KR101297359B1 - Skin agent composition containing fermented Arenaria kansuensis Maxim. extract - Google Patents

Abstract

The present invention comprises the steps of extracting the extract from the central bark stone porridge and blood gangchi outpost (全 草); Mixing the whole culture solution of the leaf mushroom (KCTC 10337BP) mycelium or leaf mushroom (KCTC 10337BP) mycelium to the extract, and then culturing the culture; After the main culture, to remove the leaf mushroom (KCTC 10337BP) mycelium from the culture medium; relates to a composition for external application for skin, comprising the filtrate obtained as an active ingredient, antioxidant effect, collagen synthesis promoting effect, It has excellent effect on skin improvement such as skin wrinkle improvement, whitening effect, moisturizing effect, skin irritation relieving effect, acne prevention effect, atopy improvement effect and anti-inflammatory effect.

Description

Skin agent composition containing fermented Arenaria kansuensis Maxim. extract}

The present invention relates to a composition for external application for skin, and more particularly, to a composition for external application for skin containing a filtrate obtained by fermenting an extract of the whole herbaceous plant in the blood family belonging to the genus Pinus japonica.

Human skin is constantly changing with age. Skin aging is largely divided into natural aging (or endogenous aging) and external aging (EB Doris, Cosmetics & Toiletories, 111, 31-37, 1996), and natural aging (endogenous aging) is artificial because it is influenced by genetic factors. While it is difficult to control, aging is relatively easy because it is influenced by environmental factors. Representative external aging factors include ultraviolet rays, and the most prominent external aging phenomenon is wrinkle formation (HW Daniell, Ann Intern). Med , 75, 873-880, 1971, GL Grove, J Am Acad Dermatol , 21, 631-637, 1989, CE Griffiths et al, Arch Dermatol , 128, 347-351, 1992).

On the other hand, the skin exposed to ultraviolet rays generates free radicals and reactive oxygen species, causing 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 is regulated in vivo, but its synthesis decreases as aging progresses, and the expression of matrix metalloproteinase (MMP), an enzyme that breaks down collagen, promotes the expression of skin elasticity. It is lowered and wrinkles are formed.

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. Ascorbic acid (JP-A 4-9320), hydroquinone (JP-A 6-192062), kojic acid (JP-A-56-7710), arbutin (JP-A 4-93150), and plant extracts have already inhibited tyrosinase. It has been used as a whitening cosmetic due to its activity, but its stability in cosmetic formulations is deteriorated, resulting in coloration, off-flavor, unclear efficacy and effects at the biological level, or safety issues, which limit its use. .

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

Recently, in order to reduce skin irritation caused by various chemicals and the like, much attention has been focused on functional cosmetics having functions such as antioxidant, wrinkle reduction, whitening, and itching relief obtained from natural extracts. For example, US Pat. No. 5,972,341 relates to the wrinkle improvement effect of Commiphora mukul extract, and Japanese Patent Application Laid-open No. Hei 9-672662 relates to seaweed extracts having a hyaluroronidase inhibitory effect. Japanese Patent Application Laid-Open No. Hei 10-85905 relates to the skin texture improvement effect of Fucoidan extracted from Wakame, and Japanese Patent Application Laid-open No. Hei 2-245087 relates to the antioxidant effect of Sargassum genus extract. Republic of Korea Patent No. 0431076 is a cosmetic composition for improving the healing of atopy skin, including white powder, purple, echinacea and fermented soybean extract, Korean Patent No. 0511494 is an extract for the treatment of atopic dermatitis, including sewage, leaf, illite and Korean Patent Application Publication No. 2004-0011584 discloses a composition by mixing and mixing substances of natural ingredients such as licorice, changja, safflower and pork (pork). Generated discloses about the excellent composition for the treatment of atopic dermatitis.

On the other hand, in the case of extracts simply extracted herbal herbal medicines show an effective efficacy in vitro, there is a problem that the effect is not satisfactory in the case of external preparations containing them.

Accordingly, an object of the present invention is to provide a composition for external application for skin, characterized in that it contains the blood-rich fermented extract obtained by fermenting the blood-stained ground extract with leaf mycelium mycelium as an active ingredient.

It is an object of the present invention to provide an external preparation composition for skin containing fermented extract of blood vertebrae that solves problems such as discoloration due to poor stability in cosmetic formulations, odor, odor, unclear efficacy and safety at the biological level. To provide.

It is also an object of the present invention to provide a blood-rich fermented extract having an effect on skin whitening, anti-aging, skin irritation and atopic skin improvement when applied to cosmetics or pharmaceuticals.

In order to achieve the above object, the present invention comprises the steps of extracting the extract from the bloody field outpost (全 草) of the main body stone porridge; Mixing the extract with the preculture of the leaf mushroom (KCTC 10337BP) mycelium or leaf mushroom (KCTC 10337BP) mycelium, and then culturing the culture; After the main culture, removing the leaf mushroom (KCTC 10337BP) mycelium from the main culture solution; found that the purpose can be achieved by using the fermented extract obtained from the blood age as an active ingredient as a cosmetic or skin external preparation The invention was completed.

Hereinafter will be described in detail for an external preparation composition for skin containing the blood-rich fermentation extract of the present invention.

The present inventors confirmed that the main ingredient exhibiting an efficacy effect in the extract extracted from the blood group is a flavonoid. Plant-rich phenolic compounds such as flavonoids, tannins and anthocyanins are excellent antioxidant candidates for protecting skin from UV damage (Shibamot, American Chem. Soc., 564: 247). -256, 1994), in particular, flavonoids or derivatives thereof are not only known for their pharmacological effects such as anti-inflammatory, anti-allergic, anti-cancer and diabetes (Middleton et al., Pharmacol. Rev., 52: 673, 2000 ) It has been reported to be excellent in antioxidant activity such as radical scavenging activity caused by ultraviolet light and inhibition of lipid peroxidation and tyrosinase activity which is a whitening effect (Nijveldt et al., Am. J. Clin. Nutr., 74: 418, 2001) .

In general, hydrophobic substances are more effective for skin penetration than hydrophilic substances, and the components distributed in the stratum corneum are hydrophobic substances called ceramides, which are more effective for interacting with hydrophobic substances than hydrophilic substances, and hydrophobic substances are more easily skin outermost. Because it can pass through layers.

Flavonoids, on the other hand, are mostly glycosides in which more than one molecule of sugar is combined. They are present in nature, have a relatively high molecular weight, and have some water-soluble properties. This is difficult and there is a problem that the absorption is difficult.

Therefore, the present invention comprises the steps of extracting the blood source to provide a skin external composition for easy skin permeability to play an effective role as a skin external preparation; Mixing the whole culture solution of the leaf mushroom (KCTC 10337BP) mycelium or leaf mushroom (KCTC 10337BP) mycelium to the extract, and then culturing the culture; After the main culture, to remove the leaf mushroom (KCTC 10337BP) mycelium from the culture medium; the filtrate obtained from, that is, the blood-rich fermentation extract as an active ingredient, the blood-rich fermentation extract obtained through the above step It was confirmed in the present experimental example that the total phenol content and the total flavonoid content was increased than the blood extract extracted by the conventional method.

In addition, in the manufacturing process of the blood-stained fermentation extract containing the active ingredient in the present invention, after mixing the whole culture solution of leaf mushrooms (KCTC 10337BP) mycelium or leaf mushrooms (KCTC 10337BP) mycelium to the blood group extract, the main culture Characterized in that it comprises a step. Here, the pre-culture refers to a culture for increasing the amount of mycelial leaves (KCTC 10337BP) mycelium before the main culture, and the main culture is a blood culture extract using the leaf mushrooms (KCTC 10337BP), the amount of mycelia increased through the pre-culture. The fermentation process is smooth.

On the other hand, the term "included as an active ingredient" in the present invention means that the filtrate is added to the external preparation composition for skin to the extent that it can exhibit the effect of preventing and improving skin diseases from the external preparation composition for skin of the present invention, drug delivery and It is meant to include the formulation in various forms by adding various components as subcomponents for stabilization and the like.

In addition, the present invention is the filtrate contained as an active ingredient, the antioxidant effect, intracellular free radical removal effect, substrate metalloproteinase (MMP-1) activity inhibitory effect, substrate metalloproteinase (MMP-1) expression inhibitory effect , Procollagen biosynthesis promoting effect, elastin production promoting effect, whitening effect, inhibitory effect on inflammation-inducing enzyme (hyaluronidase) activity, cytotoxic effect by UV irradiation, inflammatory cytokine expression inhibitory effect by UV irradiation, acne bacteria Antibacterial effect, anti-inflammatory effect against atopic dermatitis, characterized by excellent moisture hygroscopicity.

In addition, the present invention relates to an external preparation composition for skin containing a blood-rich fermentation extract, characterized in that the content of the filtrate is 0.001 to 30.0% by weight based on the whole external preparation composition. When the filtrate content is less than 0.001% by weight, there is almost no skin improvement effect, and when the content of the filtrate is more than 30.0% by weight, the effect of increasing the content is not economical.

In addition, the present invention relates to an external preparation composition for skin, characterized in that using an aqueous solution containing 85 ~ 99 (v / v) of ethanol as the extraction solvent.

In addition, the present invention relates to an external composition for skin, characterized in that the addition of a reduced pressure concentration or freeze-drying process to the extract.

The present invention also relates to a composition for external application for skin, wherein the extract under reduced pressure or lyophilization is dissolved in at least one solvent selected from purified water, ethanol, butylene glycol, and propylene glycol.

The present invention also relates to a composition for external application for skin, characterized in that the reduced-pressure concentrated or lyophilized extract is dissolved in 0.001 to 30.0% by weight in at least one solvent selected from purified water, ethanol, butylene glycol and propylene glycol.

In addition, the present invention after adjusting the pH of the culture medium at the time of the culture to 5.0 ~ 6.0, inoculated soybean mushroom (KCTC 10337BP) mycelium 4 ~ 6% (v / v), the temperature 22 ~ 32 ℃, rotation speed 120 It relates to an external preparation composition for skin, characterized in that incubated for 5-7 days at ~ 180rpm, aeration conditions 1.2-1.8vm conditions.

In addition, the present invention relates to an external preparation composition for skin showing the skin whitening efficacy containing the blood-rich fermentation extract.

In addition, the present invention relates to an external preparation composition for skin, which exhibits anti-aging efficacy, which contains the blood-rich fermented extract.

In addition, the present invention relates to an external preparation composition for skin, which exhibits a skin irritation-releasing effect containing a blood-rich fermented extract.

In addition, the present invention relates to an external preparation composition for skin, which exhibits atopic dermatitis improving efficacy, which contains the blood-rich fermented extract.

In addition, the present invention relates to an external preparation composition for skin, which exhibits an effect of preventing acne containing the blood-rich fermentation extract.

In addition, the present invention is a composition for external application of the skin, such as plasters, lotions, liniments, liquids and solutions, aerosols, extracts, ointments ( pharmaceutical composition selected from ointments, fluid extracts, emulsions, suspensions, capsules, creams, soft or hard gelatin capsules, patches and sustained release preparations It is characterized by using as.

In addition, the pharmaceutical composition of the present invention is a pharmacologically acceptable base; Carrier; Excipients; Binders comprising starch, tragacanth gum, gelatin, molasses, polyvinyl alcohol, polyvinylether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose and carboxymethyl cellulose; Grinding agents including agar, starch, vellatin powder, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, crystalline cellulose, calcium carbonate, sodium bicarbonate and sodium alginate; Lubricants including magnesium stearate, talc and hydrogenated vegetable oils; And 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. Pharmaceutical compositions containing the extract of Seolchoen fermented extract as an active ingredient may be formulated in an injection form or a coating agent by a conventional method by combining with a carrier generally acceptable in the pharmaceutical field. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries (eg, preservatives, stabilizers, wetting or emulsifier solution promoters, salts or buffers for controlling osmotic pressure). In addition, they may contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be parenterally, that is, topical application as desired, and the dose may be administered in a daily dose of 0.001 μg-10 mg / kg divided into several doses. have. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, severity of the disease, and the like. In addition, the standard dosage of the topical skin preparation composition of the present invention may vary depending on the individual characteristics of the patient, and a substantially skilled clinician will be able to determine the ideal dosage and dosing schedule for the skin topical composition of the present invention for patients. For example, the most appropriate treatment strategy can be determined in terms of specific needs and the overall condition of the patient. In determining the appropriate dosage for the external preparation composition for skin of the present invention, reference may be made to a number of references known to those skilled in the art.

In addition, appropriate dosages of the topical skin composition of the invention may also be determined generally in vitro or in animal models. For example, appropriate concentrations may be determined by adding various concentrations of the topical skin composition of the invention to target cells in vitro.

In addition, the coating agent as a formulation of the external preparation for skin of the present invention can be easily prepared in any form according to a conventional manufacturing method. As an example, in preparing a cream coating agent, the fermented extract of the present invention is contained in a cream base of a general oil-in-water type (O / W) or water-in-oil type (W / O), and includes perfume, chelating agent, pigment, and antioxidant. While preservatives and the like are used as necessary, synthetic or natural materials such as proteins, minerals and vitamins can be used in combination for the purpose of improving the properties.

In addition, the blood stream fermentation extract of the present invention can be used as a cosmetic, by adding a pH adjuster, fragrance, emulsifier, preservatives, etc. as needed, in a conventional cosmetic preparation method, lotion, gel, water-soluble powder, fat-soluble powder, water-soluble liquid , Lotions, creams, essences, shampoos, cleansers, rinses, hair conditioners and the like.

In addition, the present invention, the skin external composition is a cosmetic composition consisting of a lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / O) type; It is characterized in that the cosmetic composition selected from oil-in-water or water-in-oil makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, teak color and eyebrow pencils.

The composition containing the blood plasma fermentation extract of the present invention exhibits antioxidant, collagen synthesis promotion, skin whitening, skin aging prevention, skin irritation relief, acne prevention, atopic improvement and anti-inflammatory effect.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be noted, however, that these examples are only illustrative examples of the present invention, and the scope of the present invention is not limited to these examples.

Comparative example  One : Blood place  Extract Preparation (Simple Extraction)

10 kg of dry blood plasma was shredded and refluxed three times for 5 hours with 95% (v / v) ethanol aqueous solution, and the resultant was cooled and then filtered through Whatman # 5 filter paper. The filtered extract was concentrated under reduced pressure and freeze-dried at 50 ° C. or lower, and then dissolved in purified water: butylene glycol = 1: 1 mixed solvent to contain 15% by weight of the obtained freeze-dried product.

Example 1 Preparation of Blood Spirit Fermentation Extract

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 the mycelium subcultured in the slope medium to prepare an optimized synthetic medium (Korean patent registration: 10-0461960) mixed with the blood-prickly water extract obtained in Comparative Example 1 and sugar, adjusted to pH 5.5 After inoculating soybean mushroom mycelium 5% (v / v) to the liquid medium, and incubated for 6 days under conditions of temperature 27 ℃, rotation speed 150rpm, aeration volume 1.5vvm in the fermenter. After cultivation, the leaf mushroom mycelium was removed from the culture medium, and then a filtrate (hereinafter referred to as 'blood rot fermentation extract') was obtained.

Experimental Example  One : Blood place  Determination of Components and Contents of Fermented Extracts

In Experimental Example 1, qualitative analysis of phenolic substances, flavonoids, flavonoid glycosides, and phenolic contents and total flavonoid contents among the main components of the blood-puff extract prepared in Comparative Example 1 and the blood-puff fermentation extract prepared in Example 1 Compared.

First, qualitative analysis of phenolic substances, flavonoids, flavonoid glycosides, etc. was performed as follows.

(1) Qualitative analysis of phenolic substances and flavonoids

When 1 to 2 drops of 2.5% FeCl ₃ ethanol solution was added to Comparative Example 1 (Spirit of Blood Extract by Simple Extraction) and Example 1 (Stained Blood of Fermented Extract), respectively, the color of dark green or the formation of precipitates was determined. Observed.

(2) Qualitative analysis of flavonoid glycosides

5 ml of concentrated sulfuric acid was added dropwise to Comparative Example 1 and Example 1 using a concentrated sulfuric acid method to observe the formation of yellow and yellow-red precipitates.

(3) Qualitative analysis of sugar

After adding 2-3 drops of 5% a-naphthol alcohol solution to Comparative Example 1 and Example 1, respectively, it was observed whether reddish violet appeared at the interface when concentrated sulfuric acid was added through the base wall.

As a result of qualitative analysis, it was confirmed that Comparative Example 1 and Example 1 contained a phenolic substance, flavonoid flavonoid glycosides.

Total phenol content and total flavonoid content measurements were performed as follows.

(4) Determination of total phenolic content

10 ml of distilled water was added to 1 ml of Comparative Example 1 and Example 1, 2 ml of Folin-Ciocalteu phenol reagent (Sigma) was added, mixed, and reacted at room temperature for 5 minutes. 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).

(5) Determination of total flavonoid content

Comparative Example 1 and Example 1 After mixing 1.5% of 2% AlCl ₃ H ₂ O dissolved in the same amount of methanol, and reacted for 10 minutes at room temperature, the absorbance was measured at 367nm. In this case, catechin was used as a marker.

Name of sample Total phenol content
(Μg / mg of extract) Total flavonoid content
(Μg / mg of extract) Comparative Example 1
(Blood clot extract by simple extraction) 401.5 47.4 Example 1 (blood place fermentation extract) 483.4 73.4

As a result of measuring the total phenol content and the total flavonoid content (Table 1), it was confirmed that Example 1 has a higher total phenol content and total flavonoid content than Comparative Example 1, in particular, the flavonoid content was found to increase about 1.5 times or more. .

From the above results, it was confirmed that the total phenolic content and the total flavonoid content could be increased by fermenting the blood-prickly ground extract prepared by simple extraction with leaf mushroom mycelium, and based on this, the blood-grain fermentation fermented by the leaf mushroom mycelium. The external preparation composition for skin of the present invention is characterized by containing an extract.

Experimental Example  2 : NBT Antioxidant effect measurement

In order to confirm the antioxidant level of the blood-rich fermentation extract obtained in Example 1, BHT (Butylated hydroxytoluene), which is well known as an antioxidant, was used as a comparative sample, and the antioxidant measurement was performed using NBT method.

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

2.4 ml of 0.05 M Na ₂ CO ₃ , 0.1 ml of 3 mM xanthine solution, 0.1 ml of 3 mM EDTA solution, 0.1 ml of BSA solution, and 0.1 ml of 0.72 mM NBT solution were added to the Bayer bottle. , It was left for 10 minutes at 25 ℃. After standing, 0.1 ml of xanthine oxidase solution was added and stirred rapidly, followed by incubation at 25 ° C. for 20 minutes, and 0.1 ml of 6 mM CuCl ₂ solution was added thereto to stop the reaction, and the absorbance at 560 nm was measured. It was. In the blank test, distilled water was used instead of the sample solution, and the absorbance (Bt) was measured in the same manner as above. Blank of the sample solution was measured for absorbance (Bo) in the same manner as above using distilled water instead of xanthine oxidase solution.

The measurement result was computed by the formula (1).

Name of sample Free radical scavenging rate (antioxidative effect;%) Blood Spirit Fermented Extract 0.1% by weight 96 Blood Spirit Extract 0.1 wt% 77 BHT 0.1 wt% 89

As a result of measuring the antioxidant effect using the NBT method (Table 2), it was confirmed that the blood-rich fermented extract exhibited more excellent antioxidant effects at the same concentration as the blood-rich extract and BHT.

From the above results, it was possible to confirm the excellent antioxidant effect of the blood-rich fermentation extract of the active ingredient of the present invention, from which it was possible to deduce the excellent antioxidant effect of the present invention composition.

Experimental Example  3: DPPH Antioxidant effect measurement

In Experimental Example 3, BHT, which is well-known as an antioxidant, was used as a comparative sample to confirm the antioxidant level of the blood-rich fermented extract obtained in Example 1, and the antioxidant measurement was used for the DPPH method.

The DPPH method is to measure free radical scavenging activity by reducing force using a free group called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical). The degree of reduction of the absorbance by reduction of DPPH by the test substance is compared with the absorbance of the blank test solution and the free radical scavenging ratio is measured at a wavelength of 560 nm.

After the preparation of 0.1% concentration of blood sterilized extract, blood sterilized extract, BHT, each sample was put in 96-well plate. DPPH prepared in 100 μm methanol solution was added thereto to have a total volume of 200 μl. After leaving it at 37 ° C. for 30 minutes, the absorbance (St) was measured at 560 nm.

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

Name of sample Free radical scavenging rate (antioxidant effect;%) Blood Spirit Fermented Extract 0.1% by weight 95 Blood Spirit Extract 0.1 wt% 74 BHT 0.1 wt% 85

As a result of measuring the antioxidant effect using the DPPH method (Table 3), it was confirmed that the blood-rich fermented extract exhibited better antioxidant effects than the blood-rich extract and BHT.

From the above results, it was possible to confirm the excellent antioxidant effect of the blood-rich fermentation extract, from which the excellent antioxidant effect of the composition of the present invention could be deduced.

Experimental Example  4 : Intracellular  Measurement of free radical scavenging effect

In Experimental Example 4, EGCG (Epigallocatechin gallate) was used as a comparative sample in order to measure the scavenging effect of free radicals generated in the cells by ultraviolet light of the blood sterilized fermentation extract obtained in Example 1, using a fluorescent material The following experiment was performed.

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

After 24 hours of incubation with the test sample, the remaining medium was removed by washing with HCSS (HEPES-buffered control salt solution) and DCFH-DA (2 ', 7'-dichlorodihydro-fluorescein diacetate, Molecular Probes prepared with 20μM in HCSS). , USA) was added thereto, and 37 ° C, 5% CO ₂ After incubation for 20 minutes under conditions, washed with HCSS. Since 100 μl of HCSS treated for each concentration was added, the fluorescence of DCF (dichlorofluorescein) oxidized with active oxygen initially was measured by a fluorescence plate reader (Ex; 485 nm, Em; 530 nm). After UVB (20mJ / ㎠) was irradiated and each sample was processed, the fluorescence was measured by a fluorescence plate reader (Ex; 485nm, Em; 530nm).

Name of sample Free radical scavenging effect (%) 0.01% by weight of blood spirit fermented extract 50 0.01% by weight of blood clot extract 34 EGCG 0.01% by weight 46

As a result of measuring the scavenging effect of the active oxygen (Table 4), it was confirmed that the blood oxygen fermentation extract was higher than the blood immersion extract at the same concentration, and showed an active oxygen scavenging rate similar to that of EGCE.

From the above results, it was confirmed that the excellent active oxygen scavenging effect of the extract of the blood-rich fermentation.

Experimental Example  5: in vitro ( in vitro )in Metalloproteinases ( MMP -1) activity inhibition rate measurement

As aging progresses, the synthesis of extracellular matrix, such as collagen, is reduced in vivo, and the expression of metalloproteinase (MMP-1), an enzyme that degrades collagen, is promoted, which not only reduces skin elasticity but also wrinkles. Is formed.

In Experimental Example 5, green tea extract was used as a comparative sample to measure the inhibition rate of the in vitro metalloproteinase (MMP-1) activity of the blood-derived fermentation extract obtained in Example 1, using fluorescence analysis. It was.

The substrate used in the experiment was a fluorescently labeled DQ-collaken and an enzyme (collagenase) using a product commercially available from Molecular Probe (Eugene, OR, USA), and reaction buffer (0.5M Tris-HCl, 1.5M NaCl). , 50mM CaCl ₂ , 2mM sodium azide, pH7.6) was used after 10-fold dilution. 20 μl of DQ collagen dissolved in the reaction buffer at 40 μl of the buffer solution and 40 μl of the fermented extract (0.02%) of blood-serial paper were added and 40 μl of collagenase diluted to 0.5 units was added. After 20 minutes at room temperature, the fluorescence value was measured by using a fluorescence spectrophotometer (Perkin Elmer, UK) at an absorption wavelength of 495 nm and an emission wavelength of 515 nm. In the control group, the reaction buffer solution was added in the same amount as the enzyme solution instead of the enzyme solution, and the fluorescence value was measured.

Name of sample % Inhibition of metalloproteinase (MMP-1) activity Blood Spirit Fermented Extract 0.02% by weight 91 Blood Spirit Extract 0.02 wt% 51 Green Tea Extract 0.2 wt% 69

As a result of measuring the rate of inhibition of metalloproteinase (MMP-1) activity in vitro (Table 5), the blood-protein fermentation extracts inhibited the activity of metalloproteinase (MMP-1) than the blood-shell extract of the same concentration. It was excellent, it was confirmed that effectively inhibit the activity of MMP-1 even at low concentrations compared to green tea extract.

From the above results, it was confirmed that the effect of inhibiting the activity of the metalloproteinase (MMP-1) of the blood-rich fermentation extract, based on this, the composition for external application of the present invention effectively inhibits the metalloproteinase, thereby making the skin elastic It can be deduced that degradation and wrinkle formation can be prevented.

Experimental Example  6: after ultraviolet irradiation Metalloproteinases ( MMP -1) Expression inhibition level measurement

Metalloproteinase (MMP-1), which degrades collagen and affects skin elasticity and wrinkle formation, is also activated by UV irradiation.

In Experimental Example 6, retinol was used as a comparative sample to measure the degree of inhibition of expression of metalloproteinase (MMP-1) after UV irradiation of the blood-derived fermentation extract obtained in Example 1.

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

Human dermal fibroblasts were irradiated with UVA at an energy of 6.3 J / cm 2 using a UV chamber, and UV irradiation dose and incubation time were preliminary experiments to establish the condition of maximal MMP-1 expression in fibroblasts. The negative control was wrapped in tinfoil and kept at the same time in the environment of UVA, UVA emission was measured using a UV radiometer. Cells during UVA irradiation were intact with previously dispensed medium and irradiated with UVA, exchanged with medium containing the sample for 24 hours, followed by culture recovery and coating on 96-well plates. The primary antibody (MMP-1 (Ab-5) monoclonal antibody) was treated and reacted at 37 ° C. for 60 minutes. After reacting the secondary antibody anti-mouse IgG (whole mouse, alkaline phosphatase conjugated) for about 60 minutes, the alkaline phosphatase substrate solution (1 mg / -nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes, and then the microplate reader was used. Absorbance was measured at 405 nm. As a negative control, the one without addition of the sample was used.

Test group MMP-1 expression inhibition rate (%) Negative control group - Blood Spirit Fermented Extract 0.02% by weight 72 Blood Age Extract 0.02% by weight 38 Retinol 34

As a result of measuring the inhibition of expression of metalloproteinase (MMP-1) after UV irradiation (Table 6), the blood control fermentation extract showed over 72% inhibition compared to the negative control without treatment. The results were also considerably better than the inhibition rates of the used blood extract and retinol.

From the above results, it was confirmed that the effect of inhibiting the expression of metal proteinase (MMP-1) of the blood-derived fermentation extract was excellent. Based on this, the external composition for skin of the present invention effectively inhibits the expression of the metalloproteinase. It can be inferred that the decrease in elasticity and wrinkle formation can be prevented.

Experimental Example  7: Measurement of Type 1 Procollagen Biosynthesis Promoting Effect

In Experimental Example 7 was used as a TGF-β positive control group in order to confirm the effect of promoting the biosynthesis of the skin matrix component of type 1 procollagen obtained in Example 1 fermentation extracts, procollagen assay (procollagen assay) Was carried out as follows.

Human dermal fibroblasts isolated from neonatal foreskin tissue (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) (3: 1) were obtained from Modern Tissue Technology (MTT, Korea). 10% Fetal Bovine Serum (FBS) was added to the culture medium and cultured at a concentration of 1 × 10 ⁴ cells / cm 2. When grown at about 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 at least 90% in 48 well plates, and then the blood-rich fermented extract and the blood-rich extract were added at 0.02% concentration, respectively, and after 24 hours, The amount was measured using the procollagen type-1 C-peptide EIA kit (MK101, Takara, Japan).

Name of sample Procollagen biosynthesis promoting effect (%) Blood Spirit Fermented Extract 0.02% by weight 34 Blood Spirit Extract 0.02 wt% 12 TGF-β001 wt% 36

As a result of measuring the effect of promoting procollagen biosynthesis (Table 7), the procollagen biosynthesis was promoted 34% when treated with 0.02% of blood-fermented fermentation extract, and showed a similar effect to TGF-β.

From the above results, it was possible to confirm an excellent procollagen biosynthesis promoting effect of the blood-rich fermented extract, and to deduce the skin-improving effect of the composition of the present invention prepared therefrom.

Experimental Example  8 : Elastase  Determination of activity inhibitory effect

In Experimental Example 8, in order to measure the effect of promoting the elastin production of the blood-derived fermented extract obtained in Example 1, blood-derived fermented extract and the blood-stained ground of 0.02% in human fibroblasts collected directly from humans or purchased commercially Experiment to measure the effect of inhibiting the elastase activity by treating the extract was performed as follows.

First, human fibroblasts were placed in a culture flask and incubated until about 70-80% growth. Subsequently, after treatment for one day with the blood-rich fermented extract, cell cultures were 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 the sample was used as a control.

Name of sample Elastase activity inhibition rate (Elastin production promoting effect;%) Blood Spirit Fermented Extract 0.02% by weight 48 Blood Spirit Extract 0.02 wt% 23 Control group 0

As a result of measuring the rate of inhibition of elastase activity (Table 8), it was confirmed that the blood suppressed fermentation extract had an excellent rate of inhibiting activity of elastase than the blood extract.

From the above results, it was confirmed that the excellent anti-aging and elasticity enhancing effect of the extract of the blood sterilization.

Experimental Example  9: Tyrosinase  Determination of activity inhibitory effect

In Experimental Example 9, a well-known arbutin as a whitening agent was used as a comparative sample in order to confirm the inhibitory effect of tyrosinase activity of the blood-rich fermented extract obtained in Example 1.

Tyrosinase is an enzyme that accelerates the oxidation process of a substance called tyrosine in vivo to produce melanin. Tyrosinase was isolated and purified from mushrooms and purchased from Sigma. Substrate L-tyrosine (Sigma) was dissolved in 0.05M sodium phosphate buffer (pH 6.8) to make a 0.1mg / ㎖ solution was used. Blood age fermented extract, blood age extract, and arbutin were used after dissolving in an appropriate concentration in 0.05M sodium phosphate buffer (pH 6.8). 0.5 ml of L-tyrosine solution was added to a test tube, and 0.5 ml of each sample solution was added thereto, and the mixture was left to stand at 37 ° C. for 10 minutes. Thereafter, 0.5 ml of 200 unit / ml tyrosinase was added to the sample solution, and the mixture was reacted at 3 ° C for 10 minutes. The reaction tube was placed on ice to quench the reaction, and the absorbance of the reaction tube was measured with a spectrophotometer 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).

Name of sample Tyrosinase activity inhibition rate (%) Control group - Blood Spirit Fermented Extract 0.02% by weight 62 Blood Spirit Extract 0.02 wt% 39 Arbutin 0.2 wt% 58

As a result of measuring the activity inhibitory effect of tyrosinase (Table 9), it was confirmed that the inhibition rate of tyrosinase activity similar to 0.2% by weight of arbutin when treated with 0.02% by weight of the fermented extract of blood age.

From the above results, it was possible to confirm the superior tyrosinase activity inhibitory effect of the blood-rich fermentation extract, from which the excellent whitening effect of the composition of the present invention could be deduced.

Experimental Example  10: Determination of melanin synthesis inhibitory effect using B16F1 melanocytes

In Experimental Example 10, arbutin, known as a whitening agent, was used as a comparative sample in order to confirm the melanin synthesis inhibitory effect of the blood sterilized fermentation extract obtained in Example 1, B16F1 melanin cells were used.

The B16F1 melanocytes used in Experimental Example 10 are cell lines derived from mice, and are cells that secrete a black 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 performed by R. Lotan and D. Lotan, Cancer Res , 40: 3345-3350, 1980). After washing the cell pellet with PBS once, 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt the cells. After dissolving the extracted melanin by adding 1N NaOH (10% DMSO (dimethyl sulfoxide)) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), the absorbance of melanin was measured at 405 nm with a microplate reader. Melanin was quantified to measure melanin production inhibition rate (%) of the sample.

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

Name of sample Melanin synthesis inhibition rate (%) Negative control group - Blood Spirit Fermented Extract 0.02% by weight 39 Blood Spirit Extract 0.02 wt% 29 Arbutin 0.2 wt% 40

As a result of measuring the melanin synthesis inhibitory effect using B16F1 melanocytes (Table 10), 0.02% by weight of blood-derived fermented extract showed melanin synthesis inhibition rate similar to that of 0.2% by weight arbutin.

From the above results, it was possible to confirm the excellent melanin synthesis inhibition rate of the blood-rich fermentation extract, from which the excellent whitening effect of the composition of the present invention could be deduced.

Experimental Example  11: inflammation-induced enzyme ( hyaluronidase ) Determination of activity inhibitory effect

In this experiment 11, in order to measure the anti-inflammatory effect of the blood-stained fermentation extract obtained in Example 1 and the blood-stained ground extract obtained in Comparative Example 1, the Comfrey extract, seesaw extract, oil-soluble licorice extract was used as a positive control, inflammation The inhibitory effect of the induced enzyme hyaluronidase activity was measured.

Hyaluronidase is an enzyme that induces inflammation as an enzyme that hydrolyzes hyaluronic acid. In this experimental example, the anti-inflammatory effect was determined by applying a method (Kakegawa Y: Japanese J. of Inflammation, 4, 437-438, 1984) to inhibit the activity of hyaluronidase. .

100 μl of sample and 50 μl of 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, 0.1 mg / ml) was added and the reaction was carried out again at 37 ° C. for 20 minutes. 250 µl of a hyaluronic acid solution (0.4 mg / ml) was added thereto and reacted 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 mg of ρ-dimethylaminobenzaldehyde solution was added thereto. The absorbance was measured at 585 nm to determine the inhibitory rate of hyaluronidase activity.

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

Name of sample Inhibitory effect of hyaluronidiases (IC50) Blood Spirit Fermentation Extract 0.23% Blood Spirit Extract 0.14% Comfrey Extract 0.25% Seesaw extract 0.51% Usefulness Licorice extract 0.24%

As a result of measuring the inhibitory effect of hyaluronidase activity (Table 11), the IC ₅₀ value of the blood-fermented fermentation extract was 0.23%, which was similar to that of the Comfrey and oil-soluble licorice extracts well known as anti-inflammatory agents.

From the above results, it was possible to confirm the excellent anti-inflammatory effect of the blood-rich fermentation extract, from which the excellent anti-inflammatory effect of the composition of the present invention could be deduced.

Experimental Example  12: Measurement of cytotoxic mitigation effect by ultraviolet irradiation

In Experimental Example 12, the cytotoxic alleviation effect by UV irradiation was measured between the blood-killed fermented extract obtained in Example 1 and the blood-killed extract obtained in Comparative Example 1.

Fibroblasts (fibroblasts) were placed in a 24-well test plate 1 x 10 ⁵ and attached for 24 hours. Each well was washed once with PBS and 500 占 퐇 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 sample to be evaluated here, 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. Lt; / RTI > After incubation, the medium was removed and 500 μl of isopropanol-HCl (0.04 N) was added thereto. After shaking for 5 minutes, the cells were lysed and 100 μl of the supernatant was transferred to a 96-well test plate, and the absorbance was measured at 565 nm using a microplate reader.

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

Name of sample Treatment concentration (%) Cytotoxic Relaxation Rate (%) Blood Spirit Fermentation Extract 0.02 42 0.01 28 0.005 9 Blood Spirit Extract 0.02 19 0.01 11 0.005 3

As a result of measuring the cytotoxicity mitigation effect by UV irradiation (Table 12), the blood-killed fermentation extract reduced the cytotoxicity of UV-42 by 42% at 0.02% concentration, and compared with blood-derived extract It can be seen that it effectively defends the toxicity.

From the above results, it was confirmed that the cytotoxicity mitigating effect of the blood-derived fermentation extract was excellent by the ultraviolet irradiation, and from there, it was possible to infer the stimulating effect of the skin by the ultraviolet rays of the composition of the present invention.

Experimental Example  13: Measurement of the Inhibitory Effect of Inflammatory Cytokine Expression by Ultraviolet Irradiation

Experimental Example 13 measured the degree of inhibition of inflammatory cytokine expression expressed by ultraviolet irradiation to measure the anti-inflammatory effect of the blood-rich fermentation extract obtained in Example 1.

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 light 10 mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), after which PBS was removed and FBS was not added to the cell culture medium (DMEM). Medium) 350 μl was added. After treatment of the blood-killed fermented extract to be evaluated here, it was incubated for 5 hours. After incubation, 150 μl of the culture supernatant was taken to quantify IL-1a to determine the inhibitory effect of inflammatory cytokine expression of the fermented extracts of blood larvae. The amount of IL-1a was quantified using Enzyme-linked Immunosorbent Assay, and the yield of IL-1a was calculated by Equation 8.

Name of sample Inhibitory rate of inflammatory cytokine expression (%) Blood Spirit Fermented Extract 0.02% by weight 45 Blood Spirit Extract 0.02 wt% 20

As a result of measuring the inhibitory effect of inflammatory cytokine expression by UV irradiation (Table 13), inhibiting 45% at a concentration of 0.02%, blood control fermentation extract can be confirmed that it effectively protects the inflammation caused by UV compared to blood control extract there was.

The anti-inflammatory effect of the composition of the present invention can be inferred from the above results.

Experimental Example  14: Determination of inhibitory effect of prostaglandin biosynthesis by UV irradiation

In Experimental Example 14, keratinocytes isolated from human epidermal tissues were evaluated to evaluate the inhibitory effect of prostaglandin (prostaglandin E _{2 ;} hereinafter referred to as PGE ₂ ) biosynthesis of the blood-derived fermented extract obtained in Example 1. 5 × 10 ⁴ were placed in 24-well test plates and attached for 24 hours. After replacement with medium without FBS, and cultured for 18 hours, prostaglandin biosynthetic enzymes (prostaglandin E ₂ synthetase) present in keratinocytes were treated with aspirin to achieve a final concentration of 50 μM on the keratinocytes. , Or cyclooxygenase (hereinafter referred to as COX) was removed. Two hours after aspirin treatment, each well containing keratinocytes was washed twice with PBS and 100 μl of PBS was added to each well. The keratinocytes were irradiated with UV 30 mJ / cm 2 using a UVB lamp (Model: F15T8, UV B 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 sample to be evaluated, it was incubated for 16 hours. An appropriate amount of the culture supernatant was taken to quantify biosynthesized PGE ₂ (prostaglandin E2) for 16 hours to determine the prostaglandin inhibitory effect of the sample. The amount of PGE ₂ was quantified using enzyme immunoassay.

Name of sample PGE2 Inhibition Rate (%) (-) UV B Control group (+) UV B - (+) UV B + Blood Age Fermented Extract 0.02 wt% 55 (+) UV B + blood spot extract 0.02% by weight 19

As a result of measuring the inhibitory effect of prostaglandin biosynthesis by ultraviolet irradiation (Table 14), it was confirmed that the blood-rich fermented extract effectively inhibited the production of prostaglandins by ultraviolet rays. Particularly, the produced PGE ₂ is known to be mainly produced by the COX-2 enzyme, and thus, it was possible to infer that the blood-fermented fermentation extract inhibited the induction or activity of the COX-2 enzyme.

From the above results, it was possible to confirm the excellent prostaglandin biosynthesis inhibitory effect of the blood sterilized extract.

Experimental Example  15: Determination of antimicrobial activity against acne

In Experimental Example 15, the Paper Disc Test was performed to check the antibacterial activity against the acne bacteria of the blood-killed fermented extract obtained in Example 1 and the blood-killed extract obtained in Comparative Example 1. First, in order to activate Propionibacterium acnes, the cause of acne, it was precultured for 48 hours in BHI liquid medium (Brain-Heart Infusion Broth; 3.7%). The bacterial cultures thus prepared were plated in 0.1 ml each of BHI solid medium (Brain-Heart Infusion Broth; 3.7%; agar 1.5%) and dried. The samples obtained in Example 1 and Comparative Example 1 were each diluted with 12% (w / v) in an aqueous 95% ethanol solution, and 50 µl each was added to a 8 mm diameter paper disk and placed on a solid medium prepared above. The cells were incubated for 3 days in a 35 µl anaerobic 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 of measuring the antimicrobial activity against acne bacteria, the size of the bacterium growth inhibiting ring of the blood sterilized extract was 20 mm, and the blood sterilized extract was 6 mm.

Experimental Example  16: compound ( Compound ) After atopic dermatitis induced by 48/80 Blood place  Determination of Anti-inflammatory Effects of Fermented Extracts

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 in the cage for 60 minutes He observed scratching behavior. In order to assay the anti-inflammatory efficacy of the blood-rich fermented extracts, blood-fermented fermented extracts were applied to the dorsal neck region of mice at 100 mg / ml at a concentration of 250 mg / ml from 5 days before the compound 48/80 injection. 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.

 Throat Scratch (5 minutes) 5 10 15 20 25 30 35 40 45 50 55 60 Blood place
Fermented extract 24 25 25 30 36 37 39 41 43 25 21 19 Blood Spirit Extract 37 41 49 52 59 61 64 59 50 45 40 29 Control group 39 40 54 63 78 90 91 75 61 50 45 39

As a result of measuring the anti-inflammatory effect of the blood-derived fermented extract after induction of atopic dermatitis (Table 15), it was confirmed that the blood-derived fermented extract inhibited the induction of atopic dermatitis (skin pruritus) by compound 48/80.

From the above results, it was possible to confirm the atopic prophylaxis or improvement effect of the blood-rich fermented extract, and to deduce the atopic prophylaxis or improvement effect of the present invention composition therefrom.

Example  2: Blood place  Containing fermented extract Cosmetics  Produce

In Example 2 was prepared a cosmetic containing the blood-rich fermentation extract obtained in Example 1. The prepared cosmetics were in the form of a cream, the composition of which is shown in Table 16.

First, the phase b) shown in Table 16 was heated and stored at 70 ° C., and the phase b) was added to the a) phase, and then emulsified uniformly with a homomixer and gradually cooled to a cream (Example 2, Comparative Example 2). ) Was prepared.

Raw material Example 2 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 mole addition) alcohol ester 3 3 Glyceryl monostearate Aster 2 2 I Blood Spirit Fermentation Extract One - Propylene glycol 5 5 Purified water Rest about 100 Rest about 100

Unit: weight%

Experimental Example  17: Cosmetic  Measurement of skin elasticity improvement effect

In order to measure the skin elasticity improving effect of Example 2 and Comparative Example 2, 20 experimenters (20-35 year old female) targets Example 2 on the right side of the face and Comparative Example 2 on the left side of the face 1 It was applied twice a day for two consecutive months.

In order to confirm the skin elasticity improvement effect was measured using a skin elasticity measuring instrument (cutometer SEM 575, C + K Electronic Co., Germany) before and after using the product for 2 months. The experimental results are described in Table 17 below as the ΔR7 value of the cutometer SEM 575, where the R7 value represents the viscoelasticity of the skin.

Experimental product Skin elasticity effect (R7) Example 2 0.25 Comparative Example 2 0.13

n = 20, p <0.05

As a result of measuring the skin elasticity improving effect of the cosmetic (Table 17), it was confirmed that the skin elasticity improving effect of the experimenter who applied Example 2 containing the blood-rich fermentation extract is superior to Comparative Example 2.

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

Experimental Example  18: Cosmetic  Skin wrinkle improvement

In order to measure the skin wrinkle improvement effect of Example 2 and Comparative Example 2, 20 experimenters (20-35 year old female) subjects Example 2 on the right side of the face, Comparative Example 2 on the left side of the face, respectively It was applied twice a day for two consecutive months.

In order to investigate the effect of skin wrinkles, a replica of silicone is made before and after using the product for 2 months, and the state of wrinkles at the designated area is measured by a visometer: SV60, C + K Electronic Co., Germany). The results are shown in Table 18 below, and Table 18 shows the average of subtracting the parameter value two months from each parameter value after two months. Negative values indicate higher wrinkle improvement.

Experimental product R1 R2 R3 R4 R5 Example 2 -0.25 -0.13 -0.12 -0.08 -0.06 Comparative Example 2 -0.12 -0.07 -0.06 -0.04 -0.01 R1: Difference between the highest and lowest fold contour
R2: Divide the wrinkle contours by 5 squares and then average the R1 values
R3: Highest 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

As a result of measuring the skin wrinkle improvement effect of the cosmetic (Table 18), it was confirmed that the skin wrinkle improvement effect of Example 2 containing the blood-rich fermentation extract is superior to Comparative Example 2.

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

Experimental Example  19: Cosmetic  Whitening effect measurement

In Experimental Example 19, in order to measure the whitening effect of Example 2 and Comparative Example 2, 20 experimenters (20-35 year old female) subjects Example 2 on the right side of the face, Comparative Example 2 on the left side of the face Were applied twice a day for 2 consecutive months.

After 2 months, the applied skin of both the left and right sides of the face was measured using an image analyzer and the color change of the skin using chromatameter (Minolta CR300) to measure the change in brightness of the color (L). Subjective visual observation was performed to determine the effect according to the following classification. The results are shown in Table 19 below, and the degree of whitening efficacy was evaluated by classifying them into 7 grades (-3: very worse, -2: worse, -1: slightly worse, 0: no change, 1: slightly improved, 2) : Improved, 3: very improved).

Change in brightness of skin color (L) Objective evaluation of the skilled person Subjective Evaluation of the Subject Example 2 Comparative Example 2 Example 2 Comparative Example 2 Example 2 Comparative Example 2 medium 4.18 2.21 3.2 2.8 2.9 1.5

As a result of measuring the whitening effect of the cosmetic (Table 19), it was confirmed that Example 2 containing the blood-rich fermentation extract is superior to the comparative example 2.

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

Experimental Example  20: Cosmetic  Measurement of improvement effect on atopic dermatitis

In Experimental Example 20, in order to compare and measure the improvement effect on atopic dermatitis of Example 2 and Comparative Example 2 was carried out as follows.

Thirty patients with atopic dermatitis aged 5 to 30 years who had atopic dermatitis for more than two years were examined to improve atopic dermatitis. To the same person, Example 2 on the left hand and Comparative Example 2 on the right hand were washed every evening, and then applied to the skin once a day for 60 days and the degree of improvement in atopic dermatitis was measured. The measurement was performed by sensory evaluation by survey. The results are shown in Table 20 below.

very good good so so Example 2 80% (24 people) 17% (5 people) 3% (1 person) Comparative Example 2 6% (2 people) 17% (5 people) 77% (23 people)

As a result of measuring the improvement effect on atopic dermatitis (Table 20), it was confirmed that Example 2 prepared by the addition of the blood sterilized fermentation extract is superior to the atopic skin improvement effect than Comparative Example 2.

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

Experimental Example  21: SLS Skin irritation

In Experimental Example 21, the stimulating alleviation effect of the cosmetics containing the blood-rich fermentation extract obtained in Example 1 was evaluated by human patch experiment.

1% SLS (sodium lauryl sulfate), which causes irritation to general cosmetic prescriptions (cream, lotion, skin, essence), and mixed with Example 2 for 24 hours, 48 hours, 72 hours, and then based on the stimulation index The effects of stimulating relaxation were evaluated.

FINN CHAMBER (FINLAND) was applied to the upper arm of 50 healthy men and women aged 20 to 50 by 0.3 mg each, 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-relieving effect by SLS, the area where SLS was applied alone showed red stimulation to the skin after 24 hours, but Example 2 containing the blood-rich fermented extract was patched for 24 hours and 48 hours. After 72 hours, no skin attack occurred.

From the above results, it was confirmed that the blood-rich fermentation extract can reduce the skin irritation by the substrate (surfactant, fragrance, alcohol) that causes irritation when used in cosmetics.

Example  3: Blood place  Preparation of lotion containing fermented extract

To 8 g of 95% ethanol, 0.05 g of polypyrrolidone, 0.1 g of oleyl alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of fragrance, 0.1 g of paraoxybenzoic acid methyl ester, a small amount of antioxidant, and a small amount of pigment were dissolved. . 0.05 g of the blood sterilized fermented extract obtained in Example 1 and 5 g of glycerin were dissolved in 85.33 g of purified water, and then the mixed solution was added thereto, followed by stirring to prepare a lotion containing the blood sterilized fermented extract.

Example  4 : Blood place  Preparation of latex containing fermented extract

1.2 g of cetyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of paraoxybenzoic acid ethyl ester, 1 g of glycerin monoesteralide, 1 g of polyoxyethylene (20 mole added) monooleate and 0.1 g of fragrance were heated and mixed at 70 ° C. Then, 0.5 g of the sterile ground fermented extract obtained in Example 1, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1500, 0.2 g of triethanolamine, and 76.2 g of purified water were dissolved by heating to 75 ° C. Both were emulsified by mixing, followed by cooling to prepare an oil-in-water (O / W) type emulsion.

Example  5: Blood place  Containing fermented extract Serum  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, The serum was prepared by mixing 1 g of the blood sterilized fermented extract obtained in Example 1 and an appropriate amount of pigment.

Claims (11)

Obtaining a blood clot extract using an aqueous solution containing 85 to 99% (v / v) ethanol as an extractant;
After mixing the whole culture solution of the leaf mushroom (KCTC 10337BP) mycelium or leaf mushroom (KCTC 10337BP) mycelium to the blood extract, adjust the pH of the culture medium to 5.0 ~ 6.0, the leaf mushroom (KCTC 10337BP) mycelium 4-6 After inoculating to% (v / v), the main culture step performed for 5 to 7 days at a temperature of 22 ~ 32 ℃, rotation speed 120 ~ 180rpm, aeration 1.2 ~ 1.8vvm conditions;
Removing the leaf mushroom (KCTC 10337BP) mycelium from the main culture after the main culture; the cosmetic composition for acne skin, characterized in that it contains 0.001 to 30.0% by weight of the filtrate obtained through the entire composition.
delete delete delete delete delete delete delete delete The method according to claim 1,
The cosmetic composition comprises a lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / O) type; Cosmetic composition for acne skin characterized in that it is selected from oil-in-water or water-in-oil type makeup base, foundation and skin cover. delete