KR101081913B1 - Skin agent composition containing the oriental-herb extracts using microbial fermentation - Google Patents

Abstract

The present invention comprises the steps of extracting the extract from the herbal mixture consisting of gilyeong, licorice, arbor, duct, gardenia, yellow white, golden, yellow lotus, jihwang, peony, cheonggung, donggi, shiho, peppermint, allium and nectarine; 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 effect, acne prevention effect, atopy improvement effect and anti-inflammatory effect.

Aging, whitening, fermentation, moisturizing, herbal medicine, atopy, acne, leaf mushroom, wrinkle, extract, skin, collagen, elasticity, antioxidant, anti-inflammatory

Description

Skin agent composition containing the oriental-herb extracts using microbial fermentation

The present invention relates to an external preparation composition for skin, and more particularly, to an external preparation composition for skin containing a filtrate obtained by fermenting a crude drug extract as an active ingredient.

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 produces free radicals and reactive oxygen species (ROS), which 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 controlled, but its synthesis decreases as aging progresses, and the expression of matrix metalloproteinase (MMP), an enzyme that breaks down collagen, is promoted, resulting in decreased skin elasticity. And wrinkles are formed.

On the other hand, melanin is transformed from tyrosine into dopa and dopaquinone by tyrosinase action present in pigment cells, and is produced 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 extracts are already tyrosinase. It is used as a whitening cosmetic because it has an inhibitory activity, but its stability in cosmetic formulations is poorly degraded and colored, or its use is limited due to the occurrence of off-flavor, efficacy at the biological level, unclear effect and safety problems.

On the other hand, atopic dermatitis is a typical disease in people with atopic dermatitis. Although the cause of atopic dermatitis has not been clarified yet, about 70% of patients with atopic dermatitis have a history of atopic dermatitis. It is called a human and is understood as one of genetic factors and immune diseases, especially with allergic rhinitis, asthma, and other allergic diseases. In the treatment of atopic dermatitis, corticosteroids such as corticosteroids are expected to have high pharmacological effects. However, side effects of corticosteroids have become a problem. It is also well known that a dramatic aggravation of lesions occurs later. As a countermeasure for such side effects, nonsteroidal anti-inflammatory agents or antihistamines, which do not contain hormones such as corticosteroids, are used, but since they are difficult to cure, it is very urgent 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. 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 hyaluroronidase inhibitory efficacy, Japanese Patent Application Laid-open No. Hei 10-85905 relates to the skin texture improvement effect of Fucoidan extracted from Wakame, 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 Pt., Jaso, Echinacea and fermented soybean extract. And the composition, Korean Patent Application Publication No. 2004-0011584 discloses the purification and mixing of substances of natural ingredients such as licorice, changja, safflower, and pork fat. It discloses about the excellent composition for the treatment of atopic dermatitis configured.

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

Accordingly, an object of the present invention is to provide a composition for external application for skin, which comprises a filtrate obtained by fermenting a mixed herbal extract with a leaf mycelium mycelium as an active ingredient in order to solve the above problems.

In order to achieve the above object, the present invention extracts an extract from a herbal mixture consisting of Giltyeong, Licorice, Aralia, Yeongyo, Gardenia, Yellowish white, Golden, Yellow lotus, Chihwang, Peony, Cheongung, Angelica, Siho, Mint, Allium and Chemistry step; 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, removing the leaf mushroom (KCTC 10337BP) mycelium from the main culture; Provided is an external composition for skin, comprising the filtrate obtained from the above as an active ingredient.

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

The simple extract from the herbal mixture consisting of Gil-Gyeong, Licorice, Arsenal, Falconia, Gardenia, Yellow white, Golden, Hwang lotus, Peony, Peony, Cheongung, Angelica, Siho, Peppermint, Allies and Natural flower shows effective efficacy in vitro. In case of applying it to an external preparation for skin, there is a problem that the effect is not satisfactory substantially.

On the other hand, the present inventors confirmed that the main component showing the efficacy effect in the extract extracted from the herbal mixture is flavonoids (Flavonoid). Plant-rich polyphenols, flavonoids, tannins and anthocyanins, are excellent antioxidant candidates for protecting skin damaged by UV light (Shibamot, American Chem. Soc). , 564: 247-256, 1994), in particular, flavonoids or derivatives thereof are known for their pharmacological effects such as anti-inflammatory, anti-allergic, anti-cancer and diabetes (Middleton et al., Pharmacol. Rev., 52: 673, 2000) It is reported that the antioxidant and whitening activity of tyrosinase activity such as radical scavenging activity and lipid peroxidation inhibiting activity generated by UV rays are excellent (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 is to provide a skin external composition for easy skin permeability and play an effective role as a skin external agent, Gil-Gil, Licorice, Gum, Yeon-Gi, Gardenia, Yellow-White, Golden, Hwangyeon, Jihwang, Peony, Cheongung, Dong-gu, Siho, Extracting the extract from the herbal mixture consisting of peppermint, allies and nectarine; 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, removing the leaf mushroom (KCTC 10337BP) mycelium from the main culture; The filtrate obtained from the above as an active ingredient, the filtrate obtained through the above step was confirmed that the total phenol content and the total flavonoid content than the extract extracted from the herbal mixture was confirmed in this experimental example.

On the other hand, in the manufacturing process of the filtrate containing the active ingredient in the present invention, after mixing the whole culture solution of leaf mushroom (KCTC 10337BP) mycelium or leaf mushroom (KCTC 10337BP) mycelium to the extract extracted from the herbal mixture, The step of culturing includes a pre-cultivation means a culture for increasing the amount of mycelium (KCTC 10337BP) mycelium before the main culture, and the main culture is a leaf mushroom (KCTC 10337BP) in which the amount of the mycelium is increased through the preculture. ) Is a process of fermenting the extract of the herbal mixture smoothly.

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 inhibiting skin diseases from the external preparation composition for skin of the present invention, the drug For the purpose of delivery and stabilization, it is meant to include a variety of ingredients (formulation) by adding a variety of ingredients as subcomponents (formulation).

On the other hand, the filtrate contained as an active ingredient in the present invention has an antioxidant effect, intracellular reactive oxygen removal effect, matrix metalloproteinase (MMP-1) activity inhibitory effect, matrix metalloproteinase (MMP-1) expression inhibitory effect , Procollagen biosynthesis promoting effect, elastin production promoting effect, whitening effect, inhibitory effect on inflammation-inducing enzyme (hyaluronidase) activity, cytotoxic effect by UV irradiation, inflammatory cytokine expression inhibitory effect by UV irradiation, acne bacteria It was confirmed that the antimicrobial effect, anti-inflammatory effect, and moisture hygroscopicity against atopic dermatitis, the skin external preparation composition of the present invention prepared from the wrinkle improvement effect, whitening effect, atopic dermatitis improvement effect, skin irritation relief effect It was also confirmed that it is excellent.

On the other hand, the herbal mixture is preferably Gilyeong, licorice, aralia, yeonja, gardenia, yellow white, golden, yellow lotus, jihwang, peony, cheongung, tangwi, shiho, peppermint, wooja and cheonhwabun are each composed of 1 to 30 parts by weight.

On the other hand, in the present invention, the filtrate is preferably contained 0.001 ~ 30.0% by weight based on the entire skin external preparation composition, when included in less than 0.001% by weight of the skin improvement effect is insignificant, when it exceeds 30.0% by weight This is because the efficiency is inferior to the material input and is not economical.

On the other hand, the extraction in the present invention preferably uses an aqueous solution containing 85 ~ 99 (v / v) of ethanol as the extraction solvent.

Meanwhile, in the present invention, the extract is preferably extracted, and then concentrated under reduced pressure or lyophilization, wherein the extract under reduced pressure or lyophilization is most preferably at least one selected from purified water, ethanol, butylene glycol and propylene glycol. It is preferable to dissolve in a solvent, wherein the concentrated under reduced pressure or lyophilized extract is most preferably dissolved in 0.001 ~ 30.0% by weight in at least one solvent selected from purified water, ethanol, butylene glycol and propylene glycol.

On the other hand, in the present invention, the culture is preferably adjusted to pH of the culture medium to 5.0 ~ 6.0, inoculated soybean mushroom (KCTC 10337BP) mycelia 4 to 6% (v / v), and then the temperature 22 ~ 32 ℃, rotation It is recommended to incubate for 5-7 days under the condition of water 120 ~ 180rpm, aeration 1.2 ~ 1.8vm.

On the other hand, the topical skin composition of the present invention is preferably a cosmetic composition, the formulation of the cosmetic composition, for example, lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / Basic cosmetic formulations of the form O); Oil-based or oil-in-water makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, teak color and eyebrow pencil formulations.

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

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

On the other hand, the pharmaceutical composition of the present invention is a pharmacologically acceptable base; Vehicle; Excipients; Binders 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 colorants and the like, and the carriers and excipients include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate and cellulose. In addition to the above, additives such as adjuvants such as stabilizers, dissolution aids, transdermal absorption accelerators, and preservatives may be further added.

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

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

On the other hand, the pharmaceutical composition is preferably for skin irritation relief, atopic skin improvement or acne prevention.

As described above, the composition for external application of the skin of the present invention ferments the extract extracted from the herbal mixture having the effects of antioxidant, promoting collagen synthesis, improving skin wrinkles, whitening, moisturizing, skin irritation, preventing acne, improving atopy and anti-inflammatory. By containing the filtrate prepared by the use to exhibit excellent skin improvement effect.

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples, and includes modifications of equivalent technical spirit.

Manufacturing example  1: Preparation of extract from herbal mixture (simple extraction)

Fine geun 30g, licorice 30g, arbor 30g, duct 30g, gardenia 30g, yellow white 30g, golden 10g, rhubarb 10g, yogurt 30g, peony 30g, cheongung 30g, donkey 30g, siho 30g, peppermint 10g, alligator 30g, Cheonhwa 30 g of the mixture was refluxed three times for 5 hours with 95% (v / v) ethanol aqueous solution, and then cooled and filtered through Whatman # 5 filter paper. The filtered extract was concentrated under reduced pressure and freeze-dried at 50 ° C. or lower, and then extracted from the herbal mixture by dissolving in a mixed solvent of purified water, ethanol, butylene glycol and propylene glycol so as to contain 15% by weight of the obtained freeze-dried product (hereinafter, Called 'medicinal herb extract').

Example  One: Manufacturing example  From 1 The obtained  Fermentation of herbal mixture extract to produce filtrate

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, an optimized synthetic medium mixed with the crude drug extract and sugar obtained in Preparation Example 1 (Korea Patent Registration: 10-0461960 was prepared, adjusted to pH 5.5) After inoculating 5% (v / v) leaf mushroom mycelium in the medium, it was incubated for 6 days in a fermenter at a temperature of 27 ° C., rotational speed of 150 rpm, and aeration rate of 1.5 vvm. Next, a filtrate (hereinafter referred to as 'fermented herbal extract') was obtained.

Experimental Example  1: Determination of Components and Contents of Fermented Herbal Extracts

In Experimental Example 1, qualitative analysis of phenolic substances, flavonoids, flavonoid glycosides, etc. among main ingredients of the herbal extracts prepared in Preparation Example 1 and the fermented herbal extracts prepared in Example 1, and phenol content and total flavonoid content were compared. It was.

First, qualitative analysis of phenolic substances, flavonoids, flavonoid glycosides, and the like were performed as follows.

(1) Qualitative analysis of phenolic substances and flavonoids

When left 1-2 drops of 2.5% FeCl ₃ ethanol solution in Preparation Example 1 (fermented herbal extracts by simple extraction) and Example 1 (fermented herbal extracts), respectively, the color of dark green or the formation of precipitates was observed. Was

(2) Qualitative analysis of flavonoid glycosides

5 ml of concentrated sulfuric acid was added dropwise to Preparation 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% α-naphthol alcohol solution in Preparation 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 Preparation Example 1 and Example 1 contained a phenolic substance, flavonoid flavonoid glycosides.

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

(1) Determination of Total Phenol Content

10 ml of distilled water was added to 1 ml of Preparation 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).

(2) Determination of Total Flavonoid Content

In Preparation Example 1 and Example 1, 1.5 ml of 2% AlCl ₃ .6H ₂ O dissolved in the same amount of methanol were respectively mixed, 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.

Sample Name Total phenolic content
(ug / mg of extract) Total Flavonoid Content
(ug / mg of extract) Production Example 1
(Medicinal Herb Extract by Simple Extraction) 447.3 35.1 Example 1 (fermented herbal extract) 520.7 55.2

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 Preparation Example 1, in particular, it was confirmed that the flavonoid content increased more than 1.5 times. .

From the above results, it was confirmed that the total phenol content and the total flavonoid content could be increased by fermenting the herbal mixture extract prepared by simple extraction with leaf mushroom mycelium, and fermented by leaf mushroom mycelium. The external preparation for skin composition of the present invention was prepared by containing a herbal extract.

Experimental Example  2: NBT Measure antioxidant effect

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

The NBT method is a method of generating active oxygen by xanthine and xanthine oxidase, and measuring blue at a wavelength of 560 nm by reacting the produced active oxygen with Nitro Blue Tetrazolium (NBT). Oxygen scavenging 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 NBT solution of 0.72 mM were added to the Bayer bottle. Then, it was left to stand at 25 degreeC for 10 minutes. After standing, 0.1 ml of xanthine oxidase solution was added and stirred rapidly, followed by incubation at 25 ° C. for 20 minutes, and 0.1 ml of 6 mM CuCl ₂ solution was added thereto to stop the reaction and absorbance (St) at 560 nm was obtained. Measured. 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) using the same method as above using distilled water instead of xanthine oxidase solution.

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

Free radical scavenging rate (%) = [1- (St-So) / (Bt-Bo)] × 100

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

Bt: absorbance at 560 nm after enzymatic reaction of blank test solution

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

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

Sample Name Free radical scavenging rate (antioxidative effect;%) Fermented herbal extract 0.25% by weight 98 Fermented herbal extract 0.1% by weight 94 Fermented herbal extract 0.05% by weight 83 Fermented herbal extract 0.025 wt% 72 BHT 0.1 wt% 85

As a result of measuring the antioxidant effect using the NBT method (Table 2), it was confirmed that the fermented herbal extracts exhibited better antioxidant effects at the same concentration as BHT.

From the above results, it was possible to confirm the excellent antioxidant effect of the fermented herbal extract as an active ingredient of the present invention, from which it was possible to deduce the excellent antioxidant effect of the present invention.

Experimental Example  3: DPPH Measure antioxidant effect

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 fermented herbal extract obtained in Example 1, and the antioxidant measurement was performed by 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 free radical scavenging rate is measured at a wavelength of 560 nm by comparing the degree to which the DPPH is reduced by the test substance to reduce the absorbance.

After preparing fermented herbal extracts of 0.25, 0.1, 0.05, and 0.025% concentrations, the fermented herbal extracts were added to 96-well plates, respectively. DPPH prepared in 100 uM 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.

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

% Free radical scavenging activity = {100- (B / A)} X100

A: absorbance of the control wells not treated with the fermented herbal extract of the present invention

B: Absorbance of Experimental Wells Treated with Fermented Herbal Extract of the Present Invention

Sample Name Free radical scavenging rate (antioxidant effect;%) Fermented herbal extract 0.25% by weight 97 Fermented herbal extract 0.1% by weight 94 Fermented herbal extract 0.05% by weight 93 Fermented herbal extract 0.025 wt% 91 BHT 0.1 wt% 82

As a result of measuring the antioxidant effect using the DPPH method (Table 3), it was confirmed that the fermented herbal extract exhibits excellent antioxidant effect even at a lower concentration than BHT.

From the above results, it was possible to confirm the excellent antioxidant effect of the fermented herbal extracts, from which it was possible to deduce the excellent antioxidant effect of the present invention.

Experimental Example  4: Intracellular  Measurement of free radical scavenging effect

In Experimental Example 4, EGCG was used as a comparative sample to measure the scavenging effect of free radicals generated in the cell by ultraviolet light of the fermented herbal extract obtained in Example 1, and the following experiment was performed using fluorescent materials. 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). plates were plated at 2.0 × 10 ⁴ per well and added to DMEM (Dulbeccos Modification of Eagles Medium, FBS 10%, Gibco, USA) with penicillin / streptomycin (37%, 5% CO _2). After culturing for 1 day under the conditions, the fermented herbal extracts were treated at 0.02, 0.01, 0.005% concentration.

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 was measured with a fluorescence plater (Ex; 485 nm, Em; 530 nm). After UVB (20mJ / cm ² ) was irradiated and treated with fermented herbal extracts, the fluorescence was measured by a fluorescent plate reader (Ex; 485 nm, Em; 530nm).

Sample Name Free radical scavenging effect (%) Fermented herbal extract 0.02% by weight 56 Fermented herbal extract 0.01% by weight 44 Fermented herbal extract 0.005% by weight 26 EGCG 0.01% by weight 42

As a result of measuring the scavenging effect of the active oxygen (Table 4), it was confirmed that the fermented herbal extracts showed higher oxygen scavenging rate than EGCG at the same concentration as EGCG.

From the above results, it was confirmed that the active oxygen scavenging effect of the fermented herbal extracts.

Experimental Example  5: in 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, the fermented herbal extract obtained in Example 1 in Green tea extract was used as a comparative sample to measure the inhibition of metalloproteinase (MMP-1) activity in vitro , and fluorescence analysis was used.

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 (0.5 M Tris-HCl, 1.5 M NaCl, 50 mM CaCl ₂ , 2 mM sodium azide, pH 7.6) was used after 10-fold dilution. To 100 μl of the reaction buffer, 20 μl of DQ collagen dissolved in the reaction buffer at 0.25 mg / ml and 40 μl of fermented herbal extract (0.02, 0.01, 0.005 wt%) were added and 40 μl of collagenase diluted to 0.5 unit. 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.

Sample Name % Inhibition of metalloproteinase (MMP-1) activity Fermented herbal extract 0.02% by weight 90 Fermented herbal extract 0.01% by weight 67 Fermented herbal extract 0.005% by weight 48 Green Tea Extract 0.2 wt% 67

in As a result of measuring the inhibition rate of metalloproteinase (MMP-1) activity in vitro (Table 5), it was confirmed that fermented herbal extracts effectively inhibit the activity of metalloproteinase (MMP-1) at low concentrations compared to green tea extract. Could.

From the above results, it was confirmed that the fermented herbal extracts had an excellent inhibitory effect on the activity of metalloproteinase (MMP-1). Based on this, the external composition for skin of the present invention effectively inhibited the metalloproteinase, thereby enhancing skin elasticity. 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 ultraviolet irradiation of the fermented herbal extract obtained in Example 1.

After UV irradiation and sample addition (fermented herbal extract, retinol), enzyme immunoassay (ELISA) was performed to measure metalloproteinase (MMP-1) concentrations.

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) 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 microplates. The absorbance was measured at 405 nm with a reader. As a control, one without addition of a sample was used.

Test group MMP-1 expression inhibition rate (%) Control group - Fermented herbal extract 0.02% by weight 74 Fermented herbal extract 0.01% by weight 37 Fermented herbal extract 0.005% by weight 24 Retinol 35

As a result of measuring the inhibition of expression of metalloproteinase (MMP-1) after UV irradiation (Table 6), the fermented herbal extract showed more than 74% inhibition rate compared to the control group without treatment. The results were considerably superior to the inhibition rate of.

From the above results, it was confirmed that the fermented herbal extracts had an excellent effect of inhibiting the expression of metalloproteinase (MMP-1). Based on this, the external composition for skin of the present invention effectively inhibited the expression of metalloproteinase, thereby 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, TGF-β was used as a positive control group in order to confirm the effect of promoting the biosynthesis of the skin substrate component of type 1 procollagen obtained in Example 1, the procollagen assay (procollagen assay) Was carried out as follows.

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 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 incubated at least 90% in 48 well plates, and then fermented herbal extracts were added at 0.02%, 0.01%, and 0.005% concentrations. The amount of prepared collagen was measured using the procollagen type-1 C-peptide EIA kit (MK101, Takara, Japan).

Sample Name Procollagen biosynthesis promoting effect (%) Fermented herbal extract 0.02% by weight 36 Fermented herbal extract 0.01% by weight 16 Fermented herbal extract 0.005% by weight 14 TGF-β0.001 wt% 36

As a result of measuring the effect of promoting procollagen biosynthesis (Table 7), when the fermented herbal extracts were treated with 0.02%, the biosynthesis of procollagen was promoted 36%, which was similar to the cell signaling substance TGF-β.

From the above results, it was possible to confirm the procollagen biosynthesis promoting effect of fermented herbal extracts, and to infer the skin improvement effect of the present invention prepared therefrom.

Experimental Example  8: Elastase  Determination of activity inhibitory effect

In Experimental Example 8, in order to measure the elastin production promoting effect of the fermented herbal extracts obtained in Example 1, elastase was obtained by treating the fermented herbal extracts with human fibroblasts collected directly from humans or purchased commercially. An activity inhibitory effect was performed as follows.

First, human fibroblasts were placed in a culture flask and cultured until about 70-80% growth. Thereafter, the fermented herbal extracts were treated for 1 day, and then the cell culture fluid 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 fermented herbal extracts was used as a control.

Sample Name Elastase activity inhibition rate (Elastin production promoting effect;%) Fermented herbal extract 0.02% by weight 36 Fermented herbal extract 0.01% by weight 19 Fermented herbal extract 0.005% by weight 12 Control group 0

As a result of measuring the inhibition rate of elastase activity (Table 8), it was confirmed that the fermentation herbal extract increases the activity inhibition rate of elastase in a concentration-dependent manner.

From the above results, it was confirmed that the excellent anti-aging and elasticity enhancing effect of the fermented herbal extracts.

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 to confirm the inhibitory effect of tyrosinase activity of the fermented herbal 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. Fermented herbal extracts were used after being dissolved in 0.05M sodium phosphate buffer (pH 6.8) at a suitable concentration. 0.5 ml of L-tyrosine solution was added to a test tube, and 0.5 ml of fermented herbal extract sample solution was added thereto, and the mixture was left to stand at 37 ° C. for 10 minutes. Thereafter, 0.5 ml of 200 unit / ml tyrosinase was added to the sample solution, and the mixture was reacted at 37 ° C for 10 minutes. After the test tube containing the reaction solution was placed on ice and quenched to stop the reaction, the spectrophotometer measured absorbance at a wavelength of 475 nm. As a control, 0.5 ml of buffer solution was used instead of the sample.

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

Inhibition rate of tyrosinase activity (%) = 100-[(comparative absorbance / control absorbance)] X100

Sample Name Tyrosinase activity inhibition rate (%) Control group - Fermented herbal extract 0.02% by weight 56 Fermented herbal extract 0.01% by weight 44 Fermented herbal extract 0.005% by weight 40 Arbutin 0.2 wt% 58

As a result of measuring the 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 the fermented herbal extracts were treated with 0.02% by weight.

From the above results, it was possible to confirm the excellent inhibitory effect of tyrosinase activity of the fermented herbal extracts, from which the excellent whitening effect of the present invention could be deduced.

Experimental Example  10: Determination of Melanin Synthesis Effect Using B16F1 Melanocytes

In Experimental Example 10, in order to confirm the melanin synthesis inhibitory effect of the fermented herbal extract obtained in Example 1, arbutin known as a whitening agent was used as a comparative sample, and 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 melanin synthesis inhibitory effect of B16F1 melanocytes was measured as follows. B16F1 melanocytes were dispensed in 6-well plates at a concentration of 2 × 10 ⁶ per well and attached to the cells, followed by incubation for 72 hours by treating the sample at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, and the cells were counted and then centrifuged to recover the cells. Quantification of intracellular melanin was performed by slightly modifying the method of Rotan (R Lotan and D Lotan, Cancer Res, 40: 3345-3350, 1980). After washing the cell pellet with PBS once, 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt the cells. After dissolving the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), the absorbance of melanin was measured at 405 nm with a microplate reader, and the melanin was quantified. The melanin production inhibition rate (%) of the sample was measured.

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

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

A: Melanin amount in wells without sample

B: Melanin amount in the wells to which the sample was added

Sample Name Melanin synthesis inhibition rate (%) Control group - Fermented herbal extract 0.02% by weight 34 Fermented herbal extract 0.01% by weight 24 Fermented herbal extract 0.005% by weight 12 Arbutin 0.2 wt% 35

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

From the above results, it was possible to confirm the excellent melanin synthesis inhibition rate of the fermented herbal extracts, from which the excellent whitening effect of the present invention could be inferred.

Experimental Example  11: inflammation-related enzymes ( hyaluronidase ) Determination of activity inhibitory effect

In this experiment 11, in order to measure the anti-inflammatory effect of the fermented herbal extract obtained in Example 1, Comfrey extract, seesaw extract, oil-soluble licorice extract was used as a positive control group, hyaluronidase activity of the proinflammatory enzyme Inhibitory effect 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 hyaluronidiase solution (type IV-S, Sigma, 400U / mL) were added and reacted at 37 ° C. for 20 minutes, followed by enzyme activation solution (compound 48/80 CaCl ₂ ㆍ 2H ₂ O, Sigma). , 0.1 mg / ml) was added to 100 µl and reacted 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 and reacted at 95 ° C. for 3 minutes, followed by cooling. Then, 3 ml of ρ-dimethylaminobenzaldehyde solution was added and reacted at 37 ° C. for 20 minutes, followed by color development. 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%.

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

 A: Enzyme activity of wells without sample

B: Enzyme Activity of Well Added Samples

Sample Name Inhibitory effect of hyaluronidiases (IC ₅₀ ) Comfrey Extract 0.23% Seesaw extract 0.54% Usefulness Licorice extract 0.20% Fermented Herbal Extract 0.24%

As a result of measuring the inhibitory effect of hyaluronidase activity (Table 11), the IC ₅₀ value of the fermented herbal extract was 0.24%, 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 fermented herbal extracts, and to deduce the excellent anti-inflammatory effect of the present invention therefrom.

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

In Experimental Example 12, the cytotoxic effect of the fermented herbal extracts obtained in Example 1 by UV irradiation was measured.

Fibroblasts (fibroblasts) were placed in a 24-well test plate 1 x 10 ⁵ and attached for 24 hours. Each well was washed once with PBS and 500 μl of PBS was added to each well. The fibroblasts were irradiated with UV 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 fermented herbal extracts to be evaluated therein and 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 incubation in a 37 ° C. CO ₂ incubator for 2 hours. After incubation, the medium was removed and 500 μl of isopropanol-HCl (0.04 N) was added thereto. After shaking for 5 minutes, the cells were lysed and 100 μl of the supernatant was transferred to a 96-well test plate, and the absorbance was measured at 565 nm using a microplate reader.

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

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

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

Bt: 565 nm absorbance of the wells that developed the colorless reaction wells

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

Reduction rate of cytotoxicity by ultraviolet ray (%) = [1- (St-Bo) / (Bt-Bo)] X 100

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

Bt: cell viability of wells that were not irradiated with UV light

St: Cell survival rate of wells treated with UV

Sample Name Treatment concentration (%) Cytotoxic Relaxation Rate (%) Fermented Herbal Extract 0.02 45 0.01 32 0.005 20

As a result of measuring the cytotoxicity mitigation effect by UV irradiation (Table 12), it was found that fermented herbal extracts effectively prevent cytotoxicity by UV rays by mitigating cytotoxicity by 45% at 0.02% concentration. Could.

From the above results, it was confirmed that the fermentation herbal extracts have an excellent cytotoxic effect by UV irradiation, and from them, it was possible to infer the skin irritation-releasing effect 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 in order to measure the anti-inflammatory effect of the fermented herbal extract obtained in Example 1.

Fibroblasts isolated from human epidermal tissue (Fibroblast) were placed in a 24-well test plate ⁵ x 10 ⁴ and attached for 24 hours. Each well was washed once with PBS and 500 μl of PBS was added to each well. The fibroblasts were irradiated with UV 10 mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), after which PBS was removed and FBS was not added to the cell culture medium (DMEM). Medium) 350 μl was added. After treating the fermented herbal extract to be evaluated here, it was incubated for 5 hours. After incubation, 150 μl of the culture supernatant was taken to quantify IL-1α to determine the inhibitory effect of inflammatory cytokine expression of the fermented herbal extract. The amount of IL-1α was quantified using Enzyme-linked Immunosorbent Assay, and the production rate of IL-1α was calculated by Equation 8.

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

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

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

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

Sample Name Treatment concentration (%) Inhibitory rate of inflammatory cytokine expression (%) Fermented Herbal Extract 0.02 38 0.01 22.6 0.005 13

As a result of measuring the inhibitory effect of inflammatory cytokine expression by UV irradiation (Table 13), it was confirmed that 38% inhibition at 0.02% concentration, fermented herbal extracts effectively prevent the occurrence of inflammation caused by ultraviolet rays at low concentrations.

The anti-inflammatory effects of the present invention can be deduced from the above results.

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

In Experimental Example 14, in order to evaluate the inhibitory effect of prostaglandin (prostaglandin E ₂ ; hereinafter PGE ₂ ) biosynthesis of the fermented herbal extracts obtained in Example 1, keratinocytes isolated from human epidermal tissue were examined. 5 x 10 ⁴ -well test plates were attached for 24 hours. After replacing with FBS-free medium and incubating for 18 hours, prostaglandin biosynthetic enzymes (prostaglandin E ₂ synthetase) present in keratinocytes were treated with aspirin to achieve a final concentration of 50 uM 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 fermented herbal extracts to be evaluated therein was incubated for 16 hours. An appropriate amount of the culture supernatant was taken to quantify biosynthesized PGE ₂ (prostaglandin E ₂ ) for 16 hours to determine the prostaglandin inhibitory effect of the herbal extract. The amount of PGE ₂ was quantified using enzyme immunoassay.

Sample Name PGE ₂ Inhibition Rate (%) (-) UV B control (+) UV B - (+) UV B + Fermented Herbal Extract 0.02% by weight 56 (+) UV B + Fermented Herbal Extract 0.01% by weight 32 (+) UV B + fermented herbal extract 0.005% by weight 13

As a result of measuring the inhibitory effect of prostaglandin biosynthesis by ultraviolet irradiation (Table 14),

Fermented herbal extract was found to effectively inhibit the production of prostaglandins by ultraviolet light. Particularly, the produced PGE ₂ is known to be mainly produced by the COX-2 enzyme, and thus, it was possible to infer that the fermented herbal extract inhibits 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 fermented herbal extract.

Experimental Example  15: Determination of antimicrobial activity against acne

In Experimental Example 15, a paper disc test was conducted to confirm the antibacterial activity against the acne bacteria of the fermented herbal extract obtained in Example 1. First, in order to activate propionibacterium acnes, a skin flora of the cause of acne, it was preincubated for 48 hours in BHI liquid medium (Brain-Heart Infusion Broth; 3.7%). The bacterial cultures thus prepared were plated in 0.1 ml each of BHI solid medium (Brain-Heart Infusion Broth; 3.7%; agar 1.5%) and dried. The fermented herbal extract obtained in Example 1 was diluted to 12% (w / v) in an aqueous 95% ethanol solution, and 50 µl each was added to an 8 mm diameter paper disc and placed on the solid medium prepared above. The cells were incubated for 3 days. The antimicrobial activity was evaluated by observing the bacterial growth inhibition zone around the 'paper disc' and measuring the size of the ring.

As a result of measuring the antimicrobial activity against the acne ring, the size of the bacterial growth inhibitory ring of fermented herbal extract was 22 mm.

Experimental Example  16: Compound  Anti-inflammatory Effects of Fermented Herbal Extracts after Induction of Atopic Dermatitis by 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 in the cage for 60 minutes He observed scratching behavior. In order to assay the anti-inflammatory efficacy of fermented herbal extracts, fermented herbal extracts were applied to the dorsal neck region of mice at a concentration of 250 μl / ml from 5 days before compound 48/80 injection. The application method is pretreatment by applying to the dorsal neck area of the mouse twice a day for 5 days for 5 days and then injecting compound 48/80. Measured pruritus degree was measured.

 Number of scratching (in 5min) 5 10 15 20 25 30 35 40 45 50 55 60 Fermented Herbal Extract 22 24 28 32 35 36 40 43 46 25 20 17 Control group 39 42 50 62 82 92 97 70 57 52 42 37

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

From the above results, it was possible to confirm the atopy prevention effect of the fermented herbal extract, and to deduce the atopy prevention effect of the present invention therefrom.

Experimental Example  17: moisture hygroscopicity measurement

In Experimental Example 17, in order to measure the moisture hygroscopicity of fermented herbal extracts, the fermented herbal extracts obtained in Example 1 and distilled water were respectively saturated and absorbed into the dried epidermal cells of humans obtained directly from humans or purchased commercially. After measuring the amount of the prepared water, the change in mass 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 with water content measured in saturated fermented herbal extracts and purified water were saturated and absorbed for 24 hours, and then the saturated and absorbed epidermal cells were taken out of the cells, weighed, and some were collected and contained in a Kaiser moisture meter. The amount of water per unit mass present was measured. Thereafter, the resultant was dried under reduced pressure for 48 hours, weighed, and then a portion of the sample was collected and measured by Kaiser method to determine the amount of water per unit mass contained therein.

Moisturizing capacity for epidermal cells (moisture mg / epidermal mass g) Moisture content Initial dry state Saturation After rebuilding Fermented Herbal Extract 400 800 500 Purified water 400 790 400

As a result of measuring moisture hygroscopicity (Table 16), it was confirmed that the epidermal cells saturated with the fermented herbal extract contained much more water in the redrying process than the epidermal cells saturated with the purified water.

From the above results, it was possible to confirm the excellent skin hygroscopicity of the fermented herbal extract, from which the excellent moisturizing effect of the present invention could be deduced.

Example  2: containing fermented herbal extract Cosmetics  Produce

In Example 2 was prepared a cosmetic containing the fermented herbal extract obtained in Example 1.

The prepared cosmetics were in the form of a cream, the composition of which is shown in Table 17.

First, phase b) recorded in Table 17 was heated and stored at 70 ° C., phase a) was added to phase a), and then emulsified uniformly with a homomixer after preemulsification and gradually cooled to cream (Example 2, Comparative Example 1). ) Was prepared.

Unit: weight% Raw material Example 2 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 mol addition) alcohol ester 3 3 Glyceryl Monostearic Acid Aster 2 2 I Fermented herbal extract One - Propylene glycol 5 5 Quantity Rest about 100 Rest about 100

Experimental Example  18: Cosmetic  Measurement of skin elasticity improvement effect

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

In order to confirm the skin elasticity improvement effect was measured using a skin elasticity measuring instrument (cutometer SEM 575, C + K Electronic Co., Germany) before and after using the product for 2 months. The experimental results are described in Table 18 below as the ΔR7 value of the cutometer SEM 575, where the R7 value represents the nature of the viscoelasticity of the skin.

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

n = 20, p <0.05

As a result of measuring the skin elasticity improving effect of the cosmetic (Table 18), it was confirmed that the skin elasticity improving effect of the experimenter who applied Example 2 containing the fermented herbal extract is superior to Comparative Example 1.

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

Experimental Example  19: Cosmetic  Skin wrinkle improvement

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

In order to examine the effect of wrinkles on the skin, a replica of silicone was made before and after using the product for two months, and the condition of wrinkles at the designated area was measured by a visometer (SV60, C + K Electronic Co. , Germany). The results are shown in Table 19 below, and Table 19 shows the average of subtracting the parameter value two months ago from each parameter value two months later. In other words, the negative the value, the higher the wrinkle improvement effect.

Experimental product R1 R2 R3 R4 R5 Example 2 -0.23 -0.17 -0.11 -0.08 -0.06 Comparative Example 1 -0.11 -0.07 -0.06 -0.04 -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

As a result of measuring the skin wrinkle improvement effect of the cosmetic (Table 19), it was confirmed that the skin wrinkle improvement effect of Example 2 containing the fermented herbal extract is superior to Comparative Example 1.

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

Experimental Example  20: Cosmetic  Whitening effect measurement

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

After 2 months, the applied skin of both the left and right sides of the face was measured using an image analyzer and the color change of the skin using chromameter (Minolta CR300) to measure the change in brightness of the color (ΔL). Subjective visual observation was conducted to determine the effect according to the following classification. The results are shown in Table 20 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 (ΔL) Objective evaluation of the skilled person Subjective Evaluation of the Subject Example 2 Comparative Example 1 Example 2 Comparative Example 1 Example 2 Comparative Example 1 medium 4.59 1.86 3.3 2.2 2.5 1.4

As a result of measuring the whitening effect of the cosmetic (Table 20), it was confirmed that Example 2 containing the fermented herbal extract is superior to the comparative example 1.

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

Experimental Example  21: Cosmetic  Measurement of improvement effect on atopic dermatitis

In Experimental Example 21, a clinical experiment was performed as follows to compare and measure the improvement effect on atopic dermatitis of Example 2 and Comparative Example 1.

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, Example 2 on the left hand and Comparative Example 1 on the right hand, was washed every evening, and then applied to the skin once a day for 60 days and the degree of atopic dermatitis improvement was measured. The measurement was performed by sensory evaluation by survey. The results are shown in Table 21 below.

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

As a result of measuring the improvement effect on cosmetic atopic dermatitis (Table 21), it was confirmed that Example 2 prepared by adding the fermented herbal extract is superior to the atopic skin improvement effect than Comparative Example 1.

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

Experimental Example  22: SLS Skin irritation

In Experimental Example 22, the stimulating alleviation effect of the cosmetic containing the fermented herbal 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 used to patch 0.3 mg of the product on the upper arm of 50 healthy men and women aged 20-50 years, and the acute irritation index was evaluated after 24 hours. After evaluation, the same amount of product was applied to the same site again, and the delayed stimulation index after 48 and 72 hours was evaluated.

As a result of measuring the skin irritation-relieving effect by the SLS, the area covered with SLS alone showed red stimulation after 24 hours, but Example 2 containing fermented herbal extracts was applied for 24 hours, 48 hours, No skin seizures occurred after 72 hours.

From the above results, it was confirmed that fermented herbal extracts can reduce skin irritation caused by a substrate (surfactant, fragrance, alcohol) that causes irritation when used in cosmetics.

Example  3: Preparation of lotion containing fermented herbal 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 fermented herbal extract obtained in Example 1 and 5 g of glycerine were dissolved in 85.33 g of purified water, and then the mixed solution was added thereto, followed by stirring to prepare a lotion containing the fermented herbal extract.

Example  4: Manufacture of milky milk containing fermented herbal extract

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 fermented herbal 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: containing fermented herbal extract 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, Fermented herbal extract obtained in Example 1 and a suitable amount of pigment was mixed to prepare a cosmetic solution.

Claims (20)

Extracting an extract from a herbal mixture consisting of Gil-Gyeong, Licorice, Aralia, Falconia, Gardenia, Yellow White, Golden, Yellow Lotus, Chihu-Yang, Peony, Cheongung, Angelica, Shiho, Peppermint, Allies and Natural Flower; 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; And After the main culture, the step of removing the leaf mushroom (KCTC 10337BP) mycelium from the culture medium; Cosmetic composition characterized in that it contains the filtrate obtained from the active ingredient. The method of claim 1, The herbal mixture, Gilkyung, licorice, bran, yeonkyo, gardenia, yellow white, golden, yellow lotus, jihwang, peony, cheongung, donkey, shiho, peppermint, mate and cheonhwabun each of the cosmetic composition, characterized in that 1 to 30 parts by weight. The method of claim 1,       The filtrate is, A cosmetic composition comprising 0.001 to 30.0% by weight based on the entire cosmetic composition. The method of claim 1, The extraction is Cosmetic composition, characterized in that using an aqueous solution containing 85 ~ 99% (v / v) of ethanol as the extraction solvent. The method of claim 1, The extract is, After extraction, it is concentrated under reduced pressure or lyophilized cosmetic composition. The method of claim 5, The reduced pressure concentrated or lyophilized extract, Cosmetic composition, characterized in that dissolved in at least one solvent selected from purified water, ethanol, butylene glycol and propylene glycol. The method of claim 6, The reduced pressure concentrated or lyophilized extract, Cosmetic composition, characterized in that dissolved 0.001 to 30.0% by weight in at least one solvent selected from purified water, ethanol, butylene glycol and propylene glycol. The method of claim 1, This culture, Adjust the pH of the culture medium to 5.0-6.0, inoculate the mycelium mushroom (KCTC 10337BP) to 4-6% (v / v), and then the temperature 22-32 ℃, rotation speed 120-180rpm, aeration 1.2-1.8 Cosmetic composition, characterized in that incubated for 5-7 days in vvm conditions. delete The method of claim 1, Formulation of the cosmetic composition, Basic cosmetic formulations consisting of lotion, gel, water soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / O) type; Color cosmetic formulations consisting of oil-in-water or water-in-oil makeup bases, foundations, skin covers, lipsticks, lip gloss, face powders, two-way cakes, eye shadows, cheek collars and eyebrow pencils; Cosmetic composition, characterized in that any one selected from. The method of claim 1, Cosmetic composition, Cosmetic composition, characterized in that for skin whitening. The method of claim 1 Cosmetic composition, Cosmetic composition, characterized in that the anti-aging The method of claim 1, Cosmetic composition, Cosmetic composition for reducing skin irritation. The method of claim 1, Cosmetic composition, Cosmetic composition, characterized in that for improving atopic skin. The method of claim 1, Cosmetic composition, Cosmetic composition, characterized in that for preventing acne. Extracting an extract from a herbal mixture consisting of Gil-Gyeong, Licorice, Aralia, Falconia, Gardenia, Yellow White, Golden, Yellow Lotus, Chihu-Yang, Peony, Cheongung, Angelica, Shiho, Peppermint, Allies and Natural Flower; 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; And After the main culture, removing the leaf mushroom (KCTC 10337BP) mycelium from the culture medium; Atopy skin and acne prevention pharmaceutical composition, characterized in that it contains the filtrate obtained as an active ingredient. The method of claim 16, The formulation of the pharmaceutical composition, PLASTERS, LOTIONS, LINIMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, SUSPENSIONS, CAPSULES, CREAMS, soft or hard gelatin capsules, patch, sustained-release agent for atopic skin and acne prevention Pharmaceutical composition. delete delete delete