KR101460672B1 - Composition for preventing hair damage containing the extract of mycoleptodonoides aitchisonii fruit body - Google Patents

Abstract

The present invention relates to a composition for external application for skin containing a fruity body extract of true needle mushroom, which comprises 0.001 to 90.0% by weight of a true needle mushroom fruiting body extract, a wrinkle improving effect, a collagen synthesis The present invention relates to a composition for external application for a functional skin, which is excellent in an effect of stimulation, whitening, alleviation of skin irritation, prevention of acne, improvement of atopy and anti-inflammation, prevention of hair damage, prevention of hair loss and hair growth.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing damage to hair comprising a fruiting body extract of true needle mushroom {

The present invention relates to a composition for external application for skin containing a fruity body extract of true needle mushroom, and more particularly to a composition for external application for skin containing a filtrate obtained from a fruity body extract of true needle mushroom.

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 a cosmetic composition using the mushroom extracts known so far, there have been proposed a cosmetic composition comprising a mixture of a mushroom (Korean Patent No. 0340185), a mushroom, a skimmer mushroom (Korean Patent No. 0681703, No. 0076537), a 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, Mycoleptodonoides It is a kind of edible mushroom which is called aurasin mushroom and is a fruit-flavored mushroom. The fruiting body has no mushroom sack. It has a soft meat quality and becomes strong when dried. Is shaped like a tooth. Mycelium is 2 hyphae of thick film and thin film. The bottom layer of fleshy layer is needle mushroom type, needle is pointed, length is 3 ~ 10 mm and becomes yellowish when dried. In addition to the pharmacological effects such as blood pressure strengthening action, blood sugar enhancing action, brain function improvement effect, anticancer and anti-diabetic action, it is expected to be excellent in nutrition. However, in Korea, . ≪ / RTI >

Such mushrooms have been studied mainly for anticancer and anti-diabetic effects until now. However, there has not yet been elucidated whether they exhibit skin anti-aging effect, whitening effect and atopy improvement effect as external preparations for skin.

Korean Patent No. 10-0809375 (health functional food including true needle mushroom and aloe) discloses a health functional food including true needle mushroom and aloe. However, it has been reported that the extract of true needle mushroom fruiting body is used as an external preparation for skin The point of use is not described. Korean Patent Laid-Open Publication No. 10-2012-0002025 (composition for preventing and treating diabetes using true needle mushroom) describes a composition for prevention and treatment of diabetes using true needle mushroom, wherein the extract of true needle mushroom fruiting body is used as an external preparation for skin There is no description about the use thereof as a composition.

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.

Another object of the present invention is to provide a composition for external application for skin, which comprises, as an active ingredient, an extract of true needle mushroom fruiting body which is a higher fungus in order to solve the above problems.

In addition, the present invention provides a hair styling composition which has a combination of antioxidant, collagen synthesis promotion, improvement of skin wrinkles, whitening, moisturizing, skin irritation mitigation, acne prevention, atopic improvement and anti-inflammatory effect, hair damage prevention effect, hair loss prevention, It is an object of the present invention to provide a method for efficiently obtaining a mushroom fruit body extract.

In order to achieve the above object, the present invention provides a composition for external application for skin, which comprises an extract of True Mushroom Fruit Body as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention relates to an anti-wrinkle effect, a whitening effect, an atopic dermatitis improvement, a prevention or treatment effect, a skin irritation mitigation effect, an inflammatory disease prevention or treatment effect, an acne improvement, a prevention or treatment effect, The present invention is characterized by containing a true needle mushroom fruity body extract as an effective ingredient in order to provide a skin external composition composition having excellent effects.

The true needle mushroom fruit body extract of the present invention can be obtained by extracting true needle mushroom fruiting body. Herein, the kind of true needle mushroom fruiting body is not limited to a specific kind but can be either natural reared or artificially cultured. In the case of artificial culture, for example, those cultured according to the methods described in Korean Patent No. 10-1063754 and Korean Patent No. 10-1110487 may be used, but the present invention is not limited thereto.

In addition, the method for extracting true needle mushroom fruiting bodies of the present invention can be performed by a method commonly used in the technical field to which the present invention belongs. For example, water of about 2 to 20 times, preferably about 2 to 5 times, water, a lower (C1-C4) alcohol such as methanol or ethanol, or the like is added to the fruit body powder obtained by lyophilizing and grinding the true needle mushroom fruiting body 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 true needle mushroom fruit body extract of the present invention may mean the polysaccharide itself contained as an active ingredient in the thus-obtained extract.

The thus-obtained true needle mushroom fruity body extract of the present invention can be used as an antioxidative substance, an inhibitor of matrix metalloproteinase (MMP-1) activity, an inhibitor of substrate metalloproteinase (MMP-1) Inhibitory effect of hyaluronidase activity, effect of alleviating cytotoxicity by ultraviolet irradiation, inhibitory effect of inflammatory cytokine expression by ultraviolet irradiation, inhibitory effect of prostaglandin biosynthesis by ultraviolet irradiation, The anti-inflammatory effect against atopic dermatitis, the skin irritation alleviating effect, the hair damage prevention effect, the hair loss prevention and the hair growth effect are excellent, so that it can be used in the multifunctional skin external composition composition. In addition, the true needle mushroom fruiting body extract of the present invention can sufficiently attain the above effect without requiring additional steps such as fermentation.

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 true needle mushroom fruity body extract with respect to the whole composition. When the composition contains less than 0.001% by weight, If it exceeds, it is not economical because efficiency is less than material input amount.

In the present invention, the term "comprising as an active ingredient" means that the extract of true mushroom fruiting body is added to the composition for external application for skin to such an extent that it can exhibit the effect of preventing and suppressing skin diseases from the composition for external application for skin of the present invention , And includes various components in a variety of formulations for the purpose of drug delivery and stabilization, and the like.

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, atopic skin improvement, acne improvement, hair damage prevention or hair loss prevention.

Meanwhile, the composition for external application for skin of the present invention is preferably a pharmaceutical composition. Examples of the formulation of the pharmaceutical composition include PLASTERS, LPTIONS, 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 hair cosmetic composition which can exhibit a combination of antioxidant, collagen synthesis promotion, improvement of skin wrinkles, whitening, moisturizing, skin irritation reduction, acne prevention, atopic improvement and anti- A fruity body extract of needle mushroom can be obtained in high yield, and a multifunctional skin external application composition can be prepared using the extract.

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  One: True needle mushroom  Preparation of fruit body extract

The fruiting body powder obtained by freeze-drying and pulverizing the M. aitchisonii fruiting body was subjected to reflux extraction three times for 5 hours in an aqueous solution containing 70% (v / v) ethanol, followed by cooling, # 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.

Experimental Example  One: NBT Antioxidant effect measurement

BHT, well known as an antioxidant, was used as a comparative sample to determine the antioxidant level of the true mushroom fruit body extract obtained in Example 1, and NBT method was used for the antioxidant measurement.

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;%) True needle mushroom fruit body extract 0.25 wt% 96 0.1% by weight of true needle mushroom fruit body extract 93 Mushroom extract of true needle mushroom 0.05 wt% 79 Mushroom extract of true needle mushroom 0.025 wt% 10 BHT 0.1 wt% 89

The results of the antioxidative effects of NBT method (Table 1) showed that the extract of true needle mushroom fruit showed better antioxidative effect at the same concentration as BHT.

From the above results, it is possible to confirm the excellent antioxidative effect of the true mushroom fruit body extract of the present invention, which is an effective ingredient of the present invention, and the excellent antioxidative effect of the present invention can be obtained therefrom.

Experimental Example  2: DPPH Antioxidant effect measurement

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

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 concentration of true needle mushroom fruity body extract was prepared, and the true needle mushroom fruit body extract according to the concentration was put into a 96-well plate, 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 well without treatment of true needle mushroom fruit body extract

B: Absorbance of experimental group well treated with true needle mushroom fruit body extract

Name of sample Free radical scavenging ratio (antioxidant effect;%) True needle mushroom fruit body extract 0.25 wt% 96 0.1% by weight of true needle mushroom fruit body extract 94 Mushroom extract of true needle mushroom 0.05 wt% 89 Mushroom extract of true needle mushroom 0.025 wt% 75 BHT 0.1 wt% 85

The antioxidative effects of DPPH were measured (Table 2), and it was confirmed that the extract of True mushroom fruiting body showed excellent antioxidative effect even at lower concentration than BHT.

From the above results, it was possible to confirm the excellent antioxidative effect of the extract of Tricholoma mushroom fruit body extract, and the excellent antioxidative effect of the present invention could be deduced therefrom.

Experimental Example  3: Intracellular  Measurement of reactive oxygen scavenging effect

In Experimental Example 3, EGCG was used as a comparative sample in order to measure the scavenging effect of the active oxygen generated in the cells by the ultraviolet rays of the true needle mushroom fruit body extract obtained in Example 1, The same experiment was carried out.

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 , And then treated with the extract of true needle mushroom fruiting body at concentrations of 0.02, 0.01, and 0.005 wt%.

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 the true needle mushroom fruit body extract.

Name of sample Effect of active oxygen scavenging (%) Mushroom fruiting body extract 0.02 wt% 55 Mushroom extract of true needle mushroom 0.01 wt% 48 True needle mushroom fruit body extract 0.005 wt% 22 EGCG 0.01 wt% 45

The results of the measurement of the active oxygen scavenging effect (Table 3) revealed that the extract of true mushroom fruiting body showed higher reactive oxygen scavenging rate than EGCG at the same concentration as EGCG.

From the above results, the excellent active oxygen scavenging effect of the present invention can be confirmed.

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

In Experimental Example 4, the green tea extract was used as a comparative sample and the fluorescence assay was used to measure the inhibition rate of metalloproteinase (MMP-1) activity in vitro of the true needle mushroom fruit body extract obtained in Example 1 .

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 and 40 μl of a true mushroom fruiting body extract (0.02, 0.01, 0.005% by weight) were added to 100 μl of the reaction buffer, and collagenase 40 ≪ / RTI > 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 (%) Mushroom fruiting body extract 0.02 wt% 88 Mushroom extract of true needle mushroom 0.01 wt% 62 True needle mushroom fruit body extract 0.005 wt% 40 Green tea extract 0.2 wt% 62

in (MMP-1) activity in vitro (Table 4). In addition, the extract of true needle mushroom effectively inhibited the activity of metalloproteinase (MMP-1) at a lower concentration than that of green tea extract .

From the above results, it was confirmed that the effect of inhibiting the metalloproteinase (MMP-1) activity of the true needle mushroom fruit body extract was excellent, and based on this, the composition for external application for skin of the present invention effectively inhibited the metalloproteinase It is possible to prevent the decrease of elasticity and the formation of wrinkles.

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

In Experimental Example 5, retinol was used as a comparative sample to measure the degree of inhibition of metalloproteinase (MMP-1) expression after UV irradiation of the true needle mushroom fruit extract obtained in Example 1 above.

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

Test group MMP-1 expression inhibition rate (%) Control group 0 Mushroom fruiting body extract 0.02 wt% 65 Mushroom extract of true needle mushroom 0.01 wt% 30 True needle mushroom fruit body extract 0.005 wt% 18 Retinol 36

The inhibition rate of metalloproteinase (MMP-1) expression after UV irradiation was measured (Table 5). In comparison with the untreated control group, the extract of true needle mushroom fruiting body showed an inhibition rate of 65% It was also remarkably superior to the inhibition rate of retinol used.

From the above results, it was confirmed that the effect of inhibiting the metalloproteinase (MMP-1) expression of the true mushroom fruit body extract was excellent. Based on this fact, the composition for external application for skin of the present invention effectively inhibited the expression of metalloproteinase It is possible to prevent skin elasticity and wrinkle formation.

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

In Experimental Example 6, TGF-beta was used as a positive control to confirm the effect of promoting the biosynthesis of type 1 procollagen, which is a skin substrate component of the true needle mushroom fruit body extract obtained in Example 1, 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, the fibroblasts were cultured in a 48-well plate at a concentration of 90% or more. Then, the concentration of 0.02%, 0.01% and 0.005% , 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 (%) Mushroom fruiting body extract 0.02 wt% 36 Mushroom extract of true needle mushroom 0.01 wt% 20 True needle mushroom fruit body extract 0.005 wt% 15 TGF-beta 0.001 wt% 36

As a result of measuring the promoting effect of procollagen biosynthesis (Table 6), when 0.02% by weight of the extract of true mushroom fruiting body was treated with 0.02% by weight, the biosynthesis of procollagen was promoted by 36%, which was similar to that of the cell signaling substance TGF-β.

From the above results, it was confirmed that the extract of true mushroom fruiting body promotes excellent procoagulant biosynthesis, and it was found that the present invention is excellent in improving skin aging.

Experimental Example  7: Elastase  Measurement of inhibition of activity

In Experimental Example 7, in order to measure the promoting effect of elastin production of the true needle mushroom fruit body extract obtained in Example 1, the extract of true needle mushroom fruit body was directly treated with human fibroblasts, Was performed.

First, human fibroblasts were placed in a culture flask and cultured until they were about 70-80% grown. Thereafter, the extract of the true mushroom fruiting body 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 instrument [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 not treated with the true needle mushroom fruit body extract.

Name of sample Elastase activity inhibition rate (%) Control group 0 Mushroom fruiting body extract 0.02 wt% 29 Mushroom extract of true needle mushroom 0.01 wt% 18 True needle mushroom fruit body extract 0.005 wt% 9

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

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

Experimental Example  8: Tyrosinase  Measurement of inhibition of activity

In Experimental Example 8, arbutin, which is well known as a whitening agent, was used as a comparative sample in order to confirm the tyrosinase activity inhibitory effect of the true needle mushroom fruit body extract obtained in Example 1 above.

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. The substrate L-tyrosine was dissolved in 0.05 M sodium phosphate buffer solution (pH 6.8) to make a 0.1 mg / ml solution. The true needle mushroom fruit body extract was dissolved in 0.05 M sodium phosphate buffer solution (pH 6.8) Respectively. 0.5 ml of the L-tyrosine solution was placed in a test tube, 0.5 ml of the sample solution of true needle mushroom fruiting body extract was added thereto, and the mixture was allowed to stand at 37 DEG 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 instead of the true needle mushroom fruit body extract.

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 0 Mushroom fruiting body extract 0.02 wt% 52 Mushroom extract of true needle mushroom 0.01 wt% 37 True needle mushroom fruit body extract 0.005 wt% 30 Arbutin 0.2 wt% 53

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

From the above results, the excellent tyrosinase activity inhibitory effect of the true mushroom fruit body extract was confirmed, and the excellent whitening effect of the present invention was confirmed.

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

In Experimental Example 9, arbutin known as a whitening agent was used as a comparative sample and B16F1 melanocyte was used for confirming the melanin synthesis inhibitory effect of the true needle mushroom fruit body extract obtained in Example 1 above.

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 0 Mushroom fruiting body extract 0.02 wt% 31 Mushroom extract of true needle mushroom 0.01 wt% 18 True needle mushroom fruit body extract 0.005 wt% 5 Arbutin 0.2 wt% 33

The inhibitory effect on melanin synthesis by B16F1 melanocytes was measured (Table 9), and 0.02% by weight of the true mushroom fruiting body extract showed similar inhibition of melanin synthesis as that of 0.2% by weight of arbutin.

From the above results, it was confirmed that the excellent inhibition of melanin synthesis of the fruity body extract of true mushroom fungus was confirmed, and the excellent whitening effect of the present invention was found therefrom.

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

In Experiment 10, Comfrey extract, Sepharose extract, and Yiliang licorice extract were used as a positive control to measure the anti-inflammatory effect of the true needle mushroom fruit extract obtained in Example 1, and the inflammatory enzyme, hyaruronidase hyaluronidase activity 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 0.25 Seiso extract 0.54 Usefulness Licorice extract 0.21 True needle mushroom fruit body extract 0.25

The results of measuring the inhibitory effect of hyaluronidase (Table 10) showed that the IC ₅₀ value of the true mushroom fruiting body extract was 0.25%, similar to that of the comfrey extract, sepharose extract and oiliness licorice extract And showed excellent effects.

From the above results, the excellent anti-inflammatory effect of the true mushroom fruit body extract was confirmed, and the excellent anti-inflammatory effect of the present invention was obtained.

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

In Experimental Example 11, the cytotoxic effect of the extract of true needle mushroom fruiting body obtained in Example 1 on ultraviolet irradiation was measured.

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 extract of true needle mushroom fruiting body was treated 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 (%)
True needle mushroom fruit body extract 0.02 40.2 0.01 30 0.005 15

As a result of measuring cytotoxic effect by ultraviolet irradiation (Table 11), the extract of true needle mushroom fruiting body effectively inhibited cytotoxicity by ultraviolet rays by reducing cytotoxicity by ultraviolet rays by 40.2% at 0.02 wt% concentration And it was found.

From the above results, it was confirmed that cytotoxic relaxation effect of the true mushrooms fruiting body extract by ultraviolet irradiation was excellent, and it was found that the skin irritation mitigation effect of the present invention was attenuated by ultraviolet rays.

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

In Experimental Example 12, the degree of inhibition of inflammatory cytokine expression expressed by ultraviolet irradiation was measured in order to measure the anti-inflammatory effect of the true needle mushroom fruit extract obtained in Example 1 above.

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 extract of true needle mushroom fruiting body was treated and cultured for 5 hours. After culturing, 150 [mu] l of the culture supernatant was taken to quantitate IL-1 [alpha], thereby determining the effect of inhibiting the expression of inflammatory cytokines in the true mushroom fruit body extract. 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 (%)
True needle mushroom fruit body extract 0.02 30.5 0.01 14.7 0.005 5.5

The inhibitory effect of inflammatory cytokine expression by ultraviolet irradiation was measured (Table 12), and it was confirmed that the extract of true needle mushroom fruiting body effectively inhibited the development of inflammation caused by ultraviolet rays at a low concentration by inhibiting the concentration of 0.02% by weight by 30.5% there was. From the above results, the anti-inflammatory effect of the present invention was found.

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

In Experimental Example 13, in order to evaluate the prostaglandin E ₂ (hereinafter, referred to as PGE ₂ ) inhibitory effect of the true needle mushroom fruit extract obtained in Example 1, keratinocyte 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). Here, the extract of true needle mushroom fruiting body was treated and cultured for 16 hours. The prostaglandin inhibitory effect of the true mushrooms fruiting body extract was determined by quantifying the biosynthesized PGE ₂ (prostaglandin E ₂ ) for 16 hours by taking an appropriate amount of the culture supernatant. The amount of PGE ₂ was quantitated using enzyme immunoassay.

Name of sample PGE ₂ inhibition rate (%) (-) UV B control (+) UV B - (+) UV B + true needle mushroom fruit body extract 0.02 wt% 54 (+) UV B + true needle mushroom Fruit body extract 0.01 wt% 29 (+) UV B + true needle mushroom fruit body extract 0.005 wt% 10

As a result of measuring the inhibitory effect of prostaglandin biosynthesis by ultraviolet irradiation (Table 13), it was confirmed that the extract of true needle mushroom fruiting body effectively inhibited the production of prostaglandin by ultraviolet rays. In particular, PGE ₂ produced is known to be produced mainly by COX-2 enzyme. Therefore, it can be deduced that the extract of true mushroom fruiting body inhibits the induction or activity of COX-2 enzyme through the above experiment.

From the above results, it was confirmed that the extract of true mushroom fruiting body inhibited the excellent prostaglandin biosynthesis.

Experimental Example  14: Antimicrobial activity against acne bacteria

In Experimental Example 14, the paper disc test was performed to confirm the antibacterial activity against the acne bacteria of the true needle mushroom fruit extract obtained in Example 1 above.

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 fruity mushroom fruit extract obtained in Example 1 was diluted to 12% (w / v) in a 95% ethanol aqueous solution, and 50 μl of the extract was placed on a paper disc having a diameter of 8 mm and placed on the solid medium prepared above. 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 antimicrobial activity against acne bacteria, the size of inhibition of bacterial growth of the true needle mushroom fruit body extract was found to be 16 ㎜, and the excellent antibacterial activity of the present invention was confirmed.

Experimental Example  15: Compound  After induction of atopic dermatitis by 48/80 True needle mushroom  Measurement of anti-inflammatory effect of fruit body extract

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 true needle mushroom fruiting body extract, 100 μl of the true needle mushroom fruiting body extract was applied to the dorsal neck of the mouse at a concentration of 250 μl / ml for 5 days before the compound 48/80 injection. 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.

 Number of scratching (in 5min) 5 10 15 20 25 30 35 40 45 50 55 60 True needle mushroom fruit body extract 23 25 29 28 30 35 37 39 42 24 15 13 Control group 39 41 50 62 88 94 98 72 59 50 40 32

The antiinflammatory effects of the extract of true needle mushroom fruiting body after atopic dermatitis were measured (Table 14), and the extract of true needle mushroom fruiting body 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 extract of the true mushroom fruiting body, and the excellent atopy improvement, prevention, or therapeutic effect of the present invention was confirmed.

Experimental Example  16: Measurement of water hygroscopicity

In Experimental Example 16, in order to measure the water hygroscopicity of the true needle mushroom fruit body extract, the true needle mushroom fruit body extract and distilled water obtained in Example 1 were added to dried epidermal cells of a person who was directly taken from a human or purchased commercially The amount of saturated water absorbed and absorbed was measured, and the mass change after drying for 48 hours was measured.

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 of 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 each of the true mushroom fruiting body extract and the purified water for 24 hours to be saturated and absorbed. Then, the epidermal cells in which the water was saturated and absorbed were taken out from the cells and weighed and a part thereof was collected and analyzed with a Kaiser moisture meter And the amount of water per unit mass contained therein was measured. Then, after drying for 48 hours under reduced pressure, the weight was added, and a part of the weight was taken. The amount of water per unit mass was measured by measuring the water content by the Kaiser method. (Mg of water / epidermal cell mass g)

Moisture content Initial dry state Saturation After re-drying True needle mushroom fruit body extract 400 810 600 Purified water 400 780 400

As a result of measuring the water hygroscopicity (Table 15), it was confirmed that the epidermal cells saturated and absorbed with the true mushroom fruit body extract contained much more water in the re-drying process than the epidermal cells saturated and absorbed by the purified water.

From the above results, excellent skin hygroscopicity of the true needle mushroom fruit body extract was confirmed, and the excellent moisturizing effect of the present invention was found therefrom.

Experimental Example  17: True needle mushroom  Combination Effect of Fruit Body Extract and Human Hair Protein

The extracts of true needle mushroom fruiting bodies obtained in Example 1 were added to 50 ㎍ of human hair keratin protein at test concentrations of 0.5, 0.25, and 0.1 wt%, respectively, and reacted at room temperature for 30 minutes. Then, 6% hydrogen peroxide and 0.5% 1: 1 mixed solution 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. Using the image master (Amersham Pharmacia Biotech.) Software, the keratin protein band appeared when the human hair keratin protein alone was electrophoresed at 50 占 퐂, and the keratin The binding effect was expressed in%.

True needle mushroom fruit body extract Control group 0.5 wt% 0.25 wt% 0.1 wt% Hair protein binding effect 0 80% 60% 55%

As shown in Table 16, it was found that the binding ability with hair protein keratin increases with increasing content of true mushroom fruiting body extract.

Experimental Example  18: Elimination of keratin by treatment with alkali and hydrogen peroxide True needles Of Fruit Fruit Extracts Protection effect

3 g of hair was added to a 1: 1 mixed solution of 10 ml of hydrogen peroxide (6%) and ammonia (1.68%), and the true mushroom fruit body extracts were added at test concentrations of 0.5, 0.25 and 0.1 wt% And treated 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 100 μg of human hair keratin protein alone was electrophoresed, and the keratin protein Was expressed in%.

True needle mushroom fruit body extract Control group 0.5 wt% 0.25 wt% 0.1 wt% Keratin protein 100% 30% 50% 75%

As shown in Table 17, it was found that the keratin protein content eluted from the hair was decreased as the content of the true mushroom fruiting body extract was increased.

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

3 g of hair was added to a 1: 1 mixed solution of 10 ml of hydrogen peroxide (6%) and ammonia (1.68%), and the extract of true mushroom fruiting body was added at test concentrations of 0.5, 0.25 and 0.1 wt% Min, 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.

True needle mushroom fruit body extract Purified water 0.5 wt% 0.25 wt% 0.1 wt% Tensile strength (gf) 112 124 120 116 Shinto (%) 46 50 49 47

As shown in Table 18, when the decolorizing agent (the hydrogen peroxide and ammonia mixed solution) was treated for 30 minutes, the decrease in hair strength was suppressed with an increase in the amount of the extract of true mushroom fruiting body. Therefore, it could be expected that the hair - protecting effect by the extract of true needle mushroom fruiting body can be expected when the bleaching agent is treated.

Experimental Example  20: Hair Growth Effect Test

A 30% strength ethanol solution containing 2% by weight of the true needle mushroom fruit body extract obtained in Example 1 was prepared and used for 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).

Elapsed days / sample 5 10 15  2% by weight of true needle mushroom fruit body extract 0.15 0.64 1.41 + 0.08 Control group 0.07 0.13 0.80 + 0.24

Experimental results show that the extract of true mushroom fruiting body has excellent hair growth promoting effect (Table 19).

Example  2 and Comparative Example  One : True needle mushroom  Containing a fruit body extract Cosmetics  Produce

In Example 2, a cosmetic preparation containing the true needle mushroom fruit body extract obtained in Example 1 was prepared.

The cosmetics prepared are in cream form and their compositions are shown in Table 20 below.

First, the b) phase recorded in Table 20 below was heated and stored at 70 캜. A) phase was added to the b), the mixture was pre-emulsified, homogeneously emulsified with a homomixer, and slowly cooled to obtain cream (Example 2, 1).

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 True needle mushroom fruit body extract 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 preparation prepared in Example 2 and Comparative Example 1, 20 patients (20 to 35 years old) were tested. Example 2 was applied to the right side of the face, 1 were applied twice a day 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 2 0.25 Comparative Example 1 0.11

As a result of measuring the skin elasticity improving effect of the cosmetic material (Table 21), it was confirmed that the skin elasticity improving effect of the applicant of Example 2 containing the true mushroom fruiting body extract was better than that of Comparative Example 1.

From the above results, it was confirmed that the skin external application composition of the present invention has a superior 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 Example 2 and Comparative Example 1, 20 subjects (20 to 35 years old) in the experiment were examined. Example 2 was applied to the right side of the face, Example 1 was applied twice a day 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 Table 22 below, and Table 22 shows the average of the parameter values after 2 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 2 -0.21 -0.17 -0.11 -0.08 -0.06 Comparative Example 1 -0.11 -0.08 -0.05 -0.05 -0.03 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

It was confirmed that the effect of improving the skin wrinkles of the cosmetic preparation (Table 22) and the skin wrinkle improving effect of Example 2 containing the true mushroom fruit body extract were superior to those of Comparative Example 1.

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

Experimental Example  23: Cosmetic  Whitening effect measurement

In Experimental Example 23, in order to measure the whitening effect of the cosmetic preparation prepared in Example 2 and Comparative Example 1, 20 subjects (20 to 35 year-old female) were examined. Example 2 was applied to the right side of the face, , And Comparative Example 1 was applied twice a day for two consecutive months.

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 Table 23 below, and the degree of whitening efficacy was classified into seven classes (-3: very bad, -2: worse, -1: slightly worse, 0: : Improvement, 3: very improvement).

Change in skin color brightness (ΔL) Objective evaluation of expert Subjective evaluation of the subject Example 2 Comparative Example 1 Example 2 Comparative Example 1 Example 2 Comparative Example 1 medium 3.49 1.92 3.0 2.0 2.7 1.4

As a result of measuring the whitening effect of the cosmetic material (Table 23), it was confirmed that Example 2 containing the true mushroom fruit body extract had better whitening effect than Comparative Example 1.

From the above results, the excellent whitening effect of the composition for external application 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.

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. Example 2 was applied to the left hand of the same person and Comparative Example 1 was applied to the right hand of the same person every evening for 60 days once a day and the degree of improvement of the atopic dermatitis condition was measured. Measurements were carried out by sensory evaluation by questionnaire. The results are shown in Table 24 below.

very good good so so Example 2 80% (24 people) 20% (6 people) 0% Comparative Example 1 10% (3 people) 20% (6 people) 70% (21 people)

As a result of measuring the improvement effect on cosmetic atopic dermatitis (Table 24), it was confirmed that Example 2 prepared by adding the true mushroom fruiting body extract had better atopic skin improving effect than Comparative Example 1.

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 stimulus relaxation effect of the cosmetic prepared in Example 2 was evaluated by a human skin patch test.

After applying SLS (sodium lauryl sulfate) 1% which causes irritation to general cosmetic prescription (cream, lotion, skin, essence) and cosmetic composition of Example 2 for 24 hours, 48 hours and 72 hours, stimulation induction index And 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 measuring the skin irritation mitigating effect by SLS, the portion where SLS was applied alone showed irritation on the skin after 24 hours. In Example 2 containing the extract of true needle mushroom fruiting body, No skin episodes occurred after 72 hours.

From the above results, it was confirmed that skin irritation caused by a stimulant base (surfactant, fragrance, alcohol) can be reduced when the extract of true mushroom fruiting body is mixed with cosmetics.

Example  3: True needle mushroom  Production of lotion containing fruit 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 true needle mushroom fruit body 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 true needle mushroom fruit body extract.

Example  4: True needle mushroom  Production 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 true mushroom fruit 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  5: True needle mushroom  Containing a fruit body extract 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 true needle mushroom fruiting body extract obtained in Example 1 and an appropriate amount of pigment were mixed to prepare a serum.

Claims (2)

A cosmetic composition for preventing damage to hair, characterized by containing a fruity body extract of true needle mushroom as an active ingredient.
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