KR20110134612A - A skin-care agent containing ophioglossum vulgatum extracts using microbial fermentation - Google Patents

Abstract

PURPOSE: An external use skin composition containing Ophioglossum vulgatum fermentation is provided to ensure excellent absorption onto the skin and to ensure anti-wrinkling and whitening. CONSTITUTION: An Ophioglossum vulgatum fermentation is prepared by adding Grifola frondosa(KCTC 10337BP) mycelium, culture or extract thereof and fermenting. The composition has anti-aging, whitening, and irritation relieving effect. The composition also treats atopic dermatitis and acne. The composition is manufactured in the form of a lotion, cream, emulsion, pack, or powder.

Description

A skin-care agent containing Ophioglossum vulgatum extracts using microbial fermentation}

The present invention relates to a composition for external application for skin containing fermented bottle isocho fermented as an effective microorganism, and more specifically, to a mycelium or leaf mushroom (Grifola frondosa KCTC 10337BP) which is a fermented strain. (Grifola fronodosa KCTC 10337BP) Mixing the whole culture solution to ferment the bottle isocho extract, the composition for external application for skin, characterized in that it contains an active ingredient of the bottle isocho fermentation increased in flavonoid content through the enzymatic reaction by fermentation will be.

Skin exposed to ultraviolet rays can produce free radicals and reactive oxygen species (ROS), which can cause skin cancer, phototoxicity, autoimmune disorders, photosensitivity and skin aging (Norins, J. Invest. Dermatol., 39). : 445, 1962; Cadenas, Ann. Rev. Biochem., 58:79, 1989). Synthesis and degradation of extracellular matrix such as collagen in vivo is properly regulated, but as aging progresses, its synthesis gradually decreases and the expression of matrix metalloproteinase (MMP), an enzyme that degrades collagen, promotes skin elasticity. This lowers and wrinkles are formed. In addition, these degrading enzymes are also activated by ultraviolet irradiation. Therefore, there is a need for the development of a substance capable of regulating MMP expression that inhibits activation or inhibiting its activity in cells.

Melanin is converted from tyrosine to dopa and dopaquinone by the action of tyrosinase present in pigment cells, and is generated via dopachrome. This melanin is present in the skin and protects the body from ultraviolet rays and has important functions in controlling hormone secretion in the body. However, over-production of melanin is known to play an important role in forming blemishes, freckles, etc., promoting skin aging, and causing skin cancer, and research and development for preventing melanin overproduction are being actively conducted.

Ascorbic acid (JP-A 4-9320), hydroquinone (JP-A 6-192062), kojic acid (JP-A 56-7710), arbutin (JP-A 4-93150), and some natural product extracts are already tyrosinase. It is used as a whitening cosmetic because it has an inhibitory activity, but its use is limited due to poor stability in cosmetic formulations due to degradation or coloring, odor generation, efficacy at the biological level, unclear effect and safety issues.

Skin disease refers to abnormalities on all skin including hair, nails and toenails of mammals including humans, and atopic dermatitis, which occurs in people with atopic allergies, is a representative disease. The cause of atopic dermatitis has not been clarified so far, but about 70% of patients with atopic dermatitis have a history of atopic dermatitis, which is common in people with allergic predispositions, especially with other allergic diseases such as allergic rhinitis and asthma. It is understood as one of the factors and immune diseases. Atopic dermatitis is mostly caused by the initial severe itching of the skin, resulting in secondary infections, and may be particularly exacerbated by the mental instability and emotional state of the patient. Atopic dermatitis suffers from a significant number of people, with 0.5 to 1 percent of the population and 5 to 10 percent of children. The onset of symptoms usually occurs within two to six months of age, especially in infants under one year of age, with 85% occurring within five years of age. Atopic dermatitis mainly occurs in infancy, but recently, the number of patients after puberty is increasing, and the history is very long. In the treatment of atopic dermatitis, drugs such as corticosteroids, which can expect high pharmacological effects, are used. However, such side effects of corticosteroids are problematic and patients are exposed to the risk of side effects during treatment. Moreover, it is also well known that rebounding (acute disease lesions that occur after the drug is suspended) is caused by discontinuation of the corticosteroids. As a countermeasure for such side effects, nonsteroidal anti-inflammatory agents or antihistamines, which do not contain hormones such as corticosteroids, are used, but they are difficult to cure. Therefore, there is a very urgent need to reduce or treat atopic dermatitis without side effects.

Recently, in order to reduce skin irritation caused by various chemicals, much attention has been focused on functional cosmetics having functions such as antioxidants, wrinkle relief, whitening, and itching relief. Natural ingredients have less side effects on the skin, and as the consumer's response to cosmetics using natural ingredients has recently increased, the development value of cosmetics as a raw material for cosmetics is increasing. For example, US Pat. No. 5,972,341 describes plants of the genus Commiphora, in particular Commiphora mukul ) describes the anti- wrinkle effect of extracts. Japanese Patent Application Laid-Open No. 9-672662 describes seaweed extracts having a hyaluroronidase inhibitory effect. Japanese Patent Application Laid-open No. Hei 10-85905 describes the skin texture improvement effect of Fucoidan extracted from Wakame. Japanese Patent Application Laid-Open No. 2-245087 describes the antioxidant effect of Sargassum genus extract. Japanese Patent Application Laid-Open No. 3-294384 describes an antioxidant composition extracted from the genus Hijikia. Japanese Patent Laid-Open No. 7-10769 describes a extract of Phaeophyceae having an astringent effect. Republic of Korea Patent No. 0431076 discloses a cosmetic composition for improving the healing of atopy skin, which contains a white powder, jasmine, echinacea and fermented soybean extract, and Korean Patent No. 0511494 is a treatment for atopic dermatitis including sewage, leaflet, illite Korean Patent Publication No. 2004-0065126 discloses an effect on the healing and prevention of atopy skin including herbal plant extracts obtained from natural Ganoderma lucidum, Rhizome vulgaris, licorice, Baekbongryeong, Baekjima and Baeknyeoncho. The herbal cosmetic composition which has is disclosed. In addition, the Republic of Korea Patent Publication No. 2004-0011584 discloses a composition excellent in the treatment of atopic skin diseases composed by mixing and mixing the natural ingredients such as licorice, changja, safflower, pig paper (pork scaffold).

On the other hand, the active ingredient of the herbal herbal medicine has a high body absorption rate and bioavailability, but the extract simply extracted from the herbal herbal medicine shows an effective effect in vitro, but in the case of an external preparation containing it, the effect is not satisfactory. .

In order to solve this problem, cosmetics using natural products have recently been developed to reduce skin irritation caused by various chemicals. In particular, the active ingredients of herbal herbal medicines have high body absorption and bioavailability, and fermented herbal medicines fermented with Chinese herbal medicines are fermented carefully with excellent seed spawn to enhance the properties of the original herbal medicines and the microorganisms used during fermentation. Express efficacy.

Thus, the present inventors studied the possibility of applying cosmetics in herbal herbal extracts for skin improvement, gilyeong, licorice, aralia, yeonkyo, gardenia, yellow white, golden, yellow lotus, jihwang, peony, cheongung, donkey, siho, mint It was confirmed that B. isocho and nectarine extracts were excellent in antioxidant and whitening activity.

Ophioglossum vulgatum is a perennial plant of fern cedar , with short root and columnar shape, with one leaf per year. Nutritive lobes are ovate, oval or optical lanceolate, and the spores are surrounded by the base of the lobes. The nut lobe is 6-12 cm long and 3-7 cm wide. Spores grow higher than leaves, linear, like a knife, 2-4 cm long. Outpost is called Bisoyicho (甁 爾 小草) and is medicinal. Collect in summer and fall, rinse clean and dry or use fresh. Medicinal effects are clear fever, sheep blood, pain, detoxification, waste heat seawater, hearth, soil, lung, jaundice , Stomach pain, abdominal pain caused by visa, imak (淋 濁), changjong chang (癰 腫 瘡毒), bite caused by snakes or insects (咬傷), bruises are treated. In addition, blood (血) to turn, the lungs (肺 火) to clear and pulmonary tuberculosis, heart pain (心痛), nervous (疼痛), breeze (風氣), cold and treat. 10-15g may be taken or powdered, or crushed for external use, or a combination of powder.

As a study related to the above-mentioned bottle vinegar extract and patents thereof, patent registration No. 0829729 "Antioxidant cosmetics containing bottle vinegar extract" has been reported, but in the case of simple extracts such as the bottle vinegar extract is effective in vitro Although it shows efficacy, flavonoids, which are an important component of efficacy and effect in the extract of the disease, have high molecular weight as glycosides, have some water-soluble properties, and thus have difficulty in absorbing skin effectively.

An object of the present invention is applicable to cosmetics, and when contained in cosmetics exhibits excellent anti-aging effects such as free radical scavenging effect, MMP-1 biosynthesis reduction effect and type 1 procollagen biosynthesis promoting effect, skin wrinkle improvement effect, anti-inflammatory effect It is to provide a cosmetic composition containing a fertilizer.

In addition, it is an object of the present invention to provide a cosmetic composition containing a bottle isocho fermented product exhibiting a whitening effect and a skin irritation-releasing effect on inflammatory mediators when applied to cosmetics.

It is also an object of the present invention to provide a cosmetic composition containing a bottle isocho fermented product exhibiting an effect on moisturizing effect and atopic skin improvement when applied to cosmetics.

It is also an object of the present invention to provide a cosmetic composition containing bottle isocho fermented product having excellent acne prevention effect when applied to cosmetics.

In addition, the present invention is to provide a method for producing an excellent cosmetic by fermenting the bottle vinegar extract mycelium mushroom mycelium fermentation culture.

Thus, the present inventors fermented medicinal mycelium extract using mycelium or mycelium extract or culture solution of the leaf mushroom or the prior patent [Patent No. 0461960] Grifola frondosa KCTC 10337BP of the applicant to achieve the above object It has been found that the flavonoid content is increased to complete the present invention.

Accordingly, the present invention solves the objection to chemicals compared to the process of recovering the polar bonoids by chemical acid / alkali treatment or conventional enzyme treatment through the bottle isocho extract using conventional purified water or organic solvent, Not only can greatly reduce the cost, but also can provide a cosmetic with excellent skin improvement activity than the simple extract of the disease. In addition, the inventors of the present invention, the bottle isocho fermented products have excellent skin safety, active oxygen scavenging effect, wrinkle improvement effect, whitening effect, moisturizing effect, skin irritation effect, acne prevention effect, atopy improvement effect and anti-inflammatory effect It has been found that efficacy can be expected and the present invention has been completed.

The bottle isocho fermented product obtained through the cultivation of leaf mycelium mycelium of the present invention has an increased flavonoid content, superior skin absorption, less side effects, active oxygen scavenging effect and MMP inhibitory effect and ultraviolet irradiation than the existing bottle isocho extract. It showed excellent anti-aging effects such as MMP expression control effect and skin wrinkle improvement, and alleviated skin irritation caused by UV irradiation.

In addition, the bottle isocho fermented product of the present invention showed a whitening effect such as melanin production inhibitory effect, which is a substance causing blemishes, freckles and skin pigmentation.

In addition, the bottle isocho fermentation of the present invention showed a skin irritation and atopic improvement effect by the inflammation mediator.

In addition, the bottle isocho fermentation of the present invention showed an antibacterial activity inhibitory effect of acne bacteria having excellent anti-acne effect.

Therefore, cosmetic composition such as cosmetic lotion, cream, milky lotion, pack, powder, etc. containing fermented soybean isobacterial active oxygen scavenging effect, collagen degrading enzyme activity regulating effect, skin wrinkle improvement effect, whitening, moisturizing, acne prevention, atopy It can be seen that it shows an excellent effect of external skin preparations, such as an improvement effect and a skin irritation relief effect.

Figure 1 shows the results of high-speed liquid chromatography of the changes in the content of the components before and after the cultivation of bottle Isocho extract and leaf mushroom mycelium fermentation. It was found that the peaks of No. 1 and No. 2 of the bottle extract were reduced with incubation time, and the peak content of No. 4 was significantly increased, and the major bioactive components were analyzed. As a result of confirming the peak 4 of the bottle isocho extract using an analyzer such as UV spectrum, LC-mass, and NMR, it was found that it was Quercetin 3-0-methylether.

In order to achieve the above object, the present inventors add a leaf mushroom fungus mycelium or leaf mushroom culture solution to the bottle isocho extract having a skin-improving effect to obtain the fermentation of the bottle ischo, the skin comprising the same as an active ingredient An external preparation composition is provided.

In addition, the composition for external application for skin according to the present invention is fermented extract of bottle isocho fermented extract with increased flavonoid content by fermenting the extract of bottle vinegar with leaf mycelia (Grifoal frondosa KCTC 10337BP, Patent No. 0461960) or leaf mushroom culture It is characterized by containing 0.0001 to 30% by weight relative to the total weight. If the bottle is less than 0.001% by weight of fermented product is almost no skin improvement effect, when more than 30.0% by weight is not economical because the degree of effect increase for the content is insignificant.

In addition, the present invention is characterized in that the topical skin composition is selected from a lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / O) type formulation.

In addition, the present invention is characterized in that the topical skin composition is a cosmetic composition or a pharmaceutical composition.

Isocho fermentation can be formulated into pharmaceutical compositions such as capsules, liquids, ointments, patches or sustained-release agents using a pharmaceutically acceptable carrier, pharmacologically acceptable bases, carriers, excipients, Binders (e.g. starch, tragacanth rubber, gelatin, molasses, polyvinyl alcohol, polyvinylether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose and carboxymethyl cellulose), grinding agents (e.g. Agar, starch, vellatin powder, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, crystalline cellulose, calcium carbonate, sodium bicarbonate and sodium alginate), lubricants (e.g. magnesium stearate, talc, hydrogenated vegetable oils) and coloring agents . 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.

The pharmaceutical composition prepared as described above may be applied to the skin once or several times daily according to the symptoms, and the application may be adjusted according to the improvement of the symptoms.

In addition, when the medicinal bottle fermented product of the present invention is used in medicine, quasi-drugs or cosmetics, the formulation can be arbitrarily selected without particular limitation as long as it can exhibit the effects of the present invention, such as tonic, lotion, and latex. ), Creams, ointments, gels, sprays, mousses and the like.

In addition, in such a preparation, in addition to the bottle isocho fermentation product which concerns on this invention, the component which can be mix | blended normally can be mix | blended in the field of a medicine, quasi drug, and cosmetics. For example, drugs, such as vitamin E and its derivative which have a blood circulation promoting action, etc. are mentioned.

In addition, the medicines, quasi-drugs, cosmetics, etc. include skin antifungal agents such as ceparantin; Antibacterial agents such as hexachlorophene, undecylenic acid, trichlorocarbanide, and bithionol; Zinc and its derivatives; Lactic acid or alkyl esters thereof; Organic acids such as citric acid; Protease inhibitors, such as a Trane samsamic acid, etc. can be mix | blended.

In addition, the medicines, quasi-drugs, cosmetics, etc., alcohols such as ethanol and isopropyl alcohol; Polyhydric alcohols such as glycerin, propylene glycol and polyethylene glycol; Oils such as higher fatty acids, higher alcohols, hydrocarbon oils, natural oils, ester oils and silicone oils; Surfactants; Spices; Chelating agents; Moisturizing agents such as 1,3-butylene glycol, hyaluronic acid and derivatives thereof, maltitol, atherocollagen and sodium lactate; Thickeners such as carboxyvinyl polymers; Antioxidants; Ultraviolet absorbers; Pigments; water; The component normally mix | blended with the wool agent, such as a stabilizer, can be mix | blended in the range which does not impair the effect of this invention.

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

Example  One: Isocho  Extract manufacturer

After distillation, 2 kg of dried bottle isocho was refluxed three times for 5 hours at 80 ° C. with purified water, and the mixture was cooled and then filtered through Whatman # 2 filter paper. The filtered extract was concentrated under reduced pressure and lyophilized at 50 ° C. or less, and the bottle extract was prepared using purified water such that the vacuum concentrate and the lyophilisate contained 0.001 to 30.0 wt%.

Example  2: Leaf mushroom  Used Isocho Fermentation product  Produce

Leaf fungus KCTC 10337BP mycelia were incubated in a test tube containing yeast-wort agar medium, stored at 4 ℃ and used subcultured every month. Aseptic homogenization of mycelial subcultured on the slope medium to obtain an optimized synthetic medium (patent registration No. 0461960) in which the bottle isocho extract obtained in Example 1 and sugar were mixed and adjusted to pH 5.5 After inoculating 5% (v / v) in, incubated for 6 days under conditions of a temperature of 27 ℃, rotation speed 150rpm, aeration amount 1.5vvm in a fermenter, the bottle isocho fermentation was removed.

Example  3: Isocho Fermented  Ingredient Identification and Content Test

In this example, qualitative analysis of phenolic substances, flavonoids, flavonoid glycosides, etc. among the main components of the bottle isocho extract and the bottle isocho fermentation obtained in Examples 1 and 2 and to determine the change in total phenolic content and total flavonoid content For the following:

<Quality Analysis>

(1) Phenolic substance, flavonoid: When 1 ~ 2 drops of 2.5% FeCl ₃ ethanol solution was added to the extract, dark green color or precipitation was observed.

(2) Flavonoid Glycosides: Concentrated sulfuric acid was added to 5 ml of the extract using a concentrated sulfuric acid method to observe the formation of yellow and yellow-red precipitates.

(3) Sugar: After adding 2-3 drops of 5% α-naphthol alcohol solution to the extract, it was observed whether reddish violet appeared at the interface when concentrated sulfuric acid was added through the wall.

<Content analysis>

(1) Total phenol content: 10 ml of distilled water was added to the extract 1, 2 ml of Folin-Ciocalteu phenol reagent (Sigma) was added thereto, followed by mixing for 5 minutes at room temperature. 2 ml of 20% sodium carbonate was added to the reaction mixture, followed by reaction at room temperature for 1 hour and then absorbance at 680 nm. The indicator used gallic acid (gallic acid).

(2) Total flavonoid content: 2% AlCl ₃ .6H ₂ O dissolved in the same amount of methanol was added to 1.5 ml of the extract, and then reacted at room temperature for 10 minutes, and the absorbance was measured at 367 nm. In this case, catechin was used as a marker.

As a result of the total phenol and flavonoid content, the bottle isocho fermentation fermented with leaf mushrooms showed the high total phenol content, especially the flavonoid content more than doubled.

Name of sample
Total phenolic content
(gallic acid equivalents)
(㎍ / mg of extract)

Total Flavonoid Content
(catechin equivalents)
(㎍ / ml of extract) Isocho extract
(Example 1)
527.1
42.6 Isocho fermentation
(Example 2)
714.3
87.5

Example  4: NBT Antioxidant Effect Measurement Experiment

In order to confirm the antioxidant effect of the bottle isocho fermentation obtained in Example 2, a comparative sample of BHT (Butylated hydroxytoluene), which is well known as an antioxidant under laboratory conditions, was compared and the antioxidant activity was measured using Nitro Blue Tetrazolium (NBT) method.

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

As a measuring method

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

     2.3mM xanthine solution

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

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

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

     6.Xanthine Oxidase Solution ------------------ 0.1ml

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

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

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

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

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

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

The effect was calculated by Equation 1, and the results are shown in Table 2.

As shown in Table 2, it can be confirmed that the bottle isocho fermented product showed better antioxidant power than BHT.

Sample Name Treatment concentration (%) Antioxidative effect(%) Isocho extract (Example 1)
0.1 83.1% Isocho fermentation (Example 2)
0.1 96.5% Retinol 0.1 87.5% BHT 0.1 85.2%

Example  5: Free radical  Scavenging activity measurement experiment

In order to measure the free radical scavenging activity of the bottle isocho fermentation obtained in Example 2, the free radical scavenging activity was measured using a DPPH method using green tea extract and an antioxidant such as vitamin E under laboratory conditions.

The DPPH method measures free radical scavenging activity by reducing power using a free group called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical). The free radical scavenging rate is measured at a wavelength of 560 nm by comparing the degree to which the DPPH is reduced by the sample to reduce the absorbance.

For the determination of DPPH free radical scavenging activity, bottle isocho fermentation of 0.1% concentration was prepared. Extracts of the above concentrations were placed in 96-well plates, respectively, and DPPH prepared in 100 μm methanol solution was added thereto so that the total volume of the solution was 200 μl. After leaving it at 37 ° C for 30 minutes, the absorbance was measured at 560 nm. Free radical scavenging activity (%) was calculated by the following equation.

As can be seen in Table 3, the bottle isocho fermented product showed a superior antioxidant effect than the green tea extract and vitamin E, which are widely used as the bottle isocho extract and antioxidant.

Name of sample
Treatment concentration (%) Antioxidative effect(%) Isocho extract (Example 1)
0.1
81.5 Isocho fermentation (Example 2)
0.1
96.2
Green Tea Extract
0.1
75.6
Vitamin E
0.1
24.1

Example  6: Intracellular  Free radical scavenging effect

In order to investigate the inhibitory effect of free radical generation generated in the cell by ultraviolet light of the bottle vinegar fermentation obtained in Example 2, that is, the active oxygen scavenging effect, EGCG (Epigallocatechin gallate) was used as a comparative sample. The same experiment was performed.

The cell line used in the experiment was a human keratinocytes HaCaT cell line distributed by Dr. Fusenig of the German Cancer Research Center, which was used per well in a 96-well black plate for fluorescence measurement. 37 ° C., 5% CO ₂ using DMEM (Dulbeccos Modification of Eagles Medium, FBS 10%, Gibco, USA) medium dispensed with 2.0 × 10 ⁴ and added penicillin / streptomycin After culturing for 1 day under conditions, bottle isocho fermentation was treated at 0.02, 0.01, 0.005% concentration.

Samples were incubated for 24 hours, washed with HCSS (HEPES-buffered control salt solution) to remove the remaining medium, and prepared in HCSS 20 μm with DCFH-DA (2 ', 7'-dichlorodihydro-fluorescein diacetate, Molecular Probes, USA) is added 100 ul 37 ℃, 5% CO ₂ After incubation for 20 minutes under conditions, washed with HCSS. Thereafter, 100 µl of HCSS treated for each concentration was added, and the fluorescence of DCF (dichlorofluorescein) oxidized with active oxygen was measured with a fluorescence plate reader (Ex; 485 nm, Em; 530 nm). After UVB (20mJ / ㎠) was irradiated and fluorescence was measured by a fluorescence plate reader (Ex; 485nm, Em; 530nm) after treatment with bottle isocho fermentation.

As can be seen in Table 4, the bottle isocho fermented product was found to exhibit an active oxygen scavenging effect superior to the free radical scavenging effect and the comparative sample EGCG on the active oxygen scavenging effect.

Sample Name Experimental concentration (%) Intracellular free radical scavenging effect (%) Isocho extract
(Example 1) 0.01 45.2 Isocho fermentation
(Example 2) 0.01 56.3 EGCG 0.01 42.5

Example  7: In vitro MMP -1 inhibitory experiment

Fluorescence assay was used to determine the effect of inhibiting MMP-1 activity. The substrate used in the experiment was a fluorescently labeled DQ-collagen, and the enzyme was used as a commercial product from Molecular Probe (Eugene, OR, USA). The reaction buffer solution (0.5M Tris-HCl, 1.5M NaCl, 50 mM CaCl ₂ , 2 mM sodium azide, pH 7.6) was used after 10-fold dilution. Collagenase 40 diluted with 0.5 unit was added to 100 μl of the reaction buffer solution, and 20 μl of DQ collagen dissolved in the reaction buffer solution at 0.25 mg / ml and 40 μl of fermented isocho fermentation (0.02, 0.01, 0.005 wt%). Add μl. After 20 minutes at room temperature, the fluorescence value was measured using a fluorescence spectrophotometer (Perkin Elmer, UK) at an absorption wavelength of 495 nm and an emission wavelength of 515 nm. The fluorescence value of the sample itself was also measured and corrected when calculating the enzyme activity.

The results are shown in Table 5, and when 0.02% treatment of bottle isocho fermentation inhibited collagen degrading activity of Clostridium collagenase 82.5%, which was superior to the inhibitory effect of bottle isocho extract and green tea extract.

        Sample Name Sample concentration (%) Inhibition rate (%) Isocho extract
(Example 1) 0.02 71.5 0.01 43.2 Isocho fermentation
(Example 2) 0.02 82.5 0.01 58.7        Green tea extract 0.2 67.2

Example  8: after UV irradiation Isocho  By fermentation MMP -1 expression inhibition evaluation

In this experimental example, the enzyme immunoassay (ELISA) was performed to measure the concentration of MMP-1 after UV irradiation and sample addition of the bottle isocho fermentation obtained in Example 2.

Human dermal fibroblasts were irradiated with UVA at an energy of 6.3 J / cm 2 using a UV chamber. UV irradiation amount and incubation time were established through preliminary experiments to maximize the expression of MMP in fibroblasts. The negative control was wrapped in tinfoil to keep 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 were irradiated with UVA, exchanged with the medium containing the sample, and then cultured for 24 hours, and the medium was recovered and coated in 96 wells. 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) again 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 405 nm absorbance was measured with a plate reader. As a control, one without addition of a sample was used.

Compared with the control group that did not process the MMP-1 expression induced by UV irradiation, the bottle isocho fermentation showed more than 71% inhibition, which was significantly higher than that of the retinol and bottle isocho extracts used as the control. Excellent results were shown.

        Sample Name Sample concentration (%) MMP-1 expression inhibition rate (%) Isocho extract
(Example 1) 0.02 56.5 0.01 32.2 Isocho fermentation
(Example 2) 0.02 71.3 0.01 41.5        Retinol 0.1 35.4

Example  9: Promoting Type 1 Procollagen Biosynthesis

In this experimental example, a procollagen assay was performed in the following manner to investigate the effect of promoting the biosynthesis of the type 1 procollagen, a skin substrate component, after the addition of the fermented herbal extract obtained in Example 2.

Human dermal fibroblasts isolated from neonatal foreskin tissue were obtained from Modern Tissue Technology (MTT, Korea) and added 10% fetal bovine serum (FBS) to DMEM / F-12 (3: 1) medium. Incubated at a concentration of 1 × 10 ⁴ cell / ㎠. When grown 70-80%, the cells were subcultured at a ratio of 1: 3, and the 3-4th passaged cells were used for the experiment.

For experiments to measure the amount of procollagen, fibroblasts were cultured in at least 90% in 48 well plates, and then the bottle isocho ferment was added at 0.02%, 0.01%, and 0.005% concentrations. The amount of free collagen was measured using the procollagen type-1 C-peptide EIA kit (MK101, Takara, Japan).

The results are shown in Table 7. Pro-collagen biosynthesis was promoted by 55% when 0.02% treatment of bottle vinegar fermentation was performed, which is similar to TGF-β, a cell signaling material.

        Sample Name Sample concentration (%) Pro collagen biosynthesis promoting effect (%) Isocho extract
(Example 1) 0.02 45.4 0.01 31.5 Isocho fermentation
(Example 2) 0.02 55.2 0.01 42.1         TGF-β 0.001 56.4

Example  10: Isocho  Elastin production promoting effect of fermented products

In order to investigate the elastin production-promoting effect of the bottle isocho fermentation, the treatment of the bottle isocho fermentation of Example 2 was directly inhibited to elastase activity by treating the fibroblasts obtained directly from human or commercially purchased human The effect was performed as follows.

First, human fibroblasts were placed in a culture flask and cultured until about 70-80% growth. Then, after treating the bottle isocho fermentation 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 elastase was measured using the absorbance, using the absorbance using the Suc- (Ala) 3 NA which is a substrate of elastase. At this time, the group not treated with bottle isocho fermentation was used as a control.

Experimental results showed that the bottle isocho fermentation significantly inhibited the activity of elastase as compared to the control group, and the activity inhibition rate was confirmed to increase as the concentration of the bottle isocho fermentation increased. Through this, it can be seen that the bottle isocho fermented product shows an excellent anti-aging and elasticity enhancing effect (Table 8).

        Sample Name Sample concentration (%) % Inhibition of substance activity Isocho extract
(Example 1) 0.02 25.4 0.01 11.5 Isocho fermentation
(Example 2) 0.02 36.1 0.01 23.2 Control group 0%

Example  11: Tyrosinase  Activity inhibition experiment

Tyrosinase was isolated and purified from mushrooms and purchased from Sigma. Substrate L-tyrosine (Sigma) was dissolved in 0.05M sodium phosphate buffer (pH 6.8) to make a 0.1mg / ㎖ solution was used. Isocho fermentation was used after dissolving in 0.05M sodium phosphate buffer (pH 6.8) at a suitable concentration. 0.5 ml of L-tyrosine solution was placed in a test tube, and 0.5 ml of bottle isocho fermented product solution was added thereto, and left for 10 minutes in a 37 ° C thermostat. Thereafter, 0.5 ml of 200 unit / ml tyrosinase was added to each sample solution, and the mixture was reacted at 37 ° C for 10 minutes. The test tube containing the reaction solution was placed on ice, quenched to stop the reaction, and the spectrophotometer measured absorbance at a wavelength of 475 nm. As a control, 0.5 ml of buffer solution was used instead of the sample.

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

The results are shown in Table 9. Tyrosinase inhibition rate was similar to that of arbutin when 0.02% of bottle isocho ferment was treated.

        Sample Name Sample concentration (%) Tyrosinase inhibition rate (%) Isocho extract
(Example 1) 0.02 35.6 0.01 21.2 Isocho fermentation
(Example 2) 0.02 53.5 0.01 36.4         Arbutin 0.2 54.0

Example  12: Melanin synthesis inhibition experiment using B16F1 melanocytes

In order to confirm the whitening effect of the bottle isocho fermentation obtained in Example 2, melanin synthesis inhibitory effect on B16F1 melanocytes was measured.

The melanin synthesis inhibitory effect of B16F1 melanocytes was measured as follows.

B16F1 melanocytes were dispensed into 6-well plates at a concentration of 2 x 10 ⁶ per well, and the cells were attached and incubated for 72 hours by treating the samples at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, the cell number was measured, and the cells were recovered by centrifugation. Determination of intracellular melanin was performed by Rotan: Cancer. Res . , 40, 3345-3350 (1980). The cell pellet was washed once with PBS, and then 1 ml of homogenization buffer (50 mM sodium phosphate, pH6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt the cells. Melt the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), and measure the absorbance of melanin at 405 nm with a microplate reader. Melanin production inhibition rate (%) was measured. Melanin production inhibition rate (%) of B16F1 melanocytes was calculated by the equation (4).

As a result of testing the melanin synthesis inhibitory effect of B16F1 melanocytes, the fermented medicinal plants showed melanin production inhibitory effect of 0.02% to 35.1% (Table 10).

        Sample Name Sample concentration (%) Melanin Synthesis Inhibitory Effect (%) Isocho extract
(Example 1) 0.02 27.3 0.01 15.5 Isocho fermentation
(Example 2) 0.02 35.1 0.01 21.6         Arbutin 0.2 35.8

Example  13: inflammation-related enzymes ( hyaluronidase Activity Inhibitory Effect Measurement Experiment

This example was determined by measuring the enzyme activity of hyaluronidase, an inflammation-associated enzyme, in order to confirm the anti-inflammatory effect of the bottle isocho fermentation obtained in Example 2.

Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid and causes inflammation. In this Example, the method for measuring the anti-inflammatory effect by inhibiting the activity of this enzyme [Kakegawa Y: Japanese J. of Inflammantion , 4, 437-438 (1984)] was applied to determine the anti-inflammatory effect.

The inhibitory effect of hyaluronidase activity was investigated using comfrey, seesaw, oil-soluble licorice extract, and bottle isocho fermentation as samples.

Inhibitory activity of each sample on hyaluronidase was performed as follows.

100 μl of sample and 50 μl of hyaluronidase 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 2H ₂ O, Sigma). 4, 0.1 mg / ml) was added thereto and reacted again at 37 ° C. for 20 minutes. 250 μl of hyaluronic acid solution (0.4 mg / ml) was added thereto, followed by reaction at 37 ° C. for 40 minutes, and 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. Absorbance was measured at 585 nm to determine the inhibition rate for hyaluronidase. Inhibition rate (%) for hyaluronidase was calculated by the equation (5), IC ₅₀ value is the concentration of a substance that inhibits 50% hyaluronidase enzyme activity.

As a result of testing the inhibitory effect of hyaluronidase enzyme activity, the IC ₅₀ value of S. fermented soybean fermentation was 0.23%, which is compared with the conventional anti-inflammatory agents such as oil-soluble licorice extract, comfrey extract, and seesaw extract which are known to be effective in inhibiting hyaluronidase. It showed an excellent or similar effect (Table 11).

sample Hyaluronidase activity inhibitory effect (IC ₅₀ ) Comfrey Extract 0.25% Seesaw extract 0.53% Usefulness Licorice extract 0.22% Isocho extract
(Example 1) 0.29% Isocho fermentation
(Example 2) 0.23%

Example  14: Mitigation effect of cytotoxicity by UV irradiation

This example was carried out to evaluate the cytotoxic alleviation effect by ultraviolet irradiation of the bottle isocho fermentation obtained in Example 2. Fibroblasts were placed in 24-well test plates 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 10mJ / 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 treating the bottle isocho fermentation to be evaluated it was incubated for 24 hours. After 24 hours, the medium was removed, and 500 μl of cell culture medium and 60 μl of MTT solution (2.5 mg / ml) were added to each well, followed by 37 ° C. CO ₂ for 2 hours. Cultured in the incubator. The medium was removed, and 500 μl of isopropanol-HCl (0.04N) 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, followed by measurement of 565 nm absorbance in a microplate reader. Cell viability (%) was measured by Equation 6, and the cytotoxicity remission rate by ultraviolet rays was calculated by Equation 7.

Experimental results showed that B. isocho fermentation effectively reduced cytotoxicity by UV at 0.02% at 45% and effectively protected against cytotoxicity by UV and at low concentrations. (Table 12).

Sample Name Treatment concentration (%) Cytotoxic Relaxation Rate (%) Isocho extract
(Example 1) 0.02 31.2 0.01 19.1 0.005 8.3 Isocho fermentation
(Example 2) 0.02 45.0 0.01 28.5 0.005 16.2

Example  15: Inhibitory effect of inflammatory cytokine expression by UV irradiation

This Example is to evaluate the inhibitory effect of inflammatory cytokine expression expressed by UV irradiation of the diseased soybean fermentation obtained in Example 2 Fibroblasts isolated from the epidermal tissue of humans in a 24-well test plate 5 x 10 ⁴ pieces were added 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 ultraviolet light 10 mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), and then the PBS was removed and the cell culture medium (FMEM was not added to DMEM). Medium) 350 μl was added. After treating the bottle isocho fermentation to be evaluated therein was incubated for 5 hours. 150 μl of the culture supernatant was taken to quantify IL-1α to determine the inhibitory effect on the inflammatory cytokine expression of diseased isocho fermentation. The amount of IL-1α was quantified using an enzyme-linked immunosorbent assay, and the inhibition rate of inflammatory cytokine expression was calculated by Equation 8.

Experimental results showed that the vinegar fermentation inhibits the production of inflammatory cytokines, IL-1α, by UV rays at a concentration of 0.02% at 35.7%, effectively protecting against UV-induced inflammation at low concentrations (Table 13). .

Sample Name Treatment concentration (%) Inhibitory rate of inflammatory cytokine expression (%) Isocho extract
(Example 1) 0.02 25.5 0.01 11.5 0.005 7.2 Isocho fermentation
(Example 2) 0.02 35.7 0.01 19.5 0.005 7.3

Example  16: Effect of inhibiting prostaglandin biosynthesis by ultraviolet irradiation

In this experimental example, to evaluate the inhibitory effect of prostaglandin biosynthesis of the bottle isocho fermentation obtained in Example 2, 5 x 10 ⁴ keratinocytes isolated from epidermal tissue of humans were placed in a 24-well test plate 24 It was attached for hours. After replacing with FBS-free medium and incubating for 18 hours, prostaglandin biosynthetic enzyme (prostaglandin E ₂ synthetase) present in keratinocytes was treated with aspirin so that the keratinocytes had a final concentration of 50 μm. , Or cyclooxygenase (hereinafter referred to as COX) activity 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 15W, 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 treating the bottle isocho fermentation to be evaluated here 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 bottle isocho fermentation. The amount of PGE ₂ was quantified by enzyme immunoassay, and the experimental results showed that the vinegar fermentation effectively inhibited the production of prostaglandins by UV light. Particularly, the produced PGE ₂ is known to be mainly produced by the COX-2 enzyme, and thus, it can be seen from the above experiment that the bottle isocho fermentation inhibits the induction or activity of the COX-2 enzyme (Table 14).

Sample Name Treatment concentration (%) PGE ₂ Inhibition Rate (%) Isocho extract
(Example 1) 0.02 34.3 0.01 17.1 0.005 6.8 Isocho fermentation
(Example 2) 0.02 56.0 0.01 29.5 0.005 14.2

Example  17: antimicrobial activity test against acne

This Example was a paper disc test (Paper Disc Test) to confirm the antibacterial activity against acne bacteria of the bottle isocho fermentation obtained in Example 2. 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 culture medium thus prepared was plated in 0.1 ml each of BHI solid medium (Brain-Heart Infusion Broth; 3.7%; agar 1.5%) and dried. The bottle isocho fermented product obtained in Example 2 was diluted to 12% (W / v) in a 95% ethanol aqueous solution, and 50 µl each was added to a 8 mm diameter paper disk, and then placed on the solid medium prepared above, which was then 35 C was incubated for 3 days in an anaerobic culture tank. The antimicrobial activity was evaluated by observing the bacterial growth inhibition area around the paper disk and measuring the size of the ring. As a result, it was found that the size of the bacterial growth inhibiting ring of the bottle isocho fermentation was 27.4 mm.

Example  18: Anti-inflammatory test after induction of atopic dermatitis by compound 48/80

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 (5wks, male) and place them in the cage for 60 minutes He observed scratching behavior. To test the anti-inflammatory effect of diseased isocho fermentation, the diseased isocho fermentation was applied to the dorsal neck area of the mouse at 100 μl / ml at a concentration of 250 μl / ml 5 days before the injection of compound 48/80. It was. The application method is pretreatment by applying to the dorsal neck area of the mouse twice a day for 5 days for 5 days, and then injecting compound 48/80, and the time when the mouse scratches the neck with hind feet at 5 minute intervals for a total of 1 hour. Measured pruritus degree was measured.

As can be seen from Table 15, when the disease-dichocho fermentation was used, it showed an effect of inhibiting the induction of atopic dermatitis (skin pruritus) by the compound 48/80.

Number of scratches in 5 minutes 5 10 15 20 25 30 35 40 45 50 55 60 Isocho
Fermented products 21 23 26 30 32 34 38 43 42 25 18 17 Control group 39 41 49 64 80 90 94 69 54 50 41 35

Example  19: moisture hygroscopicity experiment

In order to test the water hygroscopicity of the bottle isocho fermentation, the dried isocho fermentation of distilled water of Example 2 was saturated and absorbed into dry epidermal cells obtained directly from human or purchased commercially. After the measurement, the mass change after drying for 48 hours 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 in which the water content was measured were saturated and absorbed in bottle isocho fermentation (A) and purified water (B) for 24 hours, and then the epidermal cells saturated with water were taken out of the cells and weighed. The amount of moisture per unit mass contained in the Kaiser moisture meter was measured. Thereafter, the resultant was dried under reduced pressure for 48 hours and weighed, and then a portion of the sample was collected and water content was measured by Kaiser method to determine the amount of water per unit mass contained therein.

As shown in Table 16, it was confirmed that the epidermal cells saturated and soaked with bottle vinegar fermentation contained much more water than the purified water in the process of redrying the epidermal cells saturated and soaked with purified water. The excellent skin hygroscopicity of the isocho fermentation was confirmed. It can be seen that the bottle isocho fermented product exhibits an excellent moisturizing effect.

Moisture content Initial dry state Saturation after drying Isocho fermentation 400 800 565 Purified water 400 790 400

Example  20 and Comparative example  One

This Example prepared the cosmetics containing the bottle isocho fermentation product obtained in Example 2 to evaluate the skin elasticity improvement effect and wrinkle improvement effect in a comparative experiment with Comparative Example 1.

The cosmetics used in the comparative experiments are in the form of a cream, the composition of which is shown in Table 17. First, the phase b) recorded in Table 17 is heated and stored at 70 ° C. To this, a) phase is added, and after pre-emulsification, it is emulsified uniformly with a homomixer, and then cooled slowly to prepare a cream (Example 20, Comparative Example 1). For 20 experimenters (20-35 year old female), the cream prepared in Example 20 was applied to the right side of the face, and the cream prepared in Comparative Example 1 was applied to the left side of the face twice a day for 2 consecutive months.

The skin elasticity improvement effect after the completion of the experiment 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 18 as ΔR7 value of Cutometer SEM 575, where R7 value represents skin elasticity recovery. As shown in Table 18, it can be seen that the skin elasticity improvement effect of the experimenter who applied the cream containing bottle isocho fermented product was excellent.

Raw material Example 20 Comparative Example 1 end Stearyl alcohol 8 8 Stearic acid 2 2 Stearic acid cholesterol 2 2 Squalane 4 4 2-octyldodecyl alcohol 6 6 Polyoxyethylene (25 moles)
A) Alcohol ester 3 3 Glyceryl Monostearic Acid
Aster 2 2 I Isocho fermentation One - Propylene glycol 5 5 Quantity Quantity Quantity

Note) Unit: Weight%

Experimental product Skin elasticity effect (△ R7) Remarks Example 20 0.37 Comparative Example 1 0.12

n = 20, p <0.05

For 20 experimenters (20-35 year old female), the cream prepared in Example 20 was applied to the right side of the face, and the cream prepared in Comparative Example 1 was applied to the left side of the face twice a day for 2 consecutive months.

After completion of the experiment, the skin wrinkle improvement effect is produced by using a silicone replica to examine the wrinkle improvement effect of the skin before and after using the product for two months. SV60, C + K Electronic Co., Germany). The results are shown in Table 19, which shows the average of each parameter value after 2 months minus the parameter value 2 months ago. In other words, the negative the value, the higher the wrinkle improvement effect. Therefore, it could be confirmed that the skin wrinkle improvement effect of Example 20 containing the bottle isocho fermented product was greatly enhanced as shown in Table 19.

Experimental product R1 R2 R3 R4 R5 Example 20 -0.23 -0.19 -0.11 -0.11 -0.08 Comparative Example 1 -0.12 -0.06 -0.05 -0.05 -0.02 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

Example  21 and Comparative example  2

In this example, the cosmetics containing the bottle isocho fermentation product obtained in Example 2 were prepared, and the skin whitening effect of humans was evaluated by performing a comparative experiment with Comparative Example 2.

The cosmetics used in the comparative experiments are in the form of a cream, the composition of which is shown in Table 20. First, the phase b) recorded in Table 20 is heated and stored at 70 ° C. To this, a) phase is added, and after pre-emulsification, it is emulsified uniformly with a homomixer, and then cooled slowly to prepare a cream (Example 21, Comparative Example 2). 20 experimenters (women aged 20-35 years) were applied to the right side of the face with the cream prepared in Example 21 and the left side of the face with the cream prepared in Comparative Example 2 twice a day for 2 consecutive months. . After completion of the test, the skin of the applied areas on both 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 color (ΔL). Subjective visual observation was performed and the effect was measured according to the following classification. The results are shown in Table 21 below. At this time, the degree of whitening efficacy was classified into the following seven ratings.

Evaluation criteria for whitening efficacy:

-3: very worse, -2: worse, -1: slightly worse, 0: no change,

1: slightly improved, 2: improved, 3: highly improved

As shown in Table 21, it can be seen that the whitening effect is excellent in the facial skin of the experimenter who applied the cream containing bottle isocho fermentation.

Raw material Example 21 Comparative Example 2 end Stearyl alcohol 8 8 Stearic acid 2 2 Stearic acid cholesterol 2 2 Squalane 4 4 2-octyldodecyl alcohol 6 6 Polyoxyethylene (25 moles)
A) Alcohol ester 3 3 Glyceryl Monostearic Acid
Aster 2 2 I Isocho fermentation One - Propylene glycol 5 5 Quantity Quantity Quantity

Note) Unit: Weight%

Skin-colored
Change in brightness (ΔL) Skilled
Objective evaluation Subject's
Subjective evaluation Example 21 Comparative Example 2 Example 21 Comparative Example 2 Example 21 Comparative Example 2 medium 4.25 1.21 3.25 2.12 2.8 1.24

Example  22 and Comparative example  3

In this example, the cosmetic composition containing the bottle isocho fermentation product obtained in Example 2 was prepared, and the effect of improving atopic dermatitis in humans was evaluated by performing a comparative experiment with Comparative Example 3.

The cosmetics used in the comparative experiments are in the form of a cream, the composition of which is shown in Table 22. First, the phase b) recorded in Table 22 is heated and stored at 70 ° C. To this, a) phase is added, and after pre-emulsification, it is emulsified uniformly with a homomixer, and then cooled slowly to prepare a cream (Example 22, Comparative Example 3). In order to confirm the atopic skin improvement effect, the following clinical experiments were conducted. Thirty patients with atopic dermatitis, aged 5-30 years, who had atopic dermatitis for more than two years were examined for atopic dermatitis improvement. The same person was washed with cream of Example 22 on the left hand and the cream of Comparative Example 3 on the right hand 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 23 below.

As shown in Table 23, it can be seen that the cream of Example 22 prepared by adding fermented soybean vinegar has an atopic skin improvement effect than the cosmetic of Comparative Example 3 without the extract.

Raw material Example 22 Comparative Example 3 end Stearyl alcohol 8 8 Stearic acid 2 2 Stearic acid cholesterol 2 2 Squalane 4 4 2-octyldodecyl alcohol 6 6 Polyoxyethylene (25 moles)
A) Alcohol ester 3 3 Glyceryl Monostearic Acid
Aster 2 2 I Isocho fermentation One - Propylene glycol 5 5 Quantity Quantity Quantity

Note) Unit: Weight%

very good good so so Example 22 67% (20 people) 27% (8 people) 6% (2 people) Comparative Example 3 11% (3 people) 19% (6 people) 70% (21 people)

Example  23: SLS Skin irritation relief effect

In this experimental example, the stimulating alleviation effect of the cosmetic containing the bottle isocho fermentation obtained in Example 2 was evaluated by human patch experiment.

1% SLS (sodium lauryl sulfate) that causes irritation in general cosmetic prescriptions (cream, lotion, skin, essence) and the product prepared in Example 20 are mixed and patched for 24 hours, 48 hours and 72 hours The stimulation-relaxation effect was evaluated based on this.

FINN CHAMBER (FINLAND) was applied to each arm of 50 healthy men and women aged 50 to 50 with 0.3 mg of each product, 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 the test, the area covered with SLS alone showed red irritation to the skin after 24 hours, but no skin seizures occurred after 24 hours, 48 hours, and 72 hours after patching the product containing fermented soybean paste. .

The results of this evaluation showed that there was a significant effect of reducing skin irritation by the substrates (surfactants, fragrances, alcohols) that cause irritation when the medicinal herbs were mixed in cosmetics.

Other examples are shown below. That is, lotion, milky lotion and cosmetic liquid containing the bottle isocho fermented product obtained in Example 2 were prepared in Examples 24 to 26. The lotion, milky lotion and essence containing these fermented soybean pastes have antioxidant, collagen synthesis, skin wrinkle improvement, whitening effect, moisturizing effect, skin irritation effect, acne prevention effect, atopic improvement effect and anti-inflammatory effect. Excellent effect on skin improvement.

Example  24: Example  Obtained in 2 Isocho Fermented products  Containing lotion

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 are dissolved. . 0.05 g of bottled isocho fermented product obtained in Example 2 and 5 g of glycerin were dissolved in 85.33 g of purified water, and then the mixed solution was added and stirred to obtain a lotion having a skin improvement effect.

Example  25: Example  Obtained in 2 Isocho Fermented products  Latex preparation

1.2 g of cetyl alcohol, 10 g of squalane, 2 g of petrolatum, 0.2 g of paraoxybenzoic acid ethyl ester, 1 g of glycerin monoesteralide, 1 g of polyoxyethylene (20 mole added) monooleate and 0.1 g of fragrance were mixed and dissolved at 70 ° C. 0.5 g of bottle isocho fermented product obtained in Example 2, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1500, 0.2 g of triethanolamine, and 76.2 g of purified water were heated to 75 ° C to dissolve it. Both mixtures were emulsified and cooled to obtain an emulsion having an oil-in-water (O / W) type skin improvement effect.

Example  26: Example  Obtained in 2 Isocho Fermented products  Containing Essence  Produce

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 bottle isocho fermented product obtained in Example 2 and a suitable amount of pigment were mixed to obtain a cosmetic liquid having a skin improvement effect.

.

Claims (8)

A bottle isocho fermentation product obtained by adding and fermenting at least one selected from Mycelia (Grifola frondosa) mycelium, mycelium broth and mycelium extract to the bottle isocho extract.
The method according to claim 1,
The leaf mushroom is a bottle isocho fermentation, characterized in that KCTC 10337BP.
The composition for external application for skin containing 0.001 to 30.0% by weight of the bottle isocho fermented product of claim 1 based on the whole composition.
The method according to claim 3,
The composition is characterized in that it exhibits an anti-aging effect.
The method according to claim 3,
The composition is characterized in that the whitening effect.
The method according to claim 3,
The composition is characterized in that it exhibits a stimulating function.
The method according to claim 3,
The composition is characterized in that it exhibits an atopic skin improvement effect.
The method according to claim 1,
The composition is characterized in that the acne prevention effect.

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