TW200927157A - Grifola frondosa extract and composition containing thereof for promoting hyaluronic acid production - Google Patents

Abstract

A Grifola frondosa extract and a composition containing thereof are provided for more efficiently promoting hyaluronic acid production. In detail, the Grifola frondosa extract which is obtained by extracting dry Grifola frondosa with pure ethanol and the composition containing thereof are provided.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an extract of Griffola frondosa and a composition containing the same for promoting the production of hyaluronan. [Prior Art] As an ethanol extract of Maitake, it has been known so far: tyrosinase inhibitory effect, α-amylase inhibitory effect, skin moisturizing effect, and immunosuppression inhibitory effect (refer to Japanese Patent Laid-Open No. 1219〇5) Japanese Laid-Open Patent Publication No. 2000-319192, and Japanese Patent Laid-Open No. Hei No. 263732. In addition, the present inventors have filed the following patent application: The pure ethanol extract of the dried velvet fruit body body has been shown to promote sebum production and secretion in sebaceous glands in humans and hamsters; in addition, for patients with dry skin disease 'The pure ethanol extract of the dry velvet fruit body can bring about a clinically improved skin condition. Namely, the following patent application is filed: The pure ethanol extract of the dry velvet fruit body improves the skin barrier function by promoting sebum production, and is not necessarily effective for daily skin care, even for disease prevention and treatment. On the other hand, in the anti-aging of the skin, an extracellular matrix (ECM) such as collagen which is a protein for skin structure, elastin which imparts skin softness and elasticity, and hyaluronic acid which controls skin moisturizing function. The biosynthesis vocabulary of related molecules is an important factor. (See Baumann L. Skin ageing and its treatment J. Pathol. 211: 241-251, 2007). Moreover, the known cell matrices 200927157 (ECM)-related molecules are fibroblasts present in the dermis of the skin as their main producing cells. However, the pure ethanol extract of the dry velvet fruiting body is completely unaware of the regulation of the expression of extracellular matrix (ECM)-related molecules. Therefore, in normal human skin fibroblasts 47, the genetic level of the following extracellular matrix (ECM)-related molecules is used to analyze the regulation of collagen, elastin and hyaluronic acid production by the pure ethanol extract of dried velvet fruiting bodies. Surprisingly, it was found that the pure ethanol extract of the dry velvet fruit body promotes the performance of the uric acid synthase, which promotes the production of hyaluronan. SUMMARY OF THE INVENTION The present invention provides a Maitake extract which is more effective in promoting hyaluronic acid production and a composition containing the same. The present inventors have conducted studies to solve the above problems, and as a result, have found that a pure ethanol extract of dried Maitake mushroom (an ethanol extract derived from Maitake mushroom) has an excellent effect of promoting hyaluronic acid production, and has completed the present invention. ❹ That is, the present invention is as follows. J1) A Maitake mushroom extract obtained by extracting dry Maitake with pure ethyl yeast; * (2) The maitake extract 'the water of the dry maitake' as described in (1) is 8 wt. (3) The Maitake extract as described in (1) or (2), wherein the pure ethanol contains 99.0 v〇l% or more of ethanol; (4) a plant composition 'which contains as (i) a maitake extract according to any one of 200927157, and used to promote hyaluronic acid production; (5) a composition as described in (4), which is in the form of a cosmetic; (6) as ( 4) The composition described above is in the form of a pharmaceutical product; (7) a kind of lO^Rf value > 〇45 and/or 〇〇7> Rf value of hyaluronic acid production promotes the production of the eluted material The method comprises the following steps: (a) dissolving the Maitake extract as described in (1) above in chloroform _ 甲醇 methanol (19: 1); (b) using the gas-methanol (19: 1) The solution of the extract is added to the equilibrated normal phase gel column; (c) with gas-methanol (19:1), gas-methanol (9:1), gas-methanol (4:1) ), the order of methanol is dissolved; (d) developing a portion of the eluted material by gas-methanol (9:1) in a thin layer of lycopene; and (e) obtaining a value of lO^Rf > 0.45 and/or 〇.〇7>Rf Value Q dissolution; (8) - a value of lO^Rf value > 0.45 and / or 〇. 〇 7 > Rf value of hyaluronic acid production promoting dissolution 'which is manufactured by the method as described in (7) above And get. „ . [Effects of the Invention] According to the present invention, the production of hyaluronic acid can be effectively promoted, thereby effectively improving the damage, abnormality or disease of the skin caused by the decrease in hyaluronic acid, and the damage and abnormality of the joint caused by the decrease in hyaluronic acid. Or disease; 200927157 Eye damage, abnormality, and eye disease caused by a decrease in hyaluronic acid. [Embodiment] The present invention relates to a Maitake mushroom extract obtained by extracting dried Maitake mushroom with pure ethanol.

The maitake mushroom (7<Xi^) of the present invention refers to a mushroom of the genus Polyporaceae Grifola, which can be exemplified by: Grif〇la frondosa, Grifola albicans, and pork bristles. (Grif〇la umbellatus), Grifola gigantea, etc. The maitake of the present invention is preferably Grifolafrondosa. Maitake can be any of the fruiting bodies and myceliums 'but preferably the fruiting bodies. The dry maitake of the extracted raw material is a maitake having a moisture content of less than 1% by weight. The dry maitake preferably has a moisture content of 8 wt% or less, more preferably 7 wt% or less. The dried maitake can be obtained by dehydrating and drying the raw velvet by any method (for example, drying by drying, heating and drying, vacuum drying, freeze drying, etc.). The velvet is preferably in a powder form. ', ▲ Pure ethanol of the present invention means ethanol containing 98.0 v〇1% or more of ethanol at 15 °C. The pure ethanol content is preferably 99.0 vol% or more 'into 99.5 vol% or more of ethanol. The extraction of dried Maitake velvet with pure ethanol is carried out by adding pure ethanol to the dry maitake of the insects, and then disturbing at room temperature or under heating. The heating temperature is usually a temperature lower than the boiling point of ethanol, but ^: the temperature of the closed cell can be 5 〇 ° C or less. Better extraction temperature 疋 Normal temperature (room temperature). The extraction time is not particularly limited, for example, 5 200927157 to 10 hours or so. It can be extracted once or twice. The amount of pure ethanol relative to the dry maitake in the extraction, relative to 100 parts by weight of dry maitake, pure (10) is 丨 (9) ~ surface weight. Preferably 200 to 600 parts by weight, and more preferably 300~ 500 parts by weight. After the treatment, the pure ethanol extract can be obtained by separating the (four) paper money cloth by centrifugation or centrifugation. The pure ethanol extract of Maitake of the present invention may be in the state of the above ethanol extract. Alternatively, the pure ethanol extract may also be in the form of an extract which removes all or part of the alcohol from the extract by a usual method. That is, it may be a state of a draw powder substantially free of ethanol or an extract state containing 1 to 5 % by weight of ethanol. The extract containing 10 to 15% of Wt% ethanol is in a good form because of its good preservability. A pure ethanol extract was obtained in a yield of 2 to 10 wt%, more specifically 3 to 5 wt/i (as a solid component) with respect to dry maitake. The active ingredient in the pure ethanol extract is unknown, but the pure ethanol extract contains phospholipids and plant steroids. The present invention comprises a hydrazine composition for promoting hyaluronic acid production comprising the above extract. Hyaluronic acid is a kind of glycosaminoglycan (glycosaminoglycan), which has a disaccharide repeating structure formed by N-acetyl-D-glucosamine and D-glucuronic acid bond. _ can be expressed by the following formula : • [Chemical 1] 200927157

(wherein η is an integer of 1 or more) Further, hyaluronic acid is also called hyaluronan. ❹ 组成 The composition of the present invention comprises a form of a food, in particular, a form comprising a food for promoting hyaluronic acid production. The composition of the present invention can maintain the moisturizing property of the skin by promoting the production of hyaluronic acid, and can impart moistness, softness and elasticity to the skin, and can also prevent purple or repair due to the material, which can cause silk damage, and can also cause skin regeneration. . Accordingly, the present invention encompasses a composition for promoting the production of hyaluronic acid in the form of a cosmetic and the above extract. The composition of the present invention in the form of a cosmetic material is a conventional carrier or excipient for use in cosmetics, and is used for blending against skin roughness, improving skin texture, and reshaping. , can be used as a good skin bomb (four) 'improve the skin green or loose", change, change the cosmetic material. As a cosmetic dosage form, can be used for 2, use (washing cream, foam, gel cosmetics, etc.), J冼 冼 美容 美容 眼 眼 , 乳 乳 π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π π , eyeliner ^ sun, sun-drying cosmetics, shower gel, bathing cosmetics, etc.) 'warm protection · (anti-media cream == material 200927157, etc.) 'hair growth, hair care (hair growth agent, hair growth lotion, etc.) In these cosmetics, an effective amount of Maitake extract is blended, for example, in an extract (a maitake extract in a state substantially free of ethanol), and blended into α1 to 999. Preferably, it is 1 to 99 wt. The remainder is formulated into a conventional carrier, excipient, or an additive, etc. Further, the extract of the present invention has an effect of improving skin damage or skin diseases caused by a decrease in hyaluronic acid production. The extract is caused by a decrease in the production of hyaluronic acid. In addition, the extract of the present invention has an effect of improving eye damage, abnormality or eye disease caused by a decrease in hyaluronic acid. Therefore, the present invention encompasses the treatment or prevention of the above-mentioned hyaluronic acid production. A composition which is in the form of a pharmaceutical product to reduce the damage or disease caused by the damage. Among the skin damage or skin diseases caused by the decrease in hyaluronic acid production, dry skin disease (dry skin), ichthyosis, photoaging , dryness, atopic dermatitis, hemorrhoids (pressure sores) and skin dysfunction caused by burns, etc. It is better to photoage, dryness, atopic dermatitis, hemorrhoids (pressure Q treatment) and fire injury Skin dysfunction caused by the disease. In the joint damage or joint disease caused by the decrease in hyaluronic acid production, deformed joint disease and rheumatoid arthritis (rhematoid arthritis) may be mentioned. Sexual joint disease and rheumatoid arthritis. Also 'eye damage or eye disease caused by a decrease in hyaluronic acid production For example, corneal epithelial damage, eye fatigue, dry eye syndrome, etc. Good dry eye syndrome. The so-called dry eye syndrome refers to a state of insufficient tears or damage to the surface of the eye due to the surface of the eye 200927157. The tears of the package are exocytosis and dry keratoconjunctivitis. The treatment and prevention used in the present invention are mammals, such as humans, dogs, juvenile pets, cattle, pigs, chickens and the like, but especially The composition of the present invention in the form of a drug or a drug may contain a conventional carrier, an excipient, an additive, etc., which are formulated in a medical πσ towel. Examples of the pharmaceutical product include a cream. , sublingual agents, massage oils, solutions, suspensions, lotions, ointments, gels, lozenges, capsules, granules, powders, syrups, elixirs, and the like. To these pharmaceuticals, an effective amount of Maitake extract (in a state substantially free of ethanol) is blended, for example, 0.1 to 99.9 wt%, preferably 1 to 99%, of Maitake extract. The remaining portion is formulated into a conventional carrier, excipient, or additive. Among the conventional carriers, excipients, or additives to be formulated in cosmetics or pharmaceuticals, there may be mentioned solvents and vegetable oils (for example, almond oil, castor oil, cocoa butter, coconut oil, corn oil, cottonseed oil). , linseed oil, eucalyptus oil, palm oil, peanut oil, corn seed oil, rapeseed oil, sesame oil, soybean oil, sunflower oil, and tea seed oil, etc.), mineral oil, fatty oil, liquid Paraffin, buffer, preservative, wetting agent, chelating agent, antioxidant, stabilizer, emulsifier, suspending agent, gel forming agent, ointment base, suppository base, penetration enhancer, fragrance, and skin protection Agents, etc. The solvent may, for example, be water, ethanol, polyethylene glycol, propylene glycol, glycerin, liquid polyalkyl siloxane or a mixture thereof, but is not limited to the above solvent. 200927157 In the buffering agent, citric acid, acetic acid, tartaric acid, lactic acid 'hydrogen sulfate, monoethylamine, a mixture of these, and the like can be mentioned, but it is not limited to the above buffer. In the wetting agent, glycerin, propylene glycol, pentylene-glycol, sorbitol, lactic acid, urea, BG (1,3-butanediol), soybean sterol and the like Some mixtures, etc., are not limited to the above wetting agents. The chelating agent may, for example, be sodium EDTA, citric acid or a mixture thereof, but is not limited to the above chelating agent. ❻ Among the antioxidants, butyl hydroxyanisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, cysteine, and mixtures thereof may be mentioned, but are not limited thereto. The above antioxidants. In the emulsifier, 'natural rubber (for example, Acacia rubber), tragacanth, tri-sand gum; natural phospholipids (such as soybean egg fat); sorbitol monooleate derivatives; wool Fat; lanolin; sorbitanester; monoglyceride; fatty yeast (eg twenty-two alcohol); fatty acid esters (eg tris(caprylic/capric) glyceride, glyceryl stearate) A triglyceride of a fatty acid such as s^); and a mixture of these is exclusively, but not limited to, the above emulsifier. Examples of the suspending agent include cellulose and derivatives thereof (for example, carboxymethyl cellulose, ethyl cellulose, propyl cellulose, propyl methyl cellulose, etc.), and angular forks. Vegetable gum (earrageenin), gum arabic, gumabic, tragacanth, and mixtures thereof, but are not limited to the above suspending agents. In the gel base and the viscosity-increasing component, it can be mentioned as: liquid paraffin, polyacetonitrile 0 200927157 olefin: fatty oil, strontium oxide or sputum, rhodium = ^ base polymer, two polymerization one or two; = vitamin derivative), water-swellable hydrophilicity: (Example 2: column = selective chloric acid gel containing sodium chloride), alginate-acid vinegar, and mixtures thereof, etc., but not limited to On the top of the gelatin base and viscosity-enhancing ingredients. Among the ointment bases, there may be mentioned: beeswax, stone, and ste, stearyl acid, PEG_150 stearic acid vinegar, vegetable oil, fatty acid, sorbitol, polyethylene glycol, fatty acid, yam The condensation product of the sugar alcohol from the oxidized woman (for example, polyoxyethylene sorbitan monooleate) and the above-mentioned financial base are mixed, but are not limited to the above-mentioned ointment base. The soil in the hydrophobic ointment base may, for example, be paraffin, vegetable oil, tallow, synthetic glyceride, wax, lanolin, liquid polyalkyl siloxane, and the like, but is not limited to the above hydrophobic Oral ointment base. Λ The hydrophilic ointment base may, for example, be a solid polyethylene glycol or the like, but is not limited to the polyethylene glycol. Further, 'in the conventional carrier, excipient, or additive, etc., there are, for example, angle-burning, printing carbon, hydrogenation, fat, ascorbic acid tetrahexyl citrate, allantoin, glycyrrhizic acid 2K, glycosyl Trehalose (glyC0Syl trehal〇se), hydrolyzed weathered palace powder, hydrolyzed collagen, prepared. Wei extract, dimethicone, caprylyl glycol, beet test, hard 12 200927157 Lipidenylamine, wild oil, batyl alc〇hol, hydroproxyproline, and the like. The pharmaceutical composition of the invention may further comprise other agents for treating or preventing the damage caused by the reduction in the generation of the glass. The effective dose for the treatment or prevention of hyaluronic acid production in the current month is due to the degree of disease or damage and the method of administration. If it is effective in promoting the production of glass grades, , f 'but Maitake extract (substantially does not contain ethanol extract Γ: 〇 two amount is ο.1~1000 mg / body weight kg. 曰, preferably 1~500mg / body weight kg. 曰 ' and thus better It is 1〇~1〇〇咖重量# 曰0 The administration used in the present invention can be systemic administration or topical administration; systemic administration, local good can be: percutaneous injection, good oral administration Any of the forms of administration, enteral administration, intramuscular administration, subcutaneous administration, intravenous administration, eye-dropping, etc., preferably transdermal administration, and eye-dropping. MX - therefore, this The invention is also used for the manufacture of the drug composition 2. In addition, the present invention is also a method for (4) administering a pharmaceutical composition to the above-mentioned pharmaceutical composition by promoting the reduction of hyaluronic acid or acid production. The cause of the disease or injury is scheduled to be =2 over the wealth of money, but it is not limited to 1. Manufacturing of Maitake Extract 13 200927 157 Add 4 g of pure ethanol (moisture content: 99.5 vol% or more) to a sterilized dry powder of 1000 g of gazebo (moisture content: 6 wt%, Maitakes made by Hokuto Co., Ltd.) at 2 〇ΐ The mixture was extracted at a temperature for 18 hours. While stirring, the supernatant was removed by centrifugation, and the resulting supernatant was filtered using a filter paper. The ethanol was removed from the obtained liquid to be removed, and pure ethanol of Maitake was obtained. Extract (37.2 g of yield component and 6.0 g of ethanol) 2. Culture and treatment of human fibroblasts 正常 Normal human fibroblast NB1RGB (RCB0222) was purchased from the BioResources Center of the Institute of Physical Chemistry (Germany). The culture was carried out using Dulbecco's modified Eagles medium (DMEM 'purchased from Invitrogen) containing 1% (v/v) fetal bovine serum (FBS, Fetal Bovine Serum). In addition, the 'Yingsong extract was treated in the absence of Qiqing. Under the conditions of β, ie, transforming growth factor β (TGF-β) containing 20% ng/ml of maitake extract or as an extracellular matrix synthesis promoting factor (from R&a Mp;D Systems, Inc.) 〇.20/〇 (v/v) lactobumin hydrolysate (LAH, purchased from Sigma Aldrich) / DMEM, will be in 60 mm dish (dish) Cultured confluent human fibroblasts were cultured for 24 hours. • Real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) method (1) Extraction of total RNA Extracting total RNA from cells 'is using Isogen (from Nippongene 200927157 company purchased) and in accordance with the attached operating methods. Specifically, cerium (tetra) (〇 8 ml) was added to cells cultured with Maitake extract or TGF-ρ to dissolve the cells, and chloroform (〇16 ml) was added to the solution and mixed. After centrifugation (10,000 rpm, 1 Torr), the aqueous layer portion of the upper layer was recovered, and isopropanol having the same capacity as the recovered aqueous layer was added and mixed. After centrifugation (10,000 rpm, 1 Torr), the supernatant was removed and the precipitated RNA was recovered. Further, the recovered was added to 75 vol% of ethanol (〇.8 ml) and mixed. After centrifugation (1 〇〇〇〇 rpm/min, 10 minutes), the supernatant was completely removed, and the RNA was air-dried. The extracted total RNA was dissolved in sterilized distilled water containing no nuclease, and the absorbance of total RNA at 260 nm was measured using a spectrophotometer (UV-1600 ‘Shimadzu Corporation) to thereby quantify. In addition, the extraction of total RNA can be done without Isngen, but by Chomczynski P and Sacchi N (ChomcZynski, p. an(i

Sacchi, N. Single-step method of RNA isolation by acid guanidiniuni thiocyanate-phenol-chloroform extraction. Anal. ❹ Biochem. 162: 156-159 (1987)). (2) Reverse transcriptase reaction The reverse transcriptase reaction was carried out using the QuantiTectR Reverse Transcription Kit (purchased from QIAGEN) in accordance with the attached operation. Namely, 7 x g of DNA Wieout Buffer was added to total RNA (1 pg) to make the total amount of 14 μb genomic DNA removal reaction at 42 ° C for 2 minutes. Next, add 5 X Quantiscript RT Buffer, RT Primer Mix, and 15 200927157 to all of the genomic DNA removal reaction solution.

Quantiscript Reverse Transcriptase (RT) allowed a total of 20 μΐ to perform a 15 minute reverse transcription reaction at 42 °C. Next, a single-stranded cDNA was synthesized by performing heat treatment at 95 ° C for 3 minutes to stop the reaction. In addition, the reverse transcription enzyme reaction may be carried out according to the following method without using the QuantiTeetR Reverse Transcription Kit. That is, the DNA present in the extracted RNA solution can be mixed with the DNase reaction solution (40 mM Tris-HCl, 2 mM MgCl 2 » 0.3 mM β CaCl 2 , pH 7.9 ) with deoxyribonuclease (deoxyribonuclease). '5 unit) reacts to remove it. In addition, to 11 μΐ of an aqueous solution containing the extracted RNA (1 pg), 4 μΐ of 5 X reaction solution (250 mM Tris-HCl, 200 mM KC1, 30 mM MgCl 2 , 50 mM dithiothreitol) was added. ), pH 8.3), 2 μΐ deoxyribonucleotide mixture solution (1 mM dATP, dGTP, dCTP, dTTP), 1 μΐ oligo dT primer (50 pmol/μΐ) ), 1 μΐ of RNase inhibitor (20 units/μΐ), 1 μΐ of reverse transcriptase (RT' Reverse Transcriptase, 50 units/μΐ), so that the total amount reaches 2〇μb and then at 37 Recombinant DNA (cDNA) can be synthesized by reacting at °C for 1 hour. (3) Instant Polymerase Chain Reaction Using single-stranded cDNA synthesized by RT reaction as a template, human hyaluronic acid synthase (HAS) primer was used. Kits (QuantfTect (registered trademark) Primer Assays 'Bet from QIAGEN), and primers specific for human workers 16 200927157 collagen αΐ, primers specific for human elastin, and human glyceraldehyde _ 3_phosphorus Acid dehydrogenase (GAPDh) is a specific primer' for immediate polymerase chain reaction. In other words, the RT reaction solution was diluted 40 times with sterilized distilled water, and 3 μΐ of sterilized distilled water and 6.25 μl of SYBR (registered trademark) premix Ex TaqTMI [(purchased from Takara Bi〇) were added to 2 μl of the diluted solution. The 25 μl HAS primer set was used to make a total amount of 12.5, and 45 cycles were performed using ThermalCyderDice (registered trademark) Real time System (manufactured by Takara Bio Co., Ltd.) (1 cycle: 5 seconds at 94 ° C; at 6 〇 PCR reaction at °C for 30 seconds). For human type I-derived protein α human human elastin or human glycerate-3-hetery (four) (GApDH), each of the synonymous primers (10 μΜ each; serial number is 3, 5) and 0.625 μΐ The antisense primers (each ΙΟμΜ; SEQ ID NO: 2, 4, 6) 〇 625 μ1 in place of the HAS primer set and proceed in the same manner. As a result, GAPDH was used as an internal standard gene, and the comparative α method was relatively quantitative. The so-called _ method refers to the calculation of the branch of the mu theory based on the number of cycles required for the product of ’ and 曰曰 to reach a certain amount. The amount of complementary surface of the wire modulo (4) can be expressed as the number of cycles. In this method, the difference of the gene amount (2 iron) which is twice the difference in the detection of the secondary coating is used. In comparison with the gene (internal standard gene) which is the basis of the second, the increase of the 1 ring slit (4) Quantification of the base of the unknown sample 17 200927157 In addition to the purchased human HAS (catalog number QT00027510) introduction set, The primers are as follows. Human type I collagen αΐ (accession number Accession No. NM-000088.3): sense primer (SEQ ID NO: 1), 5'-CAGCAGATCGAGAACATCCG-3' and antisense primer (SEQ ID NO: 2), 5'-TCTTGAGGTCACGGCAGGTG-3'; Human Elastin (Accession No. M36860.1): Synonymous primer (SEQ ID NO: 3), 5, -TGGACTTGGAGTTGGTGTCG-3' and antisense 引 primer (SEQ ID NO: 4), 5'-CAGGCACTGCTGCTCCATATTT -3'; human GAPDH (Accession No. M33197.1): synonymous primer (SEQ ID NO: 5), 5'-GGTGAAGGTCGGAGTCAACG-3' and antisense bow | sub (SEQ ID NO: 6), 5, -GGCAACAATATCCACTTTAC CAGA-3, . These primers were made based on the synthesis of Operon Biotechnologies Co., Ltd. Further, the instant polymerization enzyme chain reaction may be carried out according to the PCR method described below without using QuantiTect® (registered trademark) Primer Assays. That is, 5 μΐ of 10 X PCR reaction solution (100 mM Tris-HC, 15 mM MgCl 2 , 500 mM KC1, pH 8·3), 2 was added to the cDNA solution (2 μΐ) after the RT reaction. A mixture of μΐ deoxyribonucleotide mixture (10 mM dATP, dGTP, dCTP, dTTP), 2 μΐ synonymous primer (10 μΜ), 2 μΐ antisense primer (1 μμΜ) 0.5 μΐ of Taq DNA polymerase (100 units/μΐ), 36.5 μΐ of sterilized steaming chamber 7jc, and the total amount of 18 200927157 reached 50 μΐ' and the pcR method was implemented. The PCR reaction was carried out in a single cycle of 92° C. for 40 seconds, 60° C. for 40 seconds, and 72° C. for 60 seconds, and 35 to 45 cycles were performed. The synthesized DNA 'is separated by electrophoresis using a 2 wt% agarose gel containing 0.5 pg/mi ethidium bromide, and synthesized by ultraviolet irradiation The DNA was compared for quantification. In addition, the 'Hua Hyaluronic Acid Synthetase (HAS) primer can be implemented using the following primers in place of the primer: HAS synonym (SEQ ID NO: 7): 5'-GTGTTATACATGTCGAGTTTACTTCC-3, HAS antisense primer (SEQ ID NO: 8 ): 5'-GTCATATTGTTGTCCCTTCTTCCGC-3, (see Yamada, Y., Itano, N., Hata, K., Ueda, M., and Kimata, K. Differential regulation by IL-1 beta and EGF of expression of three different Hyaluronan synthases in oral mucosal epithelial cells and fibroblasts and dermal fibroblasts: quantitative analysis using real-time RT-PCR. J. Invest. Dermatol. 122: 631-639, 2004.). (4) Statistical processing A significant difference check between the respective processes was carried out by Fisher's multivariate dispersion analysis method. 4. Experimental results

(1) It has been clarified that in the normal human fibroblasts, the Maitake extract promotes the expression of HAS mRNA in a concentration-dependent manner (Fig. 即, ie, the amount of HAS 200927157 mRNA is 5.4 in the Maitake extract of 100 pg/ml, respectively. The maitake extract at 200 pg/ml was significantly increased by 23.0 times at 22.4 times and at 400 pg/ml. In addition, TGF-β (20 ng/ml) was also promoted 9.3 times. HAS mRNA expression. (2) As shown in Fig. 2, it can be judged that Maitake extract has no effect on the gene expression of type I collagen αΐ bond. On the other hand, TGF-β (20 ng/ml) is significantly promoted. Its mRNA expression (2.4 times). (3) As shown in Figure 3, it is clear that Maitake extract has no effect on the expression of elastin® mRNA. On the other hand, TGF-β (20 ng/ml) significantly promotes it. mRNA expression (4.9 times). 5. It was first discovered that in normal human skin fibroblasts, Maitake extract has no effect on the gene expression of collagen and elastin in skin structure proteins, but can promote hyaluronic acid imparting moisturization to the skin. The synthetic enzyme is the expression of the HAS gene. Therefore, it is powerful to explain: Maitake The skin is moistened by promoting the biosynthesis of hyaluronic acid (refer to Carruthers J, 〇Carruthers A. Hyaluronic acid gel in skin rejuvenation. J. Drugs Dermatol. 5: 959-964. 2006.) as an endogenous moisturizing factor. Sexual hyaluronic acid production-promoting substance (hyaluronic acid production promotion 0). In addition, 'the maitake extract is expected to exert the following anti-aging effect: by acting on fibroblasts to activate it, the hyaluronic acid which decreases from the internal supplement with increasing age Furthermore, it is expected that the hyaluronic acid production-promoting action of the maitake extract can be effective in preventing and treating dementia, rheumatoid arthritis, and dry eye caused by hyaluronic acid reduction. 20 200927157 [Examples 2] Experimental method ❹ 鲁 1. Preparation of the separated component from the Maitake extract prepared in Example 1 using a normal phase Shijiao (BW-300, manufactured by Fuji Silysia Chemical Co., Ltd.; using an inner diameter of 35 mm x 280 mm The glass blast open column was subjected to column chromatography, and the Maitake extract (8.88 g) prepared in Example 1 was dissolved in 15 ml. In the gas imitation-sterol (19:1), the obtained solution was added to the normal phase tannin extract, and then the mobile phase was changed sequentially (chloroform-methanol (19:1, using 1 ml) of chloroform-methanol (19:1). 9 : 1, using 800 ml) - chloroform-methanol (4: 1, using 800 ml) - only methanol (using 1000 ml) - while separating, respectively, about ml. The first dissolved fraction taken out was taken as prl, and all 6 aliquots of the fraction were separated. Then, 'take a part of all the separated fractions (Frl~Fr6〇) from the thin layer tannin (TLC) plate (1 〇cmx 1 〇cm, SiHca gel 6〇F254 # 1.05715.0009 'Merck) Development was carried out using a developing solvent (chloroform 'alcohol (9:1)) (expansion distance: 8 cm). The plate was subjected to UV (254 nm) irradiation and then 1% by weight of a sulfuric acid reagent was sprayed and heated to thereby detect the separated components. As a result, the following five parts (M1 to milk) composed of the =-component group were obtained (the table of the movement degree (Rf value) was 0.98, and the dissolution of the residue was: Γ (4) (10) said New 29 The dissolved fraction is classified as, for example, the dissolved fraction having a value of .28^Rf is classified as M3, the dissolved fraction of r is classified as M4, and the fraction having the Rf value of _Rf = 〇 is classified as M5. , 21 200927157 is the yield after drying under reduced pressure, and is calculated by the wind bag elimination method. In addition, each component is adjusted to 400 mg/ml with 100% EtOH, and then added to the cell culture system. Table 1 Maitake extract The separation components of the five separate components correspond to the yield of the dissolved fraction (mg) Ml 卩1*1~?15 (0.982 financial value 20.50) 6115 M2 price 6~卩1*13 (0.45211 value 20.29) 481 M3 Frl4~Fr22 ( 0.282 Rf value 20.16) 255 M4 Fr23 to Fr47 (0.07 Rf value 0.07) 425 M5 Fr48 to Fr60 (〇.〇7> Rf value) 1260 Example 3 Experimental method 1. Dance prepared in Example 2 Preparation of the separated component of the velvet extract separation portion 5 (M5) 制造 by using a normal phase silicone (BW-300, manufactured by Fuji Silysia Chemica Co., Ltd.; The tube of the velvet knives prepared by the squid 2 towel was dissolved in the 2G (1.2 g) by the column chromatography method of the internal control 4 〇mm x 3 〇〇mm glass. In ml of chloroform methanol X (4 1 · 〇·ι ), the solution was added to the normal phase stone gel, and the mobile phase was changed sequentially after the polymerization (chloroform-sterol_water (4: :, use) - Only methanol (using 3 (K) ml)) is separated, ^ 1. The first dissolved fraction taken out is taken as (4), fine

Fr38W 38 dissolving parts. Next, take out all the dissolved fractions (Frl~ take a part, in a thin layer of Shijiao (TLC) plate 〇〇cmxl〇cm 22 200927157

Silica gel 60F254#1.05715.〇〇〇9' manufactured by Merck) was developed using a developing solvent (chloroform-methanol-water (4:1: (U)) (expansion distance: 8 cm). UV was applied to the TLC plate. After irradiation with 254 nm), 10% sulfuric acid reagent was sprayed and further heated to detect the separated components. As a result, the following eight separated fractions (M5-1 to M5-8) composed of the same component group were obtained. (Table 2). That is, the relative mobility (Rf value) is 〇.32^Rf value ^0.12, and the dissolved fraction is classified as M5. The Rf value is 〇24 2Rf value 20.11, and the dissolved fraction is classified as M5-2' The Rf value is 〇.39^Rf value ^〇.11 The dissolved fraction is classified as]\45-3', and the 1^ value is 〇.39 21^ value 2〇.15 The dissolved fraction is classified as M5-4 'Rf The dissolved fraction with a value of 〇.2〇gRf value of 2〇15 is classified as M5-5, and the dissolved fraction having an Rf value of 〇.19gRf value of 20.05 is classified as M5_6, and the dissolved fraction of Rf value of O.llgRf value of ^0.05 is returned. For M5_7, the eluted fraction having the Rf value of 0 is classified as M5-8. In addition, for each of the separated components, the yield of hydrazine after drying under reduced pressure is calculated by the windbag elimination method. Alternatively, 100% EtOH will be used. Adjust the ingredients to 2〇〇mg/ml and add In the cell culture system, the separated fraction of the velvet extract separation part 5 (M5) corresponds to the fraction yield (mg) Frl~Fr3 (0.32gRf value 〇·ΐ2) 91 Fr4, Fr5 (0.242Rf value 20.11) 129 Avoid 3 \yr?^--- Fr6~Fr8 (0.392Rf value 20.11) 152 丄V15-4 -- Fr9, FrlO (0.39gRf value > 0.15) 47 Frll~Frl3 (0.20gRf value XU5) 46 Frl4~Fr22 C 〇.19^Rf value g〇.〇5) 77 M5-7——Fr23~Fr33(0.112Rf^g〇.〇5) 122 Fr34~Fr38 (Rf value=0) 476 . 23 200927157 Example 4 Human fiber Culture and treatment of mother cells Normal human fibroblasts NB1RGB (RCB0222) were purchased from the Bioresources Institute of the Institute of Physical Chemistry, using Dulbecco's modified with 10% (v/v) fetal bovine serum (FBS). Culture in Eagle's medium (DMEM). The treatment of the Maitake extract and its separated fraction (M1 to M5, M5-1 to M5-8) was carried out in the absence of serum. That is, it contains the Maitake extract (25 to 400 pg/ml), its separated component (1〇〇 ® ~400 咫/ml), or the interleukin Ια (10 ng/ml, DS Pharma

Biomedical) 2% (v/v) lactalbumin hydrolysate (LAH) / DMEM for confluent human fibroblasts cultured in 24 well multiplate or 60 mm dish 24 Or 48 hours of processing. Furthermore, in order to investigate the regulation of HA (hyaluronic acid) biosynthesis, the HAS inhibitor 4-mercaptoacetone (4MU) (0.1~1 mM, Sigma)

The maitake extract is treated in the same manner in the presence of Aldrich. 2. Real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) method • (1) Extraction of total RNA The total RNA extracted from cells and the total RNA were quantified, which is 3 (1) of Example 1. The method described in the above. (2) Reverse transcriptase reaction Using the obtained total RNA ', a single bond cDNA was synthesized in the manner of 24 200927157 described in 3 (2) of Example 1. (3) Instant polymerase chain reaction The single-stranded cDNA synthesized by the RT reaction is used as a template. Human 钿 collagenase 1 (procollagenasel/proMMP-1 primer set - Group (QuantiTect (registered trademark) primer Assays, catalogue No. QT00014581 'purchased from QIAGEN, Inc., human tissue matrix ^ protein inhibitor _2 ( TIMP-2 ) primer set (QuantiTect (note)

Registered trademark) Primer Assays, catalog number QT00017759, purchased from QIAGEN), and primers specific for human glycerol 3_can acid dehydrogenase (gapdh) (SEQ ID NO: 5, 6), as in Example 1 3 (3) The method described in 'According to SYBR (registered trademark) premix Εχ TaqTM Ε (purchased from Takara Bio), 45 cycles (1 cycle: 5 seconds at 94 ° C, PCR was performed at 60 〇 30 seconds), thereby increasing the amount of each cDNA. As a result, GAPDH was used as an internal standard gene, and ΔΔCt method was used for relative quantification. For human procollagen enzyme j (pr〇c〇Uagenasel/❹ pr 〇MMP-1), human tissue matrix metalloproteinase inhibitor The PCR performed by _2 (TIMP-2) was carried out in the same manner as in 3 (3) of Example 1 using the above-described primer set instead of the human HAS primer set in 3 (3) of Example 1. In addition, 0.625 μΐ of the following synonymous primers (each ίο μΜ; SEQ ID NO: 9, and 〇625 μ1 antisense primers (each 1 〇 μΜ; SEQ ID NO: 12) can be used instead of the primer set. This is carried out in the same manner as in 3 (3) of Example 1. The human proMMP-Ι primer can be implemented by using the primer of the following sequence instead of the 25 200927157 set primer: proMMP-l synonym (SEQ ID NO: 9): 5' -GGTGATGAAGCAGCCCAG-3· •, proMMP-l antisense primer (SEQ ID NO: 10): 5'-CAGTAGAATGGGAGAGTC-3' (Refer to Lin, N., Sato, T., Takayama, Y., Mimaki, Y.,

Sashida, Y., Yano, M., and Ito, A. Novel anti-inflammatory actions of nobiletin, a citrus polymethoxy flavonoid, on ® human synovial fibroblasts and mouse macrophages. Biochem. Pharmacol. 65: 2065-2071, 2003.) The human TIMP-2 primer was implemented using a primer from the following sequence in place of the set primer: TIMP-2 synonymous primer (SEQ ID NO: 11): 5, -GGTACCAGATGGGCTGCGAG-3, TIMP-2 antisense primer (SEQ ID NO: 12): 5'-TTGGAGGCCTGCTTA-3' (Refer to Hirata, Μ" Sato, T" Tsumagari, Μ" Shimada, A·, ❹ Nakano, H., Hashizume, K., and Ito A. Differential regulation of the expression of matrix metalloproteinases and Tissue inhibitors of metalloproteinases by cytokines and growth factors in bovine endometrial stromal cells and tropboblast cell line BT-1 in vitro. Biol. Reprod. 68: 1276 ~1281, 2003.) ° 3. DNA quantification according to the methods of Johnson Wint and Hollis (See Johnson-Wint, 26 200927157 B., and Hollis, S. A rapid in situ deoxyribonucleic acid assay for determining cell number in culture a Nd tissue. Anal. Biochem.l22: 338~344, 1982.) to determine the amount of DNA in the cells. That is, 0.25% (w/v) trypsin / 0.02% (w) per treatment. /v) The cells treated in the above 1 were recovered in an EDTA solution (1.5 ml), and the obtained cell suspension was subjected to a closed-type ultrasonic cell disrupting apparatus (manufactured by Cosmo Bio Inc.) for 5 minutes, 200 W, 6 under ice bath cooling. The ultrasonic treatment of the second is made into a sample solution. The sample solution 100 (100 μΐ) was mixed with 160 μl of a solution of 3,5-diaminobenzoic acid dihydrochloride (400 mg/ml) (manufactured by Sigma Aldrich Co., Ltd.). 60. (: The reaction was carried out for 45 minutes. After the reaction was completed, 1.5 ml of 1N hydrochloric acid was added, and the fluorescence intensity in the sample solution was measured by a microdisk analyzer (Infinite F200, manufactured by Tecan Japan) (excitation wavelength: 430 nm, fluorescence wavelength) In addition, the amount of DNA was calculated from the calibration curve prepared in the same manner using DNA obtained from the squid testis (manufactured by Wako Pure Chemical Industries, Ltd.) ❹ 4.HA quantified the above 1 after 24 or 48 hours The amount of HA in the culture solution recovered after the culture was quantified by the HA ELISA kit (purchased from Biochemical Industries, Inc.) in accordance with the attached operation method, that is, the HA in each of the recovered culture solutions was recovered. It is combined with the uric acid-binding protein (HABp) immobilized on the microtiter plate attached to the purchased kit, and then added with the peroxide enzyme in combination with HABP to react. Then, tetramethylbenzidine is added. And hydrogen peroxide, using a micro-disc ^ 27 200927157 instrument (Infinite F200, manufactured by Tecan Japan Co., Ltd.) to measure the absorbance at 45 〇 leg. Using standard HA solution (50 ~ 800 ng / ml), by the same The weighted curve made at the time is calculated. Or, each single in each process

The amount of HA produced in the cells is represented by a value (ng/pg DNA) _ obtained by dividing the amount of HA of each treatment to be measured by the amount of DNA of each treatment quantified in the above 3 (ng/pg DNA). Alternatively, the HA ELISA kit may be used instead of performing HA quantification according to the following method. Specifically, a 96-well multiplate in which a recombinant human HABP (rHABP) (manufactured by Cosmo Bio.) was immobilized using a carbonate buffer (pH 8 to 9), and a recovered culture solution was added to bind biotin to rHABP ( Cosmo Bio made) further combined with the combined HA. Next, a peroxidase is added in combination with avidin to form a biotin-avidin complex. Further, tetramethylbenzidine and hydrogen peroxide were added, and the absorbance at 450 nm was measured using a microdisk analyzer (Lifinite F200, manufactured by Tecan Japan Co., Ltd.). The amount of HA was calculated from a calibration curve prepared at the same time using a standard HA solution (50 to 800 ng/ml). 5. Western blott method A final concentration of 3.3% (w/v) of trichloroacetic acid was added to the cell culture medium recovered after 24 or 48 hours of culture of the above 1 to 4 After standing overnight at ° C, the protein was insolubilized. The insoluble protein was precipitated by centrifugation (1 Torr, rpm/min, 10 minutes), and then the precipitate was dissolved in SDS containing 1% (v/v) 2-mercaptoethanol. PAGE sample buffer, 28 200927157

Protein electrophoresis analysis (SDS-PAGE) was performed on a 12.5% (w/v) polyacrylamide gel. After the electrophoresis is finished, the protein in the gel is transcribed on the nitrocellulose membrane, and the nitrocellulose membrane is immersed in a blocking solution according to a conventional method [5% (w/v) skim milk powder (fatty-free dry milk) /80 mM Na2HP04/100 mM NaCl/0.1% (v/v) Tween-20 (pH 7.5 )], blocking for 1 hour. After rinsing with water and PBS-T buffer [80 mM Na2HPO4/100 mM NaCl/0.1% (v/v) Tween-20 (pH 7.5)], rinsing them for 2 to 3 times, then immersing them in 1%. (w/v) BSA/PBS (-) diluted primary antibody [sheep anti-(human proMMP-1) or anti-(human TIMP-2) IgG, respectively, by YONG Yu-ming of Kennedy Rheumatic Institute, King's College London The professor provided the solution and slowly oscillated at room temperature for more than 8 hours. After primary antibody binding, wash twice with distilled water and PBS-T buffer for 2 to 3 times, and dilute it at room temperature with secondary antibody diluted with 1% (w/v) BSA/PBS (-) [peroxidase-conjugated] Goat anti-(sheep IgG) IgG » Sigma ❹ manufactured by Aldrich] was immersed in the solution for 1 hour. After the secondary antibody was bound, it was immersed in ECL-Western Blotting Detection Reagents (purchased from Amersham Biosciences) of the ECL-Western Blotting Detection kit for accurate reaction for 1 minute. After the reaction was completed, the amount of proMMP-1 and TIMP-2 was quantified by using Lumino Image Analyzer LAS-1000 plus (manufactured by Fuji Flim Co., Ltd.). The western ink dot method is a well-known experimental method known to the industry, and is described in detail in, for example, the well-known experimental book such as the Shengu Michiko Protein Experiment Manual (edited by Takehiko Ayako), Yoshitosha, 29, 200927157, pp. 152-157, 2003. Alternatively, the above antibodies may be used without using the above antibodies. Primary antibody 1) Anti-human MMP-1 monoclonal antibody (product number: F_67, manufactured by Daiichi Fine Chemical Co., Ltd.) 2) Anti-human TIMP-1 monoclonal antibody (product number: f-26, manufactured by Daiichi Fine Chemical Co., Ltd.) Secondary antibody peroxidaserconjugated rabbit anti-(mouse IgG) IgG (manufactured by Sigma Aldrich Co., Ltd.) 6. Statistical processing A significant difference test between the respective treatments was carried out by Hsher's multivariate dispersion analysis method. 7. Experimental results (1) Normal human fibroblasts NB1RGB (RCB0222) produced HA in a constant manner, while Maitake extract (25 to 100 pg/ml) enhanced HA production (Fig. 4). That is, the amount of HA was 15.37±1.56 pg/pg DNA in the untreated group, and 24.79±2.75 pg/pg DNA in the Maitake extract of 25 pg/ml, at 50 pg/ml of Maitake The extract was 26.08 ± 2.19 pg/pg DNA and 20.57 ± 1.46 DNA in 100 pg/ml of Maitake extract. The HA production enhanced by Maitake extract (100 pg/ml) was inhibited in a concentration-dependent manner by the HAS inhibitor 4-methyl-4-bellumone (0.1 to 1 mM). 30 200927157 Maitake extract promotes the production of 11 people by promoting the performance of the HAS gene. (2) In order to identify the Maitake extract component which contributes to the promotion of ha production, the regulation of HA production of the separation component of Maitake extract has been studied. As a result, a concentration-dependent HA production-promoting action was detected in the Ml and M5 separated fractions (Fig. 5). In addition, it was found that the relative promoting activity of M5 was higher than that of M1. That is, the amount of HA is 580.21±70.65 ng/m in the untreated group and 883.49±159.21 ng/m in Ml at 25 pg/ml, and 1237.28±405 in M1 at 50 gg/ml. 80 ng/m b was 1488.19±527.23 ng/ml in Ml❹ at 100々g/ml; 1041.44±186.71 ng/ml in M5 at 25 gg/ml '1587.83 in M5 at 50 pg/nil 298.1〇1^/1!11' in 1〇〇 from 8/1111 of PCT 5 is 2304.25±499.97 ng/m Bu and further, the M2~M4 separation part promotes HA production at low concentration' but at high concentration Inhibition of HA production. (3) In order to study the specificity of Maitake extract to promote HA production, the known ECM metabolism-related genes, namely pr〇Mj^p_i and ❹, for the constant expression of Maitake extract and its fractions for normal human fibroblasts The role of TIMP-2 gene expression was studied. The concentration of Maitake extract relies on the expression and production of proMMP-1 mRNA (Fig. 6 and Fig. 7 A). However, Maitake extract has no effect on the performance and production of TIMP-2 mRNA (B of Figure 6 and B of Figure 7). On the other hand, in the results of the separation component of Maitake extract, the M5 fraction had no effect on the expression of ρΓ〇ΜΜΡ-1 mRNA (Fig. 6). An increase in the expression of the proMMP-Ι gene was observed in M2 to Μ4 (Fig. 6A). For the TIMP-2 mRNA expression, the M1 fraction showed a tendency to inhibit, but there was almost no change in the fraction of 31 200927157 (Fig. 6B). Therefore, it is suggested that in the promotion of HA production and the expression of proMMP-Ι gene in Maitake extract, different components of Maitake extract may be involved. (4) In order to understand in more detail the effect of promoting the production of HA in the M5 fraction of the Maitake extract, the regulation of HA production was carried out for the eight components (M5-1 to M5_8) in which the M5 fraction was further separated. As a result, an effect of promoting HA production was observed in the U5-3 to M5-8 separated portion. ❹ 8. Investigation In normal human skin fibroblasts, Maitake Extract promotes the synthesis of HA which imparts skin wetness. Therefore, it is indicated that the maitake extract promotes skin activation and improvement due to synthesis of an important wet-conditioning component, i.e., HA (e.g., improves the moisture retention function of the skin). Further, it is explained that the action of the Maitake extract to promote HA production may be adjusted by different components. The role of skin ECM in promoting enzyme metabolism plays an important role in skin re-formation such as wound healing. It is generally believed that the balance of the amount of MMP and TIMP© is important for this .ECM metabolism. Further, the decomposition of the skin ECM caused by excessive MMP production is enhanced, and skin roughening (skin aging) such as wrinkle formation is caused. It is clear for the first time that Maitake extract promotes pr〇MMP-1 mRNA and its production in human skin fibroblasts. However, it is interesting to say that the Maitake extract separation portion (M2 to M4) showing that the proMMP-1 exhibits a promoting effect is different from the separation portion (M5) in which the HA production is promoted. The identified 'shows that HA production promotes 32 200927157 3 == the fractions are Μ5_6 and Μ5_7. In other words, it is expected that the ingredients of the substance can be separated and used to reduce the skin aging effect, and the characteristics of the skin moisturizing property can be further enhanced. It is strongly demonstrated that Maitake extract is a material derived from novel natural materials that promotes skin moisturization by promoting the biosynthesis of an endogenous moisturizing factor, i.e., strontium. Further, it is expected that the Maitake extract can also exert the following anti-aging effect: φ ❹ is activated by acting on the fibroblasts to replenish HA which is reduced with age from the inside. [Example 5] A cosmetic cream containing a Maitake mushroom extract was prepared in the following amount. Formulation amount (wt%) 2.0 10.0 10.0 77.0 100.0 Ingredient name Maitake extract Olive oil III (caprylic acid/capric acid) glycerol Tocopherol 1.0 Purified water meter [Example 6] The dance was prepared with the following blending amount Formulation of medicinal cream for velvet extract (wt%) 10.0 10.0 10.0 1.0 Ingredient name Maitake extract eucalyptus oil III (octanoic acid/capric acid) glycerol tocopherol 33 200927157 69.0 100.0 Purified hydration meter [Example 7] Dance Effect of velvet extract on human dermatosis The cream containing 2 (four) Maitake extract as shown in Example 5 was used to study the effect on dry skin disease. Test method ❹ ❹ Subjects to be tested. Dry skin disease in elderly health facilities: 12 patients (76 years old to 97 years old) with symptoms of 7 (the skin is keratinous and shedding) (6 males) , female to) implementation time:, 2_year December ~ woqi month = on the same will include the Maitake extract 1 == lower leg, left lower leg, respectively, in each part to: lower left arm, right result below, table coating Cloth start, week test 1:1«) Γ Diagnostics # Price is 0 means complete cure:. Right 7 left: Arm lower right leg left lower leg was tested by the tester 1 Diagnosis, coating period 2006/12/4 ^ 2006/12/6 ! 1 2 2 200927157 ❹ ❹ 2006/12/8 2006/12/11 Subjects 2 Diagnosis, coating period 2007/1/17 2007/1/19 2007/1/22 2007/1/24 Subject 3 Diagnosis, coating period 2006/12/6 2006/12/11 2006/12 /13 Subject 4 Diagnosis, coating period 2006/12/15 2006/12/18 2006/12/20 2006/12/22 Subject 5 Diagnosis, coating period 2007/1/5 2007/1 /8 2007/1/10 1 1 Right lower arm 1 1 0 0 Right lower arm 2 2 1 Right lower arm 1 0 0 0 1 0 1 1 1 1 Left lower arm Right lower leg Left lower leg 1 2 1 1 1 1 0 1 1 〇1 1 Left lower arm Right lower leg Left lower leg 2 2 3 2 1 2 〇0 0 Left lower arm Right lower leg Left lower leg 1 1 1 1 ο 〇1 0 〇〇〇0 Right lower arm Left lower arm Right lower leg Left lower leg 2 2 1 1 0 3 2 2 2 2 35 0

200927157 2007/1/12 Subject 6 Diagnosis, coating period 2006/12/18 2006/12/20 2006/12/25 Subject 7 Diagnosis, coating period 2006/12/18 2006/12/ 20 2006/12/22 2006/12/25 Subject 8 Diagnosis, coating period 2006/12/29 2006/12/30 2007/1/4 Subjects 9 Diagnosis, coating period 2007/1/ 5 2007/1/8 2007/1/10 2007/1/12 Testee 10 0 0 2 2 Right lower arm Left lower arm Right lower leg Left lower leg 0 1 3 2 0 0 2 1 0 0 1 1 Right lower arm Left lower arm Lower right leg Lower left leg 1 1 1 1 0 0 1 1 0 0 1 1 0 0 0 1 Arm left lower arm Right lower leg Left lower leg 2 3 3 3 1 2 2 2 1 0 2 1 Right lower arm Left lower arm Right lower leg Left lower leg 0 0 2 3 0 0 2 1 0 0 2 1 0 0 1 1 36 200927157 Diagnosis, coating, right lower arm 2006/12/20 2006/12/22 2006/12/25 2006/12/26 2006/12/27 Subject 11 1 1 1 1 1 Left lower arm Right lower leg Left lower leg 1 2 2 0 2 2 0 1 1 0 1 1 0 1 1 Ο Diagnosis, coating period right γ stone lower arm lower left arm right lower leg left lower leg 2006/ 12/4 χ 2006/12/6 2006/12/8 2006/12/11 Subjects 12 0 0 0 1 1 2 1 1 0 1 1 1 0 Ο Displacement, coating date right lower arm left lower arm right lower leg Lower leg 2006/12/18 〇2006/12/20 2006/12/22 2006/12/25 0 0 0 1 0 0 0 2 1 1 1 1 1 0 As above, the subjects 1~12 after the start of coating The degree of diagnostic evaluation of the skin condition is shown to be improved. Further, a remarkable symptom-improving effect obtained by applying a cream containing Maitake extract was found to be 丨3曰 after application. Fig. 9 is a photograph of the skin of the test subject 5 containing the Maitake extract cream before and after the treatment. An improvement in dry skin symptoms was observed by applying a treatment containing a Maitake Extract cream. Moreover, these results show that a cream having a content of 2% by weight of Maitake extract has a therapeutic effect on dry skin. [Industrial Applicability] The hyaluronic acid can be promoted by the extract of the present invention, and therefore the composition of the present invention can be used as a cosmetic and a pharmaceutical for treating skin abnormalities, injuries or diseases caused by a decrease in hyaluronic acid production, and A pharmaceutical product for the treatment of abnormalities, injuries or diseases of the joints or eyes. ® [A brief description of the formula] Fig. 1 shows that the Maitake extract promotes the gene expression of hyaluronic acid synthase (has) in human skin fibroblasts relative to the untreated group (p<〇.〇5). Figure 2 shows the effect of Maitake extract on the expression of collagen alpha mRNA in human skin fibroblasts. * * * : Relative to unprocessed groups (p < 0.001 ). Figure 3 shows the effect of Maitake Extract on the expression of elastin mRNA in human skin fibroblasts. * ; relative to the unprocessed group (p < 〇 〇 5). Figure 4 shows the promotion of hyaluronic acid (HA) production by human skin fibroblasts by Maitake Extract. 4-mercaptoketone UMU, 4-methylumbelliferone, HAS inhibitor). * and * * : Relative to unprocessed groups (ρ < 0·05 and 0.01). ##和### : The treatment group (p<〇.〇l and 〇 〇〇1) is compared to the maitake extract (100 pg/ml). Fig. 5 shows the promotion of hyaluronic acid (HA) production by human skin fibroblasts in a separate fraction of mitten extract (mi~M5). 38 200927157 (interleukiM, 10 ng/ml). * and * * : Relative to unprocessed groups (p < 0·05 and 0.01). Figure 6 shows the effect of Maitake extract and its fractions on the expression of proMMP-Ι and ΤΙΜΡ-2 genes in human skin fibroblasts. Human fibroblasts were treated with Maitake extract or its fraction (Ml~M5) (n==l/treated) for 24 hours. The proMMpq (A) and TIMP-2 mRNA expression (B) in the recovered cells were quantified. Maitake extract promotes the expression of Pr〇MMP-1 mRNA, but conversely inhibits the expression of the τΐΜΡ-2 gene. M5 had little effect on proMMP-Ι and TIMP-2 mRNA expression. On the other hand, 'M2 and M3 promote proMMP-1 gene expression, and Ml inhibits TIMP-2 gene expression. Furthermore, interleukin 1 a (IL-1) (1 〇 ng/ml) promoted proMMP-1 mRNA expression but had no effect on the ΤΙΜΡ-2 gene expression. Figure 7 shows the promotion of pr〇MMP-1 (Α) and TIMP-2 (Β) in human skin fibroblasts by Maitake Extract. IW (inkerleukin Bu 10 ng/ml). * and * * : Relative to unprocessed groups (p ❹ < 0.05 and 0.01). Fig. 8 is a graph showing the amount of hyaluronic acid in a culture solution of cells in which human fibroblasts were treated for 24 hours using the Maitake extract M5 fraction (Ms., M5-8) (n 2 / treatment). In M5_3 to M5_8, an increase in the amount of hyaluronic acid was observed. IL-1 (interleukin 1, 1〇 ng/ml) ° Figure 9 is a photograph of the skin of the subject 5 before and after treatment using a cream containing Maitake extract. 39 200927157 [Explanation of main component symbols]

Claims (1)

200927157 VII. Scope of application for patents: 1. A kind of maitake extract, which is obtained by extracting dry dance ¥ with pure ethanol and obtaining the maitake extract of the second. 2. For the scope of patent application, the dry dance material contains The shirt 8 Wt is like the next 4 extract, wherein the medicinal material is applied to the maiden velvet extract described in Item 1 or Item 2, wherein the pure ethanol contains 99.0 voI% or more of ethanol. Maitake extract as described in item 1 or item 2 of the patent scope: ;. 1: a part by weight of the dry maiden velvet, which is the maiden stalk extract of the third aspect of the patent, wherein 100 parts by weight of the dried maitake, the pure ethanol is Touch ~ face weight. 6. A hyaluronic acid production promoter comprising the Maitake mushroom extract according to any one of claims 1 to 5. A composition comprising a uric acid production promoter as described in the scope of the patent application. 8. The composition of claim 7, which is in the form of a cosmetic. 9. The composition of claim 7, which is in the form of a pharmaceutical product.