KR101787393B1 - Cosmetic composition with the cultivated medium of Schizophyllum commume mycelium with fermented red ginseng - Google Patents

Abstract

The present invention relates to a culture solution ('cosmetic composition containing' red ginseng fermented product-skim milk culture solution ') obtained by inoculating and culturing mycelia of skimmed mushroom into fermented red ginseng fermented by inoculating a red ginseng powder with a mycelium of mycelia, The 'red ginseng fermented product-skimmer mushroom culture' of the present invention can be used for aquaporin-3, filaggrin, Increased expression of ceramide, increased hyaluronic acid production, reduced transdermal water loss, and increased water content resulted in excellent skin moisturizing effect and increased expression of VEGF (vascular endothelial cell growth factor) and PCNA (proliferation nuclear antigen) , Exhibits an excellent effect of increasing skin immunity, exhibits an effect of inhibiting NO production, inhibiting TNF-α production, and inhibiting PGE ₂ production, thereby exerting an excellent effect of reducing skin irritation.

Description

[0001] The present invention relates to a cosmetic composition containing a culture solution obtained by culturing a mycelia of mycelia of a skimmed mushroom in a fermented red ginseng product,

The present invention relates to a cosmetic composition containing a culture solution obtained by culturing mycelia of mycelia of skins with a fermented product of red ginseng. More specifically, the present invention relates to a cosmetic composition comprising a red ginseng fermented product obtained by inoculating a red ginseng powder with a skimmed mushroom mycelial block, And a culture medium obtained by culturing the same.

Schizophyllum commume ) belongs to the wormy mushroom throat and chrysanthemum mushroom of the basidiomycetes and can be collected in the wild, and the fruiting bodies are free from stigma. The side of the gut is attached to the substrate and its size is usually 1.0 ~ 3.0 ㎝, It is a type or clamshell type with wrinkles, irregularly split ends and fine hairs. The wrinkles are white, but when mature, they become brownish brown or brownish brown. The tissue shrinks when dry, but when it absorbs moisture, it is restored. The whiteness is white and the spores are conical in size of 4 ~ 6 × 1.5 ~ 2 ㎛ Smooth and white. Skirt mushrooms occur on bundles of stumps or stumps of broadleaved forests.

Recently, several researches related to skim mushroom have revealed that the polysaccharide called betaglucan produced by skirt mushroom has various various effects, and products using the same are being released.

However, in the past, Korean Patent Registration No. 10-0654063 (cosmetic composition for preventing skin aging containing nanoliposome stabilized by beta-1, 6-branch-beta-1,3- Patent No. 10-2006-0031329 (a composition for enhancing skin elasticity containing beta-1,3-glucan and deep sea water), a cosmetic composition containing skim mushroom beta-glucan as an effective ingredient, Korean Patent No. 10-909857 (143-1, a new strain of chlamydomonas which produce beta-1,6-branched-beta-1,3-glucan at high concentration, and beta-1,6- The method for producing beta-1, 3-glucan), beta-glucan derived from skim mushroom was produced in large quantities to enhance the skin-improving effect.

On the other hand, Korean Patent No. 10-1580107 discloses an extract of a fermented bird house fermented with mycelium of skim mushroom, which shows a skin improving effect by using a skirt mushroom. It is more necessary to excavate the material that it is.

The inventors of the present invention have made intensive studies on materials exhibiting excellent skin improving effect by using mycelia of the skins of mushrooms. As a result, they have found that inoculation of mycelium of mycelium of skins with culture of fermented red ginseng fermented by inoculating mycelium block of skimmed mushroom to red ginseng powder, The present invention also provides a cosmetic composition containing the same. The present invention also provides a cosmetic composition containing the same, which is excellent in skin irritation alleviating effect, skin immunity increasing effect and skin moisturizing effect.

In order to achieve the above object, the present invention provides a cosmetic composition comprising a culture solution obtained by inoculating and culturing mycelia of skimmed mushroom in a red ginseng fermented product obtained by inoculating a red ginseng powder with a block of mycelia of mycelium of mycelia.

The culture solution obtained by inoculating the mycelial body of mycelia with the mycelia of the red ginseng powder of the present invention and inoculating the mycelia of the skimmed mushroom into the fermented red ginseng primary fermented by the solid fermentation and culturing was found to be Aquaporine- (Filaggrin), Increased expression of ceramide, increase of hyaluronic acid production, decrease of transdermal water loss, increase of water content, and exert excellent skin moisturizing effect and increase the expression of VEGF (vascular endothelial cell growth factor) and PCNA (proliferation nuclear antigen) , Exhibits an excellent effect of increasing skin immunity, exhibits an effect of inhibiting NO production, inhibiting TNF-α production, and inhibiting PGE ₂ production, thereby exerting an excellent effect of reducing skin irritation.

On the other hand, in the present invention, the skirt mushroom mycelium block refers to a lump state in which a mycelium is inoculated on a support (carrier) on which a mycelium of mycelia of skins is conceived and can be grown. For example, some of the alginate beads or agar beads have been grown by implantation of mycelium of skimmed mushrooms.

The skimmed mushroom mycelium block can be produced by allowing the mycelia of mycelia to be congruent with alginate beads or agar beads by using alginate beads or agar beads, and adding a carbon source or a nitrogen source to grow the mycelia of skim mushrooms. More specifically, after the addition of alginate beads and agar beads, glucose, yeast extract and malt extract were added, the mycelia of the skimmed mushrooms were inoculated, and the mixture was inoculated at 23 to 27 ° C at 130 to 170 rpm For 5 to 9 days.

In the present invention, the red ginseng fermented product is preferably prepared by inoculating a red ginseng powder with a skins mushroom mycelium block and fermenting the mixture at 22 to 26 ° C for 3 to 7 days. When liquid fermentation is carried out by inoculating mycelia of skimmed mushroom into the extract of fermented red ginseng, a large amount of an ingredient such as beta-glucan is produced.

In the present invention, the red ginseng fermented product is collectively referred to as fermented product obtained by inoculating a red ginseng powder with a layer of a mycelium of mycelium of mycelia, and is a concept including a fermentation liquid containing a mycelium block or a mycelium, And a fermentation broth in which a mycelium block or a mycelium is removed from the fermentation broth, and also includes a powder prepared by drying the fermentation broth.

The fermented product of red ginseng is preferably an extract obtained by adding ethanol to fermented red ginseng fermented by inoculating a red ginseng powder with a skins mushroom mycelium block. Since the active ingredient such as ginsenosides in red ginseng and ginseng is mainly low in solubility in water, the extraction efficiency can be increased by ethanol extraction.

More preferably, it is an extract obtained by adding 60 to 80% (v / v) aqueous ethanol solution. An aqueous solution of less than 60% ethanol has a low extraction efficiency of the active ingredients and requires more time for extraction, and an aqueous solution of more than 80% of ethanol can extract unnecessary substances, resulting in the disintegration of formulations.

On the other hand, in the present invention, it is preferable to inoculate the mycelia of skins with the fermented product of red ginseng and preferably to cultivate at 23 to 27 DEG C for 5 to 9 days. At this time, the mycelia of the skim mushroom can be added in the form of a culture solution, and it is preferable to inoculate 8-12% (v / v) of the fermented red ginseng.

On the other hand, in the present invention, the culture liquid may be a filtrate from which mycelium is removed. Since mycelium exists in the culture solution, it is preferable to add it to the cosmetic composition after filtration.

On the other hand, in the present invention, the cosmetic composition may preferably be used for relieving skin irritation, for increasing skin immunity or for skin moisturizing. According to the experimental examples of the present invention, aquaporin-3, filaggrin, Increased expression of ceramide, increase of hyaluronic acid production, decrease of transdermal water loss, increase of water content, and exert excellent skin moisturizing effect and increase the expression of VEGF (vascular endothelial cell growth factor) and PCNA (proliferation nuclear antigen) And exhibited excellent skin immunity increasing effect, suppression of NO production, suppression of TNF-α production, and inhibition of PGE ₂ production, thus demonstrating excellent skin irritation mitigation.

On the other hand, the ingredients contained in the cosmetic composition of the present invention include, in addition to the 'culture solution obtained by inoculating and culturing mycelia of skimmed mushroom in a fermented product of red ginseng fermented by inoculating blocks of skimmed mushroom mycelium with red ginseng powder as an active ingredient, And include conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavoring agents, and carriers.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powdered foundation, emulsion foundation, wax foundation, pack, massage cream and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether Alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation or a surfactant-free cleansing formulation, it may be applied to the skin and then wiped off, washed or rinsed with water. The surfactant-containing cleansing formulation may be a cleansing foam, a cleansing water, a cleansing towel and a cleansing pack. The surfactant-free cleansing formulation may be a cleansing cream, , Cleansing lotion, cleansing water and cleansing gel, but is not limited thereto.

The present invention includes a culture solution ('red ginseng fermented product - skim milk culture solution') obtained by inoculating and culturing mycelia of skimmed mushroom into fermented red ginseng fermented by inoculating a red ginseng powder with a skimmed mushroom mycelial block, A cosmetic composition exhibiting an immunity increasing effect and a skin irritation alleviating effect can be provided. The 'red ginseng fermented product-skimmer mushroom culture' of the present invention can be used for aquaporin-3, filaggrin, Increased expression of ceramide, increase of hyaluronic acid production, decrease of transdermal water loss, increase of water content, and exert excellent skin moisturizing effect and increase the expression of VEGF (vascular endothelial cell growth factor) and PCNA (proliferation nuclear antigen) , Exhibits an excellent effect of increasing skin immunity, exhibits an effect of inhibiting NO production, inhibiting TNF-α production, and inhibiting PGE ₂ production, thereby exerting an excellent effect of reducing skin irritation.

FIG. 1 shows the results of confirming the expression level of aquaporin-3 and filaggrin in 'red ginseng fermented product-skimmer mushroom culture solution'.
FIG. 2 shows the results of confirming the expression level of ceramide and filaggrin in 'red ginseng fermented product-skimmer mushroom culture'.
FIG. 3 shows the results of confirming the amount of hyaluronic acid produced in the 'red ginseng fermented product-skimmed mushroom culture solution'.
FIG. 4 is a result of measuring the amount of transdermal water loss with time after application of 'red ginseng fermented product-skimmer mushroom culture' to the skin.
FIG. 5 shows the result of measuring the moisture content with time after applying the 'red ginseng fermented product-skimmed mushroom culture solution' to the skin.
FIG. 6 shows the results of confirming the expression of VEGF (vascular endothelial cell growth factor) and PCNA (proliferation nuclear antigen) in 'red ginseng fermented product-skimmer mushroom culture solution'.
FIG. 7 shows the results of confirming the NO production inhibiting ability of the 'red ginseng fermented product-skimmer mushroom culture'.
FIG. 8 shows the results of confirming the ability of the 'red ginseng fermented product-skimmer mushroom culture solution' to inhibit TNF-α production.
FIG. 9 shows the results of confirming the PGE ₂ production rate of the 'red ginseng fermented product-skimmer mushroom culture'.

Hereinafter, the structure of the present invention will be described in detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[Example 1: Preparation of fermented red ginseng-skimmer mushroom culture]

In the present invention, the term 'red ginseng fermented product-skimmer mushroom culture solution' refers to a culture solution obtained by inoculating and cultivating mycelia of skimmed mushroom into fermented red ginseng fermented by inoculating a mycelium block of skimmed mushroom to red ginseng powder.

<Manufacture of mycelia of mycelia of skull mushroom>

After adding 0.4% of glucose, 0.4% of yeast extract and 1% of malt extract, alginate beads and agar beads were added, And cultured at 25 ° C and 150 rpm for 7 days to prepare a block of mycelia of skimmed mushroom.

<Preparation of red ginseng fermented product>

Red ginseng powder was mixed with 1: 1 mixture of red ginseng powder and skimmed mushroom produced by blending red ginseng with a blender and fermented at 24 ℃ for 5 days to prepare fermented red ginseng.

A 70% ethanol aqueous solution corresponding to 10 times the weight of the fermented red ginseng was added thereto, stirred and extracted for 3 hours, and then dried under reduced pressure to evaporate all of the ethanol to recover the fermented product (= extract of fermented red ginseng) .

<Red ginseng fermented product - Preparation of culture of skimmed mushroom>

The red ginseng fermented product was inoculated with a 10% (v / v) culture medium of Rhizoma mushroom seed culture and cultured at 24 ° C and 150 rpm for 7 days to prepare 'red ginseng fermented product-skimmer mushroom culture'.

The 'red ginseng fermented product-skimmer mushroom culture solution' was centrifuged to remove cells first, and then the filter was filtered to obtain a filtrate, which was used in the following Experimental Example.

[Comparative Example 1: Preparation of 'Red ginseng-skimmer mushroom culture']

In this Example, a culture solution was prepared by culturing mycelia of skimmed mushroom in general red ginseng powder, which was not fermented by fermented red ginseng fermented by inoculating a block of mycelium of mycelia with red ginseng powder. Hereinafter, the culture solution prepared in this example was referred to as 'red ginseng-skim milk culture solution'.

[Experimental Example 1: Assessment of skin moisturizing effect of 'red ginseng fermented product-skimmed mushroom culture solution']

In this experimental example, the skin moisturizing effect of 'red ginseng fermented product-skimmer mushroom culture solution' was examined.

(1) Aquaporine-3, Filaggrin, Bow performance evaluation

In this experiment, the effect of moisturizing effect of 'red ginseng fermented product-skimmer mushroom culture solution' was evaluated.

HaCaT cells were inoculated on a 60 mm plate at a concentration of 8 × 10 ⁵ cells / ml, cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours, and then the medium was removed. Thereafter, a sample ('red ginseng fermented product-skimmer mushroom culture', 'red ginseng-skimmer mushroom culture') diluted to 1% with a serum-free medium was treated and incubated at 37 ° C in a 5% CO 2 incubator ) For 48 hours to remove all medium and wash twice with DPBS. Then, 200 μl of RIPA buffer was added, scraped with a scraper, transferred to a micro tube, and then suspended 30 times using a syringe. Then, the cells were incubated in ice for 30 minutes and centrifuged at 14,000 rpm for 30 minutes at 4 캜. Thereafter, the obtained supernatant was transferred to a new micro tube, followed by quantification of the protein, Western blotting, and immunoblotting in the following procedure to obtain aquaporin-3 (Aquaporine-3) and Filaggrin bow performance were evaluated.

① Transfer membrane was transferred into a tube and rinsed with D.W and TBS-T.

② Blocked with 5% skim milk for 2 hours.

3) Washing was performed 6 times for 10 minutes using TBS-T.

④ The primary antibody was diluted 1: 1000 in 5% BSA solution and overnight at 4 ° C.

⑤ Washed 6 times for 10 minutes using TBS-T.

⑥ Secondary antibody was diluted 1: 3000 in 5% skim milk and shaked for 1 hour.

⑦ Washed 6 times for 10 minutes using TBS-T.

⑧ The ECL solution was sufficiently wetted on the membrane, placed in a cassette, and the film was raised and pressed well.

⑨ Developed with developer and fixer.

As a result of the experiment, it was confirmed that the 'red ginseng fermented product-skimmer mushroom culture' was superior in the expression of aquaporin-3 and filaggrin than the 'red ginseng-skimmer mushroom culture solution' (FIG. FIG. 1 shows the results of confirming the expression level of aquaporin-3 and filaggrin in 'red ginseng fermented product-skimmer mushroom culture solution'.

From the above results, it can be seen that the 'red ginseng fermented product-skimmer mushroom culture' exhibits excellent skin moisturizing effect because of its excellent expression of Aquaporin-3 and Filaggrin.

(2) Assessment of expression amount of ceramide, filaggrin

The HaCaT cell line was inoculated on a 100 mm dish at 48 × 10 ⁵ cells, cultured at 37 ° C. in a 5% CO ₂ incubator for 24 hours, and then the medium was removed and the remaining medium was removed using PBS . Then, 5,000 μl of each sample ('red ginseng fermented product-skimmed mushroom culture') diluted to 1% and 2% by using a serum-free medium was treated at 37 ° C and 5 in% CO ₂ incubator (incubator) and incubated for 24 hours. Subsequently, the medium was removed, and 1 ml of Qiazole was added. Cells were collected using a scraper, transferred to a 1.7 ml tube, and then subjected to RT-PCR in the following manner.

① After vortexing, the mixture was allowed to react at room temperature for 5 minutes, and chloroform (1/5 of the Qiazol capacity) was added.

② After mixing for 15 seconds using Vortex, the mixture was reacted at room temperature for 3 minutes and centrifuged at 4 ° C and 12,000 rpm for 15 minutes.

③ Chloroform The supernatant of double volume was transferred to a new 1.7-ml tube. Iso-propanol (1/2 volume) of Qiazol was added and mixed with vortex at room temperature. Lt; / RTI &gt;

④ Centrifuge at 4 ℃ for 7 minutes at 7,500 ~ 12,000 rpm.

⑤ The supernatant was discarded and carefully pipetted with 75% cold-ethanol as much as the used amount of Qiazol.

⑥ Centrifuge at 4 ℃, 7,500 ~ 12,000 rpm for 10 minutes, discard the supernatant and dry the pellelt.

⑦ RNA was quantified using UV-phtospectrometer and cDNA was synthesized, and PCR was performed using each primer.

At this time, EGF (Epidermal growth factor) was used as a positive control group.

As a result of the experiment, it was confirmed that the ceramide and filagrrin expressions were increased with increasing the treatment concentration of 'red ginseng fermented product-skimmer mushroom culture' (FIG. 2). FIG. 2 shows the results of confirming the expression level of ceramide and filaggrin in 'red ginseng fermented product-skimmer mushroom culture'.

From the above results, it can be seen that the 'red ginseng fermented product-skimmer mushroom culture solution' exhibits excellent skin moisturizing effect by increasing the expression of ceramide and filaggrin.

(3) Hyaluronic acid assay

CCD-986SK cells were dispensed in a 6-well plate at a concentration of 1 × 10 ⁵ cells / ml in a volume of 2 ml and cultured in DMEM, 10% FBS, 1% antibody (Antibiotics) % CO2 for 24 hours. Hyaluronic acid-linked immunosorbent assay kit (Hyaluronan Enzyme-Linked Immunosorbent Assay Kit) was used to treat 0.1%, 0.5%, and 1% of each sample (red ginseng fermented product- HA) was measured.

(1) Prepare standard and sample in a yellow U-bottom plate with 100 μl per well. (Blank control is 1 × diluent diluent 150 μl per well, The HA control was diluted with 100 μl of 1X diluent per well.

② After that, 50 ㎕ of working HA detector per well was added to all wells except blank control, gently mixed by shaking, sealed, and incubated at 37 ° C for 1 hour.

(3) Then, 100 μl of each well of the Incubation plate was transferred to a plate, gently taped, sealed, and incubated at 4 ° C for 30 minutes.

④ Remove all the solution from the plate and wash 3 times with 200 μl of 1X Wash Concentrate (Wash buffer was completely removed).

⑤ 100 ㎕ of working enzyme was added to each well, shaken, sealed, and incubated at 37 캜 for 30 minutes.

⑥ Repeat step ④.

⑦ 100 ㎕ of substrate solution was added to each well and reacted under R.T.

⑧ From 15 minutes after the reaction, the absorbance was measured at 405 nm (15 minutes, 30 minutes, 45 minutes).

⑨ 50 μl of stop solution was added to each well.

EGF (Epidermal Growth Factor) was used as a control group, and the amount of HA produced was compared with the control obtained by substituting the absorbance value of the sample into the formula of the standard curve using the following equation (1).

As a result of the experiment, it was confirmed that the amount of hyaluronic acid (HA) was increased with an increase in the treatment concentration of 'red ginseng fermented product-skimmer mushroom culture' (FIG. 3). FIG. 3 shows the results of confirming the amount of hyaluronic acid produced in the 'red ginseng fermented product-skimmed mushroom culture solution'.

From the above results, it can be seen that the 'red ginseng fermented product-skim milk culture solution' increases the production of hyaluronic acid and exerts excellent skin moisturizing effect.

(4) Confirmation of transdermal water loss

The amount of transdermal water loss was measured using a tewameter (MPA system, Courage + Khazaka electronic GmbH, TM300) after applying the 'red ginseng fermented product-skimmer mushroom culture' to the skin.

As a result of the experiment, it was confirmed that when the 'red ginseng fermented product-chima mushroom culture solution' was applied, the amount of transdermal water loss with time decreased more than that of the positive control hyaluronic acid 1% treatment (FIG. FIG. 4 is a result of measuring the amount of transdermal water loss with time after application of 'red ginseng fermented product-skimmer mushroom culture' to the skin.

From the above results, it can be seen that the 'red ginseng fermented product - skim mushroom culture solution' exhibits excellent skin moisturizing effect by reducing the transdermal water loss.

(5) Checking the water content

After applying the 'red ginseng fermented product-skimmed mushroom culture' to the skin, the moisture content was measured using a coneometer (MPA system, Courage + Khazaka electronic GmbH, CM825).

As a result of the experiment, it was confirmed that when the 'red ginseng fermented product-skimmer mushroom culture solution' was applied, the moisture content with time increased more than the 1% hyaluronic acid treatment which was a positive control group (FIG. FIG. 5 shows the result of measuring the moisture content with time after applying the 'red ginseng fermented product-skimmed mushroom culture solution' to the skin.

From the above results, it can be seen that the 'red ginseng fermented product - skim milk culture solution' increases moisture content and exerts excellent skin moisturizing effect.

[Experimental Example 2: Identification of Skin Irritation Enhancement Effect of 'Red Ginseng Fermented Product-Skimmer Mushroom Culture']

In this experiment, the effect of 'red ginseng fermented product - skimmer mushroom culture' on skin immunity was examined.

Immunofluorescence staining was used to confirm the immunocompetent effect (Areca nut extract treatment down-regulates involution in normal human oral keratinocyte through P13K / AKT activation, Oral Oncology (2007) 43, 670679 Tseng. See staining).

As a result, the expression of VEGF (vascular endothelial cell growth factor) and PCNA (proliferation nucleus antigens) was higher than that of the negative control and the immunity similar to the positive control (5% Minosidil) in the case of 1% 'red ginseng fermented product - (Fig. 6). FIG. 6 shows the results of confirming the expression of VEGF (vascular endothelial cell growth factor) and PCNA (proliferation nuclear antigen) in 'red ginseng fermented product-skimmer mushroom culture solution'.

From the above results, it can be seen that the 'red ginseng fermented product-skimmer mushroom culture solution' exhibits an excellent skin immunity increasing effect by increasing the expression of VEGF (vascular endothelial cell growth factor) and PCNA (proliferation nucleus antigen).

[ Experimental Example  3: 'Red ginseng Fermentation product - Skirt mushroom  Culture solution '

In this experimental example, the effect of 'red ginseng fermented product - skimmer mushroom culture solution' on skin was examined.

(1) Confirmation of NO production inhibition (NO assay)

Excessive production of nitric oxide (NO), which is a reactive nitric species (RNS), induces an inflammatory response and stimulates cells to produce NO by stimulating LPS (lipopolysaccharide) The inhibition of NO production by the treatment of 'red ginseng fermented product - skimmed mushroom culture' was measured by the Griessreagent method.

Raw 264.7 cells were inoculated in a 24-well plate at a concentration of 1 × 10 ⁵ cells / ml and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. Thereafter, 500 μl of a sample (10, 20, 50 μg / ml 'red ginseng fermented product - skim milk culture') diluted with a medium containing no serum (containing 100 ng / ml of LPS) Cells. After culturing for 24 hours, the medium was recovered and centrifuged at 4 ° C and 12,000 rpm for 10 minutes. Then, only the supernatant was recovered, and 50 μl of the supernatant was taken. Then, the supernatant was placed in a 96-well plate, After 10 minutes of reaction with Griessreagent, the absorbance was measured at 540 nm using an ELISA reader. At this time, NMMA (NG-monomethyl-L-arginine) was used as a positive control.

As a result, it was confirmed that NO production was inhibited depending on the concentration of 'red ginseng fermented product-skimmer mushroom culture' (FIG. 7). FIG. 7 shows the results of confirming the NO production inhibiting ability of the 'red ginseng fermented product-skimmer mushroom culture'.

(2) Evaluation of anti-inflammatory activity using TNF-α immunoassay

HaCaT cells were inoculated on a 60 mm plate at a concentration of 8 × 10 ⁵ cells / ml, cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours, and then the medium was removed. Thereafter, a sample (10, 20, 50 / / ml 'fermented red ginseng-skimmer mushroom culture'') diluted with serum-free medium was treated and incubated at 37 ° C in a 5% CO ₂ incubator for 48 Time. After that, the culture medium was recovered and centrifuged at 12,000 rpm for 10 minutes at 4 ° C. Only the supernatant was recovered and subjected to TNF-α immunoassay as follows.

(1) Assay diluent (RD1F) of 50 μl was placed in a well.

② 200 μl of the supernatant was added to the well and reacted at room temperature for 2 hours.

③ Wash the wells 4 times with 400 μl of wash buffer.

④ 200 μl of TNF-α conjugate was added to the well and reacted at room temperature for 1 hour.

⑤ Wash the wells 4 times with 400 μl of wash buffer.

⑥ 200 μl of Substrate solution was added to the well and allowed to react for 20 minutes under dark condition.

⑦ 50 ㎕ of stop solution was added to each well and reacted.

⑧ The amount of TNF-α was measured by absorbance at 450 nm wavelength.

At this time, NMMA (NG-monomethyl-L-arginine) was used as a positive control.

As a result, it was confirmed that the TNF-α production was suppressed depending on the concentration of 'red ginseng fermented product-skimmer mushroom culture', and especially the same effect as that of NMMA, which is a positive control, was exerted (FIG. FIG. 8 shows the results of confirming the ability of the 'red ginseng fermented product-skimmer mushroom culture solution' to inhibit TNF-α production.

(3) Evaluation of inhibitory effect of PGE ₂ formation using 'Immunoassay test method'

HaCaT cell lines were inoculated in a 24-well plate at a concentration of 1 x 10 ⁵ cells / ml and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. Thereafter, the samples (10, 20, and 50 / / ml 'fermented red ginseng-skimmer mushroom culture'') diluted with serum-free medium were cultured in the cells for 24 hours. Thereafter, the medium was recovered and centrifuged at 12,000 rpm for 10 minutes at 4 ° C. Only the supernatant was recovered and Prostaglandin E ₂ Immunoassay was carried out according to the following procedure.

(1) 150 [mu] l of calibrator diluent RD5-56 was placed in a well.

② 150 μl of the supernatant was added to the well.

(3) 50 μl of the primary antibody solution was added to the wells and reacted at room temperature or orbital shaker (500 ± 50 rpm) for 1 hour.

④ 50 μl of PGE ₂ conjugate was added to the well and allowed to react for 2 hours at room temperature or orbital shaker (500 ± 50 rpm).

⑤ Wash the wells 4 times with 400 μl of wash buffer.

⑥ 200 μl of Substrate solution was added to the well and allowed to react for 20 minutes under dark condition.

⑦ 10 μl of stop solution was added to each well and reacted.

⑧ The amount of PGE ₂ was measured by absorbance at 450 nm wavelength.

At this time, NMMA (NG-monomethyl-L-arginine) was used as a positive control.

As a result of the experiment, it was confirmed that the production rate of PGE ₂ was decreased depending on the concentration of 'red ginseng fermented product-skimmer mushroom culture', and it was confirmed that it exerts superior effect than the positive control NMMA (FIG. 9). FIG. 9 shows the results of confirming the PGE ₂ production rate of the 'red ginseng fermented product-skimmer mushroom culture'.

Claims (10)

To the fermented red ginseng fermented by inoculating mycelium block of skimmed mushroom into red ginseng powder,
A cosmetic composition comprising a culture obtained by further inoculating and culturing mycelia of skimmed mushrooms.
The method according to claim 1,
The skirt mushroom mycelium block comprises:
Wherein the alginate beads or agar beads are implanted with a mycelium of mycelia.
The method according to claim 1,
The red ginseng fermented product,
Wherein the red ginseng powder is inoculated with a mycelium block of skimmed mushrooms and fermented at 22 to 26 DEG C for 3 to 7 days.
The method according to claim 1,
The red ginseng fermented product,
Wherein the extract is an extract obtained by adding ethanol to fermented red ginseng fermented by inoculating mycelium block of skimmed mushroom powder to red ginseng powder.
The method according to claim 1,
The above-
Wherein the fermented red ginseng product is inoculated with mycelia of skimmed mushrooms and cultured at 23 to 27 DEG C for 5 to 9 days.
6. The method of claim 5,
Wherein the mycelia of the skim-
(V / v) to the fermented red ginseng in the form of a culture.
The method according to claim 1,
Preferably,
Wherein the filtrate is a filtrate from which mycelium is removed.
The method according to claim 1,
In the cosmetic composition,
Wherein the cosmetic composition is for skin inflammation relief.
delete The method according to claim 1,
In the cosmetic composition,
Wherein the composition is for moisturizing the skin.

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