KR101208013B1 - A skin-care agent containing mycoleptodonoides aitchisonii mycelium extract - Google Patents

Abstract

PURPOSE: An external use skin composition containing Mycoleptodonoides aitchisonii mycelium extract is provided to remove active oxygen and promote collagen synthesis, and to relieve atopic dermatitis and skin irritation. CONSTITUTION: A cosmetic composition for skin whitening contains 0.001-90 wt% of Mycoleptodonoides aitchisonii mycelium extract as an active ingredient. The extract is prepared by fermenting Mycoleptodonoides aitchisonii myclium and collecting the liquid. A cosmetic composition for antiaging contains Mycoleptodonoides aitchisonii mycelium extract as an active ingredient. The cosmetic composition is manufactured in the form of a lotion, gel, water soluble liquid, essence, O/W type and W/O type base cosmetic formulation, or makeup cosmetic formulation.

Description

A skin-care agent containing Mycoleptodonoides aitchisonii mycelium extract}

The present invention relates to an external preparation composition for skin containing a needle mushroom mycelium extract, and more particularly, to an external preparation composition for skin containing a filtrate obtained by fermenting a true needle mushroom mycelium.

Skin aging can be divided into intrinsic, chronological aging and phtoaging [Gilchrest BA: J. Am . Acad . Dermatol ., 21, 610-613 (1989). Endogenous aging is a naturally occurring aging phenomenon caused by deterioration of physiological functions of living organisms with increasing age [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 may be caused by the activation of free radicals with UV rays, stress, disease conditions, environmental factors, wounds, age, and when this condition is intensified, destroys the antioxidant defenses in vivo, cells and Damage to tissues promotes adult disease 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 is caused by severe cuts in the skin's connective tissues such as collagen, hyaluronic acid, elastin, proteoglycans, and fibronectin, resulting in severe hyper-inflammatory reactions, which affect the elasticity of the skin. This can lead to mutations, cancer, and reduced immune function.

Therefore, it protects the cell membrane by eliminating reactive oxygen species, which are mediated by the body's metabolic process, UV irradiation, or inflammatory reactions.Also, damaged cells should be regenerated by active metabolism to proliferate the skin. Can recover and maintain healthy skin. Aging involves not only reactive oxygen species but also an enzyme called matrix metallopretease (MMP) .In vivo, the synthesis and degradation of extracellular matrix such as collagen is properly regulated, but its synthesis decreases as aging progresses. Expression of matrix metalloproteinases (MMPs) is promoted, reducing the elasticity of the skin and forming wrinkles. Ultraviolet exposure also activates these degrading enzymes. Therefore, there is a need for development of a substance capable of regulating or inhibiting MMP expression in which activation is induced in cells by ultraviolet rays. Until now, most of the raw materials used as materials for cosmetics simply inhibit the activity of the enzyme (MMP).

On the other hand, melanin is transformed from tyrosine to dopa and dopaquinone by the action of tyrosinase present in pigment cells, and is generated via dopachrome. Melanin is present in the skin to protect the body from ultraviolet rays and other important functions in the body's hormone secretion control. 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. Ascorbic acid (JP-A 4-9320), hydroquinone (JP-A 6-192062), kojic acid (JP-A-56-7710), arbutin (JP-A 4-93150), and plant extracts have already inhibited tyrosinase. It is used as a whitening cosmetic due to its activity, but its stability in cosmetic formulations is poor, and it is decomposed and colored, odors occur, its efficacy and effects on the biological level are unclear, or its safety is a problem and its use is restricted. to be.

On the other hand, atopic dermatitis is a typical disease in people with atopic dermatitis. Although the cause of atopic dermatitis has not been clarified yet, 70% of patients with atopic dermatitis have a history of atopy, and in allergic people It is known to be one of the diseases caused by genetic factors and immune diseases because it is associated with allergic rhinitis, asthma, and other allergic diseases. In the treatment of atopic dermatitis, corticosteroids such as corticosteroids are expected to have high pharmacological effects. However, side effects of corticosteroids have become a problem. It is also well known that a dramatic aggravation of lesions occurs later. As a countermeasure for such side effects, nonsteroidal anti-inflammatory drugs or antihistamines, which do not contain hormones such as corticosteroids, are used, but since it is difficult to cure, there is an urgent need to develop an atopic dermatitis improving agent or treatment without side effects.

Recently, in order to reduce skin irritation caused by various chemicals, much attention has been focused on functional cosmetics (Functional cosmetics) having functions such as antioxidant, wrinkle relief, whitening, and itching relief obtained from natural extracts. For example, US Pat. No. 5,972,341 relates to the antiwrinkle effect of Commiphora mukul extract, and Japanese Patent Application Laid-open No. Hei 9-672662 relates to seaweed extract having hyaluroronidase inhibitory effect. Japanese Patent Application Laid-open No. Hei 10-85905 relates to the skin texture improvement effect of Fucoidan extracted from Wakame, and Japanese Patent Application Laid-open No. Hei 2-245087 relates to the antioxidant effect of the extract of Sargassum, Korea Patent No. 0431076 discloses a cosmetic composition for improving the healing of atopic skin, which contains white powder, jasminoide, echinacea and fermented soybean extract, and Korean Patent No. 0511494 extracts and compositions for treating atopic dermatitis, including sewage, leaf, and illite. Discussing about.

In addition, male alopecia is male hormone-dependent, and thus has a direct relationship with the amount of male hormone. Accordingly, many studies have been reported for the prevention and treatment of hair loss through the inhibition of male hormone activity. On the other hand, when the function of the sebaceous gland becomes active due to the increase in the secretion of male hormones, the scalp produced by stagnation in the hair follicles due to the hyperkeratosis of the hair follicle wall is an early stage of acne. When explaining the mechanism of hair loss and acne caused by male hormones, 5-alpha-Reductase is one of the male hormones present in testosterone-reactive tissues such as sebaceous gland, hair follicle, prostate, and epididymis. , An enzyme involved in metabolizing testosterone to dihydrotestosterone, which requires NADPH. In addition, testosterone is involved in male sexual impulses, skeletal muscle increase, male external genitalia, scrotum growth, spermatogenesis, and the like, and dehydrotestosterone is involved in tissues such as acne, sebum increase, hair loss, and prostatic hyperplasia. In particular, after puberty, excessive secretion of testosterone causes acne and hair loss, and 5-alpha-reducta to prevent overproduction of dihydrotestosterone, the active form of testosterone produced by 5-alpha-reductase. There are active studies to develop anti-hair loss and anti-acne agents using an inhibitor.

In order to solve the problem of the skin, a lot of cosmetics using natural products or medicinal and edible mushroom extracts have recently been developed to reduce skin irritation caused by various chemicals. As a cosmetic composition using the extract of the mushroom known so far, snow flower Cordyceps (Korea Patent No. 0340185), situation mushroom, skirt mushroom (Korean Patent No. 0681703, 0076537), leaf mushroom (Korean Patent No. 0438009), shiitake mushroom And the research on the anti-aging effect of these extracts has been continued.

Mushrooms used for medicinal and edible foods are numerous examples and kinds, mushrooms that are being used and studied as a cosmetic composition is still known as a part.

Meanwhile, True Needle Mushrooms ( Mycoleptodonoides aitchisonii ) is a kind of fruity edible mushroom that is called acupuncture mushroom. Fruiting body has no mushroom stalk, and it is strong when it is flexible flesh or dry, and the fungus is fan-shaped or spatula-like. Is tooth-shaped. The hyphae are two mycelial type of thick and thin film. The fruiting layer of the lower surface is needle mushroom type, needles are sharp, and yellow to orange color when dried to 3 ~ 10 mm in length. Needle mushrooms are expected to be very nutritious with pharmacological effects such as blood pressure strengthening, blood sugar strengthening, brain function improvement, anti-cancer and anti-diabetic effects, but in Korea, trees of hardwoods such as oak trees in Gangwon and Jeju It is known to occur rarely.

The true needle mushroom has not yet been identified as showing the skin anti-aging effect, whitening effect, atopy improvement effect as a skin external agent.

Republic of Korea Patent No. 10-0340185 (Cosmetic anti-wrinkle cosmetic composition containing a snow Cordyceps sinensis extract) has been described for a cosmetic composition excellent in the prevention of wrinkles by containing an extract of Snow Cordyceps sinensis, this is to use the true mushroom mycelium extract It is not described. Republic of Korea Patent No. 10-0076537 (Preparation method of beta-1,6-branch-beta-1,3-glucan derived from agaric mushroom and beta-1,6-zaja-beta-1, prepared by this method, Cosmetic composition containing 3-glucan) has been described for the cosmetic composition having the effect of promoting the activation of skin functions such as skin cell and collagen fiber proliferation effect, sunburn healing, and inhibiting skin melanogenesis by liquid culture of the mycelium of the mushrooms. In this regard, there is no description on the use of the true needle mushroom mycelium extract. Republic of Korea Patent No. 10-0438009 (cosmetic composition containing the extract of leaf mycelium mycelium) is a mycelium or mycelium of the leaf mushroom having an antioxidant effect and the effect of promoting the proliferation of skin cells, the production of collagen and the inhibitory effect of skin melanogenesis Although the cosmetic composition containing the obtained extract has been described, it has not been described in terms of using the true mushroom fungus mycelium extract.

The present invention uses natural extracts to promote antioxidant, collagen synthesis, skin fine wrinkle improvement, whitening, moisturizing, skin irritation, acne prevention, atopic improvement and anti-inflammatory effect, hair damage prevention effect, hair loss prevention and hair growth effect The purpose is to provide an external preparation composition.

In addition, an object of the present invention is to provide a topical composition for skin, characterized in that it contains a high fungus true needle mushroom mycelium extract as an active ingredient to solve the above problems.

In order to achieve the above object, the present invention provides a composition for external application to the skin, which comprises a true needle mushroom mycelium extract as an active ingredient.

Hereinafter will be described in detail with respect to the problem solving means of the present invention.

The present invention, wrinkle improvement, whitening effect, atopic dermatitis improvement, prevention or treatment effect, skin irritation effect, inflammatory disease prevention or treatment effect, acne improvement, prevention or treatment effect, hair damage prevention effect, hair loss prevention and hair growth improvement In order to provide a skin external composition having an excellent effect, it is characterized by containing the true needle mushroom mycelium extract as an active ingredient.

The extract of mycelium fungus mycelium of the present invention, antioxidant effect, matrix metalloproteinase (MMP-1) activity inhibitory effect, matrix metalloproteinase (MMP-1) expression inhibitory effect, procollagen biosynthesis promoting effect, elastin production promoting effect , Whitening effect, inhibitory effect of inflammation-inducing enzyme (hyaluronidase), cytotoxic effect by UV irradiation, inflammatory cytokine expression inhibition by UV irradiation, prostaglandin biosynthesis effect by UV irradiation, antibacterial effect against acne bacteria The anti-inflammatory effect against atopic dermatitis, skin irritation relieving effect, hair damage prevention effect, hair loss prevention and hair growth effect is very excellent and can be used in multifunctional skin external preparation composition.

The true needle mushroom mycelium extract of the present invention is obtained by extracting the true needle mushroom mycelium, the extraction method can be used a method commonly used in the art to which the present invention belongs. For example, a hot water extract of the mycelium powder obtained by freeze-drying and grinding a true needle mushroom mycelium, or an organic solvent extract extracted using methanol, ethanol, butanol or a mixed solvent thereof; The filtrate obtained by fermenting a true needle mushroom mycelium may be sufficient.

Preferably, the filtrate obtained by fermenting the true needle mushroom mycelium, specifically, after the liquid culture of the true needle mushroom mycelium, the filtrate obtained by filtering the culture filtrate removed by centrifugation of the mycelium mushroom mycelium It is good.

In addition, the true needle mushroom mycelium extract of the present invention may be a polysaccharide extracted from the hot water extract, the organic solvent extract or the filtrate.

In addition, the true needle mushroom mycelium extract of the present invention is preferably an extract of the true needle mushroom mycelium using at least one animal or plant as a substrate. Here, the animal or plant is not particularly limited, and an animal or plant commonly used in the art to which the present invention pertains may be used as a substrate.

Meanwhile, in the present invention, the true needle mushroom mycelium extract preferably contains 0.001 to 90.0% by weight of the whole skin external preparation composition, when included in less than 0.001% by weight of the skin improvement is insignificant, exceeding 90.0% by weight. In this case, it is not economical because the efficiency is inferior to the input amount.

On the other hand, the term "included as an active ingredient" in the present invention means that the extract of the true needle mushroom mycelium is added to the external preparation composition for skin to the extent that it can exhibit the effect of preventing and inhibiting skin diseases from the external preparation composition for skin of the present invention. And, for drug delivery and stabilization, etc. are added to the various components as a subcomponent means to include the formulation (formulation) in various forms.

On the other hand, the topical skin composition of the present invention is preferably a cosmetic composition, the formulation of the cosmetic composition, for example, lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / Basic cosmetic formulations of the form O); Color cosmetic formulations consisting of oil-in-water or water-in-oil makeup bases, foundations, skin covers, lipsticks, lip gloss, face powders, two-way cakes, eye shadows, cheek collars and eyebrow pencils; There is this.

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

On the other hand, the topical skin composition of the present invention is preferably a pharmaceutical composition, for example, as the formulation of the pharmaceutical composition, warning agents (PLASTERS), lotions (LPTIONS), linings (LINIMENTS), liquids (LIQUIDS AND SOLUTIONS) , AEROSOLS, EXTRACTS, Ointment, FLUIDEXTRACTS, Emulsion, Emulsion, SUSPESIONS, CAPSULES, CREAMS, Soft or Hard Gelatin capsules, patches, sustained release agents.

On the other hand, the pharmaceutical composition of the present invention is a pharmacologically acceptable base; Vehicle; Excipients; Binders including starch, tragacanth gum, gelatin, molasses, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose and carboxymethyl cellulose; Grinding agents including agar, starch, gelatin powder, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, crystalline cellulose, calcium carbonate, sodium bicarbonate and sodium alginate; Lubricants including magnesium stearate, talc and hydrogenated vegetable oils; And colorants and the like, and the carriers and excipients include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate and cellulose. In addition to the above, additives such as adjuvants such as stabilizers, dissolution aids, transdermal absorption accelerators, and preservatives may be further added.

On the other hand, for many patients, the standard dosage of the topical skin preparation composition of the present invention may vary depending on the individual characteristics of the patient, and a substantially skilled clinician would ideally administer the topical skin composition of the present invention for patients. The most appropriate treatment strategy can be determined in terms of dosage and dosing schedule, eg, specific needs and the overall conditions of the patient. For the topical dermatological compositions of the invention, reference can be made to a number of references in determining the appropriate dosage.

In addition, the appropriate dosage of the external preparation composition for skin of the present invention may be generally determined in vitro or in an animal model. For example, appropriate concentrations may be determined by adding various concentrations of the topical skin composition of the invention to target cells in vitro.

On the other hand, the pharmaceutical composition is preferably for preventing or treating inflammatory diseases, for preventing or treating atopic dermatitis or for preventing or treating acne.

As described above, the composition for external application of the skin of the present invention is effective for antioxidant, collagen synthesis, skin fine wrinkle improvement, whitening, moisturizing, skin irritation relief, acne prevention, atopy improvement and anti-inflammatory effect, hair damage prevention effect, hair loss prevention and hair growth By containing the true needle mushroom mycelium extract has an excellent skin improvement effect.

Hereinafter, the content of 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  One: Needle Mushroom  Preparation of Mycelia Extract

M. aitchisonii strains used in the experiments were directly isolated from fresh fruiting bodies, and the true mushroom mycelium was isolated from yeast-wort agar medium (3 g of yeast extract, 3 g of wort, 10 g of glucose and 5 g of peptone). , 20 g of agar, 1 L of distilled water) was inclined in a test tube and stored at 4 ° C. and used subcultured every month. After aseptic homogenization of the mycelium grown on the slope medium, it was prepared as a simple complex medium containing 3% glucose and 1.0% yeast extract and inoculated 5% (v / v) in a liquid medium adjusted to pH 5.5, The cells were incubated for 10 days under conditions of a temperature of 27 ° C., a rotational speed of 150 rpm, and an air flow rate of 1.5 vvm.

After completion of the culture, the culture filtrate from which the mycelium was removed by centrifugation was filtered through a paper filter (watman no. 4) to obtain a filtrate (hereinafter, 'true mushroom mycelium extract').

Experimental Example  One: NBT Antioxidant Effect Measurement

In order to confirm the antioxidant level of the needle mushroom mycelium extract obtained in Example 1, BHT, which is well known as an antioxidant, was used as a comparative sample, and the antioxidant measurement was performed using NBT method.

The NBT method is a method of generating active oxygen by xanthine and xanthine oxidase, and measuring blue at a wavelength of 560 nm by reacting the produced active oxygen with Nitro Blue Tetrazolium (NBT). Active oxygen scavenging rate was measured, and the measuring method is as follows.

2.4 ml of 0.05 M 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 the Bayer bottle. After each addition of the solution, the mixture was left at 25 ° C for 10 minutes. After standing, 0.1 ml of xanthine oxidase solution was added and stirred rapidly, followed by incubation at 25 ° C. for 20 minutes, and 0.1 ml of 6 mM copper chloride (CuCl ₂ ) solution was added to stop the reaction and absorbance at 560 nm. (St) was measured. In the blank test, distilled water was used instead of the sample solution, and the absorbance (Bt) was measured by the same method as described above. 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 result was calculated by the following equation.

[Equation 1]

Free radical 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 enzymatic reaction of blank test solution

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

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

Name of sample Free radical scavenging rate (antioxidative effect;%) 0.25% by weight of mycelium fungus mycelium extract 96 Needle fungus mycelium extract 0.1% by weight 92 Needle Mushroom Mycelium Extract 0.05% by weight 80 Needle Mushroom Mycelia Extract 0.025% by weight 11 BHT 0.1 wt% 89

As a result of measuring the antioxidant effect using the NBT method (Table 1), it was confirmed that the extract of mycelium fungus mycelium showed better antioxidant effect at the same concentration as BHT.

From the above results, it was possible to confirm the excellent antioxidant effect of the true needle mushroom mycelium extract of the present invention, from which the excellent antioxidant effect of the present invention was found.

Experimental Example  2: DPPH Measure antioxidant effect

In Experimental Example 2, BHT, which is well known as an antioxidant, was used as a comparative sample to check the antioxidant level of the true needle mushroom mycelia extract obtained in Example 1, and the antioxidant measurement was used as a DPPH method.

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

After preparing the true needle mycelia extract of 0.25, 0.1, 0.05, 0.025% by weight concentration, the true needle mycelium extract according to the concentration was put in 96-well plate, respectively. To this was added DPPH prepared in 100 uM methanol solution to give a total volume of 200 μl. After leaving it at 37 ° C. for 30 minutes, the absorbance (St) was measured at 560 nm.

Free radical scavenging activity (%) was calculated by the following equation.

&Quot; (2) "

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

A: Absorbance of Control Wells Untreated with Needle Mushroom Mycelia Extract

B: Absorbance of Experimental Wells Treated with Needle Mushroom Mycelia Extract

Name of sample Free radical scavenging rate (antioxidative effect;%) 0.25% by weight of mycelium fungus mycelium extract 97 Needle fungus mycelium extract 0.1% by weight 96 Needle Mushroom Mycelium Extract 0.05% by weight 94 Needle Mushroom Mycelia Extract 0.025% by weight 92 BHT 0.1 wt% 85

As a result of measuring the antioxidant effect using the DPPH method (Table 2), it was confirmed that the true mushroom mycelium extract exhibits an excellent antioxidant effect even at a lower concentration than BHT.

From the above results, it was possible to confirm the excellent antioxidant effect of the true needle mushroom mycelia extract, from which the excellent antioxidant effect of the present invention could be inferred.

Experimental Example  3: Intracellular  Measurement of free radical scavenging effect

In Experimental Example 3, EGCG was used as a comparative sample to measure the scavenging effect of free radicals generated in the cell by ultraviolet light of the extract of Mycelium fungus mycelium obtained in Example 1, The same experiment was performed.

The cell line used in Experimental Example 3 was a human keratinocytes HaCaT cell line distributed from Dr. Fusenig of the German Cancer Research Center, which was a 96-well black plate for fluorescence measurement (well black). plates were plated at 2.0 × 10 ⁴ per well and added to DMEM (Dulbeccos Modification of Eagles Medium, FBS 10%, Gibco, USA) with penicillin / streptomycin (37%, 5% CO _2). After culturing for 1 day under the conditions, the true needle mycelium extract was treated with a concentration of 0.02, 0.01, 0.005% by weight.

After 24 hours of incubation with the test sample, the remaining medium was removed by washing with HCSS (HEPES-buffered control salt solution) and DCFH-DA (2 ', 7'-dichlorodihydro-fluorescein diacetate, Molecular prepared in 20 μM in HCSS) Probes, USA) was added 100 μl and 37 ℃, 5% CO ₂ After incubation for 20 minutes under conditions, washed with HCSS. After 100 μl of HCSS treated for each concentration, the fluorescence of DCF (dichlorofluorescein) oxidized with active oxygen was measured with a fluorescence plater (Ex; 485 nm, Em; 530 nm). After UVB (20 mJ / cm ² ) was irradiated and treated with the needle mushroom mycelium extract, the fluorescence was measured using a fluorescent plate reader (Ex; 485 nm, Em; 530nm).

Name of sample Free radical scavenging effect (%) Needle Mushroom Mycelia Extract 0.02% by weight 57 0.01% by weight of mycelium fungus mycelium extract 46 Needle Mushroom Mycelium Extract 0.005% by weight 24 EGCG 0.01% by weight 45

As a result of measuring the active oxygen scavenging effect (Table 3), it was confirmed that the true mushroom mycelium extract showed a higher active oxygen scavenging rate than EGCG at the same concentration as EGCG.

From the above results, it was confirmed that the excellent active oxygen scavenging effect of the present invention.

Experimental Example  4: Metalloproteinases ( MMP -1) activity inhibition rate measurement ( in vitro )

In Experimental Example 4, green tea extract was used as a comparative sample to measure the inhibition rate of metalloproteinase (MMP-1) activity in vitro of the extract of Mycelium fungus mycelium obtained in Example 1, and fluorescence analysis was used. .

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. To 100 μl of reaction buffer, add 20 μl of DQ collagen dissolved in the reaction buffer at 0.25 mg / ml and 40 μl of true needle mycelia extract (0.02, 0.01, 0.005% by weight) and diluted collagenase 40 to 0.5 unit. Μl was added. After 20 minutes at room temperature, the fluorescence value was measured by using a fluorescence spectrophotometer (Perkin Elmer, UK) at an absorption wavelength of 495 nm and an emission wavelength of 515 nm. In the control group, the reaction buffer solution was added in the same amount as the enzyme solution instead of the enzyme solution, and then the fluorescence value was measured.

Name of sample % Inhibition of MMP-1 activity Needle Mushroom Mycelia Extract 0.02% by weight 90 0.01% by weight of mycelium fungus mycelium extract 64 Needle Mushroom Mycelium Extract 0.005% by weight 45 0.2% by weight of green tea extract 62

in As a result of measuring the inhibition rate of metalloproteinase (MMP-1) activity in vitro (Table 4), sesame mushroom mycelium extract effectively inhibits the activity of metalloproteinase (MMP-1) at low concentrations compared to green tea extract. I could confirm that.

From the above results, it was confirmed that the myofibrill mycelium extract mycelium protease (MMP-1) activity is excellent, based on this, the topical skin composition of the present invention by effectively inhibiting the metalloproteinase enzyme skin It was found that the decrease in elasticity and wrinkle formation can be prevented.

Experimental Example  5: after UV irradiation Metalloproteinases ( MMP -1) Expression inhibition level measurement

In Experimental Example 5, retinol was used as a comparative sample to measure the degree of suppression of metalloproteinase (MMP-1) expression after UV irradiation of the extract of Mycelium fungus mycelium obtained in Example 1.

After UV irradiation and sample addition (true mushroom mycelium extract, retinol), enzyme immunoassay (ELISA) was performed to measure metalloproteinase (MMP-1) concentration.

Human dermal fibroblasts were irradiated with UVA at an energy of 6.3 J / cm 2 using a UV chamber. Was established. The negative control was wrapped in tinfoil and kept at the same time in the environment of UVA, UVA emission was measured using a UV radiometer. The cells during the UVA irradiation were intact with the previously dispensed medium and irradiated with UVA, exchanged with the medium containing the sample for 24 hours, and 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 ° C for 60 minutes. After reacting the secondary antibody anti mouse IgG (whole mouse, alkaline phosphatase conjugated) for about 60 minutes, the alkaline phosphatase substrate solution (1 mg / ml ρ-nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes and then Absorbance was measured at 405 nm with a plate reader. As a control, one without addition of a sample was used.

Test group MMP-1 expression inhibition rate (%) Control group 0 Needle Mushroom Mycelia Extract 0.02% by weight 70 0.01% by weight of mycelium fungus mycelium extract 35 Needle Mushroom Mycelium Extract 0.005% by weight 21 Retinol 36

As a result of measuring the inhibition of expression of metalloproteinase (MMP-1) after UV irradiation (Table 5), the true mushroom mycelium extract showed more than 70% inhibition rate compared to the control group without treatment. The results were considerably superior to the inhibition rate of the retinol used.

From the above results, it could be confirmed that the effect of inhibiting the expression of metal protease (MMP-1) of the true mushroom fungus mycelium extract, based on this, the external preparation composition of the present invention effectively inhibits the expression of metalloproteinase. As a result, it was found that the decrease in elasticity of the skin and the formation of wrinkles can be prevented.

Experimental Example  6: Measurement of Type 1 Procollagen Biosynthesis Promoting Effect

In Experimental Example 6, TGF-β was used as a positive control group in order to confirm the effect of promoting the biosynthesis of the skin matrix component of type 1 procollagen of the extract obtained from Example 1 mycelium mycelium mycelium, procollagen analysis ( procollagen assay) was performed as follows.

Human dermal fibroblasts isolated from neonatal epidermal tissue were distributed from Modern Tissue Technology (MTT, Korea) and added 10% fetal bovine serum (FBS) to DMEM / F-12 (3: 1) medium. Incubated at 1 × 10 ⁴ cell / cm ² concentration. When grown at about 70-80%, the cells were subcultured at a ratio of 1: 3, and cells cultured in 3rd to 4th passages were used for the experiment.

For experiments to measure the amount of procollagen, fibroblasts were incubated at least 90% in a 48-well plate, and then the true mushroom mycelium extract was added at a concentration of 0.02%, 0.01%, and 0.005% by weight for 24 hours. After this, the amount of free collagen in the medium was measured using a procollagen type 1 C-peptide EIA kit (MK101, Takara, Japan).

Name of sample Pro collagen biosynthesis promoting effect (%) Needle Mushroom Mycelia Extract 0.02% by weight 37 0.01% by weight of mycelium fungus mycelium extract 19 Needle Mushroom Mycelium Extract 0.005% by weight 14 TGF-β 0.001 wt% 36

As a result of measuring the effect of promoting procollagen biosynthesis (Table 6), the procollagen biosynthesis was promoted 37% when treated with 0.02% by weight of true mushroom mycelium extract, which showed a similar effect to the cell signaling substance TGF-β.

From the above results, it was possible to confirm the procollagen biosynthesis promoting effect of the true needle mushroom mycelium extract, from which the present invention was found to be excellent in improving skin aging.

Experimental Example  7: Elastase  Determination of activity inhibitory effect

In Experimental Example 7, in order to measure the elastin production promoting effect of the true needle mushroom mycelium extract obtained in Example 1, by treating the true needle mushroom mycelium extract to human fibroblasts directly collected or commercially purchased The same experiment was performed.

First, human fibroblasts were placed in a culture flask and incubated until about 70-80% growth. Subsequently, after processing the mycelium fungus mycelium extract for 1 day, the cell culture was collected and the elastin production was measured using a commercially available elastin measuring instrument [Bieth J: Biochem. med . , 11, 350-357 (1974), Schwartz DE: J. Invest Dermatol ., 86, 63-68 (1986). That is, the activity of the elastase was measured by using the absorbance to change the color generated when the NA of the Suc- (Ala) 3 NA, which is the substrate of the elastase, was decomposed. At this time, the group was not treated with the needle mushroom mycelium extract was used as a control.

Name of sample Elastase activity inhibition rate (%) Control group 0 Needle Mushroom Mycelia Extract 0.02% by weight 31 0.01% by weight of mycelium fungus mycelium extract 17 Needle Mushroom Mycelium Extract 0.005% by weight 10

As a result of measuring the inhibition rate of elastase activity (Table 7), it was confirmed that the extract of true mushroom fungus mycelium increased the activity inhibition rate of elastase in a concentration-dependent manner.

From the above results it was confirmed that the excellent anti-aging and elasticity promoting effect of the present invention.

Experimental Example  8: Tyrosinase  Determination of activity inhibitory effect

In Experimental Example 8, Arbutin, a well-known whitening agent, was used as a comparative sample in order to confirm the inhibitory effect of tyrosinase activity of the extract of Mycelium fungus mycelium obtained in Example 1.

Tyrosinase is an enzyme that accelerates the oxidation process of a substance called 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 (pH 6.8) to make a 0.1 mg / ml solution, and the needle mushroom mycelium extract was dissolved in a controlled concentration at 0.05 M sodium phosphate buffer (pH 6.8). It was used after. 0.5 ml of L-tyrosine solution was added to a test tube, and 0.5 ml of the true mushroom fungus mycelium extract sample solution was added thereto, and the mixture was left to stand at 37 ° C. for 10 minutes. Thereafter, 0.5 ml of 200 unit / ml tyrosinase was added and reacted at 37 ° C for 10 minutes. After the test tube containing the reaction solution was placed on ice and quenched to stop the reaction, the spectrophotometer measured absorbance at a wavelength of 475 nm. As a control, 0.5 ml of buffer solution was used instead of the extract of Mycelium fungus mycelium.

The tyrosinase activity inhibition rate of each sample was calculated according to the equation (3).

&Quot; (3) "

% Inhibition of tyrosinase activity = 100-[(comparative absorbance / control absorbance)] × 100

Name of sample Tyrosinase inhibition rate (%) Control group 0 Needle Mushroom Mycelia Extract 0.02% by weight 51 0.01% by weight of mycelium fungus mycelium extract 41 Needle Mushroom Mycelium Extract 0.005% by weight 35 Arbutin 0.2 wt% 54

As a result of measuring the activity inhibitory effect of tyrosinase (Table 8), it was confirmed that the inhibition rate of tyrosinase activity similar to that of arbutin 0.2% by weight when treated with 0.02% by weight of true needle mushroom mycelium extract.

From the above results, it was possible to confirm the excellent tyrosinase activity inhibitory effect of the true mushroom mycelium extract, from which the excellent whitening effect of the present invention was found.

Experimental Example  9: Determination of Melanin Synthesis Effect Using B16F1 Melanocytes

In Experimental Example 9, arbutin, known as a whitening agent, was used as a comparative sample in order to confirm the melanin synthesis inhibitory effect of the true needle mushroom mycelia extract obtained in Example 1, and B16F1 melanocytes were used.

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

The melanin synthesis inhibitory effect of B16F1 melanocytes 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 72 hours of incubation, the cells were detached with trypsin-EDTA, and the cells were counted and then centrifuged to recover the cells. Quantification of intracellular melanin was performed by slightly modifying the method of Rotan (R Lotan and D Lotan, Cancer Res, 40: 3345-3350, 1980). After washing the cell pellet with PBS once, 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 dissolving the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), the absorbance of melanin was measured at 405 nm with a microplate reader, and the melanin was quantified. The melanin production inhibition rate (%) of the sample was measured.

Melanin production inhibition rate (%) of B16F1 melanocytes was calculated by the equation (4).

&Quot; (4) "

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

A: Melanin amount in wells without sample

B: Melanin amount in wells to which sample was added

Name of sample Melanin synthesis inhibition rate (%) Control group 0 Needle Mushroom Mycelia Extract 0.02% by weight 30 0.01% by weight of mycelium fungus mycelium extract 21 Needle Mushroom Mycelium Extract 0.005% by weight 8 Arbutin 0.2 wt% 32

As a result of measuring the melanin synthesis inhibitory effect using B16F1 melanocytes (Table 9), 0.02% by weight of mycelium mushroom mycelium extract showed a similar melanin inhibition rate when 0.2% by weight arbutin.

From the above results, it was possible to confirm the excellent melanin synthesis inhibition rate of the true mushroom fungus mycelium extract, from which the excellent whitening effect of the present invention was found.

Experimental Example  10: inflammation-associated enzyme ( hyaluronidase ) Determination of activity inhibitory effect

In this experiment 10, in order to measure the anti-inflammatory effect of the needle mushroom mycelium extract obtained in Example 1, Comfrey extract, seesaw extract, oil-soluble licorice extract was used as a positive control group, inflammatory enzymes hyaluronidase ( hyaluronidase) activity was measured.

Hyaluronidase is an enzyme that induces inflammation by hydrolyzing hyaluronic acid.

In this experimental example, the anti-inflammatory effect was determined by applying a method (Kakegawa Y: Japanese J. of Inflammation, 4, 437-438, 1984) to inhibit the activity of hyaluronidase. .

100 μl of sample and 50 μl of hyaluronidiase solution (type IV-S, Sigma, 400U / mL) were added and reacted at 37 ° C. for 20 minutes, followed by enzyme activation solution (compound 48/80 CaCl ₂ ㆍ 2H ₂ O, Sigma). , 0.1 mg / ml) was added and the reaction was continued for 20 minutes at 37 ° C. 250 μl of a hyaluronic acid solution (0.4 mg / ml) was added thereto and reacted at 37 ° C. for 40 minutes. 100 μl of 0.4N NaOH was added to terminate the reaction. 100 μl of potassium borate solution was added thereto, reacted at 95 ° C. for 3 minutes, cooled, and 3 ml of ρ-dimethylaminobenzaldehyde solution was added thereto. The absorbance was measured at 585 nm to determine the rate of inhibition of hyaluronidase activity.

The activity inhibition rate (%) of hyaluronidiases was calculated according to Equation 5, and the IC ₅₀ value is the concentration of a substance that inhibits hyaluronidiase enzyme activity by 50%.

[Equation 5]

% Inhibition of activity of hyaluronidase = [(A-B) / A] × 100

A: Enzyme Activity of Wells Without Samples

B: Enzyme Activity of Well Added Samples

Name of sample Inhibitory effect of hyaluronidase activity (IC ₅₀ ;%) Comfrey Extract 0.27 Seesaw extract 0.51 Usefulness Licorice extract 0.22 Needle Mushroom Mycelia Extract 0.26

As a result of measuring the inhibitory effect of hyaluronidase activity (Table 10), the IC ₅₀ value of true mushroom mycelium extract was 0.26%, which is similar or superior to that of the Comfrey and oil-soluble licorice extracts, which are well known as anti-inflammatory agents. Indicated.

From the above results, it was possible to confirm the excellent anti-inflammatory effect of the true needle mushroom mycelium extract, from which the excellent anti-inflammatory effect of the present invention was found.

Experimental Example  11: Measurement of cytotoxic mitigation effect by UV irradiation

In Experimental Example 11, the mitigating effect of the cytotoxic effect of the true needle mushroom mycelium extract obtained in Example 1 by ultraviolet irradiation was measured.

Fibroblasts (fibroblasts) were placed in a 24-well test plate 1 x 10 ⁵ and attached for 24 hours. Each well was washed once with PBS and 500 μl of PBS was added to each well. The fibroblasts were irradiated with UV 10 mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), and then PBS was removed and 10% FBS was added to the cell culture medium (DMEM). 1 ml) was added. After treatment with the true needle mycelium extract was incubated for 24 hours. After 24 hours, the medium was removed, 500 μl of cell culture medium and 60 μl of MTT solution (2.5 mg / ml) were added to each well, and then cultured in a 37 ° C. CO ₂ incubator for 2 hours. After incubation, the medium was removed and 500 μl of isopropanol-HCl (0.04 N) was added thereto. 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.

Cell viability (%) was measured by Equation 6, and cytotoxicity remission by UV was calculated by Equation 7.

&Quot; (6) "

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

Bo: 565 nm absorbance of wells that only reacted with cell culture media

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

St: 565 nm absorbance of the wells that reacted the sample treated wells

[Equation 7]

% Of cytotoxicity caused by UV light = [1- (St-Bo) / (Bt-Bo)] × 100

Bo: cell viability of wells without UV irradiation and no sample treatment

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

St: Cell viability of wells treated with UV

Name of sample Treatment concentration (% by weight) Cytotoxic Relaxation Rate (%)
Needle Mushroom Mycelia Extract 0.02 39.5 0.01 31 0.005 17

As a result of measuring the cytotoxicity mitigation effect by UV irradiation (Table 11), the extract of Sesame mushroom mycelium effectively prevents cytotoxicity by UV rays by reducing 39.5% of cytotoxicity at 0.02% by weight concentration. And it was found.

From the above results, it was confirmed that the mycelium mycelium mycelium extract extracts excellent cytotoxic effect by UV irradiation, and it was found that the skin irritation effect by ultraviolet rays of the present invention.

Experimental Example  12: Measurement of the Inhibitory Effect of Inflammatory Cytokine Expression by Ultraviolet Irradiation

Experimental Example 12 measured the degree of inhibition of inflammatory cytokine expression expressed by ultraviolet irradiation in order to measure the anti-inflammatory effect of the extract of the true mushroom fungus mycelium obtained in Example 1.

Fibroblasts isolated from human epidermal tissue (Fibroblast) were placed in a 24-well test plate ⁵ x 10 ⁴ and attached for 24 hours. Each well was washed once with PBS and 500 μl of PBS was added to each well. The fibroblasts were irradiated with UV 10 mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), after which PBS was removed and FBS was added to the cell culture medium (DMEM). Medium) 350 μl was added. After treatment with the true needle mycelium extract, it was incubated for 5 hours. After incubation, 150 μl of the culture supernatant was taken to quantify IL-1α, and thus the inhibitory effect of inflammatory cytokine expression of the fungus mycelium extract was determined. The amount of IL-1α was quantified using Enzyme-linked Immunosorbent Assay, and the production rate of IL-1α was calculated by Equation 8.

[Equation 8]

Inflammatory cytokine expression inhibition rate (%) = [1- (St-Bo) / (Bt-Bo)] × 100

Bo: amount of IL-1α produced in wells not irradiated with UV

Bt: amount of IL-1α produced in wells that were not irradiated with UV light

St: Production of IL-1α in Wells Treated by Ultraviolet Irradiation

Name of sample Treatment concentration (% by weight) Inhibitory rate of inflammatory cytokine expression (%)
Needle Mushroom Mycelia Extract 0.02 32.5 0.01 18.7 0.005 7.5

As a result of measuring the inhibitory effect of inflammatory cytokine expression by UV irradiation (Table 12), 32.5% inhibition at 0.02% by weight concentration, the needle mushroom mycelium extract can be confirmed that effectively protect the inflammation caused by UV rays at low concentrations there was. The anti-inflammatory effect of this invention was understood from the said result.

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

In Experimental Example 13, keratinocytes isolated from human epidermal tissue to evaluate the effect of prostaglandin (prostaglandin E ₂ ; hereinafter referred to as PGE ₂ ) biosynthesis of the true needle mushroom mycelium extract obtained in Example 1 ) Were placed in 24-well test plates and ⁴ × 10 × ⁴ were attached for 24 hours. After replacement with medium without FBS, incubation for 18 hours, prostaglandin biosynthetic enzyme (prostaglandin E ₂₎ present in keratinocytes was treated with aspirin to achieve a final concentration of 50 uM on the keratinocytes. synthetase, or cyclooxygenase, COX) was removed. Two hours after aspirin treatment, each well containing keratinocytes was washed twice with PBS and 100 μl of PBS was added to each well. The keratinocytes were irradiated with UV 30 mJ / cm 2 using a UVB lamp (Model: F15T8, UV B 15 W, Sankyo Dennki, Japan), and then PBS was removed and each cell was prepared with keratinocyte growth media, Clonetics, Biowhittacker, MD, USA) 250 μl was added. After treatment with the true needle mycelium extract was incubated for 16 hours. An appropriate amount of the culture supernatant was taken to quantify the biosynthesized PGE ₂ (prostaglandin E ₂ ) for 16 hours to determine the prostaglandin inhibitory effect of the extract of Mycelium fungus mycelium. The amount of PGE ₂ was quantified using enzyme immunoassay.

Name of sample PGE ₂ Inhibition Rate (%) (-) UV B control (+) UV B - (+) UV B + True Mushroom Mycelia Extract 0.02 wt% 51 (+) UV B + True Mushroom Mycelia Extract 0.01% by weight 27 (+) UV B + True Mushroom Mycelia Extract 0.005 wt% 8

As a result of measuring the inhibitory effect of prostaglandin biosynthesis by ultraviolet irradiation (Table 13), it was confirmed that the true mushroom mycelium extract effectively inhibits the production of prostaglandins by ultraviolet light. Particularly, the produced PGE ₂ is known to be mainly produced by the COX-2 enzyme, and thus, it can be inferred from the above experiment that the true mushroom mycelium extract inhibits the induction or activity of the COX-2 enzyme.

From the above results, it was confirmed that the excellent prostaglandin biosynthesis inhibitory effect of the extract of the mycelium fungus mycelium.

Experimental Example  14: Determination of antimicrobial activity against acne

In Experimental Example 14, a paper disc test was carried out to check the antibacterial activity of acne bacteria of the extract of Mycelium fungus mycelium obtained in Example 1.

First, in order to activate propionibacterium acnes, a skin flora of the cause of acne, it was preincubated for 48 hours in BHI liquid medium (Brain-Heart Infusion Broth; 3.7%). The bacterial cultures thus prepared were plated in 0.1 ml of BHI solid medium (Brain-Heart Infusion Broth; 3.7%; agar 1.5%) and dried. The needle mushroom mycelium extract obtained in Example 1 was diluted to 12% (w / v) in a 95% ethanol aqueous solution, and 50 μl was added to a 8 mm diameter paper disc and placed on a solid medium prepared above. Incubated for 3 days in a 35 ℃ anaerobic culture tank. The antimicrobial activity was evaluated by observing the bacterial growth inhibition zone around the 'paper disc' and measuring the size of the ring.

As a result of measuring the antimicrobial activity against acne bacteria, the size of the bacterium growth inhibiting ring of the true needle mushroom mycelium extract appeared to be 18 mm, confirming the excellent antimicrobial activity of the present invention.

Experimental Example  15: Compound  After induction of atopic dermatitis by 48/80 Needle Mushroom  Anti-inflammatory Effect of Mycelia Extract

Inject 30 μl of Compound 48/80 (Sigma, Co .; 1 mg / ml in saline) per horse into the dermis of the dorsal neck skin of BALB / c mice (5 wks, male) and place in cage for 60 minutes The mouse was observed to scratch the neck. To test the anti-inflammatory effect of the needle fungus mycelium extract, the needle fungus mycelium extract was applied to the dorsal neck region of the mouse at a concentration of 250 μl / ml from 5 days before compound 48/80 injection. The application method is pretreatment by applying to the dorsal neck part of the mouse twice a day for 5 days for 5 days and then injecting compound 48/80. Time was measured to determine the degree of pruritus.

 Number of scratching (in 5min) 5 10 15 20 25 30 35 40 45 50 55 60 Needle Mushroom Mycelia Extract 24 27 30 32 34 39 42 43 47 28 19 16 Control group 39 41 50 62 88 94 98 72 59 50 40 32

After induction of atopic dermatitis, the anti-inflammatory effect of the needle fungus mycelium extract was measured (Table 14), and it was confirmed that the extract of fungus fungus mycelium inhibited the induction of atopic dermatitis (skin pruritus) by compound 48/80.

From the above results, it was possible to confirm the atopy prevention effect of the true mushroom mycelium extract, from which the excellent atopy improvement, prevention or treatment effect of the present invention was found.

Experimental Example  16: moisture hygroscopicity measurement

In this Experimental Example 16, in order to measure the moisture hygroscopicity of the extract of the true needle mushroom mycelium, the true needle mushroom mycelium extract and distilled water obtained in Example 1 were respectively obtained from dry human epidermal cells obtained from humans or purchased commercially. After saturation absorption and the amount of water absorbed were measured, mass change after 48 hours of drying was measured.

The amount of water absorbed per unit of skin cell mass was compared from each measured change in mass. This experiment is used to compare the amount of water absorbed by epidermal cells and is used as a way to anticipate the moisturizing effect even on real human skin.

Specifically, after the human epidermal cells were dried to make the amount of water contained per unit mass constant, the weight of each epidermal cell sample and the amount of water contained therein were measured using the Kaiser method. The epidermal cells of which the water content was measured were immersed in the needle mushroom mycelium extract and purified water for 24 hours, and then saturated and absorbed.Then, the epidermal cells saturated with water were taken out of the cells, weighed, and a portion thereof was collected using a Kaiser moisture meter. The amount of water per unit mass contained was measured. Thereafter, the resultant was dried under reduced pressure for 48 hours, weighed, and then a portion of the sample was collected and measured by Kaiser method to determine the amount of water per unit mass contained therein. (Moisture mg / epidermal cell mass g)

Moisture content Initial dry state Saturation After rebuilding Needle Mushroom Mycelia Extract 400 805 550 Purified water 400 780 400

As a result of measuring the moisture hygroscopicity (Table 15), it was confirmed that the epidermal cells saturated with hygroscopic mushroom mycelium extract contained much more water in the redrying process than the epidermal cells saturated with hygroscopic water.

From the above results, it was possible to confirm the excellent skin hygroscopicity of the true needle mushroom mycelia extract, from which the excellent moisturizing effect of the present invention was found.

Experimental Example  17: Needle Mushroom  Combined Effect of Mycelium Extract and Human Hair Protein

To 50 μg of human hair keratin protein, the true mushroom mycelium extract obtained in Example 1 was added at a test concentration of 0.5, 0.25 and 0.1% by weight, respectively, and reacted at room temperature for 30 minutes, followed by 6% hydrogen peroxide and 0.5% ammonia. Treatment with a 1: 1 mixed solution was allowed to react for 30 minutes. The reaction solution was electrophoresed in a 10% SDS-polyacrylamide gel according to Laemmli's method, and the keratin protein in the gel was 0.1% Coomassie brilliant blue R 250, 10% glacial acetic acid and 40% ethanol. After dyeing for 1 hour in a mixed solution of 10% glacial acetic acid and 40% ethanol in a mixed solution was bleached and confirmed the keratin protein bands. The keratin protein band obtained when electrophoresis of only 50 μg of human hair keratin protein using the image master (Amersham Pharmacia Biotech.) Software was 100%, and the keratin-free group compared to the control group was not treated with the true mushroom mycelium extract. The binding effect is expressed in%.

Needle Mushroom Mycelia Extract Control group 0.5 wt% 0.25 wt% 0.1 wt% Hair protein binding effect 0 75% 60% 50%

As shown in Table 16, it was found that the binding ability with the hair protein keratin also increased with increasing the content of the true mushroom fungus mycelium.

Experimental Example  18: For elution of keratin by alkali and hydrogen peroxide treatment True Needle Burr Of mycelium extract Protective effect

3 g of hair was added to a 10 ml mixture of hydrogen peroxide (6%) and ammonia (1.68%) in a 1: 1 mixture, and the true fungus mycelium extract was added at a test concentration of 0.5, 0.25, and 0.1% by weight, respectively, at room temperature. Treated for 30 minutes. 0.5 ml of the reaction solution was concentrated using a Rapid-Con protein concentration kit (Elpis Biotech.), The concentrate was electrophoresed on a 10% SDS-polyacrylamide gel according to Laemmli's method, and the keratin protein in the gel was removed. The keratin protein bands were identified by staining for 1 hour in a mixed solution of 0.1% Coomassie brilliant blue R 250, 10% glacial acetic acid and 40% ethanol and decolorizing in a mixed solution of 10% glacial acetic acid and 40% ethanol. Keratin protein was compared to the control group of 100% of the keratin band which appeared when electrophoresis of only 50 μg of human hair keratin protein using the image master software (Amersham Pharmacia Biotech.). The binding effect of is expressed in%.

Needle Mushroom Mycelia Extract Control group 0.5 wt% 0.25 wt% 0.1 wt% Hair eluting keratin protein 100% 35% 60% 84%

As shown in Table 17, it was found that the keratin protein content eluted from the hair decreases as the content of the true mushroom fungus mycelium increases.

Experimental Example  19: Effect of Needle Mushroom Mycelia Extract on Hair Tensile Strength and Elongation by Alkali and Hydrogen Peroxide Treatment

3 g of hair was added to 10 ml of hydrogen peroxide (6%) and ammonia (1.68%) in a 1: 1 mixed solution, and the true mushroom mycelium extract was added at test concentrations of 0.5, 0.25 and 0.1% by weight, respectively. After treatment for minutes, washed with water and dried in the wind to measure the tensile strength (gf) and elongation (%) in the Autograph tester. Table 18 below shows the tensile strength and the elongation of the hair treated in this way.

Needle Mushroom Mycelia Extract Purified water 0.5 wt% 0.25 wt% 0.1 wt% Tensile strength (gf) 113 123 119 116 Shinto (%) 45 48 47 45

As shown in Table 18, when the decolorizing agent (the hydrogen peroxide and ammonia mixed solution) is treated for 30 minutes, it can be seen that the decrease in strength of the hair is suppressed as the amount of addition of the true mushroom fungus mycelium extract increases. Accordingly, when the decolorant was treated, it could be expected that the hair protective effect of the true needle mushroom mycelium extract could be expected.

Experimental Example  20: hair growth effect test

A 30% hydrous ethanol solution containing 2% by weight of the true needle mushroom mycelium extract obtained in Example 1 was prepared and used for the hair growth effect test. The hair growth effect test was performed using a mouse (ICR), 47-53 days of age, to remove the back hairs, and the back area was clean and 10 rats were used per material group. Applied. The length of hair and the degree of hair growth over time were compared by adding scores from 0 to 2 according to the degree of restoration after hair removal. In order to compare the degree of hair growth, as a control, 30% alcohol solution was applied to each individual to observe the growth state.

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

Elapsed Days / Sample 5 10 15  Needle Mushroom Mycelium Extract 2% by weight 0.14 0.61 1.39 ± 0.19 Control group 0.06 0.12 0.79 ± 0.24

As a result, it can be seen that the true mushroom mycelium extract has an excellent hair growth promoting effect (Table 19).

Example  2 and Comparative example  One : Needle Mushroom  Containing mycelium extract Cosmetics  Produce

In Example 2 was prepared a cosmetic containing the needle mushroom mycelium extract obtained in Example 1.

Prepared cosmetics are in the form of a cream, the composition is shown in Table 20 below.

First, the phase b) recorded in Table 20 was heated and stored at 70 ° C., and the phase b) was added to pre-emulsion, and then emulsified uniformly with a homomixer and gradually cooled to cream (Example 2, Comparative Example). 1) was prepared.

Raw material Example 2 (% 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 Needle Mushroom Mycelia Extract One - Propylene glycol 5 5 Suitable amount Rest about 100 Rest about 100

Experimental Example  21: Cosmetic  Measurement of skin elasticity improvement effect

In order to measure the skin elasticity improvement effect of the cosmetics prepared in Example 2 and Comparative Example 1, 20 subjects (20 to 35 years old) of the subjects in Example 2 on the right side of the face and Comparative Example on the left side of the face 1 was applied twice a day for two consecutive months.

In order to confirm the skin elasticity improvement effect was measured using a skin elasticity measuring instrument (cutometer SEM 575, C + K Electronic Co., Germany) before and after using the product for 2 months. The experimental results are described in Table 21 below as ΔR7 values of Cutometer SEM 575, where R7 values indicate the nature of the viscoelasticity of the skin (n = 20, p <0.05).

Experimental product Skin elasticity effect (R7) Example 2 0.24 Comparative Example 1 0.12

As a result of measuring the skin elasticity improving effect of the cosmetic (Table 21), it was confirmed that the skin elasticity improving effect of the experimenter who applied Example 2 containing the true mushroom fungus mycelium extract than Comparative Example 1.

From the above results, it was possible to confirm the excellent skin elasticity improving effect of the external composition for skin of the present invention.

Experimental Example  22: Cosmetic  Skin wrinkle improvement

In order to measure the skin wrinkle improvement effect of the cosmetics prepared in Example 2 and Comparative Example 1 20 compared to Example 2 on the right side of the face, compared to the left side of the face of 20 subjects (women aged 20 to 35 years) Example 1 was applied twice a day for two consecutive months.

In order to examine the effect of wrinkles on the skin, a replica of silicone was made before and after using the product for two months. , Germany).

The results are shown in Table 22 below, and Table 22 shows the average of subtracting parameter values from two months ago from each parameter value after two months. In other words, the negative the value, the higher the wrinkle improvement effect.

Experimental product R1 R2 R3 R4 R5 Example 2 -0.22 -0.16 -0.10 -0.07 -0.05 Comparative Example 1 -0.10 -0.07 -0.06 -0.05 -0.03 R1: Difference between the highest and lowest fold contour
R2: The wrinkle contours are divided randomly by 5 spaces, and then the average of R1 values
R3: Highest value of R1 divided by 5
R4: Baseline of the fold contour subtracted the mean of each top and valley
R5: Mean value of the baseline of the wrinkle contour subtracted each wrinkle profile

As a result of measuring the skin wrinkle improvement effect of the cosmetics (Table 22), it was confirmed that the skin wrinkle improvement effect of Example 2 containing the true mushroom fungus mycelium extract than Comparative Example 1.

From the above results, it was confirmed that the excellent wrinkle improvement effect of the external preparation composition for skin of the present invention.

Experimental Example  23: Cosmetic  Whitening effect measurement

In Experimental Example 23, in order to measure the whitening effect of the cosmetic preparations prepared in Example 2 and Comparative Example 1, Example 2 was placed on the right side of the face of 20 test subjects (women aged 20 to 35 years). Comparative site 1 was applied twice each day for two consecutive months.

After 2 months, the applied skin of both the left and right sides of the face was measured using an image analyzer and the color change of the skin using chromameter (Minolta CR300) to measure the change in brightness of the color (ΔL). Subjective visual observation was conducted to determine the effect according to the following classification. The results are shown in Table 23 below, and the degree of whitening efficacy was evaluated by classifying it into 7 grades (-3: very worse, -2: worse, -1: slightly worse, 0: no change, 1: slightly improved, 2) : Improved, 3: very improved).

Change in brightness of skin color (△ L) Objective evaluation of the skilled person Subjective Evaluation of the Subject Example 2 Comparative Example 1 Example 2 Comparative Example 1 Example 2 Comparative Example 1 medium 4.49 1.82 3.2 2.1 2.6 1.3

As a result of measuring the whitening effect of the cosmetic (Table 23), it was confirmed that Example 2 containing the true mushroom fungus mycelium extract is superior to the comparative example 1.

From the above results, the excellent whitening effect of the external preparation composition for skin of the present invention was confirmed.

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

In Experimental Example 24, clinical tests were carried out as follows to comparatively measure the improvement effect of the cosmetic preparation prepared in Example 2 and Comparative Example 1 against atopic dermatitis.

Thirty patients with atopic dermatitis aged 5 to 30 years who had atopic dermatitis for more than two years were examined to improve atopic dermatitis. The same person, Example 2 on the left hand and Comparative Example 1 on the right hand, was washed every evening, and then applied to the skin once a day for 60 days and the degree of atopic dermatitis improvement was measured. The measurement was performed by sensory evaluation by survey. The results are shown in Table 24 below.

very good good so so Example 2 70% (21 people) 30% (9 persons) 0% Comparative Example 1 10% (3 people) 20% (6 people) 70% (21 people)

As a result of measuring the improvement effect on the cosmetic atopic dermatitis (Table 24), it was confirmed that Example 2 prepared by adding the true mushroom fungus mycelium extract has a superior atopic skin improvement effect than Comparative Example 1.

The atopic dermatitis improvement effect of the skin external preparation composition of this invention was confirmed from the said result.

Experimental Example  25: SLS Skin irritation

In Experimental Example 25, the stimulating alleviation effect of the cosmetic prepared in Example 2 was evaluated by human patch experiment.

After mixing 1% SLS (sodium lauryl sulfate) that causes irritation to general cosmetic prescriptions (cream, lotion, skin, essence) and the cosmetic of Example 2, and applying for 24 hours, 48 hours, 72 hours, The stimulation-relaxation effect was evaluated based on this.

FINN CHAMBER (FINLAND) was used to patch 0.3 mg of the product on the upper arm of 50 healthy men and women aged 20 to 50 years, and the acute irritation index was evaluated after 24 hours. After evaluation, the same amount of product was applied to the same site again, and the delayed stimulation index after 48 and 72 hours was evaluated.

As a result of measuring the skin irritation-relieving effect by SLS, the area where SLS was applied alone showed red stimulation to the skin after 24 hours, but Example 2 containing Sesame needle mycelium extract was applied for 24 hours and 48 hours. After 72 hours, no skin attack occurred.

From the above results, it was confirmed that the skin fungus caused by the base material (surfactant, fragrance, alcohol), which causes irritation when the extract of mycelium fungus mycelium is mixed in cosmetics.

Example  3: Needle Mushroom  Preparation of lotion containing mycelium extract

To 8 g of 95% ethanol, 0.05 g of polypyrrolidone, 0.1 g of oleyl alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of fragrance, 0.1 g of paraoxybenzoic acid methyl ester, a small amount of antioxidant, and a small amount of pigment It was. 0.05 g of true needle mushroom mycelium extract obtained in Example 1 and 5 g of glycerine were dissolved in 85.33 g of purified water, and then the mixed solution was added thereto and stirred to prepare a lotion containing true needle mushroom mycelium extract.

Example  4: Needle Mushroom  Preparation of latex containing mycelium extract

1.2 g of cetyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of paraoxybenzoic acid ethyl ester, 1 g of glycerin monoesteralide, 1 g of polyoxyethylene (20 molar addition) monooleate and 0.1 g of fragrance at 70 ° C The mixture was heated and dissolved, and 0.5 g of the true needle mushroom mycelium extract obtained in Example 1, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1500, 0.2 g of triethanolamine, and 76.2 g of purified water were dissolved at 75 ° C. Both were emulsified by mixing, followed by cooling to prepare an oil-in-water (O / W) type emulsion.

Example  5: Needle Mushroom  Containing mycelium extract Serum  Produce

To 5 g of 95% ethyl alcohol, 1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of kitulose, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E-acetate, 0.2 g of sodium licorice, 0.1 g of paraoxybenzoic acid ethyl ester , 1 g of the true needle mushroom mycelium extract obtained in Example 1 and a suitable amount of the pigment was mixed to prepare a cosmetic solution.

Claims (18)

Cosmetic composition for skin whitening, characterized in that it contains a true mushroom fungus mycelium extract as an active ingredient.
The method of claim 1,
The true needle mushroom mycelium extract,
A cosmetic composition, characterized in that the filtrate obtained by fermenting the true needle mushroom mycelium.
The method of claim 1,
Cosmetic composition, characterized in that containing the true needle mushroom mycelium extract 0.001 ~ 90.0% by weight based on the whole composition.
The method of claim 1,
The true needle mushroom mycelium extract,
Cosmetic composition, characterized in that the extract of true needle mushroom mycelium using at least one animal or plant as a substrate.
delete delete Anti-aging cosmetic composition characterized in that it contains a true mushroom fungus mycelium extract as an active ingredient.
delete delete delete delete delete 8. The method of claim 1 or 7,
Formulation of the cosmetic composition,
Basic cosmetic formulations consisting of lotion, gel, water soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / O) type; Color cosmetic formulations consisting of oil-in-water or water-in-oil makeup bases, foundations, skin covers, lipsticks, lip gloss, face powders, two-way cakes, eye shadows, cheek collars and eyebrow pencils; Cosmetic composition, characterized in that any one selected from. delete delete delete delete delete