TWI423810B - Grifola frondosa extract and composition containing thereof for promoting hyaluronic acid production - Google Patents

Description

Maitake extract and composition containing the same for promoting hyaluronic acid production

The present invention relates to a Grifola frondosa extract and a composition therefor for promoting the production of hyaluronan.

As an ethanol extract of Maitake mushroom, tyrosinase inhibitory effect, α-amylase inhibitory effect, skin moisturizing effect, and immunosuppressive effect are known so far (refer to Japanese Patent Laid-Open No. 2-121905, Japanese Patent) JP-A-2000-319192, and JP-A-H11-263732.

Further, the present inventors have filed the following patent application: a pure ethanol extract of a dry velvet fruit body exhibits a role in promoting sebum production and secretion in sebum glands in humans and hamsters; in addition, for patients with ecdysis The pure ethanol extract of the dry Maitake fruit body can bring about a clinically improved skin condition. That is, the following patent application is filed: The pure ethanol extract of the dry velvet fruit body improves the skin barrier function by promoting the production of sebum, and is not necessarily effective for daily skin care, and is effective 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 regulation of related molecules is an important factor. (See Baumann L. Skin ageing and its treatment. J. Pathol. 211: 241-251, 2007). Moreover, the extracellular matrix is known (ECM) related molecules are fibroblasts present in the dermis of the skin as their main producing cells.

However, the pure ethanol extract of the dried velvet fruiting body is completely unaware of the regulation of the expression of extracellular matrix (ECM)-related molecules. Therefore, in normal human skin fibroblasts, the genetic level of the following extracellular matrix (ECM)-related molecules is used to analyze the regulation of collagen, elastin and hyaluronic acid by the pure ethanol extract of the dried velvet fruit body. It was found that the pure ethanol extract of the dry velvet fruit body can promote the performance of hyaluronic acid synthetase, that is, promote the production of hyaluronan.

The present invention provides a Maitake mushroom extract which more effectively promotes hyaluronic acid production and a composition containing the same.

In order to solve the above problems, the inventors of the present invention 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.

That is, the present invention is as follows.

(1) A maitake extract obtained by extracting dry maitake with pure ethanol; (2) the maitake extract of (1), wherein the dry maitake has a moisture content of 8 wt% or less; (3) The Maitake mushroom extract according to (1) or (2), wherein the pure ethanol contains 99.0 vol% or more of ethanol; (4) a composition containing any one of (1) to (3) item The velvet extract is used for promoting hyaluronic acid production; (5) the composition according to (4), which is in the form of a cosmetic; (6) the composition according to (4), It is in the form of a pharmaceutical product; (7) a method for producing a hyaluronic acid production-promoting elution product having a 1.0 ≧Rf value of >0.45 and/or a 0.07>Rf value ≧0, comprising: (a) as described above (1) The Maitake extract is dissolved in chloroform-methanol (19:1); (b) the above solution of the Maitake extract is added to the equilibrated normal phase rubber column using chloroform-methanol (19:1) (c) elution in the order of chloroform-methanol (19:1), chloroform-methanol (9:1), chloroform-methanol (4:1), methanol; (d) in a thin layer of tantalum, using chloroform - methanol (9:1) to develop a portion of the dissolution; and (e) to obtain 1.0 ≧Rf value > 0.45 and / or 0.07 > Rf value ≧ 0 of the dissolution; (8) a 1.0 ≧ Rf value > 0.45 and / or 0.07>the hyaluronic acid production-promoting extract having an Rf value of ≧0, which is obtained by the production method as described in the above (7).

[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; damage, abnormality or disease of the joint caused by the decrease in hyaluronic acid; Eye damage, abnormalities and eye diseases caused by a decrease in hyaluronic acid.

The present invention relates to a Maitake mushroom extract obtained by extracting dried Maitake with pure ethanol.

Maitake of the present invention ( ) refers to the mushroom of the Polyporaceae Grifola, which can be exemplified by Grifola frondosa, Grifola albicans, Grifola umbellatus, and Grifola gigantea. )Wait. The maitake of the present invention is preferably a mushroom (Grifola frondosa). Maitake can be any of the fruiting bodies and mycelium, but is preferably a fruiting body.

The dry maitake that extracts the raw material refers to the maitake with a moisture content of 10% by weight or less. The dry maitake preferably has a moisture content of 8 wt% or less, more preferably 7 wt% or less. The dried maitake is obtained by dehydrating and drying the raw velvet by any method (for example, drying in the sun, heating and drying, vacuum drying, freeze drying, etc.). Dry maitake is preferably in powder form.

The pure ethanol of the present invention means ethanol containing 98.0 vol% or more of ethanol at 15 °C. The pure ethanol preferably contains 99.0 vol% or more, more preferably 99.5 vol% or more of ethanol.

The extraction of dried Maitake velvet with pure ethanol is carried out by adding pure ethanol to dry maitake as a raw material, followed by stirring at room temperature or under heating for a certain period of time. The heating temperature is usually a temperature equal to or lower than the boiling point of ethanol, but it may be set to a temperature of 120 ° C or lower in a closed container. A preferred extraction temperature is room temperature (room temperature). The extraction time is not particularly limited, for example, 5 minutes. ~10 hours or so. It can be extracted once or twice.

The amount of pure ethanol relative to the dry maitake in the extraction is 100 to 1000 parts by weight, preferably 200 to 600 parts by weight, and more preferably 300 to 100 parts by weight of dry maitake. 500 parts by weight.

After the extraction treatment, a pure ethanol extract can be obtained by a separation means such as filtration using a filter paper or a filter cloth 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 ethanol from the extract by a usual method. That is, it may be an extraction powder state containing substantially no ethanol or an extract state containing 1 to 50% by weight of ethanol. The extract containing 10-15% by weight of ethanol is in a good form because of its good preservation property. A pure ethanol extract is obtained in a yield of 2 to 10% by weight, more specifically 3 to 5% by weight (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 composition for promoting hyaluronic acid production comprising the above extract.

Hyaluronic acid is a kind of glycosaminoglycan (glycosaminoglycan), which has a disaccharide repeating structure in which N-acetyl-D-glucosamine is bonded to D-glucuronic acid, and can be expressed by the following formula: 1] (where n is an integer of 1 or more)

In addition, hyaluronic acid is also known as hyaluronan.

The composition of the present invention comprises a form of a food, in particular, a form of a food for promoting the production of hyaluronic acid.

Since the composition of the present invention promotes hyaluronic acid production, it can maintain moisturizing properties of the skin, impart moisturization, softness and elasticity to the skin, and can also prevent or repair epidermal damage caused by ultraviolet rays, and can also cause skin regeneration. Accordingly, the present invention encompasses a composition for promoting hyaluronic acid production in the form of a cosmetic and containing the above extract.

The composition of the present invention in the form of a cosmetic material may contain a conventional carrier, an excipient, an additive or the like formulated in a cosmetic. It can be used as a cosmetic for preventing rough skin, improving skin texture, improving skin softness, improving skin elasticity, improving skin wrinkles or sagging, and improving skin moisturization. Examples of the cosmetic preparation include facial cleansing (washing cream, foam, gel cosmetic, etc.), skin conditioning (lotion, beauty liquid, etc.), protection (cream, moisturizer, etc.), mask, and mask. Oil, massage and cosmetics, foundation makeup (foundation, white powder, sunscreen, sunscreen, etc.), topical makeup (lipstick, eye shadow, eyeliner, etc.), bath (soap, shower gel, bathing cosmetics, etc.), Sunscreen (sunscreen, sunburn) Etc.), hair growth, hair care (hair restorer, hair care, etc.). An effective amount of Maitake extract is blended into the cosmetics, for example, in an amount of 0.1 to 99.9 wt%, preferably 1 to 1 in terms of an extract (a maitake extract in a state substantially free of ethanol). 99wt%. The remainder is formulated into conventional carriers, excipients, or additives.

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. Further, the extract of the present invention has an effect of improving joint damage or joint disease caused by a decrease in hyaluronic acid production. Further, the extract of the present invention has an effect of improving eye damage, abnormality or eye disease caused by a decrease in hyaluronic acid. Accordingly, the present invention encompasses a composition for the treatment or prevention of a form of a pharmaceutical product which is accompanied by a damage or a disease caused by a decrease in the hyaluronic acid production described above.

Among the skin damage or skin diseases caused by the decrease in hyaluronic acid production, ecdysis (dry skin), ichthyosis, photoaging, phlegm, atopic dermatitis, acne (pressure sores) and Caused by skin dysfunction and the like. Preferred are photoaging, sputum, atopic dermatitis, acne (pressure sores), and skin dysfunction caused by fire injuries.

Further, in the joint damage or joint disease caused by the decrease in hyaluronic acid production, deformable joint disease and rheumatoid arthritis may be mentioned. Preferred are osteoarthritis and rheumatoid arthritis.

Further, in the case of eye damage or eye disease caused by a decrease in hyaluronic acid production, corneal epithelial damage, eye fatigue, and blinking may be mentioned. Better is blinking. The so-called blinking eye refers to insufficient tears or due to the eye. A state in which tear components change to cause damage on the surface of the eye, which includes tear secretion reduction and spastic keratoconjunctivitis.

The subject to be treated and prevented in the present invention is a mammal, for example, a pet such as a human, a dog or a cat, a livestock animal such as a cow, a pig or a chicken, but particularly preferably a human.

The composition of the present invention which is in the form of a pharmaceutical product may contain a conventional carrier, an excipient, an additive or the like which is formulated in a pharmaceutical product. Examples of the pharmaceutical preparations include creams, sublingual agents, massage oils, solutions, suspensions, lotions, ointments, gels, lozenges, capsules, granules, powders, syrups, suppositories, 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% by weight, 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, olive oil, palm oil, peanut oil, poppy seed oil, rapeseed oil, sesame oil, soybean oil, sunflower oil, and tea seed oil, etc.), mineral oil, fatty oil, liquid paraffin, buffer Preservatives, wetting agents, chelating agents, antioxidants, stabilizers, emulsifiers, suspending agents, gel forming agents, ointment bases, suppository bases, penetration enhancers, fragrances, and skin protectants.

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.

The buffering agent may, for example, be citric acid, acetic acid, tartaric acid, lactic acid, hydrogen phosphate, diethylamine or a mixture thereof, but is not limited to the above buffer.

Examples of the wetting agent include glycerin, propylene glycol, pentylene glycol, sorbitol, lactic acid, urea, BG (1,3-butylene glycol), soybean sterol, and mixtures thereof. However, it is not limited to the above wetting agent.

The chelating agent may, for example, be sodium EDTA, citric acid or a mixture thereof, but is not limited to the above chelating agent.

Examples of the antioxidant include butylated hydroxyanisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, cysteine, and mixtures thereof, but are not limited thereto. Antioxidants.

Among the emulsifiers, natural rubber (for example, Acacia rubber), tragacanth, tricyon; natural phospholipids (for example, soybean lecithin); sorbitol monooleate derivatives; lanolin; ; lanolin; sorbitan ester; monoglyceride; fatty alcohol (eg behenyl alcohol); fatty acid ester (eg tris(caprylic/capric) glyceride, glyceryl stearate (SE) A triglyceride of a fatty acid such as a compound; and a mixture of these, etc., but is not limited to the above emulsifier.

Among the suspending agents, cellulose and derivatives thereof (for example, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc.), carrageenan (for example) Carrageenin), gum arabic, gum arabic, tragacanth, and mixtures thereof, but are not limited to the above suspending agents.

Among the gel base and the tackifying component, liquid paraffin, polyethyl b Alkene, fatty oil, colloidal cerium oxide or aluminum, zinc soap, glycerin, propylene glycol, tragacanth, carboxyvinyl polymer, magnesium citrate-aluminum, hydrophilic polymer (eg starch, carboxymethyl cellulose, Cellulose derivatives such as hydroxyethyl cellulose and other cellulose derivatives), water-swellable hydrophilic colloids, carrageenan, hyaluronic acid (for example, hyaluronic acid gel optionally containing sodium chloride), alginic acid ester (for example) The propylene glycol alginate), a mixture of these, etc., are not limited to the above-mentioned gel base and a thickening component.

Among the ointment bases, beeswax, paraffin, cetyl alcohol, cetyl palmitate, palmitol, polyglyceryl-10 stearate, stearic acid, PEG-150 a condensate product of a stearate, a vegetable oil, a sorbitan ester of a fatty acid, a polyethylene glycol, a sorbitan ester of a fatty acid, and ethylene oxide (for example, polyoxyethylene sorbitan monooleate) and the like A mixture of ointment bases, etc., but is not limited to the above ointment base.

Examples of the hydrophobic ointment base include paraffin, vegetable oil, tallow, synthetic glyceride, wax, lanolin, liquid polyalkyl siloxane, and mixtures thereof, but are not limited to the above hydrophobicity. Ointment base.

The hydrophilic ointment base may, for example, be a solid polyethylene glycol or the like, but is not limited to the polyethylene glycol. Further, examples of the conventional carrier, excipient, or additive include squalane, lecithin, hydrogenated lecithin, tetrahexyl ascorbate, allantoin, glycyrrhizic acid 2K, and glycosyl trehalose. (glycosyl trehalose), hydrolyzed hydrogenated starch, hydrolyzed collagen, rose extract, dimethicone, caprylyl glycol, betaine, hard Sodium glutamate glutamate, wild rose oil, batyl alcohol, hydroproxyproline, and the like.

The pharmaceutical composition of the present invention may further contain other agents for treating or preventing diseases or damage caused by a decrease in hyaluronic acid production.

The effective administration amount used in the present invention for treating or preventing a disease or damage caused by a decrease in hyaluronic acid production varies depending on the degree of the disease or damage and the administration method, and is effective for promoting hyaluronic acid production. The amount is not limited, but the amount of Maitake extract (the form of the extract powder which does not substantially contain ethanol) is 0.1 to 1000 mg / body weight kg. Day, preferably 1~500mg/body weight kg. Day, and then preferably 10~100mg/body weight kg. day.

The administration used in the present invention may be systemic administration or topical administration; systemic administration and topical administration may be: transdermal administration, sublingual administration, oral administration, enteral administration, intramuscular administration. Any of the administration forms such as administration, subcutaneous administration, intravenous administration, nasal administration, and eye-dropping. Preferably, transdermal administration, sublingual administration, and eyedropping are preferred.

Accordingly, the present invention is directed to the use of the Maitake Extract for the manufacture of the above pharmaceutical compositions. Further, the present invention relates to a method comprising administering a pharmaceutical composition to a mammal, and treating or preventing a disease or damage caused by a decrease in hyaluronic acid production by promoting hyaluronic acid production.

[Example 1]

Hereinafter, the present invention will be described by way of examples, but the invention is not limited to the examples.

1. Manufacture of Maitake Extract

4000 g of pure ethanol (ethanol content: 99.5 vol% or more) was added to a sterilized dry powder of 1000 g of maitake (water content: 6 wt%, Maitake Co., Ltd.), and stirred at a temperature of 20 ° C for 18 hours. Extract while performing. The residue was removed by centrifugation, and the resulting supernatant was filtered using a filter paper. Ethanol was evaporated and removed from the obtained filtrate to obtain a pure ethanol extract of Maitake mushroom (yield 43.2 g, wherein solid content was 37.2 g and ethanol was 6.0 g).

2. Culture and treatment of human fibroblasts

Normal human fibroblast NB1RGB (RCB0222) was purchased from the BioResource Center of the Institute of Physical Chemistry (Germany) using Dulbecco's modified Eagle's medium (DMEM, containing 10% (v/v) fetal bovine serum (FBS, Fetal Bovine Serum). Culture was carried out from Invitrogen. In addition, Maitake extract treatment is carried out in the absence of serum. That is, 0.2% (v/v) of transforming growth factor β (TGF-β) (20 ng/ml, purchased from R&D Systems) containing Maitake extract or as an extracellular matrix synthesis-promoting factor. Confluent human fibroblasts cultured in 60 mm dish were cultured for 24 hours in lactobumin hydrolysate (LAH, purchased from Sigma Aldrich)/DMEM.

3. Real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) method (1) Extraction of total RNA

Total RNA extracted from cells is using Isogen (from Nippongene The company purchases) and follows the attached operating methods. Specifically, Isogen (0.8 ml) was added to cells cultured with Maitake extract or TGF-β to dissolve the cells, and chloroform (0.16 ml) was added to the solution and mixed. After centrifugation (10,000 rpm, 10 minutes), the upper aqueous layer portion was recovered, and isopropanol having the same capacity as the recovered aqueous layer was added and mixed. After centrifugation (10,000 rpm, 10 minutes), the supernatant was removed and the precipitated RNA was recovered. The recovered RNA was further added to 75 vol% ethanol (0.8 ml) and mixed. After centrifugation (10,000 rpm, 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, manufactured by Shimadzu Corporation) to thereby quantify. In addition, the extraction of total RNA may be performed without the use of Isogen, but by Chomczynski P and Sacchi N (Chomczynski, P. and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. The method described in Anal. Biochem. 162: 156-159 (1987)) was carried out.

(2) Reverse transcriptase reaction

The reverse transcriptase reaction was carried out using the QuantiTect ^R Reverse Transcription Kit (purchased from QIAGEN) in accordance with the attached procedure. Namely, 7 x g of DNA Wipeout Buffer was added to total RNA (1 μg) to a total amount of 14 μl, and a genomic DNA removal reaction was carried out at 42 ° C for 2 minutes. Next, 5 x Quantiscript RT Buffer, RT Primer Mix, and Quantiscript Reverse Transcriptase (RT) were added to all of the genomic DNA removal reaction solution to make a total amount of 20 μl, and a reverse transcription reaction was carried out at 42 ° C for 15 minutes. ). Next, heat treatment was performed at 95 ° C for 3 minutes to stop the reaction, and single-stranded cDNA was synthesized.

In addition, the reverse transcriptase reaction may be carried out according to the following method without using the QuantiTect ^R Reverse Transcription Kit. That is, the DNA present in the extracted RNA solution can be mixed with DNase reaction solution (40 mM Tris-HCl, 2 mM MgCl ₂ , 0.3 mM CaCl ₂ , pH 7.9) with deoxyribonuclease (deoxyribonuclease, 5unit) is removed by reaction. Further, to 11 μl of an aqueous solution containing the extracted RNA (1 μg), 4 μl of a 5 x reaction solution (250 mM Tris-HCl, 200 mM KCl, 30 mM MgCl ₂ , 50 mM dithiothreitol) was added, and the pH was 8.3. 2 μl of deoxyribonucleotide mixture (10 mM dATP, dGTP, dCTP, dTTP), 1 μl of oligo dT primer (50 pmol/μl), 1 μl of ribonuclease inhibitor (RNase) The inhibitor, 20 units/μl), 1 μl of reverse transcriptase (RT, Reverse Transcriptase, 50 units/μl) was brought to a total amount of 20 μl, and then reacted at 37 ° C for 1 hour, whereby complementary DNA (cDNA) was synthesized.

(3) Instant polymerization enzyme chain reaction

The single-stranded cDNA synthesized by the RT reaction was used as a template, and a human hyaluronic acid synthase (HAS) primer set (QuantiTect (registered trademark) Primer Assays, purchased from QIAGEN) and a human type I collagen α1 were used. Sex primers, primers specific for human elastin, and primers specific for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for immediate polymerase chain reaction. That is, the RT reaction solution was diluted 40-fold with sterilized distilled water, 3 μl of sterilized distilled water, 6.25 μl of SYBR (registered trademark) Premix Ex Taq ^TM II (purchased from Takara Bio Co., Ltd.), and 1.25 μl of HAS primer were added to 2 μl of the diluted solution. The total amount of the kit was 12.5 μl, and 45 cycles were performed using Thermal Cycler Dice (registered trademark) Real time System (manufactured by Takara Bio) (1 cycle: 5 seconds at 94 ° C; 30 seconds at 60 ° C) PCR reaction.

For human type I collagen alpha 1, human elastin or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), add 0.625 μl of each synonymous primer (10 μM each; serial number 1, 3, 5) and 0.625 μl The antisense primers (10 μM each; serial number 2, 4, 6) 0.625 μl were used instead of the HAS primer set and were carried out in the same manner.

As a result, GAPDH was used as an internal standard gene, and relative quantification was performed by a comparative Ct method (ΔΔCt method). The ΔΔCt method is a method for calculating the initial amount based on the number of cycles required to increase the amount of DNA product to a certain amount, and the initial amount of complementary DNA (cDNA) to be a PCR template can be expressed as a loop. number. In this method, the difference of the gene amount (2 ^{- ΔΔCt} ) which is twice as large as the detection difference of one cycle is used. Therefore, compared with the gene (internal standard gene) which is the standard in the ΔΔCt method, the amount of the unknown sample can be relatively quantified based on the increase or decrease in the number of cycles of the unknown sample.

Except for the purchased human HAS (catalog number QT00027510) primer set, the primers used are as follows.

Human type I collagen alpha 1 (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 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 primer (SEQ ID NO: 6), 5'-GGCAACAATATCCACTTTACCAGA-3'.

These primers were made based on the synthesis of Operon Biotechnologies Co., Ltd.

Further, the immediate polymerase chain reaction may be carried out according to the PCR method described below without using QuantiTect (registered trademark) Primer Assays. That is, 5 μl of 10 x PCR reaction solution (100 mM Tris-HCl, 15 mM MgCl ₂ , 500 mM KCl, pH 8.3) and 2 μl of deoxyribonucleoside were added to the cDNA-containing solution (2 μl) after the RT reaction. Deoxyribonucleotide mixture solution (containing 10 mM dATP, dGTP, dCTP, dTTP), 2 μl of synonymous primer (10 μM), 2 μl of antisense primer (10 μM), 0.5 μl of Taq DNA polymerase (100 units/μl), 36.5 μl of sterilized distilled water was brought to a total amount of 50 μl, and a PCR method was carried out. 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 was separated by electrophoresis using a 2 wt% agarose gel containing 0.5 μg/ml of ethidium bromide, and the synthesized DNA was subjected to ultraviolet irradiation. Compare quantitative.

In addition, the human hyaluronic acid synthetase (HAS) primer can be implemented using the following sequence of primers instead of the set of primers:

HAS synonymous primer (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 various treatments was performed by Fisher's multivariate dispersion analysis.

4. Experimental results

(1) It has been clarified that in normal human fibroblasts, Maitake extract promotes HAS mRNA expression in a concentration-dependent manner (Fig. 1). Ie, HAS The amount of mRNA was significantly increased by 5.4-fold in the Maipu extract of 100 μg/ml, 22.4-fold in the Maitake extract of 200 μg/ml, and 23.0-fold in the Maitake extract of 400 μg/ml. In addition, TGF-β (20 ng/ml) also promoted HAS mRNA expression by 9.3-fold.

(2) As shown in Fig. 2, it can be judged that the Maitake extract has no effect on the gene expression of the type I collagen α1 chain. On the other hand, TGF-β (20 ng/ml) significantly promoted its mRNA expression (2.4-fold).

(3) As shown in Fig. 3, it can be understood that Maitake extract has no effect on the expression of elastin mRNA. On the other hand, TGF-β (20 ng/ml) significantly promoted its mRNA expression (4.9-fold).

5. Inspection

For the first time, in normal human skin fibroblasts, Maitake Extract has no effect on the gene expression of collagen and elastin in skin structure proteins, but it can promote the expression of the hyaluronic acid synthase, the HAS gene, which imparts moisturization to the skin. Therefore, it is strongly stated that the Maitake extract is a bioreactor that promotes hyaluronic acid as an endogenous moisturizing factor (refer to Carruthers J, Carruthers A. Hyaluronic acid gel in skin rejuvenation. J. Drugs Dermatol. 5:959-964.2006.) A hyaluronic acid production promoting substance (hyaluronic acid production promoter) synthesized to enhance skin moisturization. Further, it is expected that the Maitake extract has an anti-aging effect which is activated by acting on the fibroblasts to exert hyaluronic acid which is reduced from the internal supplement with an increase in age. Furthermore, it is expected that the hyaluronic acid production promoting action of the Maitake mushroom extract can be effective for the prevention and treatment of osteoarthritis, rheumatoid arthritis, and blinking caused by reduction of hyaluronic acid.

[Embodiment 2] experimental method 1. Preparation of separated components from the Maitake extract prepared in Example 1.

The Maitake extract prepared in Example 1 was extracted by column chromatography using a normal phase tantalum gel (BW-300, manufactured by Fuji Silysia Chemical Co., Ltd.; using an open tubular column made of glass having an inner diameter of 35 mm × 280 mm). The material (8.88 g) was dissolved in 15 ml of chloroform-methanol (19:1), and the resulting solution was added to the normal phase tantalum gel, and then the mobile phase was changed sequentially (chloroform-methanol (19:1, using 1000 ml) → Chloroform-methanol (9:1, using 800 ml) → chloroform-methanol (4:1, using 800 ml) → methanol alone (using 1000 ml) was separated, and about 60 ml was taken out. The first dissolved fraction taken out was taken as Fr1, and all 60 dissolved fractions were separated. Next, a part of all the separated fractions (Fr1 to Fr60) were taken out, and a developing solvent (chloroform-) was used in a thin layer of tannin (TLC) plate (10 cm × 10 cm, Silica gel 60 F254 # 1.05715.0009, manufactured by Merck). Methanol (9:1)) was spread (expansion distance: 8 cm). The separated components were detected by subjecting the TLC plate to UV (254 nm) irradiation, spraying 10% sulfuric acid reagent, and further heating. As a result, the following five parts (M1 to M5) composed of the same component group were obtained (Table 1). That is, the eluted fraction having a relative mobility (Rf value) of 0.98 ≧ Rf value ≧ 0.50 is classified as M1, and the eluted fraction having an Rf value of 0.45 ≧ Rf value ≧ 0.29 is classified as M2, and the Rf value is 0.28 ≧ Rf value ≧ The dissolved fraction of 0.16 was classified as M3, the eluted fraction having an Rf value of 0.13 ≧Rf value ≧0.07 was classified as M4, and the eluted fraction having an Rf value of 0.07>Rf value ≧0 was classified as M5. In addition, for each separate part, It is calculated as a yield after drying under reduced pressure by a wind bag elimination method. Further, each component was adjusted to 400 mg/ml with 100% EtOH and added to the cell culture system.

Example 3 experimental method 1. Preparation of Separation Component of Maitake Extract Separation Part 5 (M5) Prepared in Example 2

The maitake prepared in Example 2 was obtained by column chromatography using a normal phase tantalum gel (BW-300, manufactured by Fuji Silysia Chemical Co., Ltd.; using an open tubular column made of glass having an inner diameter of 40 mm × 300 mm). The extract fraction 5 (M5) (1.2 g) was dissolved in 20 ml of chloroform-methanol-water (4:1:0.1), and the resulting solution was added to the normal phase gel, and then the mobile phase was changed sequentially (chloroform). - Methanol-water (4:1:0.1, using 1700 ml) - only methanol (using 300 ml) was separated, and about 50 ml was taken out separately. The first dissolved fraction taken out was taken as Fr1, and all 38 dissolved fractions were separated. Next, a part of all the separated fractions (Fr1 to Fr38) was taken out and placed on a thin layer of tannin (TLC) plate (10 cm × 10 cm, Silica gel 60F254 #1.05715.0009 (manufactured by Merck) was developed using a developing solvent (chloroform-methanol-water (4:1:0.1)) (expansion distance: 8 cm). The TLC plate was irradiated with UV (254 nm), and then sprayed with 10% sulfuric acid reagent, followed by heating, thereby detecting 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 elution fraction having a relative mobility (Rf value) of 0.32 ≧ Rf value ≧ 0.12 is classified as M5-1, and the elution fraction having an Rf value of 0.24 ≧ Rf value ≧ 0.11 is classified as M5-2, and the Rf value is 0.39. The dissolved fraction of ≧Rf value ≧0.11 is classified as M5-3, the eluted fraction having Rf value of 0.39≧Rf value ≧0.15 is classified as M5-4, and the dissolved fraction having Rf value of 0.20≧Rf value ≧0.15 is classified as M5- 5. The dissolved fraction having an Rf value of 0.19 ≧Rf value ≧0.05 is classified as M5-6, and the dissolved fraction having an Rf value of 0.11 ≧Rf value ≧0.05 is classified as M5-7, and the solvating fraction having an Rf value of 0 is classified as M5-8. Further, the respective separated components were calculated as a yield after drying under reduced pressure by a wind bag elimination method. Further, each component was adjusted to 200 mg/ml with 100% EtOH and added to the cell culture system.

Example 4 Culture and treatment of human fibroblasts

Normal human fibroblasts NB1RGB (RCB0222) were purchased from the BioResource Center of the Institute of Physical Chemistry (German) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS). The treatment of the Maitake extract and its fractions (M1 to M5, M5-1 to M5-8) were carried out in the absence of serum. That is, 0.2% (v/v) containing Maitake extract (25-400 μg/ml), its separated component (100-400 μg/ml), or interleukin 1α (10 ng/ml, manufactured by DS Pharma Biomedical) Confluent human fibroblasts cultured in 24 well multiplate or 60 mm dish were treated for 24 or 48 hours in lactobumin hydrolysate (LAH)/DMEM.

Further, in order to investigate the regulation of HA (hyaluronic acid) biosynthesis, the Maitake extract treatment was carried out in the same manner in the presence of the HAS inhibitor 4-methylumbelliferone (4MU) (0.1 to 1 mM, manufactured by Sigma Aldrich Co., Ltd.).

2. Real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) (1) Extraction of total RNA

The quantification of total RNA and total RNA extracted from the cells was carried out in the manner described in 3 (1) of Example 1.

(2) Reverse transcriptase reaction

Using the total RNA obtained, it is recorded in 3(2) of Example 1. The single-stranded cDNA was synthesized in a manner.

(3) Instant polymerization enzyme chain reaction

The single-stranded cDNA synthesized by the RT reaction was used as a template, and human procollagenase 1 (procollagenase1/proMMP-1 primer set (QuantiTect (registered trademark) Primer Assays, catalog number QT00014581, purchased from QIAGEN), human Tissue Matrix Metalloproteinase Inhibitor-2 (TIMP-2) primer set (QuantiTect (registered trademark) Primer Assays, catalog number QT00017759, purchased from QIAGEN), and human glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) (SEQ ID NO: 5, 6) is a specific primer, which is attached to SYBR (registered trademark) Premix Ex Taq ^TM II (purchased from Takara Bio Co., Ltd.) in the manner described in 3 (3) of Example 1. In the operation method, PCR was carried out for 45 cycles (1 cycle: 5 seconds at 94 ° C and 30 seconds at 60 ° C), thereby increasing the respective cDNAs. As a result, GAPDH was used as an internal standard gene, and ΔΔ was used. The Ct method is used for relative quantification.

For the PCR performed on human procollagenase 1 (proMMP-1) and human tissue matrix metalloproteinase inhibitor-2 (TIMP-2), the above primer set was used instead of the 3 of Example 1 ( The human HAS primer set in 3) was carried out in the same manner as in 3 (3) of Example 1. In addition, 0.625 μl of the following synonymous primers (10 μM each; SEQ ID NO: 9, 11) and 0.625 μl of antisense primers (10 μM each; SEQ ID NO: 10, 12) can be added instead of the primer set. 3(3) of Example 1 was carried out in the same manner.

Human pro MMP-1 primer, which can be replaced with the following sequence of primers The implementation of the set of primers:

proMMP-1 synonymous primer (SEQ ID NO: 9): 5'-GGTGATGAAGCAGCCCAG-3'

proMMP-1 antisense primer (SEQ ID NO: 10): 5'-CAGTAGAATGGGAGAGTC-3'

(See 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 the following sequence of primers instead 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'

(See Hirata, M., Sato, T., Tsumagari, M., Shimada, A., 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 method of Johnson-Wint and Hollis (see Johnson-Wint, B., and Hollis, S. A rapid in situ deoxyribonucleic acid assay for determining cell number in culture and tissue. Anal. Biochem. 122: 338-344, 1982.) to determine the amount of DNA in cells. Namely, at each treatment, the cells treated in the above 1 were recovered using 0.25% (w/v) trypsin/0.02% (w/v) EDTA solution (1.5 ml). The obtained cell suspension was ultrasonically treated with a closed ultrasonic cell disrupting apparatus (manufactured by Cosmo Bio Co., Ltd.) for 200 minutes and for 6 seconds in an ice bath to prepare a sample solution. The sample solution (100 μl) was mixed with 160 μl of a solution of 3,5-diaminobenzoic acid dihydrochloride (400 mg/ml) (manufactured by Sigma Aldrich Co., Ltd.) at 60 ° C. The reaction was carried out for 45 minutes. After completion of the reaction, 1.5 ml of 1N hydrochloric acid was added, and the fluorescence intensity (excitation wavelength: 430 nm, fluorescence wavelength: 535 nm) in the sample solution was measured by a microdisk analyzer (Infinite F200, manufactured by Tecan Japan). In addition, the amount of DNA was calculated from a calibration curve prepared in the same manner using DNA obtained from a squid testis (manufactured by Wako Pure Chemical Industries, Ltd.).

4. HA quantification

The amount of HA in the culture solution recovered after 24 or 48 hours of culture of the above 1 was quantified by an HA ELISA kit (purchased from Biochemical Industries, Inc.) in accordance with the attached operation method. That is, the HA in the culture solution of each of the recovered treatments was combined with hyaluronic acid-binding protein (HABP) immobilized on the microtiter plate attached to the purchased kit, and then peroxidase was added in combination with HABP to cause a reaction. Next, add tetramethylbenzidine and hydrogen peroxide, using microdisk analysis The instrument (Infinite F200, manufactured by Tecan Japan Co., Ltd.) measured the absorbance at 450 nm. 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). Alternatively, the amount of HA generated per unit cell in each treatment is expressed as a value (ng/μg 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/μg DNA).

Alternatively, the HA ELISA kit may be used instead of performing HA quantification according to the following method. In other words, the recovered culture solution was added to 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) to bind biotin to rHABP (Cosmo). Bio manufactured by Bio) is further combined with the combined HA. Next, a peroxidase is added to bind avidin to form a biotin-avidin complex. Further, tetramethylbenzidine and hydrogen peroxide were added, and the absorbance at 450 nm was measured by a microdisk analyzer (Infinite 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

Trichloroacetic acid having a final concentration of 3.3% (w/v) was added to the cell culture solution recovered after 24 or 48 hours of the above culture, and allowed to stand overnight at 4 ° C to insolubilize the protein. The insoluble protein was precipitated by centrifugation (10,000 rpm, 10 minutes), and then the precipitate was dissolved in an SDS-PAGE sample buffer containing 1% (v/v) 2-mercaptoethanol. Protein electrophoresis analysis (SDS-PAGE) was performed in a 12.5% (w/v) polyacrylamide gel. After the completion of the electrophoresis, the protein in the gel was transcribed on the nitrocellulose membrane, and the nitrocellulose membrane was immersed in a blocking solution [5% (w/v) skim milk powder according to a conventional method ( In a fatty-free dry milk/80 mM Na ₂ HPO ₄ /100 mM NaCl/0.1% (v/v) Tween-20 (pH 7.5)], blocking was performed for 1 hour. After washing with distilled water and PBS-T buffer [80 mM Na ₂ HPO ₄ /100 mM NaCl/0.1% (v/v) Tween-20 (pH 7.5)] for 2 to 3 times, it was immersed in 1% (w/ v) BSA/PBS(-) diluted primary antibody [sheep anti-(human proMMP-1) or anti-(human TIMP-2) IgG, respectively, provided by Prof. Yongsui Xiu Ming from the Kennedy Rheumatic Institute of King's College London] In the solution, slowly shake 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] The goat anti-(sheep IgG) IgG, manufactured by Sigma Aldrich Co., Ltd. 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 Co., Ltd.) of the ECL-Western Blotting Detection kit for accurate reaction for 1 minute. After the reaction, the amount of proMMP-1 and TIMP-2 was quantified using Lumino Image Analyzer LAS-1000plus (manufactured by Fuji Flim Co., Ltd.).

The Western blotting method is a well-known experimental method of the industry, and is described in detail in, for example, the Kotani Michiko Protein Experiment Manual (edited by Takehisa Josuke). In pages 152-157, 2003 and other well-known experimental books.

Further, it is also possible to carry out the above antibody using a commercially available product without using the above antibody.

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

Peroxidase-conjugated rabbit anti-(mouse IgG) IgG (manufactured by Sigma Aldrich)

6. Statistical processing

A significant difference check between the various treatments was performed by Fisher's multivariate dispersion analysis.

7. Experimental results

(1) Normal human fibroblasts NB1RGB (RCB0222) constantly produced HA, while Maitake extract (25-100 μg/ml) enhanced HA production (Fig. 4). That is, the amount of HA was 15.37±1.56 μg/μg of DNA in the untreated group, whereas 24.79±2.75 μg/μg of DNA in the 25 μg/ml of Maitake extract, and the Maitake extract at 50 μg/ml. The medium was 26.08 ± 2.19 μg / μg DNA, and it was 20.57 ± 1.46 μg / μg DNA in 100 μg / ml of Maitake extract. The HA production enhanced by the Maitake extract (100 μg/ml) was inhibited in a concentration-dependent manner by the HAS inhibitor 4-methylumbelliferone (0.1 mM mM). Maitake extract promotes HA production by promoting HAS gene expression.

(2) In order to identify the elements of Maitake extract which contribute to the promotion of HA production, the regulation of HA production of the separation component of Maitake extract was studied. As a result, a concentration-dependent HA production promoting action was detected in the M1 and M5 separated portions (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 was 580.21±70.65 ng/ml in the untreated group, and 883.49±159.21 ng/ml in M1 at 25 μg/ml, and 1237.28±405.80 ng/ml in M1 at 50 μg/ml. , 1488.19±527.23 ng/ml in 100 μg/ml of M1; 1041.44±186.71 ng/ml in 25 μg/ml of M5, and 1587.83±298.10 ng/ml in 50 μg/ml of M5, at 100 μg/ml The M5 is 2304.25 ± 499.97 ng / ml. Further, the M2 to M4 separation portion promotes HA production at a low concentration, but inhibits HA production at a high concentration.

(3) In order to study the specificity of Maitake extract to promote HA production, the known ECM metabolism-related genes, namely proMMP-1 and TIMP-2, which are consistently expressed in the normal human fibroblasts of Maitake extract and its fractions. The role of gene expression was studied. Maitake extract promoted the expression and production of proMMP-1 mRNA in a concentration-dependent manner (A of Figure 6 and A of Figure 7). However, Maitake extract has no effect on the expression 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 proMMP-1 mRNA (Fig. 6A). An increase in the expression of the proMMP-1 gene was observed in M2 to M4 (Fig. 6A). For TIMP-2 mRNA expression, the M1 fraction showed a tendency to inhibit, but There is almost no change in some of the separated parts (B of Figure 6). Therefore, it is suggested that in the promotion of HA production and the expression of proMMP-1 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 Maitake extract, the HA component was adjusted for the eight components (M5-1 to M5-8) in which the M5 fraction was further separated. the study. As a result, an effect of promoting HA production was observed in the M5-3 to M5-8 separated portion.

8. Inspection

In normal human skin fibroblasts, Maitake Extract promotes the synthesis of HA that imparts moisturization to the skin. Therefore, it is indicated that the Maitake extract promotes skin activation and improvement due to synthesis of an important moist component, that is, HA, for example, improving the moisture retention function of the skin. Further, it is explained that the action of the Maitake extract to promote the production of HA may be regulated 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 due to excessive MMP production is enhanced, and skin roughening (skin aging) such as wrinkle formation is caused. For the first time, Maitake extract promotes proMMP-1 mRNA and its production in human skin fibroblasts.

However, it is interesting to prove 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. Approved to show that HA production is promoted The M5 fractions that proceeded were M5-6 and M5-7. In other words, it is expected that the components of the Maitake extract can be separated and used to reduce the skin aging effect, and the characteristics of the skin moisturization can be further enhanced.

It is strongly suggested that Maitake extract is a material derived from novel natural materials that enhances skin wettability by promoting the biosynthesis of an endogenous moisturizing factor, HA. Further, it is expected that the Maitake extract can also exert the following anti-aging effect: by acting on the fibroblasts to activate them, the HA which is reduced with age is replenished from the inside.

[Example 5]

A cosmetic cream containing Maitake extract was prepared in the following amount.

[Embodiment 6]

A medical cream containing Maitake extract was prepared in the following amount.

[Embodiment 7] Effect of Maitake Extract on human ecdysis

The effect on ecdysis was studied using a cream containing 2% by weight of Maitake extract as shown in Example 5.

experiment method

Subjects: 12 patients (76 to 97 years old) with 6 patients (76 to 97 years old) with symptoms of desquamation (symptoms of skin becoming horny and falling off) in ecchymosis in the elderly health facility "Goodwell" (6 males and 6 females) )

Implementation time: December 2006 ~ January 2007

Method: The skin condition of the subject was evaluated in three stages (3: severe, 2: moderate, 1: mild) based on desquamation (the symptom that the skin became horny and shedding), and The cream of the Maitake extract is applied to the skin of the subject in an appropriate amount. The application site was the right lower arm, the left lower arm, the lower right leg, and the lower left leg, and evaluation was performed at each site.

result

In the following, the degree of diagnostic evaluation of the skin condition of the subject from one week after the application (3: severe illness, 2: moderate illness, 1: mildness) is shown. In addition, a diagnostic evaluation degree of 0 indicates complete cure.

Testee 1

Testee 2

Testee 3

Testee 4

Testee 5

2007/1/12 0 0 2 2

Testee 6

Testee 7

Testee 8

Testee 9

Subject 10

Testee 11

Subject 12

As described above, the degree of diagnosis evaluation of the skin condition of the subjects 1 to 12 after the start of application was improved. In addition, the remarkable symptom-improving effect obtained by applying the cream containing the Maitake mushroom extract was found 1 to 3 days after the application.

Fig. 9 is a photograph of a skin of a subject 5 containing a Maitake extract cream before and after treatment. By applying a cream containing Maitake extract Treatment, and found improvement in suede symptoms.

Moreover, these results show that a cream having a content of 2% by weight of Maitake extract has a therapeutic effect on ecdysis.

[Industrial availability]

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 or a medicament for treating skin abnormalities, injuries or diseases caused by a decrease in hyaluronic acid production, and for treating joint or eye abnormalities, A medicine for injury or disease.

Figure 1 shows the promotion of the gene expression of hyaluronic acid synthase (HAS) in human skin fibroblasts by Maitake Extract. *: Relative to the untreated group (p < 0.05).

Figure 2 shows the effect of Maitake extract on the expression of type I collagen alpha 1 mRNA in human skin fibroblasts. ***: Relative to the untreated group (p < 0.001).

Figure 3 shows the effect of Maitake extract on the expression of elastin mRNA in human skin fibroblasts. *; relative to untreated population (p < 0.05).

Figure 4 shows the promotion of hyaluronic acid (HA) production by human skin fibroblasts by Maitake Extract. 4-methylumbelliferone (4MU, 4-methylumbelliferone, HAS inhibitor). * and **: relative to untreated groups (p < 0.05 and 0.01). ##和## #: The treatment group (p<0.01 and 0.001) relative to Maitake extract (100 μg/ml).

Fig. 5 shows the promotion of hyaluronic acid (HA) production by human skin fibroblasts in the separated fraction (M1 to M5) of Maitake extract. IL-1 (interIeukin 1, 10 ng/ml). * and **: relative to untreated groups (p < 0.05 and 0.01).

Figure 6 shows the effect of Maitake extract and its fraction on the expression of proMMP-1 and TIMP-2 genes in human skin fibroblasts. Human fibroblasts were treated with Maitake extract or its fraction (M1~M5) (n=1/treated) for 24 hours. The proMMP-1 (A) and TIMP-2 mRNA expression (B) in the recovered cells were quantified. Maitake extract promotes proMMP-1 mRNA expression, but conversely inhibits TIMP-2 gene expression. M5 had little effect on proMMP-1 and TIMP-2 mRNA expression. On the other hand, M2 and M3 promoted the expression of the proMMP-1 gene, and M1 inhibited the expression of the TIMP-2 gene. Furthermore, interleukin 1α (IL-1) (10 ng/ml) promoted proMMP-1 mRNA expression but had no effect on TIMP-2 gene expression.

Figure 7 shows the promoting effect of Maitake extract on the production of proMMP-1 (A) and TIMP-2 (B) in human skin fibroblasts. IL-1 (inkerleukin 1, 10 ng/ml). * and **: relative to untreated groups (p < 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 (M5-1 to M5-8) (n = 1/treatment). In M5-3 to M5-8, an increase in the amount of hyaluronic acid was observed. IL-1 (interleukin 1, 10 ng/ml).

Fig. 9 is a photograph of a skin of a subject 5 using a cream containing a Maitake extract before and after treatment.

Claims (9)

A Maitake mushroom extract characterized in that: the Maitake extract is extracted from dried maitake with pure ethanol, and developed in a thin layer of silicone using chloroform-methanol (9:1) to obtain 0.98 ≧. Maitake extract with Rf value ≧0.50 and/or 0.07>Rf value ≧0. The maitake extract according to claim 1, wherein the dry maitake has a moisture content of 8 wt% or less. The Maitake mushroom extract according to claim 1 or 2, wherein the pure ethanol contains 99.0 vol% or more of ethanol. The maitake extract according to claim 1 or 2, wherein the pure ethanol is 100 to 1000 parts by weight with respect to 100 parts by weight of the dried maitake. The Maitake mushroom extract according to claim 3, wherein the pure ethanol is 100 to 1000 parts by weight with respect to 100 parts by weight of the dried Maitake. A hyaluronic acid production promoter comprising the Maitake mushroom extract according to any one of claims 1 to 5. A composition comprising the hyaluronic acid production promoter according to item 6 of the patent application. The composition of claim 7, which is in the form of a cosmetic. The composition according to claim 7 of the patent application, which is in the form of a pharmaceutical product.