KR20140081138A - A skin-care agent containig Antrodia camphorata fruit body extract or Antrodia camphorata mycelium extract - Google Patents

Abstract

The present invention relates to a skin external application composition containing a culture medium liquid obtained by culturing an Antrodia camphorata fruit body extract, an Antrodia camphorata mycelium extract, or an Antrodia camphorata mycelium. In particular, the present invention relates to a functional skin external application composition containing 0.001-90.0 wt% of a culture medium liquid obtained by culturing an Antrodia camphorata fruit body extract, an Antrodia camphorata mycelium extract, or an Antrodia camphorata mycelium, wherein the functional skin external application composition has effects in removing active oxygen, preventing aging, reducing wrinkles, promoting collagen synthesis, moisturizing the skin, whitening the skin, alleviating skin irritation, preventing pimples, alleviating atopy, resisting inflammation, preventing hair damage, preventing hair loss, and promoting hair growth.

Description

[0001] The present invention relates to a skin-care agent containing Antrodia camphorata fruit body extract or Antrodia camphorata mycelium extract containing a fungus body extract,

The present invention relates to a composition for external application for skin containing extracts of Fusarium oxysporum fusiformis or Fusarium oxysporum fusiformis, and more specifically, to a composition for external application for skin comprising a filtrate obtained by fermenting a filtrate obtained from Fusarium oxysporum fucus extract or a fusarium mushroom mycelium To a composition for external application for skin.

Skin aging can be divided into two types: intrinsic (chronological aging) and phtoaging (Gilchrest BA: J. Am . Acad . Dermatol ., 21, 610-613 (1989)]. Endogenous aging is a naturally occurring aging phenomenon that is accompanied by decreased physiological function of the body as age increases [Braverman IM et al . : J. Invest . Dermatol ., 78, 434-443 (1982)]. Photoaging means that the skin is repeatedly exposed to light to change the appearance or function of the skin [Ridder GM et al . : J. Am . Acad . Dermatol ., 25, 751-760 (1991)]. In addition, skin aging can be caused by active oxygen species activated by ultraviolet rays, stress, disease states, environmental factors, wounds, and aging. When such conditions are deepened, the antioxidant defense network existing in the living body is destroyed, It damages tissues and promotes adult diseases and aging. More specifically, lipids, proteins, polysaccharides and nucleic acids, which are major constituents of the skin, are oxidized to destroy skin cells and tissues, resulting in skin aging. In particular, the oxidation of proteins causes severe hyperinflammation of collagen, hyaluronic acid, elastin, proteoglycans, and fibronectin, which are connective tissues of the skin, which interferes with skin elasticity. Mutation, cancer induction, and immune function.

Therefore, it is necessary to protect the cell membranes by abolishing reactive oxygen species that are mediated by the body's metabolic processes, ultraviolet irradiation, and inflammatory reactions, and regenerate already damaged cells by active metabolism to proliferate the cells Can be restored and healthy skin can be maintained. In aging, not only active oxygen species but also MMP (Matrix metallopretease) is involved. In vivo, the synthesis and degradation of extracellular matrix such as collagen are properly regulated, but the synthesis is reduced as aging progresses and the collagen- Expression of matrix metalloproteinases (MMPs) is promoted, and skin elasticity is lowered and wrinkles are formed. These degrading enzymes are also activated by exposure to ultraviolet light. Therefore, there is a demand for development of a substance capable of controlling the activation of MMP induced in the cell by ultraviolet light or inhibiting its activity. Until now, the raw materials used as cosmetic materials mostly inhibited only the activity of degrading enzyme (MMP).

On the other hand, melanin is converted from tyrosine to dopa and dopaquinone by the action of tyrosinase existing in pigment cells, and is generated by dopachrome. Melanin is present in the skin, protects the body from ultraviolet light and has an important function in regulating hormone secretion in the body. However, when melanin is overproduced, it is known that it forms spots and freckles, promotes skin aging, and plays an important role in skin cancer induction. Therefore, research and development are actively conducted to prevent melanin overproduction. (Japanese Patent Laid-open No. 4-93050) and plant extracts inhibit tyrosinase (Japanese Patent Laid-open Publication No. 4-9320), hydroquinone (Japanese Patent Laid Open No. 6-192062), kojic acid Activity and is used as a whitening cosmetic, but its stability is poor in cosmetic formulations, it is decomposed to cause coloration, odor, or efficacy at a biological level, its effect is unclear, or its safety is a problem. to be.

Atopic dermatitis is a common disease in people with atopic allergies. Although the cause of atopic dermatitis has not yet been clarified, atopic dermatitis has a history of atopy in 70% of the patients, And it is understood to be one of diseases caused by genetic factors or immune diseases because it is accompanied by allergic diseases such as allergic rhinitis and asthma. In the treatment of atopic dermatitis, medicines such as corticosteroids, which can expect high pharmacological effects, are used, but side effects of corticosteroids are problematic, and the use of corticosteroids causes the rebound It is also well-known that a later severe worsening of the affected part occurs. As a countermeasure for such side effects, a non-steroidal anti-inflammatory agent or an antihistamine agent which does not contain a hormone such as adrenocortical hormone is used but it is very difficult to develop a remedy or therapeutic agent for atopic dermatitis without side effects because it is difficult to cure.

Recently, much attention has been focused on functional cosmetics having functions such as antioxidation, wrinkle reduction, whitening, and itching relief obtained from natural extracts in order to reduce skin irritation caused by various chemical substances and the like. For example, U.S. Patent No. 5,972,341 discloses a wrinkle-reducing effect of an extract of Commiphora mukul. Japanese Patent Application Laid-Open No. 9-672662 discloses an extract of seaweed having a hyaluronidase inhibitory effect, Japanese Patent Application Laid-Open No. Hei 2-245087 discloses an antioxidative effect of Sargassum sp. Extract on the skin texture improvement effect of Fucoidan extracted from Wakame, Patent No. 0431076 discloses a cosmetic composition for improving the healing of atopic skin including a white, black, echinacea, and fermented soybean extract, Korean Patent No. 0511494 discloses an extract and composition for treating atopic dermatitis including at least one of Hashuo, .

In addition, male-pattern alopecia are male hormone-dependent and have a direct relationship with the amount of male hormone. Accordingly, many studies for prevention and treatment of alopecia through inhibition of male hormone activity have been reported. On the other hand, when the function of the sebaceous gland is activated by the increase of the secretion of the male hormone, the excess sebum produced in the hair follicle is stagnated in the hair follicle due to the overgrowth of the hair follicle wall. 5-alpha-Reductase is one of the male hormones present in male hormone reactive tissues such as sebaceous glands, hair follicles, prostate, and epididymis, as a result of male hormone-induced hair loss and acne. , An enzyme involved in the metabolism of testosterone into dihydrotestosterone, and requires NADPH for its conversion. In addition, testosterone is involved in male sexual dysfunction, skeletal muscle increase, male external genitalia, scrotum growth, spermatogenesis and the like, and dihydrotestosterone is involved in the relevant tissues such as acne, sebum increase, hair loss and enlargement of the prostate. In particular, after puberty, excessive secretion of male hormones causes acne and hair loss. To prevent excessive production of dihydrotestosterone, an active form of male hormone produced by 5-alpha-reductase, 5-alpha-reductase Studies have been actively conducted to develop anti-hair loss and anti-acne agents using an azide inhibitor.

In order to solve such skin problems, many cosmetics using natural products or medicinal and edible mushroom extracts have recently been developed to reduce skin irritation caused by various chemical substances and the like. As the cosmetic compositions using the mushroom extracts known so far, there have been proposed various cosmetic compositions such as snow flake (Korean Patent No. 0340185), Situ mushroom, Chima mushroom (Korean patent No. 0681703, No. 0295623), Leaf mushroom (Korean patent No. 0438009) And the research on the anti-aging effect of these extracts has been continued.

Mushrooms which are used for medicinal and edible purposes are many examples and kinds, but mushrooms which are used and studied as cosmetic composition are still known as a part.

On the other hand, Antrodia camphorata is a rare mushroom which is parasitic on camphor tree that has survived for over 100 years on the altitude of 1,000 m or more above sea level in Taiwan and is called " In China, it is traditionally known to be effective in hypertension, liver cancer, inflammation, itchy skin, abdominal pain and diarrhea.

In addition to the anti-aging effect, the whitening effect, the skin irritation alleviation effect, the atopy improvement effect, the acne improvement effect, the hair damage prevention effect, the hair loss prevention and hair growth improvement effect, Has never been identified.

Korean Patent No. 10-0438009 (a cosmetic composition containing an extract of mycelia of leaf mushrooms) has been reported to be useful as an antioxidant, an antioxidant, an antioxidant, an antioxidant, an antioxidant, The cosmetic composition containing the obtained extract has been described, but no description has been made on the use of the extract of Fusarium oxysporum fusiformis or the culture of Fusarium oxysporum fusiformis. Korean Patent No. 10-0340185 (a cosmetic composition for preventing wrinkles containing an extract of snow flower Cordyceps) contains cosmetic composition excellent in prevention of wrinkles by containing an extract of Snow Flower Cordyceps, however, There is no description about the use of a mycelium culture solution.

The present invention relates to the use of natural extracts for the treatment of skin having antioxidation, promoting collagen synthesis, improving skin wrinkles, whitening, moisturizing, alleviating skin irritation, preventing acne, improving atopic appearance and antiinflammation, preventing hair damage, And an object of the present invention is to provide a composition for external application.

The present invention also provides a composition for external application for skin, which comprises, as an active ingredient, a culture solution obtained from a culture of a higher mushroom fruit body extract, a mushroom mycelial extract or a mushroom mycelium extract, The purpose is to provide.

The present invention also relates to a composition for preventing or treating hair loss, which comprises a combination of antioxidant, collagen synthesis promotion, skin wrinkle improvement, whitening, moisturizing, skin irritation mitigation, acne prevention, atopic improvement and anti- It is an object of the present invention to provide a method for efficiently obtaining a mushroom fruit body extract or a mushroom mycelium extract.

In order to achieve the above object, the present invention provides a composition for external application for skin, which comprises, as an active ingredient, a culture solution obtained from the cultivation of a mushroom extract of Fusarium oxysporum, a mycelium extract of Fusarium oxysporum, or a mycelium of Fusarium oxysporum.

Hereinafter, the present invention will be described in detail.

The composition for external application of the present invention is effective for preventing or treating atopic dermatitis, skin irritation mitigation effect, prevention or treatment of inflammation, antioxidative effect, anti-aging effect, wrinkle improvement effect, skin moisturizing effect, collagen synthesis promotion effect, whitening effect , An effect of preventing or treating acne, prevention of hair damage, prevention of hair loss, and improvement of hair growth, comprising the step of cultivating a composition for external application for skin, which is obtained from the cultivation of a mushroom fruiting body extract or a mushroom mycelium extract or a mushroom mycelium Characterized by containing a culture medium as an effective ingredient.

The extract of the mushroom fruiting body of the present invention can be obtained by extracting the mushroom fruiting body of the fruiting body. The kind of the fruiting body of the mushroom is not limited to a specific kind but can be either natural collected or artificially cultured.

In addition, the method for extracting the fruiting body extract of the mushroom of the present invention may be a method commonly used in the technical field to which the present invention belongs. For example, it is preferable to add about 2 to 20 times, preferably about 2 to 5 times, water, a lower (C1-C4) alcohol such as methanol or ethanol to the fruity body powder obtained by freeze- A polar solvent or a mixed solvent thereof at an extraction temperature of 10 캜 to 30 캜, such as cold extraction, reflux cooling extraction, hot water extraction, ultrasonic extraction and ultrahigh pressure extraction.

Further, preferably, the thus obtained extract can be filtered, concentrated under reduced pressure, or lyophilized to be soluble in a polar solvent. For example, the obtained extract is filtered and then transferred to a concentration tank and concentrated under reduced pressure and freeze-dried at 50 DEG C or lower. To obtain the thus obtained reduced pressure concentrate and the lyophilized product in an amount of 0.001 to 70.0 wt%, purified water, ethanol, Propylene glycol, and the like can be used to produce an extract.

In addition, the fruity body extract of the mushroom of the present invention may mean the polysaccharide itself contained as an active ingredient in the thus obtained extract.

On the other hand, the extract of the mushroom mycelium of the present invention is obtained by extracting the mycelium of mushroom, and the extraction method can be a method commonly used in the technical field to which the present invention belongs. For example, it may be a hot-water extract of the mycelial powder obtained by lyophilizing and pulverizing the mycelium of mushroom, or an organic solvent extract obtained by using methanol, ethanol, butanol or a mixed solvent thereof.

On the other hand, the culture liquid obtained from the cultivation of the mycelia of the mushroom can be used as it is, or dried (or lyophilized) and used in powder form. Preferably, the culture solution may be used by filtration. Specifically, the mycelia can be removed by centrifugation after liquid cultivation of mycelium of mushroom, and the filtrate obtained by filtering the culture filtrate with a paper filter can be used. At this time, the culture means 'fermentation'.

In addition, the culture solution obtained from the cultivation of the mycelium extract of mycelia or mycelia of the present invention may be a polysaccharide extracted from the hydrothermal extract, the organic solvent extract or the filtrate.

The culture solution obtained from the cultivation of the mycelia of mycelium of mushroom mycelia of the present invention or the culture of mycelia of mushroom of mushroom of the present invention is preferably an extract of mushroom mycelium of mushroom using at least one animal or plant or herb medicine as a substrate. Herein, the animal, plant or herb medicine is not particularly limited, and an animal, a plant or a herbal medicine commonly used in the technical field of the present invention can be used as a substrate.

The culture solution obtained from the cultivation of the thus obtained extract of Fusarium oxysporum fumarum or Fusarium oxysporum fungus extract or Fusarium oxysporum fusatus of the present invention has antioxidant activity, inhibitory effect on substrate metalloproteinase (MMP-1) activity, The effect of suppressing the expression of the enzyme (MMP-1), the promoting effect of pro-collagen biosynthesis, the promoting effect of elastin production, the whitening effect, the inhibitory effect of hyaluronidase activity, the cytotoxic relaxation effect by ultraviolet irradiation, The anti-inflammatory effect against atopic dermatitis, the skin irritation alleviation effect, the hair damage prevention effect, the hair loss prevention effect and the hair growth effect are excellent, so that the multi-functional It can be used in a composition for external application for skin.

On the other hand, the composition for external application for skin of the present invention preferably contains 0.001 to 90.0% by weight of a culture solution obtained from the cultivation of the fungus extract of Fusarium oxysporum f. Mushroom, the fungus extract of Fusarium oxysporum If it is included, the effect of improving the skin is insignificant. If it exceeds 90.0% by weight, the efficiency with respect to the amount of the material is inferior, which is not economical.

In the present invention, the term "included as an active ingredient" means that the composition for external application for skin can be applied to the composition for external application for skin to show the effect of prevention and inhibition of skin disease from the composition for external application for skin of the present invention, Means the addition of a culture solution obtained from the culture of the extract or the mushroom mycelium, and includes various formulations in which various components are added as a sub ingredient for drug delivery and stabilization.

The composition for external application for skin of the present invention is preferably a cosmetic composition. Examples of the cosmetic composition include cosmetics, gels, water-soluble liquids, creams, essences, oil-in-water (O / W) O) type; A color cosmetic formulation consisting of an underwater type or a water-in-oil type makeup base, a foundation, a skin cover, a lipstick, a lip gloss, a face powder, a two way cake, an eye shadow, a cheek color and an eyebrow pencil; .

On the other hand, the cosmetic composition is most preferably used for skin whitening, anti-aging, skin irritation, skin moisturizing, wrinkle improvement, atopic skin improvement, acne improvement, hair damage prevention or hair loss prevention.

Meanwhile, the external composition for skin of the present invention is preferably a pharmaceutical composition. Examples of the pharmaceutical composition include PLASTERS, LOTIONS, LINIMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, SUSPESIONS, CAPSULES, CREAMS, Gelatin capsules, pastes, and sustained release preparations.

On the other hand, the pharmaceutical composition of the present invention comprises a pharmacologically acceptable base; Carrier; Excipients; Binders comprising starch, tragacanth gum, gelatin, molasses, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, ethylcellulose and carboxymethylcellulose; A pulverizing agent comprising agar, starch, gelatin powder, carboxymethyl cellulose sodium, carboxymethyl cellulose calcium, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate and sodium alginate; Lubricants including magnesium stearate, talc and hydrogenated vegetable oils; And a coloring agent. Examples of the carrier and excipient include lactose, glucose, sucrose, mannitol, potato starch, cornstarch, calcium carbonate, calcium phosphate, and cellulose. In addition to the above, additives such as stabilizers, solubilizers, perfume fragrances such as transdermal absorption accelerators, and preservatives may be further added.

The standard dose of the composition for external application for skin of the present invention can be varied depending on the individual characteristics of the patient, and a practitioner skilled in the art will be able to make an ideal administration Amount and dosage regimen, for example, the most appropriate treatment strategy in terms of the specific needs and the overall condition of the patient. For the dermal topical composition of the present invention, a number of references can be referred to in determining an appropriate dosage.

In addition, a suitable dose of the external skin application composition of the present invention may be generally determined in vitro or in an animal model. For example, various concentrations of the inventive skin topical composition may be added to the target cells in vitro to determine their proper dosage.

On the other hand, the pharmaceutical composition is preferably used for preventing or treating an inflammatory disease, for preventing or treating atopy, or for preventing or treating acne.

According to the present invention, it is possible to provide a cosmetic composition for preventing or treating hair loss, which comprises a combination of antioxidant, collagen synthesis promotion, aging prevention, skin wrinkle improvement, whitening, moisturizing, skin irritation mitigation, acne prevention, atopic improvement and anti- A multifunctional dermal external composition composition can be obtained by using the mushroom fruiting body extract or the mushroom mycelium culture medium of the present invention.

Hereinafter, the present invention will be described in more detail in 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 fruiting body extract

Antrodia The vitreous body powder obtained by lyophilizing and grinding the camphorata fruiting body was refluxed three times for 5 hours in an aqueous solution containing 70% (v / v) ethanol, cooled down, and filtered through a Whatman # 5 filter paper . The filtered extract was concentrated under reduced pressure and freeze-dried at 50 DEG C or lower, and the lyophilized product thus obtained was dissolved in a mixed solvent of purified water, ethanol, butylene glycol and propylene glycol to contain 15 wt% Fruit body extract ") was prepared.

Example  2: Preparation of mycelium extract

The antioxidant mushroom ( Antrodia camphorata) strain was isolated directly from fresh fruit bodies, right middle finger the mushroom mycelium Yeast-Malt Extract agar (yeast extract 3 g, wort 3 g, glucose 10 g, peptone 5 g, agar 20 g, distilled water 1 ℓ) is The slides were incubated at 4 ° C and subcultured every month. The mycelia grown on the slope medium were aseptically homogenized and inoculated in a liquid medium adjusted to pH 5.5 to 5% (v / v) as a simple complex medium containing 3% glucose and 1.0% yeast extract, The mixture was incubated in a fermenter at a temperature of 27 ° C, a rotation speed of 150 rpm, and aeration amount of 1.5 vvm for 10 days.

After completion of the cultivation, the mycelium was recovered through centrifugation, and then extracted with the method described in Example 1 to prepare a mycelial extract (hereinafter, referred to as "mycelium extract").

Example  3: Preparation of culture liquid from cultivation of mycelium of mung bean japonica

The antioxidant mushroom ( Antrodia camphorata) strain was isolated directly from fresh fruit bodies, right middle finger the mushroom mycelium Yeast-Malt Extract agar (yeast extract 3 g, wort 3 g, glucose 10 g, peptone 5 g, agar 20 g, distilled water 1 ℓ) is The slides were incubated at 4 ° C and subcultured every month. The mycelia grown on the slope medium were aseptically homogenized and inoculated in a liquid medium adjusted to pH 5.5 to 5% (v / v) as a simple complex medium containing 3% glucose and 1.0% yeast extract, The mixture was incubated in a fermenter at a temperature of 27 ° C, a rotation speed of 150 rpm, and aeration amount of 1.5 vvm for 10 days.

After completion of the cultivation, the mycelium was removed by centrifugation, and the culture filtrate was filtered with a paper filter (watman no. 4) to obtain a filtrate (hereinafter, referred to as "cultured Mycorrhizae mycelium").

Experimental Example  One: NBT Antioxidant effect measurement

To confirm the antioxidant levels of the Fusarium oxysporum fucanus extract obtained in Example 1 and the Fusarium oxysporum fusarium cultivar obtained in Example 3, BHT, which is well known as an antioxidant, was used as a comparative sample. Respectively.

The NBT method is a method in which active oxygen is generated by xanthine and xanthine oxidase and blue light generated by the reaction of the generated active oxygen with Nitro Blue Tetrazolium (NBT) is measured at a wavelength of 560 nm, The active oxygen scavenging rate was measured and the measurement method was as follows.

2.4 ml of sodium carbonate (Na ₂ CO ₃ ), 0.1 ml of 3 mM xanthine solution, 0.1 ml of 3 mM EDTA solution, 0.1 ml of BSA solution and 0.1 ml of 0.72 mM NBT solution were added to Bayer's disease, Solution, respectively, and left at 25 캜 for 10 minutes. After incubation, 0.1 ml of xanthine oxidase solution was added, and the mixture was rapidly stirred. Then, the mixture was incubated at 25 ° C for 20 minutes, and then 0.1 ml of a 6 mM copper chloride (CuCl ₂ ) solution was added to stop the reaction. (St). The blank test was performed using distilled water instead of the sample solution, and the absorbance (Bt) was measured in the same manner as described above. The blank of the sample solution was measured for absorbance (Bo) in the same manner as above using distilled water instead of xanthine oxidase solution.

The measurement results were calculated by the following equation (1).

[Equation 1]

Active oxygen scavenging rate (%) = [1- (St-So) / (Bt-Bo)] 100

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

Bt: Absorbance at 560 nm after enzyme reaction of blank test solution

So: absorbance at 560 nm before enzymatic reaction of enzyme solution-free sample solution

Bo: Absorbance at 560 nm before enzymatic reaction of enzyme-free blank test solution

Name of sample Active oxygen scavenging rate (antioxidant effect;%) Wujiji mushroom fruit body extract 0.25 wt% Wujiji mushroom fruit body extract 0.1 wt% Fruit of mushroom mushroom Extract of 0.05% Woojang mushroom Fruit body extract 0.025 wt% BHT 0.1 wt%

Name of sample Active oxygen scavenging rate (antioxidant effect;%) Cultured mycelia of rice bran mushroom 0.25 wt% Lt; tb > ______________________________________ < tb > 0.0 > 0.05% < / RTI > 0.025% BHT 0.1 wt%

The results of the antioxidative effects of NBT method (Table 1 and Table 2) showed that both the extracts of Fusarium oxysporum mushroom and Fusarium oxysporum showed excellent antioxidative effects at the same concentration of BHT.

From the above results, it is possible to confirm the excellent antioxidative effect of the extracts of Wujiao mushroom fruit bodies and the mushroom mycelium cultivars of the present invention.

Experimental Example  2: DPPH Antioxidant effect measurement

In Experimental Example 2, BHT, which is well known as an antioxidant, was used as a comparative sample in order to confirm the antioxidant levels of the fruiting body mushroom extract obtained in Example 1 and the fruiting body mushroom culture obtained in Example 3, The DPPH method was used for the measurement.

The DPPH method is a method of measuring free radical scavenging activity by reducing power using a free radical called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical) The degree of decrease of the absorbance was compared with the absorbance of the blank test solution, and the free radical scavenging rate was measured at a wavelength of 560 nm. The measurement method is as follows.

0.25, 0.1, 0.05, and 0.025% by weight of Fusarium oxysporum fucanum and Fusarium oxysporum were prepared. Then, the Fusarium oxysporum fusiformis extract and the Fusarium oxysporum fusiform cell culture were added to 96-well plates, respectively. To this was added DPPH prepared from 100 uM methanol solution so that the total volume of the solution became 200 μl. The absorbance (St) was measured at 560 nm after being left at 37 캜 for 30 minutes.

The free radical scavenging activity (%) was calculated by the following equation (2).

&Quot; (2) "

Free radical scavenging activity (%) = {100- (B / A)} 100

A: Absorbance of control wells not treated with Fusarium oxysporum fumarate extract or Fusarium mushroom culture

B: Absorbance of the experimental group well treated with the mushroom mycelium extract or the mushroom mycelium extract

Name of sample Free radical scavenging ratio (antioxidant effect;%) Wujiji mushroom fruit body extract 0.25 wt% Wujiji mushroom fruit body extract 0.1 wt% Fruit of mushroom mushroom Extract of 0.05% Woojang mushroom Fruit body extract 0.025 wt% BHT 0.1 wt%

Name of sample Free radical scavenging ratio (antioxidant effect;%) Cultured mycelia of rice bran mushroom 0.25 wt% Lt; tb > ______________________________________ < tb > 0.0 > 0.05% < / RTI > 0.025% BHT 0.1 wt%

The results of the antioxidative effects of DPPH assay (Table 3 and Table 4) show that both the mushroom extract and the mushroom mycelium extract showed excellent antioxidative effects even at lower concentrations than BHT.

From the above results, it was possible to confirm the excellent antioxidative effect of the mushroom extract of Wujiao mushroom and the culture of the mushroom mycelium, and the superior antioxidative effect of the present invention was deduced therefrom.

Experimental Example  3: Measurement of reactive oxygen scavenging effect in cells

In Experimental Example 3, EGCG was used to measure the scavenging effect of intracellular reactive oxygen species produced by the ultraviolet rays of the Fusarium oxysporum fucus extract obtained in Example 1 and the Fusarium oxysporum fusarium cultivar obtained in Example 3 As a comparative sample, the following experiment was performed using a fluorescent substance.

The cell line used in Experimental Example 3 was a human keratinocyte HaCaT cell line (human keratinocytes HaCaT cell line) distributed from Dr. Fusenig of the German Cancer Research Center, which was cultured in a 96-well black plate for fluorescence measurement plate) in per well 2.0 × 10 ⁴ pieces busy and penicillin / streptomycin (10% penicillin / streptomycin) are added to the DMEM (Dulbeccos Modification of Eagles medium, FBS, 37 ℃ using the Gibco, USA) medium, 5% CO ₂ After culturing for 1 day under the conditions, 0.02, 0.01, and 0.005 wt% concentration of the mushroom extract of Fusarium oxysporum and Fusarium oxysporum were treated, respectively.

The test samples were incubated for 24 hours and then washed with HCSS (HEPES-buffered control salt solution) to remove the remaining medium. DCFH-DA (2 ', 7'-dichlorodihydro-fluorescein diacetate, Probes, USA) was added, and the mixture was incubated at 37 ° C in 5% CO ₂ Lt; 0 > C for 20 min, and then washed with HCSS. After the addition of 100 μl of the treated HCSS, the fluorescence of DCF (dichlorofluorescein) initially oxidized to active oxygen was measured with a fluorescent plate reader (Ex: 485 nm, Em: 530 nm). Then, UVB (20 mJ / cm ² ) was irradiated and the fluorescence intensity was measured using a fluorescence plate reader (Ex (485 nm, Em: 530 nm) after treating each of the mushroom fruiting body extract and the mushroom mycelium culture .

Name of sample Effect of active oxygen scavenging (%) Wang Jiji mushroom fruit body extract 0.02 wt% Extract of Fusarium oxysporum fumarum mushroom 0.01 wt% Fruit of mushroom fungus extract of woojoo 0.005% EGCG 0.01 wt%

Name of sample Free radical scavenging ratio (antioxidant effect;%) Cultured mycelia of rice bran mushroom 0.25 wt% Lt; tb > ______________________________________ < tb > 0.0 > 0.05% < / RTI > 0.025% BHT 0.1 wt%

As a result of measuring the effect of active oxygen scavenging (Table 5, Table 6), it was confirmed that both the mushroom fruiting body extract and the mushroom mycelial body culture showed higher ROS than EGCG at the same concentration as EGCG.

From the above results, it can be concluded that the extract of Wujangji mushroom fruit body extract, It was confirmed that the active oxygen scavenging effect of the culture broth was excellent and that the excellent active oxygen scavenging effect of the present invention was confirmed.

Experimental Example  4: Metalloproteinase ( MMP -1) Activity inhibition rate measurement ( in vitro )

The experiment of Example 4, the right middle finger mushroom mycelium culture obtained in the right middle finger mushroom fruit body extract as in Example 3 obtained in Example 1 in Green tea extract was used as a comparative sample and fluorescence assay was used to measure inhibition rate of metal protease (MMP-1) activity in vitro .

A commercially available collagenase was used in the Molecular probe (Eugene, OR, USA) and the reaction buffer (0.5 M Tris-HCl , 1.5 M NaCl, 50 mM CaCl ₂ , 2 mM sodium azide, pH 7.6) was used after 10-fold dilution. 20 μl of DQ collagen dissolved in the reaction buffer at a concentration of 0.25 mg / ml was added to each 100 μl of the reaction buffer, and 40 μl of the culture solution of the mushroom mycelium extract and the mushroom mycelium (0.02, 0.01, 0.005% by weight) 40 [mu] l of collagenase was added. After 20 minutes at room temperature, the cow was measured for fluorescence with an absorption wavelength of 495 nm and an emission wavelength of 515 nm using a fluorescence spectrophotometer (Perkin Elmer, UK). In the control group, fluorescence value was measured after addition of enzyme buffer in the same amount as the enzyme buffer, and the fluorescence value of the sample itself was also measured to calculate the enzyme activity.

Name of sample MMP-1 activity inhibition rate (%) Wang Jiji mushroom fruit body extract 0.02 wt% Extract of Fusarium oxysporum fumarum mushroom 0.01 wt% Fruit of mushroom fungus extract of woojoo 0.005% Green tea extract 0.2 wt%

Name of sample MMP-1 activity inhibition rate (%) 0.02 wt% The mushroom mycelium culture solution of 0.01% 0.005 wt% Green tea extract 0.2 wt%

in (MMP-1) activity in vitro (Table 7, Table 8). The inhibition rate of metalloproteinase (MMP-1) activity in vitro was lower than that of green tea extract in both the mushroom extract of Fusarium oxysporum f -1) was inhibited effectively.

From the above results, it was confirmed that the effect of inhibiting the metalloproteinase (MMP-1) activity of the mushroom extract of Fusarium oxysporum fucus and the fungal mycelia of Fusarium oxysporum mushroom was excellent. Based on this fact, It was found that skin elasticity and wrinkle formation can be prevented by effectively inhibiting the enzyme.

Experimental Example  5: After UV irradiation Metalloproteinase ( MMP -1) Measurement of inhibition of expression

In Experimental Example 5, in order to measure the degree of suppression of metalloproteinase (MMP-1) expression after ultraviolet irradiation of the fungus body extract obtained in Example 1 and the fungus body extract obtained in Example 3 Retinol was used as a comparative sample.

Enzyme immunoassay (ELISA) was performed to measure the concentration of metalloproteinase (MMP-1) after UV irradiation and sample addition (right fungus mushroom fruiting body extract or right fungus body mycelium culture, retinol).

UVA was irradiated to human dermal fibroblasts at an energy of 6.3 J / cm 2 using a UV chamber, and ultraviolet irradiation dose and incubation time were measured by preliminary experiments to determine the maximum amount of metalloproteinase (MMP-1) Respectively. The negative control was wrapped in silver foil and kept in the UVA environment for the same time, and the UVA emission was measured using a UV radiometer. The cells while UVA was irradiated were irradiated with UVA, and then cultured for 24 hours in a medium containing the sample. Then, the medium was recovered and coated on a 96-well plate. The primary antibody {MMP-1 (Ab-5) monoclonal antibody} was treated and reacted at 37 DEG C for 60 minutes. After incubating for 60 minutes with a secondary antibody, anti mouse IgG (alkaline phosphatase conjugated), the alkaline phosphatase substrate solution (1 mg / ml ρ-nitrophenyl phosphate in diethanolamine buffer) was reacted at room temperature for 30 minutes, Absorbance was measured at 405 nm with a plate reader. As a control group, a sample to which no sample was added was used.

Name of sample MMP-1 expression inhibition rate (%) Control group Wang Jiji mushroom fruit body extract 0.02 wt% Extract of Fusarium oxysporum fumarum mushroom 0.01 wt% Fruit of mushroom fungus extract of woojoo 0.005% Retinol

Name of sample MMP-1 expression inhibition rate (%) Control group 0.02 wt% The mushroom mycelium culture solution of 0.01% 0.005 wt% Retinol

The inhibition rate of metalloproteinase (MMP-1) expression after UV irradiation was measured (Table 9, Table 10). As a result, the inhibition rate of both mushroom fruiting body extract and mushroom mycelial body was superior to that of untreated control , Which was significantly better than the inhibition rate of retinol used as a comparative sample.

From the above results, it was confirmed that the effect of inhibiting the expression of metalloproteinase (MMP-1) in the extracts of Fusarium oxysporum fucus and Fusarium oxysporum was excellent. Based on this fact, It was found that the skin elasticity and wrinkle formation can be prevented by effectively inhibiting the expression of the degrading enzyme.

Experimental Example  6: Measuring the promoting effect of type 1 procollagen biosynthesis

In Experimental Example 6, in order to confirm the effect of promoting the biosynthesis of type 1 procollagen, which is a skin matrix component, of the culture of the mushroom fruit extract obtained in Example 1 and the mushroom mycelium obtained in Example 3, TGF -β was used as a positive control, and procollagen assay was performed as follows.

Human dermal fibroblasts isolated from the epidermal tissue of newborns were purchased from Modern Tissue Technology (MTT, Korea) and supplemented with 10% fetal bovine serum (FBS) in DMEM / F-12 (3: And cultured at a concentration of 1 × 10 ⁴ cells / cm ² . When 70-80% of the cells were grown, the cells were subcultured at a ratio of 1: 3, and cells in the third to fourth subculture were used for the experiment.

For the measurement of the amount of procollagen, fibroblasts were cultured in a 48-well plate at a concentration of 90% or more, and then cultured in a concentration of 0.02%, 0.01%, and 0.005% by weight of the extracts of Fusarium oxysporum f . After 24 hours, the amount of procollagen liberated in the medium was measured using a procollagen type-1 C-peptide EIA kit (MK101, Takara, Japan) .

Name of sample Promoting effect of procollagen biosynthesis (%) Wang Jiji mushroom fruit body extract 0.02 wt% Extract of Fusarium oxysporum fumarum mushroom 0.01 wt% Fruit of mushroom fungus extract of woojoo 0.005% TGF-beta 0.001 wt%

Name of sample Promoting effect of procollagen biosynthesis (%) 0.02 wt% The mushroom mycelium culture solution of 0.01% 0.005 wt% TGF-beta 0.001 wt%

As a result of measuring the promoting effect of procollagen biosynthesis (Table 11, Table 12), the extracts of Fusarium oxysporum fusiformis and Fusarium oxysporum showed similar effects to the cell signaling substance TGF-β.

From the above results, it was confirmed that the extract promoted the excellent procoagulant biosynthesis of the Fusarium oxysporum fucus extract and the Fusarium oxysporum fusiform mycelia, and that the present invention is superior in improving the skin aging.

Experimental Example  7: Elastase  Measurement of inhibition of activity

In Experimental Example 7, in order to measure the effect of promoting the production of elastin by the fungus body extract obtained in Example 1 and the fungus body extract obtained in Example 3, The following experiment was carried out by treating each of the fibroblasts of Wujiao mushroom fruiting body extract and the mushroom mycelium culture broth respectively.

First, human fibroblasts were placed in a culture flask and cultured until they were about 70-80% grown. After that, each of the mushroom extract of Fusarium oxysporum mushroom and the fusarium mushroom mycelium was treated for 1 day, and the cell culture fluid was collected and the degree of elastin production was measured using a commercially available elastin measuring apparatus [Bieth J: Biochem med . , 11, 350-357 (1974), Schwartz DE: J. Invest Dermatol ., 86, 63-68 (1986)]. That is, the activity of elastase was measured using the absorbance of a color change caused by the decomposition of NA of Suc- (Ala) 3 NA which is a substrate of elastase. At this time, the control group was treated with the Fusarium oxysporum fructus extract and the Fusarium mushroom mycelial culture.

Name of sample Elastase activity inhibition rate (%) Control group Wang Jiji mushroom fruit body extract 0.02 wt% Extract of Fusarium oxysporum fumarum mushroom 0.01 wt% Fruit of mushroom fungus extract of woojoo 0.005%

Name of sample Elastase activity inhibition rate (%) Control group 0.02 wt% The mushroom mycelium culture solution of 0.01% 0.005 wt%

As a result of measuring the inhibition rate of the elastase activity (Table 13, Table 14), it was confirmed that the inhibitory rate of the elastase activity was increased in a concentration-dependent manner in both the mushroom fruiting body extract and the mushroom mycelium culture solution.

From the above results, it was confirmed that the extract of Fusarium oxysporum fumarum and Fusarium oxysporum fusarium was superior in the inhibition of elastase activity, and the excellent anti-aging and elasticity-enhancing effect of the composition for external application for skin of the present invention was confirmed.

Experimental Example  8: Tyrosinase  Measurement of inhibition of activity

In Experimental Example 8, arbutin, well known as a whitening agent, was used as a comparative sample in order to confirm the inhibitory effect of tyrosinase activity of the mushroom extract obtained in Example 1 and the mushroom mycelium obtained in Example 3 .

Tyrosinase is an enzyme that stimulates the oxidation of tyrosine in vivo to produce melanin.

Tyrosinase was isolated and purified from mushrooms and purchased from Sigma. L-tyrosine, a substrate, was dissolved in 0.05 M sodium phosphate buffer solution (pH 6.8) to make a 0.1 mg / ml solution. The culture broths of the mushroom extract and the mushroom mycelium were 0.05 M sodium phosphate buffer solution (pH 6.8) And then the solution was used. 0.5 ml of L-tyrosine solution was added to a test tube, and 0.5 ml of the Fusarium oxysporum fusiformis extract and the culture solution of the mycelia of the fungus mushroom were added thereto, respectively, and the mixture was allowed to stand at 37 ° C for 10 minutes. Thereafter, 0.5 ml of 200 units / ml tyrosinase was added, and the mixture was reacted at 37 占 폚 for 10 minutes. The reaction tube was placed on ice to quench the reaction, and the absorbance at 475 nm was measured by a spectrophotometer. As a control, 0.5 ml of buffer solution was used in place of the culture solution of Fusarium oxysporum fusiformis and Fusarium oxysporum fusiformis.

The inhibition rate of tyrosinase activity of each of the above samples was calculated according to Equation (3).

&Quot; (3) "

Tyrosinase activity inhibition rate (%) = 100 - [(comparative group absorbance / control group absorbance)] x 100

Name of sample Inhibition rate of tyrosinase (%) Control group Wang Jiji mushroom fruit body extract 0.02 wt% Extract of Fusarium oxysporum fumarum mushroom 0.01 wt% Fruit of mushroom fungus extract of woojoo 0.005% Arbutin 0.2 wt%

Name of sample Inhibition rate of tyrosinase (%) Control group 0 0.02 wt% 51 The mushroom mycelium culture solution of 0.01% 41 0.005 wt% 35 Arbutin 0.2 wt% 54

The inhibitory effect of tyrosinase was measured (Table 15, Table 16), and the culture of the mushroom fruiting body extract and the mushroom mycelial body showed inhibition rates of tyrosinase activity similar to arbutin.

From the above results, excellent tyrosinase activity inhibitory effect of the mushroom extract of Fusarium oxysporum fungus and the mycelia of Fusarium oxysporum was confirmed, and it was found that the excellent whitening effect of the composition for external application for skin of the present invention was confirmed.

Experimental Example  9: Measurement of melanin synthesis inhibitory effect using B16F1 melanocytes

In Experimental Example 9, albutin, which is known as a whitening agent, was used as a comparative sample in order to confirm the melanin synthesis inhibitory effect of the fruiting body extract obtained in Example 1 and the fruiting body obtained in Example 3, B16F1 melanocytes were used.

The B16F1 melanocyte used in Experimental Example 9 is a cell line derived from a mouse and is a cell that secretes a melanin pigment called melanin.

The inhibitory effect of B16F1 melanin on melanin synthesis was measured as follows. B16F1 melanocytes were plated at a concentration of 2 x 10 ⁶ per well in a 6-well plate, and the cells were adhered, treated at a concentration not causing toxicity, and cultured for 72 hours. After culturing for 72 hours, cells were detached with trypsin-EDTA, the number of cells was measured, and the cells were recovered by centrifugation. Quantification of intracellular melanin was carried out with a slight modification of the method of Rotan (R Lotan and D Lotan, Cancer Res, 40: 3345-3350, 1980). Cell pellets were washed once with PBS and then 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added and vortexed for 5 minutes to disrupt the cells. After centrifugation (3,000 rpm, 10 min), 1N NaOH (10% DMSO) was added to the cell filtrate to dissolve the extracted melanin, and the absorbance of melanin was measured at 405 nm using a microplate reader. Melanin was quantified The percent inhibition of melanin formation in the sample was measured.

The inhibition rate (%) of melanin formation of B16F1 melanocytes was calculated by the following equation (4).

&Quot; (4) "

Melanin synthesis inhibition rate (%) = [(A-B) / A] 100

A: Amount of melanin in wells to which no sample was added

B: Amount of melanin in the well to which the sample was added

Name of sample Melanin synthesis inhibition (%) Control group Wang Jiji mushroom fruit body extract 0.02 wt% Extract of Fusarium oxysporum fumarum mushroom 0.01 wt% Fruit of mushroom fungus extract of woojoo 0.005% Arbutin 0.2 wt%

Name of sample Melanin synthesis inhibition (%) Control group 0.02 wt% The mushroom mycelium culture solution of 0.01% 0.005 wt% Arbutin 0.2 wt%

The inhibitory effect of melanin on B16F1 melanocytes was measured (Table 17, Table 18). The results showed that melanin synthesis inhibition rate similar to that of arbutin was shown in the cultures of Fusarium oxysporum fusiformis extract and Fusarium spp.

From the above results, it was confirmed that the melanin synthesis inhibition rate of the mushroom fruit body extract and the mushroom mycelium culture medium were excellent, and the excellent whitening effect of the composition of the present invention was confirmed based on the above results.

Experimental Example  10: Inflammation-inducing enzymes hyaluronidase ) Measurement of inhibitory effect

In Experimental Example 10, in order to measure the anti-inflammatory effect of the Fusarium oxysporum fucus extract obtained in Example 1 and the Fusarium oxysporum fusarium cultivar obtained in Example 3, Compressi extract, Sepharose extract, And the inhibitory effect of hyaluronidase activity, which is an inflammation inducing enzyme, was measured.

Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid and causes inflammation.

In this experimental example, anti-inflammatory effect was evaluated by the method of inhibiting the activity of hyaluronidase and measuring the anti-inflammatory effect (Kakegawa Y: Japanese J. of Inflammation, 4, 437-438, 1984) .

Sample 100 ㎕ and hyaluronic ruro nidiah agent solution (type Ⅳ-S, Sigma, 400U / ㎖) was introduced into a 50 ㎕ at 37 ℃ 20 minutes of reaction, the enzyme solution active (compound 48/80 CaCl ₂ and 2H ₂ O, Sigma , 0.1 mg / ml) was added and reacted at 37 캜 for 20 minutes. 250 μl of hyaluronic acid solution (0.4 mg / ml) was added, reacted at 37 ° C for 40 minutes, and 100 μl of 0.4 N NaOH was added to terminate the reaction. 100 μl of potassium borate solution was added, reacted at 95 ° C for 3 minutes, cooled, added with 3 ml of? -Dimethylaminobenzaldehyde solution, reacted at 37 ° C for 20 minutes, and developed. The absorbance was measured at 585 nm to measure the inhibition rate of hyparonidase activity.

The activity inhibition (%) of the hyaruronidase is calculated by the equation (5), and the IC ₅₀ value is the concentration of the substance that inhibits the enzyme activity of the hyaruronidase by 50%.

&Quot; (5) "

(%) = [(A-B) / A] x 100

A: Enzyme activity of well without addition of sample

B: Enzyme activity of the well to which the sample was added

Name of sample Inhibitory effect of hyaluronidase (IC ₅₀ ;%) Comfrey extract Seiso extract Usefulness Licorice extract Extract of Wujiji mushroom fruit body

Name of sample Inhibitory effect of hyaluronidase (IC ₅₀ ;%) Comfrey extract Seiso extract Usefulness Licorice extract Culture medium of mushroom mycelium

IC ₅₀ values of the mushroom fruiting body extract and IC ₅ of the mushroom mycelium culture solution were measured by the results of measuring the inhibitory effect of hyaluronidase (Table 19, Table 20) The extracts showed similar or superior effects to the licorice extract.

From the above results, it is possible to confirm the excellent anti-inflammatory effect of the mushroom fruit body extract of Wujiao mushroom and the culture medium of the mushroom mycelium, and the excellent anti-inflammatory effect of the composition for external application for skin of the present invention was confirmed based on this.

Experimental Example  11: Measurement of mitigation effect of cytotoxicity by ultraviolet irradiation

In Experimental Example 11, the mitochondrial cytotoxic effect of the fruiting body extract obtained in Example 1 and the fruiting body obtained in Example 3 was measured by ultraviolet irradiation.

Fibroblasts were placed in 24-well test plates at 1 × 10 ⁵ cells and adhered for 24 hours. Each well was washed once with PBS and 500 占 퐇 of PBS was added to each well. After irradiating the fibroblasts with 10 mJ / cm 2 of ultraviolet light using an ultraviolet B (UVB) lamp (Model: F15T8, UVB 15W, Sankyo Dennki, Japan), PBS was removed and the cell culture medium (DMEM supplemented with 10% FBS 1 ml) was added. Here, the extracts of Fusarium oxysporum fusiformis and Fusarium oxysporum fungi were treated respectively and cultured for 24 hours. After 24 hours, the medium was removed, and 500 μl of the cell culture medium and 60 μl of MTT solution (2.5 mg / ml) were added to each well, followed by culturing in a 37 ° C. CO ₂ incubator for 2 hours. After the culture, the medium was removed and 500 μl of isopropanol-HCl (0.04 N) was added. After shaking for 5 minutes, the cells were lysed and 100 μl of the supernatant was transferred to a 96-well test plate, and the absorbance was measured at 565 nm using a microplate reader.

The cell viability (%) was measured by the equation (6) and the cytotoxic relaxation rate by ultraviolet was calculated by the formula (7).

&Quot; (6) "

Cell survival rate (%) = [(St-Bo) / (Bt-Bo)] 100

Bo: Absorbance at 565 nm of wells reacting with cell culture medium

Bt: Absorbance at 565 nm of a well that underwent chromogenic reaction in a sample not treated with the sample

St: absorbance at 565 nm of wells treated with the sample

&Quot; (7) "

(%) = [1- (St-Bo) / (Bt-Bo)] x 100

Bo: Cell survival rate of wells not irradiated with ultraviolet light and not treated with sample

Bt: Cell viability of wells irradiated with ultraviolet light and not treated with the sample

St: Cell viability of well treated with ultraviolet light and treated with sample

Name of sample Treatment concentration (% by weight) Cytotoxic Relaxation Rate (%)
Extract of Wujiji mushroom fruit body 0.02 0.01 0.005

Name of sample Treatment concentration (% by weight) Cytotoxic Relaxation Rate (%)
Culture medium of mushroom mycelium 0.02 0.01 0.005

The cytotoxic relaxation effect of ultraviolet irradiation was measured (Table 21, Table 22), and the culture of the mushroom fruiting body extract and the mushroom mycelial culture solution of the mushroom mushroom excellently mitigated cytotoxicity by ultraviolet rays, .

From the above results, it was confirmed that the cytotoxic relaxation effect of the fungus extract of Fusarium oxysulatus mushroom and the culture of Fusarium oxysporum mushroom mycelium by UV irradiation was excellent, and based on this, it was confirmed that the skin irritation mitigation effect of UV- I could.

Experimental Example  12: Measurement of inhibitory effect of inflammatory cytokine expression by ultraviolet irradiation

Experimental Example 12 was conducted in order to measure the anti-inflammatory effect of the Fusarium oxysporum fucus extract obtained in Example 1 and the Fusarium mushroom mycelium obtained in Example 3, and the inhibitory effect of the inflammatory cytokine expressed by ultraviolet irradiation Were measured.

Fibroblasts isolated from human epidermal tissue were placed in a 24-well test plate in an amount of ⁵ × 10 ⁴ and adhered for 24 hours. Each well was washed once with PBS and 500 占 퐇 of PBS was added to each well. The fibroblasts were irradiated with ultraviolet rays at 10 mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: F15T8, UVB 15W, Sankyo Dennki Co., Japan), and PBS was removed. Cell culture medium ≪ / RTI > medium). Here, the extracts of Fusarium oxysporum fusiformis and Fusarium oxysporum were treated respectively and then cultured for 5 hours. After culturing, 150 μl of culture supernatant was taken to quantitate IL-1α, and thus the inhibitory effect of inflammatory cytokine expression inhibition was determined on cultures of Fusarium oxysporum fusiformis extract and Mycobacterium tuberculosis mycelium. The amount of IL-1.alpha. Was quantitated using Enzyme-linked Immunosorbent Assay, and the production rate of IL-1.alpha. Was calculated by Equation (8).

&Quot; (8) "

Inhibitory rate of inflammatory cytokine expression (%) = [1- (St-Bo) / (Bt-Bo)] 100

Bo: IL-1? Production in wells without UV irradiation

Bt: IL-1? Production in wells irradiated with ultraviolet light and not treated with the sample

St: IL-1α production in wells irradiated with ultraviolet light and treated with the sample

Name of sample Treatment concentration (% by weight) Inhibitory rate of inflammatory cytokine expression (%)
Extract of Wujiji mushroom fruit body 0.02 0.01 0.005

Name of sample Treatment concentration (% by weight) Inhibitory rate of inflammatory cytokine expression (%)
Culture medium of mushroom mycelium 0.02 0.01 0.005

As a result of measuring the inhibitory effect of inflammatory cytokine expression by ultraviolet irradiation (Table 23, Table 24), it was confirmed that the culture of Fusarium oxysporum fusiformis extract and Fusarium oxysporum fusiformis effectively inhibited the development of inflammation caused by ultraviolet light at a low concentration . From the above results, it was confirmed that the effect of inhibiting the expression of inflammatory cytokine by ultraviolet irradiation of the fungus body extract of Fusarium oxysporum mushroom and the fungal mycelium culture was excellent, and the anti-inflammatory effect of the composition for external application for skin of the present invention was found based on this .

Experimental Example  13: Measurement of inhibitory effect of prostaglandin biosynthesis by ultraviolet irradiation

In Experimental Example 13, the inhibitory effect of the prostaglandin E ₂ (hereinafter referred to as PGE ₂ ) biosynthesis inhibitory effect of the Fusarium oxysporum fructus extract obtained in Example 1 and the Fusarium oxysporum fusiform mycelia obtained in Example 3 was evaluated Keratinocytes isolated from human epidermal tissue were placed in a 24-well test plate in an amount of 5 × 10 ⁴ and adhered for 24 hours. After replacing the medium with FBS-free medium for 18 hours, the keratinocytes were treated with aspirin to a final concentration of 50 uM to remove prostaglandin biosynthesis enzyme (prostaglandin E ₂ synthetase, or cyclooxygenase, hereinafter referred to as COX). Two hours after aspirin treatment, each well containing keratinocytes was washed twice with PBS, and 100 占 퐇 of PBS was added to each well. The keratinocyte was irradiated with ultraviolet rays of 30 mJ / cm 2 using a UVB lamp (Model: F15T8, UVB 15 W, Sankyo Dennki, Japan), and PBS was removed. Keratocyte growth media Clonetics, Biowhittacker, MD, USA). The extracts of the mushroom fruiting body and the mushroom mycelium were treated for 16 hours. The prostaglandin inhibitory effect of the mugwort fruiting body extract and the mushroom mycelium culture was evaluated by quantifying the biosynthesized PGE ₂ (prostaglandin E ₂ ) by taking an appropriate amount of the culture supernatant for 16 hours. The amount of PGE ₂ was quantitated using enzyme immunoassay.

Name of sample PGE ₂ inhibition rate (%) (-) UV B (+) UV B (+) UV B + Fungus mushroom Fruit body extract 0.02 wt% (+) UV B + Fungus mushroom Fruit body extract 0.01 wt% (+) UV B + Fungus mushroom Fruit body extract 0.005 wt%

Name of sample PGE ₂ inhibition rate (%) (-) UV B (+) UV B (+) UV B + 0.02 wt% (+) UV B + 0.01% (w / w) (+) UV B + Raw mushroom Mycelial culture 0.005 wt%

As a result of measuring the inhibitory effect of prostaglandin biosynthesis by ultraviolet irradiation (Table 25, Table 26), it was confirmed that both the mushroom extract of Fusarium oxysporum and the culture of Fusarium oxysporum effectively inhibit the production of prostaglandin by ultraviolet rays. In particular, it is known that PGE ₂ produced is mainly produced by COX-2 enzyme. Therefore, it can be inferred from the above experiment that the culture of Fusarium oxysporum fusiformis extract and Fusarium oxysporum fusiformis mycelia suppress the induction or activity of COX-2 enzyme there was.

From the above results, it was confirmed that the excellent inhibitory effect on the prostaglandin biosynthesis of the mushroom extract of Fusarium oxysporum f. Mushroom and the Fusarium oxysporum f. Mushroom culture was confirmed, and it was confirmed that the composition for external application for skin of the present invention has excellent inhibitory effect on prostaglandin biosynthesis.

Experimental Example  14: Antimicrobial activity against acne bacteria

In Experimental Example 14, a paper disc test was conducted to examine the antimicrobial activity of the fungus extract obtained in Example 1 and the fungus extract obtained in Example 3 against acne bacteria. Respectively.

First, for activation of Propionibacterium acnes, a skin overgrowth caused by acne, 48 hours pre-culture was carried out in a BHI liquid medium (Brain-Heart Infusion Broth; 3.7%). The bacterial culture thus prepared was plated in 0.1 ml of BHI solid medium (Brain-Heart Infusion Broth; 3.7%; agar 1.5%) and dried. The mushroom extract of Fusarium oxysporum mushroom and Fusarium oxysporum mushroom culture were diluted to 12% (w / v) in 95% ethanol aqueous solution, and 50 μl was added to a paper disc having a diameter of 8 ㎜, placed on the previously prepared solid medium, And cultured in an anaerobic incubator at 35 ° C for 3 days. The antimicrobial activity was evaluated by observing the growth inhibition zone around the 'paper disc' and measuring the size of the inhibition zone.

As a result of measuring the antibacterial activity against acne bacterium, the size of the bacterial growth inhibition of the fungus body extract of Fusarium oxysporum mushroom and Fusarium oxysporum fusiformis was 16 ㎜, and the excellent antibacterial activity of the composition for external application for skin of the present invention was confirmed.

Experimental Example  15: Compound  Anti-inflammatory effects of fruiting body extracts and mushroom mycelium extracts induced by atopic dermatitis induced by 48/80

30 μl of compound 48/80 (Sigma, Co .; 1 mg / ml in saline) was injected into the dermis of the dorsal skin of BALB / c mice (5 wks, male) Mice were observed to scratch their necks. In order to test the anti - inflammatory efficacy of the mushroom extract of Fusarium oxysporum mushroom and Fusarium oxysporum mushroom, the culture of Fusarium oxysporum fusiformis and Fusarium oxysporum fusiformis was added at a concentration of 250 μL / Mu] L of each mouse. After applying Compound 48/80 for 5 days, the mice were pretreated at the dorsal neck of the mouse for 10 times for 5 days. The time was measured and the degree of pruritus was measured.

Name of sample  Number of scratching (in 5min) 5 10 15 20 25 30 35 40 45 50 55 60 Extract of Wujiji mushroom fruit body Control group

Name of sample  Number of scratching (in 5min) 5 10 15 20 25 30 35 40 45 50 55 60 Culture medium of mushroom mycelium Control group

The antiinflammatory effects of Fusarium oxysporum fusiformis extract and Fusarium oxysporum mycelium cultures after atopic dermatitis induction were measured (Table 27 and Table 28) (Skin pruritus).

From the above results, it was confirmed that the atopic prevention effect of the mushroom extract of Fusarium oxysporum f. Mushroom and the fungus mushroom culture was excellent.

Experimental Example  16: Measurement of water hygroscopicity

In Experimental Example 16, in order to measure the water hygroscopicity of the mushroom extract of Fusarium oxysporum mushroom and the mycelia of Fusarium oxysporum mushroom, the extracts of mushroom fruiting body or mushroom mycelium The amount of water absorbed by the culture solution and distilled water was measured, and the mass change after drying was measured for 48 hours.

The amount of water absorbed per skin cell unit mass was compared from each measured mass change. This experiment is a method for comparing the amount of water absorbed to the epidermal cells, and is used as one method for predicting the moisturizing effect even in the human skin.

Specifically, human epidermal cells were dried to make the amount of water contained per unit mass constant, and then the weight of each epidermal cell sample and the amount of water contained were measured using the Kaiser method. The epidermal cells, in which the water content was measured, were carried on the mushroom fruiting body extract of mushroom mushroom or the mycelia of mushroom mycelium and the purified water for 24 hours, respectively, and the skin cells were saturated and absorbed. And the amount of water per unit mass contained in the Kaiser moisture meter was measured. Then, after drying for 48 hours under reduced pressure, the weight was added, and a part of the weight was measured. The water content was measured by the Kaiser method and the amount of water per unit mass was measured (mg / epidermal cell mass g).

Name of sample Moisture content Initial dry state Saturation After re-drying Extract of Wujiji mushroom fruit body Purified water

Name of sample Moisture content Initial dry state Saturation After re-drying Culture medium of mushroom mycelium Purified water

As a result of measuring the water hygroscopicity (Table 29, Table 30), it was found that the epidermal cells that were saturated and absorbed with the mushroom fruiting body extract and the mushroom mycelial culture of the mushroom mushroom showed much more water in the re-drying process than the saturated moisture- As shown in Fig.

From the above results, it was possible to confirm excellent skin hygroscopicity of the Fusarium oxysporum fucanum and Fusarium oxysporum fungi cultures, and the excellent moisturizing effect of the present invention was confirmed.

Experimental Example  17: Combination of human hair protein and cultured mycelium of fruiting body and mushroom of Woojangji mushroom

To 50 사람 of human hair keratin protein were added 0.5, 0.25, and 0.1 wt% of the test extracts of the mushroom extract and the mushroom mycelium extract obtained in Example 1 or 3, respectively, and the reaction was carried out at room temperature for 30 minutes , Treated with a mixed solution of 6% hydrogen peroxide and 0.5% ammonia 1: 1, and reacted for 30 minutes. The reaction solution was electrophoresed on a 10% SDS-polyacrylamide gel according to the method of Laemmli and the keratin protein in the gel was dissolved in 0.1% Coomassie brilliant blue R 250, 10% glacial acetic acid and 40% ethanol For 1 hour, decolorized in a mixed solution of 10% glacial acetic acid and 40% ethanol, and the keratin protein band was identified. 100% of the keratin protein band appeared when 50 μg of human hair keratin protein was electrophoresed using the image master (Amersham Pharmacia Biotech.) Software, and the culture of the mushroom fruiting body extract or the mushroom mycelium was not treated Were compared as control and the keratin binding effect was expressed in%.

Extract of Wujiji mushroom fruit body Control group 0.5 wt% 0.25 wt% 0.1 wt% Hair protein binding effect

Culture medium of mushroom mycelium Control group 0.5 wt% 0.25 wt% 0.1 wt% Hair protein binding effect

The results of the experiment (Table 31, Table 32) showed that the binding capacity of hair protein keratin increased with increasing contents of both the mushroom extract of Fusarium oxysporum and the mycelium of Fusarium mushroom.

Experimental Example  18: Extraction of keratin mushroom fruiting body and fermented mushroom mycelium for keratin leaching by treatment with alkali and hydrogen peroxide Protection effect

3 g of hair was placed in a 1: 1 mixed solution of 10 ml of hydrogen peroxide (6%) and ammonia (1.68%), and the culture solution of the mushroom extract and the mushroom mycelium obtained in Example 1 or Example 3 Were added at test concentrations of 0.5, 0.25, and 0.1 wt%, respectively, and then treated at room temperature for 30 minutes. 0.5 ml of the reaction solution was concentrated using a Rapid-Con protein concentration kit (Elpis Biotech.), And the concentrate was electrophoresed on a 10% SDS-polyacrylamide gel according to Laemmli's method. The mixture was dyed in a mixed solution of 0.1% Coomassie brilliant blue R 250, 10% glacial acetic acid and 40% ethanol for 1 hour and discolored in a mixed solution of 10% glacial acetic acid and 40% ethanol to identify a keratin protein band. Using the image master software (Amersham Pharmacia Biotech.), The keratin band appeared when 50 μg of human hair keratin protein alone was electrophoresed, and 100% of the keratin band was not treated with the mugwort fruiting body extract or the mushroom mycelium culture The binding effect of the keratin protein was expressed as% in comparison with the control group.

Extract of Wujiji mushroom fruit body Control group 0.5 wt% 0.25 wt% 0.1 wt% Keratin protein

Culture medium of mushroom mycelium Control group 0.5 wt% 0.25 wt% 0.1 wt% Keratin protein

The results of the experiment (Table 33, Table 34) showed that the content of keratin protein eluted from the hair decreased with increasing contents of the mushroom extract of fungus mushroom and the mushroom mycelium culture.

EXPERIMENTAL EXAMPLE 19 Effect of Fruit Extracts of Fungus mushroom and Fungus mushroom Mycelium on Tensile Strength and Elongation of Hair by Treatment with Alkali and Hydrogen Peroxide

3 g of hair was placed in a 1: 1 mixed solution of 10 ml of hydrogen peroxide (6%) and ammonia (1.68%), and the culture solution of the mushroom extract and the mushroom mycelium obtained in Example 1 or Example 3 (0.5, 0.25, and 0.1 wt%, respectively), treated at room temperature for 30 minutes, washed with water, and dried in air to measure tensile strength (gf) and elongation (%) in an autograph tester. Table 18 below shows the tensile strength and elongation of the hair thus treated.

Extract of Wujiji mushroom fruit body Purified water 0.5 wt% 0.25 wt% 0.1 wt% Tensile strength (gf) Shinto (%)

Culture medium of mushroom mycelium Purified water 0.5 wt% 0.25 wt% 0.1 wt% Tensile strength (gf) Shinto (%)

As a result of the experiment (Table 35, Table 36), when the decolorizing agent (the hydrogen peroxide and ammonia mixed solution) was treated for 30 minutes, the decrease in hair strength was suppressed as the addition amount of both the mushroom fruiting body extract and the mushroom mycelium can see. Therefore, it could be expected that the hair - protecting effect can be expected when the decolorant is treated with the extract of Fusarium oxysporum f.

Experimental Example  20: Hair Growth Effect Test

The 30% ethanol solution containing 2% by weight of the mushroom extract and the mushroom mycelium extract obtained in Example 1 or Example 3 were respectively prepared and used for the hair growth effect test. Hair growth test was carried out by using mouse (ICR), 47 ~ 53 days after birth, removing the hairs from the dorsal area, and selecting the back area clean. Using 10 animals per group, 100 ml of test substance per day Respectively. The length of hair and hair growth according to time were removed and the scores were added from 0 to 2 according to the degree of restoration. In order to compare the degree of hair growth, a 30% alcohol solution was applied to each individual as a control group and the growth state was observed.

The degree of hair growth promoting effect was classified into three grades (0: no growth, 1: slight growth, 2: much growth).

Sample / elapsed days 5 10 15  Wujiji mushroom fruit body extract 2% Control group

Sample / elapsed days 5 10 15  Mushroom mycelium culture medium 2 wt% Control group

Experimental results (Table 37, Table 38) confirmed the excellent hair growth promoting effect of the Fusarium oxysporum fructus extract and the Fusarium oxysporum fusiform mycelia.

Example  4 and Comparative Example  1: Fungus extract containing fruiting body Cosmetics  Produce

In Example 4, a cosmetic preparation containing the Fusarium oxysulfurum mushroom extract obtained in Example 1 was prepared.

The cosmetic preparation was in the form of a cream, and its composition was as shown in Table 39 below.

First, the b) phase recorded in Table 39 below was heated and stored at 70 캜, and a) phase was added to b) to emulsify uniformly with a homomixer after preliminary emulsification and gradually cooled to obtain cream (Example 3, Comparative Example 1).

Raw material Example 4 (% by weight) Comparative Example 1 (% by weight) end Stearyl alcohol 8 8 Stearic acid 2 2 Stearic acid cholesterol 2 2 Squalane 4 4 2-octyldodecyl alcohol 6 6 Polyoxyethylene (25 mole addition) alcohol ester 3 3 Glyceryl monostearate Aster 2 2 I Extract of Wujiji mushroom fruit body One - Propylene glycol 5 5 Purified water Until it is 100 Until it is 100

Example  5 and Comparative Example  2: a culture containing the culture medium of the mushroom mycelium Cosmetics  Produce

In Example 5, a cosmetic containing the cultured mycelia of mushroom mycelium obtained in Example 3 was prepared.

The cosmetics prepared are in the form of a cream, and the composition thereof is shown in Table 40 below.

First, the b) phase recorded in Table 40 below was heated and stored at 70 占 폚, and a) phase was added to b) to emulsify uniformly with a homomixer after preliminary emulsification and then slowly cooled to obtain a cream (Example 4, 2).

Raw material Example 5 (% by weight) Comparative Example 2 (% by weight) end Stearyl alcohol 8 8 Stearic acid 2 2 Stearic acid cholesterol 2 2 Squalane 4 4 2-octyldodecyl alcohol 6 6 Polyoxyethylene (25 mole addition) alcohol ester 3 3 Glyceryl monostearate Aster 2 2 I Culture medium of mushroom mycelium One - Propylene glycol 5 5 Purified water Until it is 100 Until it is 100

Experimental Example  21: Cosmetic  Measuring skin elasticity improvement effect

In order to measure the skin elasticity improvement effect of the cosmetic preparations prepared in Examples 4 to 5 and Comparative Examples 1 and 2, 20 patients (20 to 35 years old) 5 on the left side of the face, and Comparative Example 1 or Comparative Example 2 on the left side of the face for 2 consecutive months.

To evaluate the effect of skin elasticity improvement, the skin elasticity was measured before use and after 2 months of use with a skin elasticity meter (SEM 575, C + K Electronic Co., Germany). The experimental results are shown in Table 21 as ΔR7 values of Cutometer SEM 575, where R7 values are indicative of viscoelasticity of the skin (n = 20, p <0.05).

Experimental product Skin elasticity effect (R7) Example 4 Comparative Example 1

Experimental product Skin elasticity effect (R7) Example 5 Comparative Example 2

The skin elasticity improvement effects of the cosmetic compositions (Table 41, Table 42), the improvement of the skin elasticity of the experiment 4 in which the Example 4 containing the Fusarium oxysporum fucanthus extract and the Example 5 containing the Fusarium aeruginosa culture solution were applied It was confirmed that the effect was better than those of Comparative Examples 1 to 2. [

From the above results, it was confirmed that the skin external-application composition of the present invention has an excellent skin elasticity-improving effect.

Experimental Example  22: Cosmetic  Measurement of wrinkle improvement effect

In order to measure the effect of improving the skin wrinkles of the cosmetic preparations prepared in Examples 4 to 5 and Comparative Examples 1 and 2, 20 subjects (20 to 35 years old) 4 on the left side of the face and Comparative Example 1 or Comparative Example 2 on the left side of the face for 2 consecutive months.

To evaluate the effect of wrinkles on the skin, a replica of silicone material was prepared before use and after 2 months of use, and the state of the wrinkles at the designated area was measured with a visiometer (SV60, C + K Electronic Co.) using an image analyzer. , Germany).

The results are shown in Tables 42 and 43, and Tables 42 and 43 show the average of the parameter values after two months, minus the parameter values two months ago. That is, the more negative the value, the higher the effect of wrinkle improvement.

Experimental product R1 R2 R3 R4 R5 Example 4 Comparative Example 1 R1: Difference between the maximum value and the minimum value of the wrinkle contour line
R2: The wrinkle contour line is arbitrarily divided into 5 squares, and the average of R1 values
R3: Maximum value of R1 divided by 5
R4: Mean value of the baseline of the wrinkle contour minus the value of each apex and valley
R5: Mean value of the value obtained by subtracting the outline of each wrinkle from the base line of the wrinkle contour line

Experimental product R1 R2 R3 R4 R5 Example 5 Comparative Example 2 R1: Difference between the maximum value and the minimum value of the wrinkle contour line
R2: The wrinkle contour line is arbitrarily divided into 5 squares, and the average of R1 values
R3: Maximum value of R1 divided by 5
R4: Mean value of the baseline of the wrinkle contour minus the value of each apex and valley
R5: Mean value of the value obtained by subtracting the outline of each wrinkle from the base line of the wrinkle contour line

The results of measurement of the skin wrinkle-improving effect of cosmetics (Table 43, Table 44) show that the effect of improving the skin wrinkles of Example 4 containing the fungus extract of Fusarium oxysporum mushroom and Example 5 containing the culture of the Fusarium oxysporum fungus was comparable to that of Comparative Examples 1, It was confirmed that it was superior to Comparative Example 2.

From the above results, it was confirmed that the wrinkle-improving effect of the composition for external application for skin of the present invention is excellent.

Experimental Example  23: Cosmetic  Whitening effect measurement

In Experimental Example 23, in order to measure the whitening effect of the cosmetic compositions prepared in Examples 4 to 5 and Comparative Examples 1 to 3, 20 subjects (20 to 35 years old) Or Example 5 was applied to the left side of the face, and Comparative Example 1 or Comparative Example 2 was applied to the left side of the face for 2 consecutive months, twice a day.

Two months later, the color of the applied area of both sides of the face was analyzed with an image analyzer and color change (ΔL) of the color was measured using a Minolta CR300, and visual observation by a plurality of experts and observation And the effects were measured according to the following classification. The results are shown in the following Tables 44 and 45 and the degree of whitening efficacy was classified into seven grades (-3: very bad, -2: worse, -1: slightly worse, 0: Improvement, 2: improvement, 3: very improvement).

Change in skin color brightness (ΔL) Objective evaluation of expert Subjective evaluation of the subject Example 4 Comparative Example 1 Example 4 Comparative Example 1 Example 4 Comparative Example 1 medium

Change in skin color brightness (ΔL) Objective evaluation of expert Subjective evaluation of the subject Example 5 Comparative Example 2 Example 5 Comparative Example 2 Example 5 Comparative Example 2 medium

The results of measuring the whitening effect of cosmetics (Table 45, Table 46), Example 4 containing the mushroom fruiting body extract of Example 1 and Example 5 containing the mushroom mycelium culture culture had better whitening effect than Comparative Examples 1 and 2 .

From the above results, it was confirmed that the skin whitening agent composition of the present invention has excellent whitening effect.

Experimental Example  24: Cosmetic  Measurement of improvement effect on atopic dermatitis

In Experimental Example 24, clinical experiments were conducted as follows to compare and evaluate the improvement effects of the cosmetic compositions prepared in Examples 4 to 5 and Comparative Examples 1 and 2 on atopic dermatitis.

We evaluated the improvement of atopic dermatitis in 30 patients with atopic dermatitis for 2 years or more as a 5 to 30 year old atopic patient. The same person applied Example 4 or Example 5 on the left hand and Comparative Example 1 or Comparative Example 2 on the right hand every evening after washing the skin every day for 60 days once a day to measure the degree of improvement of the atopic dermatitis condition. Measurements were carried out by sensory evaluation by questionnaire. The results are shown in Tables 47 and 48 below.

very good good so so Example 4 80% (24 people) 20% (6 people) 0% Comparative Example 1 10% (3 people) 20% (6 people) 70% (21 people)

very good good so so Example 5 70% (21 people) 30% (9 people) 0% Comparative Example 2 10% (3 people) 20% (6 people) 70% (21 people)

The results are shown in Table 47 and Table 48. The results are shown in Table 47. The results are shown in Table 47. The results are shown in Table 47, It was confirmed that the effect of improving atopic skin is excellent.

From the above results, it was confirmed that the composition for external application for skin of the present invention is excellent in improving atopic dermatitis.

Experimental Example  25: SLS Of skin irritation mitigation

In Experimental Example 25, the effects of stimuli relaxation of the cosmetic compositions prepared in Examples 4 to 5 were evaluated by a human skin patch test.

1% of SLS (sodium lauryl sulfate) which stimulates general cosmetic prescription (cream, lotion, skin, essence) and the cosmetic materials of Example 4 or 5 were mixed for 24 hours, 48 hours and 72 hours, Based on the stimulation index, the effect of stimulation relaxation was evaluated.

Fifty milliliters of healthy men and women aged 20 to 50 years were exposed to FINN CHAMBER (FINLAND) using 0.3 mg of the product, and the acute irritation index was evaluated after 24 hours. After the evaluation, the same amount of product was applied again to the same site again, and the delayed irritation index after 48 hours and 72 hours was evaluated.

As a result of the measurement of the skin irritation mitigation effect by SLS, the portion where SLS was applied alone showed irritation to the skin from 24 hours later. However, in Example 4 containing the Fusarium oxysporum fructus extract and the culture solution of the fusarium mushroom mycelium , Did not cause any skin seizure even after 24 hours, 48 hours, and 72 hours.

From the above results, it was confirmed that skin irritation caused by stimulants (surfactant, fragrance, alcohol) can be reduced when the culture of the mushroom fruiting body extract and the mushroom mycelium culture liquid are mixed with the cosmetics.

Example  6: Manufacture of lotion containing fruity body extract

0.05 g of polypyrrolidone, 0.1 g of oleyl alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of perfume, 0.1 g of p-hydroxybenzoic acid methyl ester, a small amount of antioxidant and a small amount of dye were mixed and dissolved in 8 g of 95% Respectively. 0.05 g of the Fusarium oxysporum fructus extract obtained in Example 1 and 5 g of glycerin were dissolved in 85.33 g of purified water. The mixture was added to the mixture and stirred to prepare a lotion containing the Fusarium oxysporum fructus extract.

Example  7: Production of cosmetic lotion containing cultured mycelium culture

0.05 g of polypyrrolidone, 0.1 g of oleyl alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of perfume, 0.1 g of p-hydroxybenzoic acid methyl ester, a small amount of antioxidant and a small amount of dye were mixed and dissolved in 8 g of 95% Respectively. 0.05 g of the cultured mycelia of the mushroom mycelium obtained in Example 3 and 5 g of glycerin were dissolved in 85.33 g of purified water. The mixture was added to the mixture, followed by stirring to prepare a lotion containing the cultured mycelia of the mushroom mycelium.

Example  8: Manufacture of emulsion containing fruity body extract

1.2 g of cetyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of p-hydroxybenzoic acid ethyl ester, 1 g of glycerin monoestearaide, 1 g of polyoxyethylene (20 mols) monooleate and 0.1 g of perfume were mixed at 70 캜 0.5 g of the fungus extract obtained in Example 1, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1,500, 0.2 g of triethanolamine and 76.2 g of purified water were dissolved by heating at 75 占 폚. The mixture was emulsified by mixing them and then cooled to prepare oil-in-water (O / W) type emulsion.

Example  9: Preparation of emulsion containing cultured mycelia

1.2 g of cetyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of p-hydroxybenzoic acid ethyl ester, 1 g of glycerin monoestearaide, 1 g of polyoxyethylene (20 mols) monooleate and 0.1 g of perfume were mixed at 70 캜 0.5 g of the cultured mycelia of mushroom mycelium obtained in Example 3, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1,500, 0.2 g of triethanolamine and 76.2 g of purified water were dissolved by heating at 75 캜. The mixture was emulsified by mixing them and then cooled to prepare oil-in-water (O / W) type emulsion.

Example  10: Fungus extract containing fruiting body Serum  Produce

To 5 g of 95% ethyl alcohol, 1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of chitoolose, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E-acetate, 0.2 g of sodium permanganate and 0.1 g of p-hydroxybenzoic acid ethyl ester , 1 g of the Fusarium oxysporum fucanthus extract obtained in Example 1 and an appropriate amount of pigment were mixed to prepare a serum.

Example  11: Culture medium containing cultured mycelia Serum  Produce

1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of chitoolose, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E-acetate, 0.2 g of sodium permanganate, 0.1 g of p-hydroxybenzoic acid ethyl ester , 1 g of the cultured mycelia of the mushroom mycelia obtained in Example 3, and the appropriate amount of pigment were mixed to prepare a serum.

Claims (18)

A composition for external application for skin, which comprises an extract of Fusarium oxysporum fusiformis as an active ingredient.
The composition for external application for skin according to claim 1 or 2, wherein the composition comprises a mushroom mycelium extract as an active ingredient.
A composition for external application for skin, which comprises as an active ingredient a culture solution obtained from the cultivation of mycelium of mushroom,
The method according to any one of claims 1 to 3,
The composition for external application for skin,
Wherein the composition contains 0.001 to 90.0% by weight of a culture solution obtained from the cultivation of a fruiting body extract of Fusarium oxysporum f. Mushroom or an extract of Fusarium oxysporum mushroom or a fungal mycelium.
The method according to any one of claims 1 to 3,
The composition for external application for skin,
A composition for external application for skin, which is a cosmetic composition.
6. The method of claim 5,
In the cosmetic composition,
A composition for external application for skin, which is used for skin whitening.
6. The method of claim 5,
In the cosmetic composition,
The composition for external application for skin is characterized in that it is for preventing aging.
6. The method of claim 5,
In the cosmetic composition,
Wherein the composition is for wrinkle improvement.
6. The method of claim 5,
In the cosmetic composition,
A composition for external application for skin, which is used for moisturizing the skin.
6. The method of claim 5,
In the cosmetic composition,
A composition for external application for skin, which is used for relieving skin irritation.
6. The method of claim 5,
In the cosmetic composition,
A composition for external application for skin, which is for improving atopic skin.
6. The method of claim 5,
In the cosmetic composition,
The composition for external application for skin is characterized in that it is for improving acne.
6. The method of claim 5,
In the cosmetic composition,
Wherein the composition is for preventing hair damage.
6. The method of claim 5,
In the cosmetic composition,
Wherein the composition is for preventing hair loss and for improving hair growth.
The method according to any one of claims 1 to 3,
The composition for external application for skin,
A composition for external application for skin, which is a pharmaceutical composition.
16. The method of claim 15,
The pharmaceutical composition may contain,
Wherein the composition is for preventing or treating an inflammatory disease.
16. The method of claim 15,
The pharmaceutical composition may contain,
A composition for external application for skin, which is characterized in that it is for prevention or treatment of atopy.
16. The method of claim 15,
The pharmaceutical composition may contain,
A composition for external application for skin characterized by being for preventing or treating acne.