KR100438009B1 - A cosmetic composition containing an extract of Grifola frondosa - Google Patents

Abstract

The present invention relates to a cosmetic composition having an anti-aging effect and a whitening effect. The present invention relates to a mycelium or a culture medium of a leaf mushroom ( Grifola frondosa ) having an antioxidant effect, a skin cell proliferation and a collagen production promoting effect, and a skin melanogenesis inhibitory effect. It is characterized by containing an extract of molecular weight 12000 or more obtained in an amount of 0.00001 to 30% by weight based on the total amount of the cosmetic composition.

Description

A cosmetic composition containing an extract of leaf mushroom mycelium {A cosmetic composition containing an extract of Grifola frondosa}

The present invention utilizes the mycelium extract obtained by the liquid culture of the leaf mushroom ( Grifola frondosa ) or the polysaccharide of the extracellular polymer material prepared from the culture medium thereof, the effect of promoting skin cell proliferation and skin function activity, the inhibitory effect of skin melanogenesis and the antioxidant effect It is to provide a cosmetic composition having an excellent anti-aging effect and whitening effect of the leaf extract mushrooms.

Skin is the largest tissue in the human body and is a primary protective film that protects against stimulation of the external environment and prevents the loss of biological components while preventing the invasion of harmful substances into the skin. When chemical force is applied from the outside, the sebaceous membrane is first protected, and the stratum corneum is secondary, and in severe cases, the collagen fiber of the dermis is a system to protect. However, if the external physical, chemical stimulation, stress, nutritional deficiency, etc. are severe, the normal function of the skin is lowered, the aging of the skin is promoted and the skin is damaged. Efforts have been made to effectively suppress skin aging by maintaining existing skin functions and activating skin cells by adding existing bioactive materials using animals and plants and microorganisms to cosmetics.

As part of this research, industrial applications of polysaccharides produced by higher fungi mushrooms have been applied as functional additives for moisturizing and skin improvement (Critical Rev in Immun 19, 65-96). Because mushrooms absorb and use nutrients from organic substances containing various nutrients, the mushrooms contain a wide variety of substances, and most of them are composed of polysaccharides, which are functional substances such as biopolymers. These polysaccharides act as a protective barrier against toxic substances and heavy metals, and also maintain moisture in dry areas. Bioactive substances such as Cordyceps sinensis, Ganoderma lucidum, Shiitake, Cloud mushroom, and Situation mushroom are known as medicinal mushrooms. It is known to have excellent whitening or antioxidant function by blocking function.

Cosmetic compositions using the extracts of mushrooms known so far include Snow Cordyceps sinensis (Korean Patent No. 2000-0018909), Situary Mushroom, Skirt Mushroom (Korean Patent No. 2000-0055724, 1999-0076537), Shiitake Mushroom, etc. Research on the anti-aging effect has been continued.

Although mushrooms used for medicinal and edible foods have numerous examples and kinds, mushrooms that are being used and studied as cosmetic compositions are still known as a part. However, until now, it has not been identified which substance using the leaf mushroom extract has a skin anti-aging effect and a whitening effect as a cosmetic.

Therefore, the inventors of the present invention, while researching on natural substances with low irritation to skin and excellent skin cell activity and collagen synthesis and proliferative effect, mycelia extract of Grifola frondosa , which has excellent anticancer and antibacterial effects, has an excellent effect on preventing skin aging. The present invention has been completed.

In addition, the present inventors have found that in producing a polysaccharide obtained by liquid culture of the mycelium of leaf mushrooms, it is possible to produce polysaccharides using a simple complex medium, and to produce even higher polysaccharides using an optimized synthetic medium. The invention was completed.

Accordingly, it is an object of the present invention to provide a cosmetic composition having a whitening effect by inhibiting skin cell activation, collagen synthesis proliferation and melanogenesis, which have an anti-aging effect on the skin containing leaf mushroom mycelium extract.

In order to achieve the above object, the cosmetic composition having an anti-aging effect and a whitening effect according to the present invention is characterized in that it contains an extract obtained from the leaf mycelium mycelium or its culture solution.

Another object of the present invention is to provide a method for preparing a cosmetic composition from a polysaccharide obtained by liquid culture of the mycelium of leaf mushrooms.

In the present invention, leaf mycelium mycelium extract or extract obtained from mycelium culture medium is obtained by centrifugation of the precipitate obtained by adding alcohol to the hydrothermal extract or mycelium from which hot water extract is obtained by adding distilled water to the mycelium at high temperature. It is obtained by filtering low molecular material.

Hereinafter, the configuration of the present invention in more detail.

Leafy mushrooms ( Grifola frondosa Fr) are taxonomically belonging to Aphllophorales, Polyporaceae , and Grifola . The fungus nucleus of leaf mushroom is used as a medicinal herb because of antipyretic, edema, anti-tumor, etc. In addition to the necessary ingredients, it contains abundant flavor ingredients and is excellent in its taste and form. In addition, the chemical structure of beta glucan, the major polysaccharide of leaf mushrooms, has been reported to be the most potent component of stimulating cellular immune responses, unlike the structure of beta glucan found in other medicinal mushrooms (Chem Pharm Bull, 34 (4), 1709-1715, Nippon Shokuhin Kogyo Gakkaishi, 41 (10), 733-740). In addition, leaf mushrooms are known to be effective in lowering blood pressure, diabetes, obesity, reducing blood cholesterol, antibacterial, diuretic, tonic, anti-anemia, and pulmonary bleeding as well as anti-cancer activity (Critical Review in Immunology, 19, 65-96).

Leaf mycelium mycelium having the pharmacological effect in the present invention can be obtained by incubating the mycelium obtained by germinating spores obtained from the fruiting body of the leaf mushroom in a solid medium or a liquid medium. However, mycelial culture by solid medium has a long culture period and low mycelial yield. In addition, in the case of using a solid medium, the culture of the mycelium is not uniformly performed, and there is a possibility that the media components are mixed when the mycelium is extracted.

Referring to the liquid medium culture method of the leaf mycelium mycelium of the present invention.

That is, after washing the spores several times with sterile distilled water, smeared in yeast-wheat agar medium (3 g of yeast extract, 3 g of wort, glucose 10 g, peptone 5 g, agar 20 g, distilled water 1 L) The mycelium is obtained by incubating at 30 DEG C, preferably 25 DEG C for 5 to 15 days, preferably 7 to 8 days. Mycelium is inclined in a test tube containing yeast-wort agar medium, stored at low temperature, preferably 4 ~ 10 ℃ and subcultured every month to use.

Aseptic homogenize mycelia grown on slope media and inoculate them into liquid media. Preferably inoculated at a concentration of 3 to 10% (v / v).

In the 1) step of culturing mycelia grown on a slope medium into a liquid medium, the liquid medium of the simple complex medium (PMP) is 1-5% of potato-glucose medium (Potato dextrose broth), preferably 2.4%, and malt (Malt). A nutrient medium containing 0.5-2%, preferably 1.0%, and 0.1-0.5%, preferably 0.1%, of peptone is preferred in terms of mycelial growth and polysaccharide production.

In the liquid culture step 2) of the leaf mycelium mycelium, as a liquid medium, glucose is 3-6%, preferably 5%, yeast extract 0.5-1.0%, preferably 0.6%, polypeptone 0.1-0.5%, preferably 0.2 %, Magnesium sulfate (MgSO ₄ 7H ₂ O) 0.01 to 0.10%, preferably 0.05%, potassium monophosphate (KH ₂ PO ₄ ) 0.01 to 0.10%, preferably 0.05%, potassium diphosphate (K ₂ HPO ₄ ) Optimal nutrient medium containing 0.01 to 0.10%, preferably 0.05%, and manganese sulfate (MgSO ₄ 5H ₂ O) 0.01 to 0.05%, preferably 0.02%, is a simple complex culture method in terms of mycelial growth and polysaccharide production. It is more preferable than when performed.

Liquid culture of the mycelium is carried out in the medium of the optimum nutrient medium and the simple complex medium at a temperature of 20 to 28 ℃, preferably 25 ℃, pH 2.0 to 9.0, preferably pH 5.5, rotational speed 100 to 300 rpm, preferably 150 rpm, barrel The culture is carried out for 7 to 12 days under the same fermentation tank conditions of 1.0 to 3.0 vvm, preferably 2.0 vvm.

The culture solution from which the mycelium is removed is left overnight at 4 ° C. with 3-7 times the amount of alcohol, preferably ethyl alcohol. The resulting precipitate is collected by centrifugation for 5 to 60 minutes at 7000 to 20,000 rpm. The collected precipitate is dissolved in distilled water and filtered using a high molecular weight ultrafiltration membrane to remove low molecular weight material, followed by drying, preferably lyophilization, to obtain samples GPC-1 and GPS-1. Here, GPC-1 refers to a sample obtained by ethyl alcohol precipitation of leaf mushroom mycelium culture medium using a simple complex medium, and GPS-1 refers to a sample obtained by ethyl alcohol precipitation of leaf mushroom mycelium culture medium using an optimized synthetic medium. .

The mycelium obtained by filtration of the culture solution is washed with water, 1 to 5 times the amount of distilled water is added, hot water is extracted for several hours at a high temperature, and the filtration process is repeated several times. The filtrate, from which the residue was removed, was filtered using a high molecular weight ultrafiltration membrane to remove the low molecular substance, and then left overnight at 4 ° C. with 3-7 times the amount of alcohol, preferably ethyl alcohol. The resulting precipitate is collected by centrifugation at 7,000-20,000 rpm for 5 to 60 minutes and dried, preferably lyophilized to obtain samples GPC-2 and GPS-2. Here, GPC-2 refers to a sample obtained by extracting a mycelium by cultivating leaf mushrooms using a simple complex medium, and GPS-2 refers to a sample obtained by extracting mycelium by cultivating leaf mushrooms using an optimized synthetic medium.

Thus obtained mycelial extracts of leaf mushrooms GPC-1, GPS-1 and extracts of the culture medium GPC-2, GPS-2 has excellent skin cell proliferation effect, collagen production effect and antioxidant effect and melanin production inhibitory effect It contains a large amount of polysaccharide.

Leaf mushroom mycelium obtained by the above method or the extract obtained from the culture solution thereof may be contained in an amount of 0.00001 to 30% by weight relative to the total weight of the composition in the cosmetic composition having the skin anti-aging and whitening effect of the present invention. If it is less than 0.00001% by weight of the total weight can not exhibit the function of anti-aging and whitening, etc., if it exceeds 30% by weight, the degree of improvement of the function against the increase of the amount of the extract is insignificant.

The formulation of the cosmetic composition having the skin anti-aging effect and the whitening effect is not particularly limited, and may be easily formulated with basic cosmetics such as supple cosmetics, nourishing cosmetics, massage creams, nourishing creams, and color cosmetics.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited only to the description of these examples.

Example 1

The leaf mycelium mycelia were incubated in a test tube containing yeast-wort agar medium (yeast extract 3 g, wort juice 3 g, glucose 10 g, peptone 5 g, agar 20 g, distilled water 1 l) and stored at 4 ° C for 1 month. Subculture was used every time.

Aseptic homogenization of the mycelia grown on the slope medium with a simple complex medium (PMP) containing 2.4% potato-glucose medium (Potato dextrose broth), 1.0% malt extract, 0.1% peptone After inoculating 5% (v / v) in the liquid medium adjusted to pH 5.5, and incubated in a fermenter for 10 days under the conditions of temperature 25 ℃, rotation speed 150rpm, aeration rate 2vvm.

After completion of the culture, the culture solution from which the mycelium was removed was added to 4 times the amount of ethyl alcohol and left overnight at 4 ° C. The resulting precipitate was collected by centrifugation at 12,000 rpm for 30 minutes. The collected precipitate was dissolved in distilled water and filtered using an ultrafiltration membrane having a molecular weight of 12,000 to remove low molecular weight substances having a molecular weight of 12,000 or less, followed by lyophilization to obtain GPC-1 (GPC-1; leaf mushroom mycelium culture medium using a simple complex medium). Sample obtained by precipitation of ethyl alcohol).

Example 2

Aseptic homogeneous mycelia of leaf mushrooms subcultured on slope medium, 5% glucose, 0.6% yeast extract, 0.2% polypeptone, 0.05% magnesium sulfate (MgSO ₄ 7H ₂ O), potassium monophosphate (KH ₂ PO ₄ ) Optimized synthetic medium containing 0.05%, potassium diphosphate (K ₂ HPO ₄ ) 0.05%, and manganese sulfate (MgSO ₄ 5H ₂ O) 0.02%. 5% (v / v) inoculated, and then incubated in the fermenter for 10 days under conditions of temperature 25 ℃, rotation speed 150rpm, aeration rate 2vvm.

After completion of the culture, the culture solution from which the mycelium was removed was obtained according to the same method as in Example 1 (GPS-1; a sample obtained by precipitating ethyl alcohol with the leaf mushroom mycelium culture medium using an optimized synthetic medium).

Example 3

After filtering the culture solution in Examples 1 and 2, the remaining mycelium was washed with water and 4 times of distilled water was added, followed by hot water extraction at 100 ° C. for 4 hours, followed by filtration three times. The filtrate, from which the residue was removed, was filtered using an ultrafiltration membrane having a molecular weight of 12,000 to remove the low molecular weight substance, and then 4 times of ethyl alcohol was added thereto and left at 4 ° C. overnight. The resulting precipitate obtained samples GPC-2 and GPS-2 (GPC-2; sample obtained by extracting the mycelium by cultivating leaf mushroom using simple complex medium, GPS-2; cultivating leaf mushroom using optimized synthetic medium Sample obtained by extracting mycelium).

Test Example 1 Antioxidant Effect of Leaf Extract

In this test example, the antioxidant activity of the leaf mushroom mycelium extract obtained in Examples 1 to 3 was measured using NBT method under laboratory conditions.

In order to measure the antioxidant effect, the active oxygen produced by xanthine and xanthine oxidase is measured by the NBT method, and the effect of the test substance removing the active oxygen, that is, the active oxygen scavenging effect, is evaluated. Free radicals are produced by xanthine and xanthine oxidase. This active oxygen reacts with Nitro Blue Tetrazolium (NBT) to measure the active oxygen scavenging rate by measuring the blue color produced by this at a wavelength of 560 nm.

As the reagent used

1) 0.05M (50mM) Na ₂ CO ₃ buffer (MW = 105.99): 5.25g (50mM) of sodium carbonate (pure photochemical) is dissolved in purified water and 50mM NaHCO ₃ (MW = 84.01) solution is mixed to pH 10.2. .

2) 3mM xanthine solution: 45.6 mg of xanthine (MW = 152.11)) is dissolved in water to make 10 ml.

3) 3mM EDTA solution: Sodium ethylenediamine tetraacetate (MW = 60.1) is dissolved in distilled water to make 3mM.

4) 0.15% BSA solution: Dissolve 15mg of BSA (Fraction V, powder, Sigma) in distilled water to make 10ml.

5) 0.75 mM NBT solution: 61.32 mg of nitroblue tetrazolium (MW = 817.65, Tokyo University) is dissolved in distilled water to make 100 ml.

6) xanthine oxidase solution: Dilute xanthine oxidase (Boehringer) with distilled water about 100-fold and make the absorbance of the blank test within the range of 0.2-0.23 in the measurement operation. In other words, dilute with purified water so that the change in absorbance is A ₅₆₀ = 0.3 / 20 minutes.

7) 6mM CuCl ₂ solution: Dissolve 102.29mg of copper chloride (CuCl ₂ -2H ₂ O, MW = 134.45) in distilled water to make 100ml.

As a measuring method

0.05M Na ₂ CO ₃ ------------------------------------ 2.4ml

2.3mM xanthine solution ---------------------------------- 0.1ml

3.3mM EDTA Solution ------------------------------------ 0.1ml

4.BSA solution ---------------------------------------- 0.1ml

5.0.72 mM NBT solution ---------------------------------- 0.1ml

6. Xanthine oxidase solution ---------------------------- 0.1ml

7.6mM CuCl ₂ solution ------------------------------------ 0.1ml

① Add 1,2,3,4,5 to Bayer's bottle and add 0.1ml of sample solution to it and leave at 25 ℃ for 10 minutes.

② Add 6 and stir quickly, and start incubation for 20 minutes at 25 ℃.

③ After that, add 7 to stop the reaction and measure the absorbance St at 560nm.

④ For the blank test, measure the absorbance Bt by using distilled water instead of the sample solution as above.

⑤ Also, the blank of the sample solution is operated in the same manner using distilled water instead of 6 to measure the absorbance Bo.

The result of the effect was calculated by Equation 1, the results are shown in Table 1.

% Inhibition = [1- (St-So) / (Bt-Bo)] X 100

St: absorbance at 560 nm after enzyme reaction of sample solution

Bt: absorbance at 560 nm after enzymatic reaction of blank test solution

So: absorbance at 560 nm before reaction without enzyme addition of sample solution

Bo: absorbance at 560 nm before reaction without enzyme in blank test solution

The sample used for the test is the result of experimenting with 3% aqueous solution of each leaf mushroom extract. The results are shown in Table 1.

Leaf Mushroom Extract Antioxidative effect(%) GPC-1 84 GPS-1 96 GPC-2 75 GPS-2 80

Test Example 2 Measurement of Cell Activity Effect of Leaf Mushroom Extract (MTT assay)

The cell line was Hs-68, a mouse-derived fibroblast, and the cells were dispensed in 12-well plates at 2 x 10 ⁵ cells per well and incubated for 24 hours. Each raw material is treated at a concentration that does not cause cytotoxicity, and after 24 hours of treatment with the sample, the medium is removed, and 500ul fresh medium and 60ul MTT solution are added to each well, followed by incubation in a 37 ° C CO ₂ incubator for 2 hours. Lysate the cells and transfer 100ul of each to a 96 well plate.

Measure absorbance at an ELISA reader 565 nm. As a control, absorbance was measured by the same method as above except that GPC-1, GPS-1, GPC-2, and GPS-2 prepared in Examples were not added. The absorbance of the experimental group and the control group containing the leaf mushroom extract is shown in Table 2.

Leaf Mushroom Extract Cell growth rate (%) GPC-1 125 GPS-1 130 GPC-2 115 GPS-2 125 Control 100

Test Example 3 Measurement of Collagen Production Promoting Effect of Leaf Mushroom Extract

The cell line used in the experiment was human fibroblast (Human Dermal Fibroblast, HDF), and ELISA was performed to measure the concentration of collagen, which is a major component of the extracellular matrix, after the addition of the sample. Samples were exchanged with added serum (serum free), incubated for 12 hours, and washed three times with HDBS with PBS-T (0.02% tween-20 in PBS). 3% formaldehyde cells were fixed for 20 minutes and then blocked by treating with 5% skim milk for 1 hour. The primary antibody, monoclonal anti-collagen type IV, is reacted at 37 ° C. and 5% CO ₂ for 40 minutes. After washing three times with PBS-T, the secondary antibody (anti-mouse IgG) is diluted to 1 / 1,000 in PBS-T and reacted for another 40 minutes, and then with PBS-T Wash 5 times. Alkaline phosphatase substrate solution (1 mg / ml ρ-nitrophenyl phosphate in diethanolamine buffer) dissolved in a diethanolamine buffer as a substrate is added and reacted for 30 minutes, and then absorbance is measured at 405 nm. As a control, absorbance was measured by the same method as above except that GPC-1, GPS-1, GPC-2, and GPS-2 prepared in Examples were not added. The absorbance of the experimental group and the control group containing the leaf mushroom extract is shown in Table 3.

Leaf Mushroom Extract Collagen synthesis rate (%) GPC-1 140 GPS-1 150 GPC-2 120 GPS-2 130 Control 100

Test Example 4 Experiment of Measuring Tyrosinase Inhibitory Effect Using Mushroom Tyrosinase

This test example is to determine the whitening effect by looking at the degree of inhibition of the function of the enzyme tyrosinase (tyrosinase) to confirm the whitening effect of the leaf mushroom extract obtained in Examples 1-3.

Tyrosinase is an enzyme that accelerates the oxidation process of tyrosine in vivo and helps melanin production. In this test example, the method of inhibiting the function of this enzyme to inhibit the oxidation of tyrosine to form a black polymer called melanin (Pomerantz SH, J. Biochem., 24: 161-168, 1966) is applied. The sun whitening effect was determined.

The inhibitory effect of tyrosinase activity was investigated by using GPC-1, GPS-1, GPC-2, and GPS-2 as a sample of lettuce extract, arbutin and leaf mushroom extract.

The inhibitory activity against tyrosinase of each sample was determined by placing 15 μl of the sample into a 96 well plate, adding 150 μl of 50 mM phosphate buffer (pH6.5), 25 μl of 1.5 mM L-tyrosine solution, and then using mushroom tyrosinase (1,500 units / ml, Sigma) 10 μl was added and reacted at 37 ° C. for 20 minutes, and then the absorbance was measured at 490 nm using a microplate reader (ELx800, USA) to measure the inhibition rate against tyrosinase. Inhibition rate for tyrosinase (%) was calculated according to the following equation, IC ₅₀ value is the concentration of a substance that inhibits the tyrosinase enzyme activity by 50%.

% Inhibition = {[(D-C)-(B-A)] / (D-C)} × 100

A: Absorbance before reaction of the well containing the sample

B: Absorbance after reaction of the well containing the sample

C: Absorbance before reaction of the well without sample

D: Absorbance after reaction of well without sample

As a result of testing the inhibitory effect of tyrosinase activity, the IC ₅₀ value of the GPS-1 of the leaf mushroom extract was 0.12%, which was much better than the conventional whitening agents arbutin and lettuce extract, which are known to be excellent in inhibiting tyrosinase (Table 4). Reference).

sample Mushroom tyrosinase inhibitory effect (IC ₅₀ ) Arbutin 0.4% Lettuce extract 1.0% GPC-1 1.0% GPS-1 0.12% GPC-2 0.2% GPS-2 0.15%

[Test Example 5] Experiment for measuring melanin production inhibitory effect using B16F1 melanocytes

This test example was to determine the whitening effect by looking at the degree of melanin inhibition on B16F1 melanocytes in order to confirm the effect of the leaf mushroom extract obtained in Examples 1-3.

The B16F1 melanocytes used in this test example are cell lines derived from mice and secrete a black pigment called melanin. Samples were treated during the artificial culture of these cells to evaluate the degree of melanin black pigment reduction. The B16F1 melanosite used in this test example was used after being sold from ATCC (American Type Culture Collection, Accession No .: 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocytes was measured as follows. B16F1 melanocytes were dispensed in 6-well plates at a concentration of 2 × 10 ⁶ per well and attached to the cells, followed by incubation for 72 hours by treating the sample at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, and the number of cells was measured.

Intracellular melanin quantification was performed by slightly modifying the method of Lotan (Cancer Res., 40: 3345-3350, 1980). The cell pellet was washed once with PBS, and then 1 ml of homogeneous buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt the cells. Melt the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), and measure the absorbance of melanin at 405 nm with a microplate platen. Percent inhibition of production was measured. Melanin inhibition rate (%) of B16F1 melanocytes was calculated by the following equation, IC ₅₀ value is the concentration of a substance that inhibits melanin production by 50%.

% Inhibition = [(A-B) / A] × 100

A: Melanin amount in wells without sample

B: Melanin amount in the wells to which the sample was added

As a result of testing the melanin production inhibitory effect of B16F1 melanocytes, the IC ₅₀ value of the GPS-1 of the leaf mushroom extract was 0.1%, which is superior to the conventional whitening agents arbutin and lettuce extract.

sample Melanin production inhibitory effect (IC ₅₀ ) Arbutin 0.45% Lettuce extract 0.5% GPC-1 0.5% GPS-1 0.1% GPC-2 0.2% GPS-2 0.5%

Test Example 6 Whitening Efficacy Test on Human Skin

A cosmetic containing the leaf mushroom extract obtained in Examples 1 to 3 was prepared and the skin whitening effect of humans was evaluated by performing a comparative experiment with Comparative Example 1. The cosmetics used in the comparative experiments are in the form of a cream, the composition of which is shown in Table 6. First, the phase b) recorded in Table 6 is heated and stored at 70 ° C. To this, a) phase is added, and after pre-emulsification, it is emulsified uniformly with a homomixer and then cooled slowly to prepare a cream (Preparation Example 1, Comparative Example 1). For 20 experimenters (20-35 year old female), the cream prepared in Preparation Example 1 was applied to the right side of the face, and the cream prepared in Comparative Example 1 was applied to the left side of the face twice a day for 2 consecutive months. After completion of the experiment, the skins of the application areas on the left and right sides of the face were compared by using an image analyzer and a color difference meter, and the darkest color was set to 5, the middle color to 3, and the brightest color to 1. Table 6 compares the facial skin color of the experimenter using the cream prepared in Preparation Example 1 with the facial skin color of the experimenter using the cream made in Comparative Example 1.

As shown in Table 7, it can be seen that the whitening effect is excellent in the facial skin of the experimenter applying the cream containing leaf mushroom extract.

Raw material name Preparation Example 1 (%) Comparative Example 1 (end) Stearyl alcohol 8 8 Stearic acid 2 2 Stearic Acid Cholesterol 2 2 Squalane 4 4 2-octyldodecyl alcohol 6 6 Polyoxyethylene (25 molar addition)-alcohol ester 3 3 Glyceryl Monostearic Acid Ester 2 2 (I) Leaf Mushroom Extract 0.1 - Propylene glycol 5 5 Purified water Quantity Quantity

Product used Preparation Example 1 Comparative Example 1 Experiment No. Right skin color Left skin color One 2 3 2 2 3 3 2.5 3.5 4 3 3 5 1.5 2.5 6 2 3 7 2.5 3 8 3 4 9 3 3 10 2 3 11 3 3 12 1.5 3 13 2 3.5 14 1.5 2.5 15 2.5 3.5 16 2 3 17 2.5 3 18 4 4 19 2.5 3 20 2.5 3

As shown in the above test example, it can be seen that the extract of leaf mushroom mycelium contained in the cosmetic composition of the present invention is a substance having skin cell proliferation, antioxidant effect and collagen production promotion, skin aging prevention and whitening effect.

Test Example 7 Comparison of Skin Wrinkle Improvement Effects of Subjects

Thirty-five subjects with facial wrinkles aged 35 to 45 years were evaluated to evaluate the skin wrinkle improvement effect of the following nutritional creams (Glycobacterial mycelium cultures were obtained from GPC-1 and GPS-1 obtained in Examples 1-3. Or GPC-2 and GPS-2).

Raw material name Preparation Example 2 Comparative Example 2 Leaf Mushroom Mycelium Culture 2.0 - Beeswax 10.0 10.0 Polysorbate 60 1.5 1.5 PIO 60 Hardened Castor Oil 2.0 2.0 Solbitan Sesquin Olate 0.5 0.5 Liquid paraffin 10.0 10.0 Squalene 5.0 5.0 Caprylic / Capric Triglycerides 5.0 5.0 glycerin 3.0 3.0 Butylene glycol 3.0 3.0 Triethanolamine 0.2 0.2 Preservative, coloring, flavoring Quantity Quantity Purified water Remaining amount Remaining amount

Experimental Example

The cream of manufacture example 2 was applied to the face left part of the subject, and the cream of the comparative example 2 was applied to the right part for 3 months. The skin condition was measured on both sides of the face before the use of the cream, and after 3 months of the use of the cream, the same area was remeasured to measure the change in skin wrinkles. Wrinkles at the tail of the eye area were replicated, and skin wrinkles were measured with a Visiometer system (C + K). The amount of change in skin wrinkles was calculated according to the following equation.

Change Amount (Δ%) = (Tdi-Tdo) / Tdo × 100

Tdo ; Value of the measuring part in Do

Tdi ; Value of the measuring part at D ₉₀

As a result of the calculation according to the above formula, in the case of Comparative Example 2 cream containing no leaf mushroom mycelium culture, the skin wrinkle showed a decrease of 4.5 ± 5.0% (mean ± standard deviation), whereas the leaf mushroom mycelium culture was prepared. The cream of Example 2 showed a good reduction effect of 22 ± 10%. Therefore, it can be seen that the cosmetic composition containing the leaf mushroom mycelium culture of the present invention is effective in preventing skin aging.

Examples of specific formulations of the skin cosmetic composition containing the leaf mycelium mycelium culture medium of the present invention are as follows. On the other hand, leaf mycelium mycelium culture medium refers to GPC-1 and GPS-1 or GPC-2 and GPS-2 obtained in Examples 1-3.

Formulation Example 1: Softener (Skin Lotion)

Raw material name weight% Leaf Mushroom Mycelium Culture 2.0 glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Carboxy Vinyl Polymer 0.1 Nonylphenyl Ether 0.2 Polysorbate 80 0.4 ethanol 10.0 Triethanolamine 0.1 Preservative, coloring, flavoring Quantity Purified water Remaining amount

Formulation Example 2: Nutrients (Milk Lotion)

Raw material name weight% Leaf Mushroom Mycelium Culture 2.0 Squalene 5.0 Beeswax 4.0 Polysorbate 60 1.5 Solbitan Sesquioleate 1.5 Liquid paraffin 0.5 Caprylic / Capric Triglycerides 5.0 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxy Vinyl Polymer 0.1 Triethanolamine 0.2 Preservative, coloring, flavoring Quantity Purified water Remaining amount

Formulation Example 3: Massage Cream

Raw material name weight% Leaf Mushroom Mycelium Culture 2.0 Beeswax 10.0 Polysorbate 60 1.5 PIO 60 Hardened Castor Oil 2.0 Sorbitan sesquioleate 0.8 Liquid paraffin 40.0 Squalene 5.0 Caprylic / Capric Triglycerides 4.0 glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservative, coloring, flavoring Quantity Purified water Remaining amount

As described above, a cosmetic composition effective in preventing skin aging and whitening by containing the extract from the leaf mycelium mycelium or its culture medium having the effect of proliferation, antioxidant effect and collagen production and melanin production inhibitory effect of skin cells Can be provided.

Claims (5)

An anti-aging cosmetic composition containing an extract having a molecular weight of 12000 or more in an extract obtained from a culture solution of a leaf mushroom ( Grifola frondosa Fr) or a leaf mushroom mycelium. The method according to claim 1, wherein the extract from the leaf mycelium mycelium extract or the extract obtained from the culture of the leaf mycelium mycelium contains a leaf mycelium mycelium extract, characterized in that the extract of molecular weight 12000 or more in an amount of 0.00001 to 30% by weight based on the total weight of the composition Skin anti-aging cosmetic composition. The mycelium or the extract obtained from the culture medium is obtained by centrifugation of the precipitate obtained by adding distilled water to the mycelium and adding hydrothermal extract for several hours at high temperature or by adding alcohol to the culture medium from which the mycelium is removed. Dissolving it in distilled water and filtered with a high molecular weight ultrafiltration membrane to remove the low-molecular substance skin aging cosmetic composition containing a leaf mycelium mycelium extract. The anti-aging cosmetic composition according to claim 1 or 2, wherein the cosmetic composition has a formulation of softening cream, nutrient cream, nutrition cream or massage cream. A whitening cosmetic composition containing an extract having a molecular weight of 12000 or more in an extract obtained from a leaf mushroom fungus mycelium extract or a culture solution of leaf mushroom mycelium.