraceae, is obtained by extraction from the ground with an ethanol solution or a 1,3-butylene glycol solution. The extract is known to have mucopolysaccharide fragmentation inhibition, antioxidant action (JP-A-6-24937), elastase activity inhibitory action (JP-A 2000-159627), etc. Is not known about.

  Saponaria officinalis Linne: An extract of Savonsou, a kind of Nadesicoaceae, is obtained by extracting from its leaves with propylene glycol, 1,3-butylene glycol or an aqueous solution thereof. Although the extract is known to have a catalase protecting action or the like (Japanese Patent Laid-Open No. 2001-114634), it is not known about a basement membrane stabilizing action.

  Shobu (scientific name: Acorus calamus Linne Besser): Shobu, a member of the taro family, is extracted from its rhizome with an ethanol solution. Although the extract is known to have an active oxygen scavenging action or the like (Japanese Patent Laid-Open No. 2000-247897), it is not known about a basement membrane stabilizing action.

  Honeysuckle (scientific name: Lonicera japonica Thumberg): An extract of honeysuckle, a member of the honeysuckle family, is obtained by extracting water, propylene glycol, 1,3-butylene glycol or a mixture thereof from its flowers, leaves or stems. It can be obtained in the same manner from other plants of the same genus. The extract is known to have a dermal collagen fiber bundle normalizing action and the like (Japanese Patent Laid-Open No. 10-330222), but is not known about a basement membrane stabilizing action.

  Achillea millefolium Linne; an extract of Achillea millefolium Linne, which is a member of the Asteraceae family, is water, ethanol, propylene glycol, 1,3-butylene glycol, or a mixture of these or 1% urea. It is obtained by extraction with an ethanol solution containing 1, 1-butylene glycol solution containing 1% urea. The extract is known to have a catalase protecting action (Japanese Patent Laid-Open No. 2001-114634), a hyaluronic acid production promoting action (Japanese Patent Laid-Open No. 2001-114636), etc., but is not known about a basement membrane stabilizing action. .

  Tounin: Tounin is the seed of a peach (Prunus persica Batsch or Prunus persica Batsch var. Davidiana Maximowicz), a species of the family Rosaceae, and its extract contains water, ethanol, 1,3-butylene glycol or Obtained by extraction with a mixture of these. The extract is known to have a fibroblast proliferation promoting action and the like (Japanese Patent Laid-Open No. 10-36279), but is not known about a basement membrane stabilizing action.

  Tomato (scientific name: Lycopersicon esculentum Miller (Solanum lycopersicum Linne)): An extract of tomato, a member of the solanaceous family, is extracted from its fruit (raw) with water, propylene glycol, 1,3-butylene glycol or a mixture of these. Obtained. Although the extract is known to have a catalase action or the like (Japanese Patent Laid-Open No. 9-291023), it is not known about a basement membrane stabilizing action.

  Garlic (scientific name Allium scorodoplasum L. or Allium sativum L.): Extract of garlic, a member of the lily family, is purified from its bulb with purified water, absolute ethanol, propylene glycol, 1,3-butylene glycol or a mixture of these. Obtained by extraction. Although the dermal tightening action and the like are known (Japanese Patent Laid-Open No. 2001-181169), the basement membrane stabilizing action is not known.

  Mukuroji (scientific name: Sapindus mukurossi Gaertner): An extract of Mukuroji, a kind of Mukuroji family, is obtained by extracting water, ethanol or a mixture of these from the pericarp. Although the extract is known to have a Maillard reaction inhibitory action or the like (Japanese Patent Laid-Open No. 2003-221770), it is not known about a basement membrane stabilizing action.

  Lettuce (scientific name: Lactuca sativa Linne): An extract of lettuce, a member of the Asteraceae family, is obtained by extracting water (raw) with water, 1,3-butylene glycol, or a mixture thereof. Although the extract is known to have a dermal tightening action or the like (Japanese Patent Laid-Open No. 2003-277227), it is not known about a basement membrane stabilizing action.

  Yeast: Extracts of yeasts of the genus Saccharomyces are concentrated or dried after filtering the liquid obtained by autolysis or acid hydrolysis or without filtering. Although the extract is used as a cell activator (Japanese Patent Laid-Open No. 7-277939), the basement membrane stabilizing action is not known.

  Hydrolyzed conchiolin: An aqueous solution obtained by hydrolyzing the hard protein conchiolin contained in the nacreous layer of the pearl oyster, Pinctada fucata. Conchiolin hydrolyzate is known to have a histamine-inhibiting action and the like (Japanese Patent Laid-Open No. 6-211625), but is not known about a basement membrane stabilizing action.

  Therefore, the present invention provides a basement membrane stabilizer, an extracellular matrix production promoter comprising any one or more of the above-described extracts as an active ingredient, a production method thereof, and an artificial using the above-described extract and the like A method for producing a skin is provided.

  It is also known for anti-inflammatory action and wound healing promotion effects for pearl millet, hazelnut, cornflower, perilla, barley, edible persimmon, hydrolyzed concholin, kyounin, geno shochu, scorpion, shobu, honeysuckle, tomato, mugwort, and lettuce. Absent.

  The basement membrane stabilizer according to the present invention promotes the production of extracellular matrices such as laminin 5, type IV collagen, type VII collagen and the like, which are important constituents of the basement membrane, by including the extract and the like. Moreover, the said extract etc. also have an anti-inflammatory action and a wound healing action. Although the exact mechanism is not clear, it is thought that all of these actions are due to the restoration and stabilization of the basement membrane as a result of the accelerated production of laminin and the like. Therefore, the basement membrane stabilizer of the present invention is also a symptom of epidermolysis bubbling caused by instability of the basement membrane, such as thawing of basement membrane cells, exfoliation of the basement membrane from the epidermis, and type VII collagen abnormality. It can contribute to improvement. In addition, it should be particularly noted regarding the effect of the present invention that all of the above-mentioned extracts promote the production of all of laminin 5, type IV collagen and type VII collagen, not alone.

Basement membrane stabilizer As used herein, “basement membrane stabilization” refers to a basement membrane that exists between the epidermis and the dermis that is deeply involved in maintaining the homeostasis of the skin, and whose skeleton is stabilized. Means to be repaired. More specifically, it is intended to mean an overall effect on the basement membrane caused by the promotion of production of important components constituting the basement membrane, particularly the extracellular matrix.

Extracellular matrix production promoter The present invention further includes an extract of pearl millet, hazelnut, cornflower, barley, scotch, cypress, ginseng, scorpion, shobu, honeysuckle, yarrow, corn, tomato, garlic, mugwort, lettuce, yeast And / or an extracellular matrix production promoter comprising any one or more of hydrolyzed conchiolin as an active ingredient. Here, “extracellular matrix” means those generally recognized in the art, and particularly important constituents in the basement membrane including laminin type 5, type IV or type VII collagen. means.

  In addition, the extract and the like not only increase the production of extracellular matrix and stabilize the basement membrane, but also have an anti-inflammatory effect, a wound healing promoting effect, and further an epidermolysis bullosa amelioration effect. However, as described above, the exact mechanism of these actions is not clear. As used herein, “anti-inflammatory action” and “wound healing promoting action” are relative to the non-application site when the biological extract of the present invention is applied to an inflammation site and a wound site, respectively. It is to be decided. The wound healing promoting effect obtained by the present invention is considered to be due to the stabilization and repair of the basement membrane as a result of the promotion of extracellular matrix production. However, it is clear that this action is not necessarily limited to that resulting from increased extracellular matrix production. It should be noted that an anti-inflammatory action and a wound healing promoting action have been reported for extracts of Achillea millefolium, tonin, garlic, and yeast.

  As used herein, “epidermolysis bullosa ameliorating action” means only alleviation and healing of general epidermolysis bullosa classified into simple epidermolysis bullosa, junctional epidermolysis bullosa and dystrophic epidermolysis bullosa Rather, it is intended to prevent such symptoms. This effect is brought about by stabilizing the structure of the basement membrane by promoting the production of collagen, which is an important component of the basement membrane.

  In the case of plants, the extract and the like used in the present invention are obtained by extracting from the roots, leaves, stems, flowers, buds, bark, fruits, etc. of each plant, and in the case of yeast, Saccharomyces (Saccharomyces The liquid obtained by self-digesting or acid-hydrolyzing the yeast belonging to) is filtered, concentrated or dried, and in the case of conchiolin, conchiolin, which is a hard protein contained in the pearl oyster pearl layer Is obtained by hydrolyzing.

  The extraction method of the starting material such as the plant may be performed by solvent extraction. In the case of solvent extraction, the material is dried as necessary, and further chopped or pulverized as necessary, then an aqueous extractant, For example, it is extracted by using cold water, hot water, hot water having a boiling point or lower than that, or an organic solvent such as methanol, ethanol, 1,3-butanediol, ether or the like at normal temperature or by heating. However, the extraction method is not limited to solvent extraction, and may be a conventional method known in the art. A commercially available extract may be used. The form of the extract may include not only the extract itself, but also those that are appropriately diluted or concentrated or dried by conventional techniques.

  The basement membrane stabilizer or extracellular matrix production promoter of the present invention may contain the above extract alone or a combination of a plurality of extracts. In yet another embodiment, the basement membrane stabilizer or extracellular matrix production promoter of the present invention may further comprise a serine protease inhibitor. As described in Examples below, the basement membrane stabilizer or extracellular matrix production promoter of the present invention contains a serine protease inhibitor, thereby inhibiting collagen degradation and keeping the basement membrane in a better state. . This result is considered to work well for the anti-wrinkle action and the wound healing promoting action obtained by the present invention, as well as the action for improving epidermolysis bullosa. Examples of the serine protease inhibitor include extracts of karin, jujube, and wild rose, and other than that, yew, hypericum, mint, arnica, hawthorn, sunflower, rose, grape, button, hop, Extracts such as raspberries and roman chamomile are included. These can be extracted by solvent extraction or the like in the same manner as the organism extraction method used in the present invention. Further, the serine protease inhibitors include aprotinin, tranexamic acid, ε-aminocaproic acid and the like.

  The extract or the like used in the present invention achieves an amount necessary for achieving a basement membrane stabilizing action, in other words, an increase in the production amount of an extracellular matrix such as laminin 5, type IV or type VII collagen. The required amount is blended. The blending amount varies depending on various factors such as the dosage form to be blended. Although not limited, the said extract etc. are the density | concentration of 0.0001-10 mass% with respect to the whole mass of the said basement membrane stabilizer or extracellular matrix production promoter, Preferably 0.001-1 mass% May be blended at a concentration of

  In the basement membrane stabilizer or extracellular matrix production promoter of the present invention, in addition to the above extract and the like, components that can be usually added to cosmetics and pharmaceuticals within a range that does not impair the basement membrane stabilizing action, such as liquid fats and oils , Solid fats, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, silicones, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, humectants, water-soluble polymers Compounds, thickeners, coating agents, UV absorbers, sequestering agents, lower alcohols, polyhydric alcohols, sugars, amino acids, organic amines, pH adjusters, skin nutrients, vitamins, antioxidants, fragrances , Powder, coloring material, water and the like can be appropriately blended as necessary. Further, L-ascorbic acid and its salts, L-ascorbic acid phosphate ester, ester derivatives of L-ascorbic acid such as L-ascorbic acid sulfate, and salts thereof, and L-ascorbic acid such as L-ascorbic acid glucoside Glycosides and salts thereof, alkoxysalicylic acids such as 4-methoxysalicylic acid and salts thereof, hydroquinone glycosides such as hydroquinone β-D-glucose and hydroquinone α-D-glucose and salts thereof, tranexamic acid and tranexam Tranexamic acid derivatives such as acid methylamide hydrochloride, resorcin derivatives such as 4-n-butylresorcin, kojic acid, ellagic acid, linoleic acid, chamomile extract, retinoic acid, retinol, retinol acetic acid, retinol palmitic acid, glycyrrhizic acid and derivatives thereof Etc., Rhizophosphatiji Lucolin, lysophosphatidic acid, laminin 5 production promoters such as soybean preparations, sugars such as glucose, fructose, mannose, sucrose, trehalose, nicotinic acid benzyl ester, nicotinic acid β-butoxyethyl ester, capsaicin, gingerone, Cantharis tincture, ictamol, caffeine, tannic acid, α-borneol, tocopherol nicotinate, inositol hexanicotinate, cyclandrate, cinnarizine, trazoline, acetylcholine, verapamil, cephalanthin, γ-oryzanol and other blood circulation promoters, sulfur, Antiseborrheic agents such as thianthol, for various purposes, hinokitiol, iris extract, asenyaku extract, allantoin, aloe extract, yakiyaku extract, ibujakaku extract, turmeric extract, o Extract, Auren extract, Hypericum extract, Ononis extract, Hydrolyzed casein, Hydrolyzed yeast extract, Cuckoo extract, Xylitol, Clara extract, Rice extract hydrolyzed solution, Psycho extract, Saffron extract , Zinc oxide, winged bean extract, lion extract, peony extract, ginger extract, silica-coated zinc oxide, sage extract, mallow extract, sensu extract, assembly extract, chimpi extract, chili extract, Toki extract, Tonin extract, thiotaurine, carrot extract, garlic extract, Hiragi extract, Hipotaurine, birch extract, loquat extract, grape extract, beech bud extract, loofah extract, marjoram extract, Lily extract, Yokuinin extract, Rosemary extract, Arginine and its hydrochloride, Amino acids and derivatives thereof, such as calcium and its hydrochloride, pantothenic acids such as calcium pantothenate, D-pantothenyl alcohol, pantothenyl ethyl ether, acetyl pantothenyl ethyl ether, vitamin Ds such as ergocalciferol and cholecalciferol, nicotine Nicotinic acids such as acid, nicotinic acid amide and benzyl nicotinate, vitamin E such as α-tocopherol, tocopherol acetate, DL-α-tocopherol nicotinate, DL-α-tocopherol succinate, vitamin K, vitamin P, biotin, etc. Vitamins, coenzymes such as ubiquinone, and the like can be appropriately blended.

  The dosage form of the basement membrane stabilizer or extracellular matrix production promoter of the present invention is not particularly limited, and examples thereof include solution systems, solubilization systems, emulsion systems, powder dispersion systems, water-oil bilayer systems, Arbitrary dosage forms such as a water-oil-powder three-layer system, an ointment, a gel, and an aerosol can be taken. Further, the usage form is not particularly limited, and can take any form such as lotion, emulsion, cream, essence, jelly, gel, ointment, pack, foundation, and the like.

Skin external preparation Furthermore, this invention provides the skin external preparation containing the said extract etc. Here, the external preparation for skin of the present invention is intended to have an anti-inflammatory action, a wound healing promoting action, and further an action for improving epidermolysis bullosa. However, the topical skin preparation of the present invention is not limited to these purposes, and may be used to improve various symptoms of the skin caused by the basement membrane stabilizing action of the extract and the like, in other words, the extracellular matrix production promoting action. Conceivable.

  The substances to be blended in the external preparation for skin of the present invention are the same as those described above for the basement membrane stabilizer, but this is appropriately determined by those skilled in the art according to the desired purpose. Can be done. For example, when aiming at an anti-inflammatory action and a wound healing promotion action, the extract is at a concentration of 0.0001 to 10% by mass, preferably 0.001 to 1% by mass, with respect to the mass of the whole external preparation for skin. May be blended at a concentration of

In another embodiment of the method for producing a basement membrane stabilizer and an extracellular matrix production promoter , the present invention comprises a step of adding the above extract or the like to an aqueous phase or an oil phase. A method for producing a matrix production promoter is provided.

Method for producing artificial skinFurthermore, the present invention relates to an extract of pearl millet, hazelnut, cornflower, barley, edible perilla, leopard, gentian, salamander, ginger, honeysuckle, yarrow, corn, tomato, garlic, lettuce, yeast And / or adding one or more of hydrolyzed conchiolin to an artificial skin formation medium.

  As the basal medium used for the production of artificial skin in the present invention, any medium conventionally used for the production of artificial skin can be used, and these media include Dulbecco's modified eagle containing 10% fetal calf serum. Medium (DMEM): DMEM-Ham'sF12 (3: containing 10% fetal bovine serum, transferrin 5 μg / ml, insulin 5 μg / ml, tri-iodothyronine 2 nM, cholera toxin 0.1 nM, hydrocortisone 0.4 μg / ml 1); a medium in which keratinocyte growth medium (KGM) and DMEM containing 10% fetal bovine serum are mixed at 1: 1, and the like. Although the said extract etc. which are added to these basal media differ according to the kind, they are about 0.001 mass%-about 1 mass%. In addition to the extract, various substances used in the embodiment of the basement membrane stabilizer may be added together. In particular, the above-mentioned serine protease inhibitor inhibits collagen degradation and stabilizes the basement membrane. Therefore, it is considered preferable.

In the production of the artificial skin of the present invention, first, a contracted type I collagen gel containing human fibroblasts is placed on a wire mesh. A contracted type I collagen gel containing human fibroblasts can be prepared, for example, as follows. After preparing a fibroblast-suspended collagen solution on ice, collagen is gelled in a Petri dish. Thereafter, the gel is peeled from the wall surface of the Petri dish, and the collagen gel is contracted in a CO ₂ incubator.

Next, epidermal cells such as human normal epidermal keratinocytes are cultured on the collagen gel to form the epidermis. Formation of the epidermal layer by culturing skin cells can be performed as follows. A contracted collagen gel is placed on a wire mesh, and a glass ring is placed on the gel. An epidermis keratinocyte suspension derived from human foreskin is placed in the glass ring so as not to leak. Adhere keratinocytes in a CO ₂ incubator and remove the ring. The medium is filled up to the boundary of the epidermal layer, and the culture is continued while the epidermal layer is exposed to air to form a stratum corneum.

  According to this method, artificial skin close to natural skin in which a basement membrane component is sufficiently deposited between the dermis layer composed of contracted type I collagen gel containing fibroblasts and the epidermis layer can be obtained. The extract and the like used in the present invention are not only contained in a new medium when the medium is changed several days after seeding of epidermal cells, but are not limited to this stage, and are appropriately added as necessary. be able to.

In another aspect of the cosmetic method , the present invention relates to an extract of pearl millet, hazelnut, cornflower, barley, edible perilla, leopard, gentian, salamander, ginger, honeysuckle, yarrow, corn, tomato, garlic, lettuce, yeast And / or applying one or more of hydrolyzed conchiolin to the skin. The cosmetic method is intended for use in ameliorating various skin conditions, such as epidermolysis bullosa, that require the production of extracellular matrix and thus the stabilization of the basement membrane. In the cosmetic method, the subject to which the extract or the like is applied is generally a mammal, and in particular, application to humans is conceivable.

  Next, the present invention will be described more specifically with reference to the following examples.

(Experiment Method-1)
Test method for laminin 5 production promoting effect (1) Culture of epidermal keratinocytes Epidermal keratinocytes were isolated from human foreskin and cultured in epidermal cell growth medium (KGM) with low calcium concentration. The bovine pituitary extract and EGF were added to this medium. The cells were cultured in KGM until the fourth generation, then the adherent cells were suspended by trypsin-EDTA treatment, the cell aggregates were removed by filtration, and a uniform cell suspension was obtained. Cells were collected by centrifugation and resuspended in DMEM-F12 (2: 1) -0.1% BSA to 8 × 10 ⁴ / ml. This cell suspension was added to 0.5 ml of 0.5 ml of the same medium containing a double concentration of the drug. The culture was performed at 37 ° C. for 24 hours using a 24-well plate. At the end of the culture, the culture supernatant was transferred to an Eppendorf tube, centrifuged at 10,000 rpm for 5 minutes, the supernatant transferred to a new tube, and stored at −20 ° C. until the day of laminin 5 measurement. In addition, in order to solubilize laminin 5 bound to the inside of the cell and the culture plastic, Tris-HCl buffer (pH 7.4) containing various surfactants was added to each well and stored at −20 ° C. overnight. The next day, it was sonicated and frozen again. The next day, after dissolving again, the mixture was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was transferred to a tube and stored at −20 ° C. until the day of measurement of laminin 5.

(2) Measurement of laminin 5 by sandwich ELISA method Laminin 5 present in the culture supernatant and cell layer was measured by sandwich ELISA method. A BM165 monoclonal antibody against laminin α3 chain of laminin 5 was bound to a solid layer of a 96-well ELISA plate. In order to sandwich and measure laminin 5, 6F12, which is a monoclonal antibody against laminin β3 chain, was biotinylated (b-6F12) in advance as another type of antibody. In this method, only the heterotrimeric body (α3β3γ2) capable of exhibiting the function is measured, and the heterodimer (β3γ2) is not detected. Samples are added to each well previously filled with 3% gelatin / phosphate buffer solution containing b-6F12. The final dilution ratio in the sample hole was 1/4 for the culture solution and 1/10 for the cell layer. The antigen-antibody reaction was carried out at 37 ° C. for 2 hours. After washing the plate, an avidin HRP (horseradish peroxidase) solution was added, and the reaction was further continued for 30 minutes to 1 hour. After washing, an ABTS solution which is a substrate for HRP was added, and the absorbance at 405 nm was measured with an ELISA plate reader. A calibration curve was prepared in the range of 0 to 40 ng / ml.

  The amount of laminin 5 produced was calculated as the sum of the amount released in the medium and the amount remaining in the cell layer, and indicated as a relative value to the sample (control) to which no biological extract or the like was added. Table 1 below summarizes the rate of increase in laminin 5 production for each extract and the like.

(Experiment Method-2)
Test Method for Promoting Production Effect of Type IV Collagen and Type VII Collagen (1) Culture of human fibroblasts Human fibroblasts cultured in DMEM medium containing 10% FBS were seeded in a 24-well plate, and the cells were adhered. The medium was replaced with DMEM medium containing 25% FBS and 250 μM ascorbic acid glucoside, and the drug was added. One day later, the supernatant of the medium was collected and centrifuged, and the type IV and type VII collagens in the obtained supernatant were measured, and the amount of DNA was measured for the cells, which was used as an indicator of the number of cells.

(2) DNA quantification The amount of DNA was measured by a fluorescence measurement method using Hoechst H33342.

(3) Measurement of type IV and type VII collagen by sandwich ELISA
Type IV and type VII collagen were measured by sandwich ELISA. The antibodies used in this example are as follows.
Type IV collagen specific antibody; monoclonal antibody JK-199 and polyclonal antibody MO-S-CLIV
Type VII collagen specific antibody; monoclonal antibody NP-185 and monoclonal antibody NP-32

  When the amount of type IV and type VII collagen per DNA of the sample to which the drug is not added (control) is 100, the amount of type IV and type VII collagen per DNA of the drug added sample is expressed as type IV and type VII collagen. Production promotion rate. Table 2 below summarizes the collagen production promotion rates.

(Experiment Method-3)
Artificial skin production method Collagen gel is a gel solution of human dermis-derived fibroblasts (1 × 10 ⁵ cells / ml) suspended on ice and then gelled at 37 ° C. in a 60 mm Petri dish. did. Thereafter, the gel was peeled off from the petri dish wall surface, the collagen gel was placed on the metal, and a glass ring (inner diameter 12 mm) was placed on the gel. A KGM-DMEM (1: 1) mixed medium containing a human foreskin-derived epidermal keratinocyte suspension was added so as not to leak into the glass ring. Incubate overnight to allow epidermal cells to adhere and remove the ring the next day. A skin model having a multi-layered epidermis showing formation of the stratum corneum was prepared while filling the medium up to the boundary of the epidermal layer and exposing the epidermal layer to air.

  From the 4th day after seeding of epidermal cells, (1) No drug added, (2) Tokasenka extract 0.3%, (3) Tokasenka extract 0.3% + Karin extract 0.5%, (4) Hazelnut Oil 0.3%, (5) Hazelnut oil 0.3% + zelkova extract 0.5%, (6) Cornflower extract 0.3%, (7) Cornflower extract 0.3% + Novara extract 0.5%, 8) Barley extract, (9) Odorikosou extract, (10) Kyonin extract, (11) Gentian pepper extract, (12) Soybean extract, (13) Perilla extract, (14) Shobu extract, (15) Honeysuckle extract, (16) Achillea millefolium extract, (17) Tonine extract, (18) Tomato extract, (19) Garlic extract, (20) Mukuroji extract, (21) Lettuce , (22) yeast extract, (23) hydrolyzed conchiolin (0.3% each for (8)-(23)), and then contain the same type and concentration of drugs every 2-3 days The culture medium was replaced with a medium to be cultured for another 2 weeks.

  The formed artificial skin was stained with hematoxylin-eosin staining and immunostaining (anti-IV collagen antibody and anti-VII collagen antibody). The degree of staining of type IV and type VII collagen was scored from 5 to 1-5 in order from the lowest to the highest.

  As described above, in the control (1), type IV collagen was weakly stained, but almost no type VII collagen was observed. In contrast, laminin-5, type IV collagen, type VII collagen production promoting action (2)-(23) all have been confirmed in artificial skin that both enhance the staining of type IV and type VII collagen It was done. Furthermore, it has been clarified that the serine protease inhibitors Karin, Juyaku, and Novara extract further enhance the dyeability.

(Experiment Method-4)
Anti-inflammation, wound healing promotion test Inflammation or wounds were artificially formed in 8-week-old HR-1 mice. (1) No drug added, (2) Tokinsenka extract, (3 ) Hazelnut oil, (4) Cornflower extract, (5) Barley extract, (6) Odori extract, (7) Kyonin extract, (8) Ginseng extract, (9) Salmon extract, (10) Perilla extract, (11) Shobu Extract, (12) Honeysuckle extract, (13) Achillea millefolium extract, (14) Tonine extract, (15) Tomato extract, (16) Garlic extract, (17) Mukuroji extract, (18) Lettuce extract, (19) Yeast extract, (20) 0.5 g of 1,3-butylene glycol containing hydrolyzed conchiolin (5% each) was applied twice a day for 5 days It was observed the state of the site of inflammation or wound site on day 5. The anti-inflammatory action and the wound healing promoting action were scored in 5 stages of 1-5 in order from the lowest to the highest.

  As described above, in control (1), the degree of improvement of inflammation and wound was low. On the other hand, it was confirmed in vivo that each of the drugs (2) to (20) has an anti-inflammatory action and a wound healing promoting action.

(Example 1-Cream)
(1) Prescription (Phase A)
Stearic acid 10.0% by weight
Stearyl alcohol 4.0
Butyl stearate 8.0
Stearic acid monoglycerin ester 2.0
Vitamin E acetate 0.5
Vitamin A palmitate 0.1
Macadamia nut oil 1.0
Preservative Appropriate amount Perfume Appropriate amount (Phase B)
Glycerin 4.0
1,2-pentanediol 3.0
Potassium hydroxide 0.4
Magnesium ascorbate phosphate 0.1
L-arginine hydrochloride 0.01
Edetate trisodium 0.05
Toukensen 1,3-butylene glycol extract 0.5
Ion exchange water

(2) Manufacturing method The oil phase part of A and the aqueous phase part of B are each heated to 70 ° C. and completely dissolved. Add Phase A to Phase B and emulsify with an emulsifier. The emulsion is cooled using a heat exchanger.

(Example 2-Cream)
(1) Prescription stearic acid 5.0% by weight
Stearyl alcohol 4.0
Isopropyl myristate 18.0
Glycerol monostearate 3.0
Propylene glycol 10.0
Hazelnut methanol extract 0.01
Caustic potash 0.2
Sodium bisulfite 0.01
Preservative Appropriate amount of perfume Appropriate amount of ion-exchanged water

(2) Manufacturing method Propylene glycol, hazelnut methanol extract and caustic potash are added to ion-exchanged water, dissolved, heated and maintained at 70 ° C. (aqueous phase). The other ingredients are mixed, dissolved by heating and kept at 70 ° C. (oil phase). The oil phase is gradually added to the aqueous phase, and after the addition is complete, the temperature is maintained for a while to cause the reaction. Thereafter, the mixture is uniformly emulsified with a homomixer and cooled to 30 ° C. while stirring well.

(Example 3-Wrinkle prevention cream)
(1) Prescription stearic acid 2.0% by weight
Stearyl alcohol 7.0
Hydrogenated lanolin 2.0
Squalane 5.0
2-Octyldodecyl alcohol 6.0
Polyoxyethylene (25 mol)
Cetyl alcohol ether 3.0
Glycerin monostearate ester 2.0
Propylene glycol 5.0
Cornflower ethanol extract 0.05
Turmeric ethanol extract 0.05
Sodium bisulfite 0.03
Ethylparaben 0.3
Perfume Appropriate amount of ion-exchanged water

(2) Manufacturing method Propylene glycol is added to ion-exchanged water and heated to maintain at 70 ° C. (aqueous phase). The other ingredients are mixed, dissolved by heating and kept at 70 ° C. (oil phase). Preliminarily emulsify by adding an oil phase to the aqueous phase, uniformly emulsify with a homomixer, and then cool to 30 ° C. while stirring well.

Example 4 Anti-Aging Cream
(1) Prescription solid paraffin 5.0 wt%
Beeswax 10.0
Vaseline 15.0
Liquid paraffin 41.0
Glycerin monostearate ester 2.0
Polyoxyethylene (20 mol)
Sorbitan monolaurate 2.0
Soap powder 0.1
Borax 0.2
Barley acetone extract 0.05
Psychoethanol extract 0.05
Sodium bisulfite 0.03
Ethylparaben 0.3
Perfume Appropriate amount of ion-exchanged water Residue (2) Production method Add soap powder and borax to ion-exchanged water, dissolve by heating and maintain at 70 ° C (aqueous phase). The other ingredients are mixed, heated and melted, and kept at 70 ° C. (oil phase). The reaction is gradually added while stirring the oil phase in the aqueous phase. After completion of the reaction, the mixture is uniformly emulsified with a homomixer and cooled to 30 ° C. while stirring well after emulsification.

Example 5-Emulsion
(1) Prescription stearic acid 1.0 wt%
Vaseline 5.0
Liquid paraffin 10.0
Polyoxyethylene (10 mol)
Monooleate 2.0
Polyethylene glycol 1500 3.0
Triethanolamine 1.0
Carboxyvinyl polymer 0.2
(Product name: Carbopol 941, BFGoodrich Chemical company)
Adriatic ethyl acetate extract 0.01
Sodium bisulfite 0.01
Ethylparaben 0.3
Perfume Appropriate amount of ion-exchanged water

(2) Production method A carboxyvinyl polymer is dissolved in a small amount of ion-exchanged water (phase A). Polyethylene glycol 1500 and triethanolamine are added to the remaining ion-exchanged water, dissolved by heating and maintained at 70 ° C. (aqueous phase). The other ingredients are mixed, heated and melted, and kept at 70 ° C. (oil phase). Add the oil phase to the water phase, preliminarily emulsify, add the A phase, uniformly emulsify with a homomixer, and cool to 30 ° C. while stirring well after emulsification.

(Example 6-Emulsion)
(1) Prescription microcrystalline wax 1.0 wt%
Beeswax 2.0
Lanolin 2.0
Liquid paraffin 10.0
Squalane 5.0
Sorbitan sesquioleate 4.0
Polyoxyethylene (20 mol)
Sorbitan monooleate 1.0
Propylene glycol 7.0
Kyonin 1,3-butylene glycol extract 10.0
Sodium bisulfite 0.01
Ethylparaben 0.3
Perfume Appropriate amount of ion-exchanged water

(2) Manufacturing method Propylene glycol is added to ion-exchanged water and heated to maintain at 70 ° C. (aqueous phase). The other ingredients are mixed, heated and melted and kept at 70 ° C. (oil phase). While stirring the oil phase, the aqueous phase is gradually added thereto and uniformly emulsified with a homomixer. Cool to 30 ° C. while stirring well after emulsification.

Example 7-Emulsion
(1) Prescription (Phase A)
Squalane 5.0 wt%
Oleolate 3.0
Vaseline 2.0
Sorbitan sesquioleate ester 0.8
Polyoxyethylene oleyl ether
(2OEO) 1.2
Evening primrose oil 0.5
Preservative Appropriate amount Perfume Appropriate amount (Phase B)
1,3-butylene glycol 4.5
Gennosho ethanol extract 1.5
Ethanol 3.0
Carboxyvinyl polymer 0.2
Potassium hydroxide 0.1
L-Arginine L-Aspartate 0.01
Cascon ethanol extract 1.5
Erythritol 0.5
Sodium hexametaphosphate 0.05
Ion exchange water

(2) Manufacturing method The oil phase part of A and the aqueous phase part of B are each heated to 70 ° C. and completely dissolved. Add Phase A to Phase B and emulsify with an emulsifier. The emulsion is cooled using a heat exchanger.

Example 8-Jelly
(1) Formulation 95% ethyl alcohol 10.0% by weight
Dipropylene glycol 15.0
Polyoxyethylene (50 mol)
Oleyl alcohol ether 2.0
Carboxyvinyl polymer 1.0
(Product name: Carbopol 940, BFGoodrich Chemical company)
Caustic soda 0.15
L-Arginine 0.1
Soybean ethanol extract 7.0
Sodium 2-hydroxy-4-methoxybenzophenone sulfonate 0.05
Ethylenediamine tetraacetate
Trisodium 2 water 0.05
Methylparaben 0.2
Perfume Appropriate amount of ion-exchanged water

(2) Manufacturing method Carbopol 940 is uniformly dissolved in ion-exchanged water, while sorghum ethanol extract and polyoxyethylene (50 mol) oleyl alcohol ether are dissolved in 95% ethanol and added to the aqueous phase. Next, after adding other components, the mixture is neutralized and thickened with caustic soda and L-arginine.

(Example 9-Cosmetic liquid)
(1) Prescription (Phase A)
Ethyl alcohol (95%) 10.0% by weight
Polyoxyethylene (20 mol)
Octyldodecanol 1.0
Pantotenyl ethyl ether 0.1
Shobu methanol extract 1.5
Methylparaben 0.15
(Phase B)
Potassium hydroxide 0.1
(Phase C)
Glycerin 5.0
Dipropylene glycol 10.0
Sodium bisulfite 0.03
Carboxyvinyl polymer 0.2
(Product name: Carbopol 940, BFGoodrich Chemical company)
Ion exchange water

(2) Manufacturing method A phase and C phase are each melt | dissolved uniformly, A phase is added to C phase, and it solubilizes. Next, filling is performed after adding phase B.

Example 10-Pack
(1) Prescription (Phase A)
Dipropylene glycol 5.0% by weight
Polyoxyethylene (60 mol)
Hardened castor oil 5.0
(Phase B)
Honeysuckle extract 0.01
Olive oil 5.0
Tocopherol acetate 0.2
Ethylparaben 0.2
Fragrance 0.2
(Phase C)
Sodium bisulfite 0.03
Polyvinyl alcohol (degree of saponification 90, degree of polymerization 2,000) 13.0
Ethanol 7.0
Ion exchange water

(2) Manufacturing method A phase, B phase, and C phase are each uniformly dissolved, and B phase is added to A phase to solubilize. Next, this is added to phase C and then filled.

(Example 11-lotion)
(1) Prescription (Phase A)
Ethanol 5.0 wt%
POE oleyl alcohol ether 2.0
Oleyl alcohol 0.1
2-Ethylhexyl P-dimethylaminobenzoate 0.18
Perfume appropriate amount (phase B)
1,3-butylene glycol 9.5
Glycerin 2.0
Sodium pyrrolidonecarboxylate 0.5
Nicotinamide 0.3
Achillea millefolium 1,3-butylene glycol extract
0.1
β-cyclodextrin 1.0
Erythritol 0.05
Ion exchange water

(2) Production method The alcohol phase of A is added to the aqueous phase of B and solubilized to obtain a lotion.

Example 12-Solid Foundation
(1) Prescription talc 43.1% by weight
Kaolin 15.0
Sericite 10.0
Zinc flower 7.0
Titanium dioxide 3.8
Yellow iron oxide 2.9
Black iron oxide 0.2
Squalane 8.0
Isostearic acid 4.0
Monooleic acid POE sorbitan 3.0
Isocetyl octoate 2.0
Tonin ethanol extract 0.5
Preservative Appropriate amount Fragrance Appropriate amount

(2) Manufacturing method Thoracic to black iron oxide powder components are thoroughly mixed in a blender, and oily components of squalane to isocetyl octoate, tonin ethanol extract, preservatives and fragrances are added and kneaded well, then filled into a container. , Molding.

(Example 13-emulsified foundation (cream type))
(1) Formula (powder part)
Titanium dioxide 10.3% by weight
Sericite 5.4
Kaolin 3.0
Yellow iron oxide 0.8
Bengala 0.3
Black iron oxide 0.2
(Oil phase)
Decamethylcyclopentasiloxane 11.5
Liquid paraffin 4.5
Polyoxyethylene-modified dimethylpolysiloxane 4.0
(Water phase)
Ion exchange water 50.0
1,3-butylene glycol 4.5
Tomato ethanol extract 1.5
Sorbitan sesquioleate 3.0
Preservative Appropriate amount Fragrance Appropriate amount

(2) Manufacturing method After heating and stirring the aqueous phase, a sufficiently mixed and pulverized powder part is added and homomixed. Furthermore, after adding the heat-mixed oil phase and carrying out a homomixer process, a fragrance | flavor is added with stirring and it cools to room temperature.

Claims (9)

  Extracts of pearl millet, hazelnuts, cornflowers, barley, dolphins, ginger, ginseng, sorghum, ginger, honeysuckle, yarrow, corn, tomato, garlic, mungoji, lettuce, yeast, and / or hydrolyzed conchiolin A basement membrane stabilizer comprising one or more active ingredients.   The basement membrane stabilizer of claim 1, further comprising one or more serine protease inhibitors. Extracellular matrix production promoter , extract of pearl millet, hazelnuts, cornflower, barley, edible perilla, leopard, ginseng, bonito, ginger, honeysuckle, yarrow, yarrow, tomato, garlic, mugwort, lettuce, yeast, and / or hydrolysis Ri comprising any one or more as an active ingredient of the conchiolin, components of the extracellular matrix is facilitated production is selected from the group consisting of laminin 5, IV collagen and type VII collagen An extracellular matrix production promoter , which is one kind or two or more kinds . The extracellular matrix production promoter according to claim 3 , which promotes production of all of laminin 5, type IV collagen and type VII collagen. The extracellular matrix production promoter according to claim 3 or 4 , further comprising one or more serine protease inhibitors. The extracellular matrix production promoter according to any one of claims 3 to 5 , wherein the serine protease inhibitor is an extract of karin, jujube and / or Novara. A method for producing an extracellular matrix production promoter, comprising pearl millet, hazelnut, cornflower, barley, edible perilla, leopard, gentian, salamander, ginger, honeysuckle, yarrow, corn, tomato, tomato, garlic, lettuce, yeast extract, and / or any one or more of the hydrolysis conchiolin Ri comprising the step of adding to the aqueous phase or oil phase, construction of extracellular matrix promoted produced by the extracellular matrix production enhancer A method for producing an extracellular matrix production promoter, wherein the component is one or more selected from the group consisting of laminin 5, type IV collagen and type VII collagen .   Extracts of pearl millet, hazelnuts, cornflowers, barley, dolphins, ginger, ginseng, sorghum, ginger, honeysuckle, yarrow, corn, tomato, garlic, mungoji, lettuce, yeast, and / or hydrolyzed conchiolin A method for producing artificial skin, comprising the step of adding one or more to an artificial skin formation medium. The method for producing artificial skin according to claim 8 , further comprising a step of adding one or more serine protease inhibitors.