KR101810825B1 - Cosmetics compositions containing fermentation extract from leaf Daphniphyllum macropodum Miq. - Google Patents

Abstract

The present invention relates to a cosmetic composition containing a ginkgo tree leaf culture, and more particularly, to a cosmetic composition containing a filtrate as an active ingredient by fermenting leaf extract of ginkgo tree with Jeju Nuruk And is excellent in skin absorption, and has an excellent antioxidative effect, collagen synthesis promoting effect, wrinkle improving effect, whitening effect, moisturizing effect, and skin irritation alleviating effect.

Description

[0001] The present invention relates to a composition for external application for skin containing a gabbro leaf culture,

The present invention relates to a cosmetic composition, and more particularly, to a cosmetic composition comprising a filtrate obtained by fermenting a leaf extract of a barberry tree belonging to the evergreen tree branch tree.

Skin exposed to ultraviolet rays generates free radicals and reactive oxygen species (ROS) to 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 collagen-like extracellular matrix in vivo are appropriately controlled, but as the aging progresses, the synthesis gradually decreases and the expression of matrix metalloproteinase (MMP), an enzyme that degrades collagen, is promoted, And wrinkles are formed. These degrading enzymes are also activated by ultraviolet irradiation. Therefore, it is required to develop a substance capable of controlling the activation of MMP expression in cells or inhibiting its activity.

 Skin disease refers to abnormalities appearing in all skin including nails, claws and head hair of mammals including humans. Atopic dermatitis (atopic dermatitis) in people with atopic allergy is a typical disease. The cause of atopic dermatitis has not yet been clarified. However, atopic dermatitis has a history of atopic dermatitis in about 70% of the patients, and it is common in people with allergic constitution, especially with allergic diseases such as allergic rhinitis and asthma, It is understood as one of factors or immune diseases. Atopic dermatitis can be exacerbated by mental instability and emotional state of the patient, most of which is caused by scratching the skin and causing secondary infection due to severe itching at the initial stage. Atopic dermatitis is a fairly large number of people suffering from 0.5-1% of the population and 5-10% of children. Symptoms usually appear within 2-6 months of life, especially in infants less than 1 year of age, and 85% are within 5 years of age. Atopic dermatitis is frequently seen in infancy, but in recent years, the number of patients after puberty has increased, and the medical history is also getting longer. In the treatment of atopic dermatitis, medicines such as corticosteroids which can expect a high pharmacological effect are used. However, it is well known that the use of such corticosteroids results in rebound (weakness of the dramatic lesion that occurs after drug withdrawal). As a countermeasure for such side effects, a non-steroidal anti-inflammatory agent or an antihistamine agent which does not contain a hormone such as adrenocortical hormone is used but it is difficult to cure. Therefore, it is very urgent to develop a remedy or treatment for atopic dermatitis without side effects.

The skin of a person is constantly changing with age. Skin aging is largely divided into natural aging (or endogenous aging) and external aging (EB Doris, Cosmetics & Toiletries, 111, 31-37, 1996). Natural aging (endogenous aging) While control is difficult, external aging is relatively easy to manipulate because it is affected by environmental factors. Exemplary external aging factors include ultraviolet light and the most prominent external aging phenomenon is wrinkle formation (HW Daniell, Ann Intern Med, 75, 873-880, 1971, GL Grove, Jamecad Dermatol, 21, 631-637 , 1989, CE Griffiths et al, Arch Dermatol, 128, 347-351, 1992). On the other hand, skin exposed to ultraviolet rays generates free radicals and reactive oxygen species, leading to skin cancer, phototoxicity, autoimmune disorders, photosensitivity and skin aging (Norins, J. Invest. Dermatol., 39: 445, 1962; Cadenas, Ann. Rev. Biochem., 58: 79, 1989). The synthesis and degradation of extracellular matrix such as collagen in vivo is appropriately controlled, but its synthesis is reduced as aging proceeds, and the expression of matrix metalloproteinase (MMP), an enzyme that degrades collagen, is promoted, 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 protects the body from ultraviolet light and skin, and plays an important role in regulating hormone secretion in the body. However, when melanin is overproduced, it is known that it forms spots and freckles, promotes skin aging, and plays an important role in skin cancer induction. Therefore, research and development are actively conducted to prevent melanin overproduction.

(Japanese Patent Laid-open No. 4-93050) and plant extracts inhibit tyrosinase (Japanese Patent Laid-open Publication No. 4-9320), hydroquinone (Japanese Patent Laid Open No. 6-192062), kojic acid And is used as a whitening cosmetic. However, it is limited in its use due to poor stability in cosmetic formulations, resulting in decomposition, coloring, odor generation, efficacy at a biological level, unclear effects or safety problems .

Recently, attention has been focused on functional cosmetics having functions such as antioxidation, wrinkle reduction, and itching relief obtained from natural extracts in order to reduce skin irritation caused by various chemical substances and the like. In addition to low adverse effects on the skin, natural materials have recently become highly appreciated as a raw material for cosmetics due to the increasing popularity of cosmetics using natural materials. For example, U.S. Pat. No. 5,972,341 describes the wrinkle-reducing effect of the Commiphora plant, particularly the Commiphora mukuli extract. Japanese Patent Application Laid-Open No. 9-672662 describes a seaweed extract having hyaluronidase inhibitory activity. Japanese Patent Application Laid-Open No. 10-85905 describes the effect of improving the skin texture of fucoidan extracted from Wakame. Japanese Patent Laid-Open No. 2-245087 describes the antioxidative effect of Sargassum sp. Extract. Japanese Patent Laid-Open No. 3-294384 discloses an antioxidant composition extracted from a genus of Hizikia. Japanese Patent Application Laid-Open No. 7-10769 discloses Phaeophyceae extract having an astringent effect. Korean Patent No. 0431076 discloses a cosmetic composition for improving the healing property of atopic skin including a white lotus, a jasmine, an echinacea, and a fermented soybean extract, and Korean Patent No. 0511494 discloses a cosmetic composition for treating atopic dermatitis including Hashuo, Korean Patent Application Laid-Open No. 2004-0065126 discloses an extract and composition for treating and preventing atopic skin including herbal plant extracts obtained from natural gingko mushrooms, yugun pei, licorice, white bream, And the like. Also, Korean Patent Application Publication No. 2004-0011584 discloses a composition excellent in the treatment of atopic skin diseases, which is formed by mixing and mixing natural ingredients such as licorice, licorice, safflower and lard (pig scaffold).

Daphniphyllum macropodum Miq. Is distributed in Korea, China, Japan and Taiwan. It is an evergreen broad-leaved arboreous tree native to the South coast of Korea and Jeju-do in the southern coast of Korea. Its leaves are long oval-shaped. Leaf and reddish petiole than flower has higher ornamental value. It is beautiful in shape and leaves are unique, and it is planted frequently as a tree or garden in south coast or Jeju Island. After picking the ripe fruits in autumn, they obtain the seeds and then open them in the open air. Then they plant them in the spring of the following year to obtain seedlings. The leaf is like a rubber tree and has a southern flavor. In herbal or folk remedies, leaves and bark were used for acute pleurisy and peritonitis or diuretic, and leaf water was used as insect repellent. Most of the exotic species introduced into the indoor plants grow well in the shadows of the lower parts of the gully. Unlike the shade, they grow well in the hinchinji rather than in the shade. The general habitat species can winter at more than 10 ℃, while the wintering is possible at more than 5 ℃. Cold resistance is very strong.

Nuruk consists of various microorganisms such as wheat bran and rice containing starch and protein, especially microorganisms having excellent saccharification function of starch, and they secrete extracellular enzymes and produce by the process of proliferation of yeast using sugar produced by this enzyme do. When the yeast is dried, the enzyme is kept in a dry state and the yeast is in a dormant state. When water and starch are added, starch glycation and alcohol fermentation are promoted. (Homo lactic acid bacteria) and yeast (Saccharomyces coreanus, Aspergillus oryzae, Asp alopecia var. Kawachii, Asp awamori) mainly composed of yeast fungus grown in yeast (Aspergillus oryzae, Saccharomyces cerevisiae (Saccharomyces crerevisiae) coexist together and fermentation occurs. The Patent Documents on Jeju Jujube have reported antistress compositions [Korean Patent Laid-open No. 10-2014-0072421], compositions for improving wrinkles [Korean Patent Laid-open Publication No. 10-2014-0072419], etc., and cosmetic compositions containing plant extracts [Korean Patent Registration No. 10-0971629], and the like.

Accordingly, the inventors of the present invention completed the present invention by establishing an increase in the content of the active ingredient in the barnyard fermented product cultured by using the Jeju japonicum japonicum, and confirming that it has an excellent effect as a cosmetic composition without inducing skin irritation.

The present invention provides a cosmetic composition characterized by containing, as an active ingredient, a filtrate obtained by fermenting a gingko leaf extract with Jeju Nuruk.

In addition, the present invention confirms the efficacy as a raw material for a cosmetic ingredient of a ginseng leaf cultured product and has effects such as antioxidant effect, collagen synthesis promoting effect, wrinkle improving effect, whitening effect, moisturizing effect, skin irritation alleviating effect atopy and anti- The present invention also provides a cosmetic composition comprising the above-mentioned extract of the present invention.

Accordingly, the inventors of the present invention recognized the excellent efficacy of the Leaf Tree Leaf Extract, and in order to make it more efficient and variously available, the inventors of the present invention found that a cosmetic composition of Leaf Tree Leaf Culture containing the filtrate obtained by fermentation using Jeju Nuruk Lt; / RTI >

Jeju yeast is composed of homo lactic acid bacteria and yeast (Saccharomyces coreanus, Aspergillus oryzae, Asp Luchuensis mut. Kawachii, Asp awamori) Saccharomyces crerevisiae) can coexist together and be used for fermentation. In addition, the culture after inoculation of the microorganisms can be carried out at a temperature of 25 ° C to 37 ° C in consideration of the inoculated microorganisms. If the incubation temperature is lower than the above range, the fermentation rate may be slowed and undesired fermentation products may be produced. Also, if the incubation temperature is higher than the above range, the fermentation rate may be slowed down or the undesirable fermentation products It can happen. In the case of the incubation time, it is preferable to continue until the growth of the inoculated microorganism reaches a maximum. The time at which the inoculated microorganism reaches its maximum growth will depend on the incubation temperature, the amount of fermentation material, the amount of microorganism inoculated, and so on. It is possible to determine at what point in time the maximum growth of the inoculated microorganism takes place in consideration of the culture temperature, the amount of the fermentation raw material, the amount of the microorganism to be inoculated, and the like within the normal capability range.

In the present invention, "included as an active ingredient" means that the filtrate is added to the composition for external application for skin to such an extent that it can exhibit the effect of preventing and improving skin diseases from the composition for external application for skin of the present invention. And the like. The term " formulation "

The present invention also relates to a method for inhibiting the activity of matrix metalloproteinase (MMP-1), inhibiting the expression of matrix metalloproteinase (MMP-1) , A promoting effect of pro-collagen biosynthesis, a promoting effect of elastin production, a whitening effect, an inhibitory effect of hyaluronidase activity, an effect of mitigating cytotoxicity by ultraviolet irradiation, an inhibitory effect of inflammatory cytokine expression by ultraviolet irradiation, An anti-inflammatory effect against atopic dermatitis, and an excellent water hygroscopicity.

Further, the present invention relates to a cosmetic composition containing a barnyard leaf culture characterized in that the content of the filtrate is 0.001 to 30.0% by weight relative to the total amount of the cosmetic composition. When the content of the filtrate is less than 0.001% by weight, the effect of improving skin is scarcely produced. When the content of the filtrate is more than 30.0% by weight, the effect of increasing the content of the filtrate is insufficient,

Further, the present invention relates to a cosmetic composition characterized by using an aqueous solution containing 85 to 99 (v / v) of distilled water or ethanol as an extraction solvent.

Further, the present invention relates to a cosmetic composition characterized by adding a reduced-pressure concentrated or lyophilized process to the extract.

In addition, the present invention relates to a cosmetic composition composition characterized in that the reduced-pressure concentrated or lyophilized extract is dissolved in at least one solvent selected from purified water, ethanol, butylene glycol and propylene glycol.

The present invention also relates to a cosmetic composition, wherein the cosmetic composition is a cosmetic preparation, a gel, a water-soluble liquid, a cream, an essence, an oil-in-water (O / W) type and a water-in-oil (W / O) type; Wherein the composition is a cosmetic composition selected from the group consisting of a makeup base, a foundation, a skin cover, a lipstick, a lip gloss, a face powder, a two-way cake, an eye shadow, a cheek color and an eyebrow pencil.

The present invention also relates to a composition for external application for skin which exhibits skin whitening efficacy containing ginkgo tree leaf cultures.

The present invention also relates to a composition for external application for skin, which exhibits an anti-aging effect containing ginkgo biloba cultures.

The present invention also relates to a composition for external application for skin, which exhibits a skin irritation mitigating effect containing a ginkgo tree leaf culture.

In addition, the present invention relates to a composition for external application for skin which exhibits an atopic skin improving effect containing a ginkgo tree leaf culture.

In addition, the present invention relates to a composition for external application for skin, which exhibits an acne-preventing effect containing a ginkgo tree leaf culture.

The present invention also relates to a cosmetic composition, wherein the cosmetic composition contains at least one selected from the group consisting of PLASTERS, LPTIONS, LINIMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, OINTMENTS Used as a pharmaceutical composition selected from FLUIDEXTRACTS, EMULSIONS, SUSPESIONS, CAPSULES, CREAMS, soft or hard gelatine capsules, patches, and sustained release preparations. .

The pharmaceutical composition of the present invention may further comprise a pharmacologically acceptable base; Carrier; Excipients; Binders comprising starch, tragacanth gum, gelatin, molasses, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, ethylcellulose and carboxymethylcellulose; A pulverizing agent comprising agar, starch, bellatin powder, carboxymethylcellulose sodium, carboxymethylcellulose calcium, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate and sodium alginate; Lubricants including magnesium stearate, talc and hydrogenated vegetable oils; And a coloring agent. Examples of the carrier and excipient include lactose, glucose, sucrose, mannitol, potato starch, cornstarch, calcium carbonate, calcium phosphate, and cellulose. In addition to the above, additives such as stabilizers, solubilizers, perfume fragrances such as transdermal absorption accelerators, and preservatives may be further added.

In addition, the standard dose of the present cosmetic composition may vary depending on the individual characteristics of the patient, and a practitioner skilled in the art will be able to determine the ideal dosage and administration schedule for the skin external composition of the present invention for the patient, For example, the most appropriate treatment strategy can be defined in terms of specific needs and the overall condition of the patient. Reference to a number of references known to those skilled in the art to determine an appropriate dosage for the topical composition for skin of the present invention may be found by those skilled in the art.

In addition, suitable doses of the inventive cosmetic compositions may be routinely determined in vitro or in animal models. For example, various concentrations of the inventive skin topical composition may be added to the target cells in vitro to determine their proper dosage.

The ginseng leaf cultivar of the present invention showed excellent whitening action, such as melanin inhibitory effect, which is a cause of spots, freckles and skin pigmentation, which is superior to conventional simple extracts even without skin irritation.

In addition, the ginseng leaf culture of the present invention showed excellent anti-aging effect such as active oxygen scavenging effect, MMP inhibitory effect, and modulation of MMP expression by ultraviolet irradiation, and mitigates skin irritation caused by ultraviolet irradiation.

In addition, the ginkgo leaf cultivar of the present invention showed skin irritation mitigation and atopic improvement by inflammatory substances.

Therefore, cosmetic compositions such as lotion, cream, emulsion, pack, powder and the like containing such a ginkgo tree leaf culture can be used as an active oxygen scavenging effect and a collagenase activity controlling effect, a skin wrinkle improving effect, a whitening, Skin irritation alleviating effect and the like.

Hereinafter, the present invention will be described in more detail by way of 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.

Extract preparation (simple extraction)

Ten kilograms of dried persimmons were refluxed three times for 5 hours in 95% (v / v) distilled water or aqueous ethanol solution, cooled down, and filtered through Whatman # 5 filter paper. The filtered extract was concentrated under reduced pressure and freeze-dried at 50 ° C or less, and the extract was prepared from the barnyardgrass by dissolving the lyophilized product in a mixed solvent of purified water, ethanol, butylene glycol and propylene glycol so as to contain 15% by weight of the obtained freeze- .

Manufacture of oyster leaf cultures

To the ginseng leaf extract obtained in Example 1, 5% (v / v) of Jeju Nuruk prepared in PDB (Potato Dexrtose Broth) medium was added and fermented at 26 ° C for 7 days to prepare a ginseng leaf culture. The gruel leaf cultures were sterilized at 121 ° C for 15 minutes, concentrated under reduced pressure, and lyophilized to be used in the experiment.

Determination of the contents of components and components contained in the gravel tree leaf culture

Qualitative analysis of phenolic materials, flavonoids, flavonoid glycosides, and phenolic contents and total flavonoid contents in the major components of the ginseng leaf extracts prepared in Example 1 and the ginseng leaf cultures prepared in Example 2 were compared.

First, qualitative analysis of phenolic materials, flavonoids, flavonoid glycosides and the like was carried out as follows.

(1) Qualitative analysis of phenolic substances and flavonoids

When 1-2% of a 2.5% FeCl3 ethanol solution was left to stand in Example 1 (extract of gabbia leaf by simple extraction) and Example 2 (gavia leaf culture), it was color-changed to dark green color or formation of precipitate Respectively.

(2) Qualitative analysis of flavonoid glycosides

Using concentrated sulfuric acid method, 5 ml of concentrated sulfuric acid was added to each of Example 1 and Example 2 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 TS in each of Example 1 and Example 2, a red purple color appeared on the interface when concentrated sulfuric acid was added through the barrier wall.

As a result of the qualitative analysis, it was confirmed that Examples 1 and 2 contained a phenolic substance and a flavonoid flavonoid glycoside.

Total phenol content and total flavonoid content were measured as follows.

(1) Total phenol content measurement

10 ml of distilled water was added to 1 ml of each of Example 1 and Example 2, followed by addition of 2 ml of Folin-Ciocalteu phenol reagent (Sigma), followed by reaction at room temperature for 5 minutes. 2 ml of 20% sodium carbonate was added to the reaction mixture, and the mixture was reacted at room temperature for 1 hour and then absorbance was measured at 680 nm. At this time, gallic acid was used as the indicator material.

(2) Total flavonoid content measurement

1.5 ml of each of Example 1 and Example 2 was mixed with 2% AlCl 3 H 2 O dissolved in the same amount of methanol, and the reaction was carried out at room temperature for 10 minutes, and the absorbance was measured at 367 nm. At this time, the indicator material was catechin.

Name of sample Total phenol content
(μg / mg of extract) Total flavonoid content
(μg / mg of extract) Example 1
(Extract of Leaf Tree Leaf by Simple Extraction) 540.5 31.2 Example 2 (gravel tree leaf culture) 785.1 80.9

Total phenol content and total flavonoid content were measured (Table 1), and it was confirmed that the total phenol content and total flavonoid content were higher in Example 2 than in Example 1, and the content of flavonoid was increased more than twice .

From the above results, it was confirmed that the filtrate obtained by the simple extraction of the ginkgo tree leaf extract fermented with Jeju Nuruk can increase the total phenol content and the total flavonoid content, and based on this, A cosmetic composition of the present invention was prepared, which contained a gravel tree leaf culture containing water as an active ingredient.

Experiment to measure free radical scavenging activity

Free radical scavenging activity of green tea extracts obtained in Example 2 was measured by DPPH method using extracts having excellent antioxidant activity, such as green tea extract, under laboratory conditions.

The DPPH method measures free radical scavenging activity by reducing power using a free radical called DPPH (2,2-Di (4-tert-octylphenyl) -1-1picrylhydrazyl 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.

For the measurement of DPPH free radical scavenging activity, 0.2%, 0.1%, 0.05% and 0.005% concentration of high temperature and high pressure extracts were prepared. The extracts of the above concentrations were placed in a 96-well plate, and DPPH prepared from 100 uM methanol solution was added thereto to make the total volume of the solution 200 μl. After incubation at 37 ° C for 30 minutes, absorbance was measured at 560 nm.

A: Absorbance of control wells not treated with the gherkin tree cultures of the present invention

B: Absorbance of the experimental group well treated with gravel tree cultures of the present invention

As can be seen in Table 2, the ginseng cultivars showed better antioxidative effect than the green tea extract having excellent antioxidant activity.

Name of sample Antioxidative effect(%) Gt; 0.25 wt% < tb > 95% Gt; 0.1% < / RTI > 92% Ginkgo tree extract 0.25 wt% 85% Green tea extract 92%

Cytotoxicity of the sample

The cytotoxicity of the gravel tree cultures obtained in Example 2 to skin cells was evaluated. Fibroblast diluted in a cell culture medium (DMEM supplemented with 10% FBS) was added to each well of a 96-well test plate at 1 × 10 5 cells / mL for 24 hours. The sample obtained in Example 2 diluted to an appropriate concentration in each well was treated and cultured for 24 hours. After 24 hours, the medium was removed, and 200 μl of a cell culture medium containing MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl tetrazolium bromide) solution (2.5 mg / And incubated in a 37 ° C CO2 incubator for 2 hours. The medium was removed and 100 μl of DMSO (dimethyl sulfoxide) was added. After shaking for 5 minutes to dissolve the cells, absorbance at 565 nm was measured in a microplate reader. Cell viability (%) was determined by mathematical formula 1 and the concentration of the sample which did not affect cell survival was determined.

Bo: 565 nm absorbance value of wells that had undergone color development of the cell culture medium

Bt: 565 nm absorbance value of the well that underwent color reaction without sample treatment

St: the absorbance value at 565 nm of the sample treated and color-developed

Name of sample 100% cell viability concentration Gt; 0.25 wt% < tb > 2% Gt; 0.1% < / RTI > 2% Gt; 0.05% < / RTI > by weight & 2% 0.0 >% < / RTI > 2%

As can be seen in Table 3, the 100% survival rate of the skin fibroblasts was not more than 2% when the gravel tree cultures of Example 2 were treated by concentration, It is a safe sample that is less likely to cause irritation.

In vitro inhibition of MMP-1 activity

 Fluorescence assay was used to measure the inhibitory effect of MMP-1. The substrate used was DQ-collagen labeled with fluorophore and the collagenase was purchased from Molecular probe (Eugene, OR, USA) and incubated in reaction buffer (0.5 M Tris-HCl, 1.5 M NaCl, 50 mM CaCl2, 2 mM sodium azide, pH 7.6) was used after 10-fold dilution. 20 ㎕ of DQ collagen dissolved in the reaction buffer at 0.25 mg / ml and 40 쨉 l of the immature tomato fermented product (0.1% by weight) obtained in each of Examples 2 to 4 were added to 100 반응 of the reaction buffer, and the collagenase diluted with 0.5 units (collagenase). After 20 minutes at room temperature, the fluorescence value was measured using a fluorescence spectrophotometer (Perkin Elmer, UK) at an absorption wavelength of 495 nm and an emission wavelength of 515 nm. As a control, the fluorescence value was measured by adding the same amount of the enzyme buffer as the enzyme buffer The fluorescence value of the sample itself was measured and corrected for the enzyme activity.

The results are shown in Table 4. The inhibitory activity of metalloproteinase (MMP-1) was excellent when treated with 0.25% of ginkgo biloba cultivar of Example 2, and the inhibitory activity was also superior to that of ginkgo biloba leaf extract.

From the above results, it was confirmed that the effect of inhibiting the metalloproteinase (MMP-1) activity of the ginseng leaf cultivar was excellent, and the cosmetic composition of the present invention effectively inhibited the metalloproteinase, And wrinkle formation can be prevented.

Name of sample MMP-1 activity inhibition rate (%) ≪ tb > < sep > 0.25 wt% 88 Sorghum leaf extract 0.25 wt% 65 Green tea extract 0.2 wt% 67

Evaluation of Inhibition of MMP-1 Expression by Ultraviolet Light Irradiated Leaf-Blade Cultures

In this experiment, enzyme immunoassay (ELISA) was performed to determine the concentration of MMP-1 after UV irradiation and sample addition of the cultivar obtained from Example 2.

UVA was irradiated in human dermal fibroblasts at an energy of 6.3 J / cm < 2 > using a UV chamber. Ultraviolet irradiation dose and incubation time were established by preliminary experiment to maximize MMP expression level in fibroblasts. The negative control was wrapped in a tinfoil

The cells were incubated for 24 hours after replacing the medium with the medium containing the sample after irradiation with UVA, and the medium was recovered and coated on 96 wells. The primary antibody (MMP-1 (Ab-5) monoclonal antibody) was treated and reacted at 37 ° C for 60 minutes. After reacting the secondary antibody, anti mouse IgG (whole mouse, alkaline phosphatase conjugated) for about 60 minutes, the alkaline phosphatase substrate solution (diethanolamine buffer solution containing 1 mg / ml p-nitrophenyl phosphate) For 30 min and absorbance at 405 nm was measured with a microplate reader. As a control group, a sample to which no sample was added was used.

As shown in Table 5, in the case of MMP-1 in which the expression was induced by ultraviolet irradiation, the inhibitory activity of MMP-1 on the ginseng leaf cultured in Example 2 was increased, and more than 65% Respectively. This is an excellent result compared to the inhibition rate of retinol used as a control.

Test group MMP-1 expression inhibition rate (%) Negative control group - ≪ tb > < sep > 0.25 wt% 73 Sorghum leaf extract 0.25 wt% 42 Retinol 39

Tyrosinase activity inhibition experiment

Arbutin, well known as a whitening agent, was used as a comparative sample to confirm the tyrosinase activity inhibitory effect of the gilt-woody cultures obtained in Example 2 above.

Tyrosinase was isolated and purified from mushrooms and purchased from Sigma. L-Tyrosine (Sigma), a substrate, was dissolved in 0.05 M sodium phosphate buffer solution (pH 6.8) and used as a 0.1 mg / ml solution. The ginkgo tree extract of Example 1 and the ginkgo tree culture of Example 2 were dissolved in 0.05 M sodium phosphate buffer solution (pH 6.8) adjusted to an appropriate concentration and then used. 0.5 ml of L-tyrosine solution was added to a test tube, and 0.5 ml of the extract of the ginkgo tree and the ginkgo tree culture was added thereto, followed by reaction at 37 ° C for 25 minutes. The test tube containing the reaction solution was placed on ice, quenched to stop the reaction, and the absorbance at a wavelength of 475 nm was measured. As a control group, 0.5 ml of buffer solution was used instead of the above-mentioned sample.

The inhibition rate of tyrosinase activity of each of the above samples was calculated according to Equation (3).

The results are shown in Table 6, and the tyrosinase inhibition rate in Example 2 treated with gull tree cultures is similar to arbutin.

Name of sample Inhibition rate of tyrosinase activity (%) Control group - Gt; 0.25 wt% < tb > 74 Ginkgo tree extract 0.25 wt% 45 Arbutin 0.2 wt% 62

Melanin synthesis inhibition experiment using B16F1 melanocytes

In this experiment, the effect of inhibiting melanin synthesis on B16F1 melanocytes was examined in order to confirm the whitening effect of the barnyardgrass extract obtained in Example 1 and the barnyardgrass culture of Example 2.

Melanin synthesis inhibitory effect of B16F1 melanocytes was measured as follows. B16F1 melanocytes were plated at a concentration of 2 x 10 ⁶ per well in a 6-well plate, and after attaching the cells, the samples were treated at a concentration not causing toxicity and cultured for 72 hours. After incubation for 72 hours, the cells were detached with trypsin-EDTA, and the number of cells was measured, followed by centrifugation to recover the cells. Quantification of intracellular melanin was performed using lotan: Cancer Res. , 40, 3345-3350 (1980)]. The cell pellet was washed once with PBS, and 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF (phenylmethylsulfonyl fluoride)) was added and vortexed for 5 minutes to disrupt the cells. After centrifugation (3,00 rpm, 10 min) gkdu, 1N NaOH (10% DMSO (Dimethyl Sulfoxide)) was added to the cell filtrate to dissolve the extracted melanin and the absorbance of melanin was measured at 405 nm with a microplate reader. Was measured to determine the inhibition rate (%) of melanin formation in the sample. The inhibition rate (%) of melanin formation of B16F1 melanocytes was calculated by the following equation (4).

A: Amount of melanin in wells to which no sample was added

B: Amount of melanin in the well to which the sample was added

As shown in Table 7, the melanin synthesis inhibitory effect of B16F1 melanocytes was examined. The melanin synthesis inhibitory effect of Example 2 was 0.25% to 44%. In particular, melanin production inhibitory effect was superior to that of Example 1, and it showed a similar inhibitory effect to arbutin.

Name of sample Melanin synthesis inhibition (%) Negative control group - Gt; 0.25 wt% < tb > 44 Ginkgo tree extract 0.25 wt% 14 Arbutin 0.2 wt% 38

Cytotoxicity mitigation effect by ultraviolet irradiation

This example was carried out to evaluate the mitigation effect of cytotoxicity by ultraviolet irradiation of the ginkgo tree extract obtained in Example 1 and the ginkgo tree culture of Example 2. Fibroblasts were placed in 24-well test plates at 1x105 cells for 24 hours. Each well was washed once with PBS and 500 ul of PBS was added to each well. The cells were irradiated with 10 mJ / cm 2 of ultraviolet light using ultraviolet B (UVB) lamp (Model: F15T8, UVB 15W, Sankyo Dennki, Japan) 1 ml) was added. Herein, the gravel tree cultures to be evaluated were treated and cultured for 24 hours. After 24 hours, the medium was removed, and cell culture medium (500 μl) and MTT solution (2.5 mg / ml) (60 μl) were added to each well and incubated for 2 hours at 37 ° C in a CO2 incubator. The medium was removed and 500 μl of isopropanol-HCl (0.04 N) was added. The cells were shaken for 5 minutes to dissolve the cells, and 100 쨉 l of the supernatant was transferred to a 96-well test plate, and absorbance at 565 nm was measured in a microplate reader. The cell viability (%) was measured by the equation (5) and the cytotoxic relaxation rate by ultraviolet was calculated by the formula (6).

Bo: Absorbance at 565 nm of the well that underwent chromogenic reaction only in the cell culture medium

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

St: absorbance at 565 nm of the well that underwent chromogenic reaction in the sample-treated well

Bo: Cell survival rate of wells not irradiated with ultraviolet light and not treated with sample

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

St: Cell viability of well treated with ultraviolet light and treated with sample

As can be seen from Table 8, the results of the experiment showed that the gravel tree cultures of Example 2 showed cytotoxicity by ultraviolet light of 30% at the concentration of 0.25%. It was found that the cytotoxic relaxation was increased compared to the extract of the ginkgo tree of Example 1, and it was found that the cytotoxicity by ultraviolet rays was effectively prevented even at a low concentration.

Name of sample Cytotoxic Relaxation Rate (%) Gt; 0.25 wt% < tb > 30 Ginkgo tree extract 0.25 wt% 13

Inhibitory Effect on Inflammatory Cytokine Expression by UV Irradiation

In order to evaluate the effect of inhibiting the expression of inflammatory cytokines expressed by the ultraviolet irradiation of the ginkgo tree extract obtained in Example 1 and the ginkgo tree fermentation of Example 2, fibroblasts isolated from human epidermal tissue were treated with 24-well 5X104 pieces were placed on the test plate and adhered for 24 hours. Each well was washed once with PBS and 500 占 퐇 of PBS was added to each well. After irradiating the fibroblasts with 10 mJ / cm 2 of ultraviolet light using an ultraviolet B (UVB) lamp (Model: F15T8, UVB 15W, Sankyo Dennki, Japan), PBS was removed and the cells were cultured in DMEM Medium) was added. The extract of Cyprinus vulgaris, which is to be evaluated, was treated and cultured for 5 hours. 150 [mu] l of the culture supernatant was taken to quantitate IL-1 [alpha], and the effect of inhibiting the expression of inflammatory cytokines in the gristle wood cultures was evaluated. The amount of IL-l [alpha] was quantitated using Enzyme-linked Immunosorbent Assay and the production rate of IL-l [alpha] was calculated by Equation (7).

Bo: IL-1? Production in wells without UV irradiation

Bt: IL-1? Production in wells irradiated with ultraviolet light and not treated with the sample

St: IL-1α production in wells irradiated with ultraviolet light and treated with the sample

The results of the experiment show that barnyardgrass cultures inhibit the production of IL-1α, which is an inflammatory cytokine caused by ultraviolet rays, by 41% at a concentration of 0.25%, effectively preventing inflammation caused by ultraviolet rays at low concentrations (Table 9).

Name of sample Inhibitory rate of inflammatory cytokine expression (%) Gt; 0.25 wt% < tb > 41 Ginkgo tree extract 0.25 wt% 22

Promoting effect of type 1 procollagen biosynthesis

Procollagen assay was performed as follows to examine the effect of promoting the biosynthesis of type 1 procollagen, which is a skin substrate component, after addition of the gravel tree leaf culture obtained in Example 2.

Human dermal fibroblasts isolated from neonatal foreskin tissues were purchased from Modern Tissue Technology (MTT, Korea) and supplemented with 10% fetal bovine serum (FBS) in DMEM / F-12 (3: And cultured at a concentration of 1 × 10 4 cells / cm 2. When 70-80% of the cells were grown, the cells were subcultured at a ratio of 1: 3, and cells in the third to fourth subculture were used for the experiment. For the measurement of the amount of procollagen, the fibroblasts were cultured in a 48-well plate at a concentration of 90% or more, and then the extracts of the ginkgo tree leaf extract of Example 1 and the ginkgo leaf cultures of Example 2 were respectively added at a concentration of 0.25% And the amount of procollagen liberated in the medium after 24 hours was measured using a procollagen type-1 C-peptide EIA kit (MK101, Takara, Japan).

The results are shown in Table 10. The treatment of 0.25% of the ginkgo biloba cultivar of Example 2 promoted 54% of the biosynthesis of procollagen and the promotion of the biosynthesis of procollagen was increased as compared with that of Example 1, This indicates that the cell signaling substance has an effect similar to that of TGF-β.

Name of sample Promoting effect of procollagen biosynthesis ≪ tb > < sep > 0.25 wt% 54 Sorghum leaf extract 0.25 wt% 25 TGF-beta 50

The present example was prepared by preparing a cosmetic preparation containing the cultivar of gravel tree obtained in Example 2 and evaluating the skin elasticity improvement effect and the wrinkle improvement effect on humans by performing a comparative experiment with Comparative Example 1. [

The cosmetics used in the comparative experiment are in cream form and their composition is shown in Table 10. First, the b) image recorded in Table 11 is heated and stored at 70 ° C. To this was added a phase, which was pre-emulsified, uniformly emulsified with a homomixer, and then slowly cooled to prepare a cream (Example 13, Comparative Example 1). The cream prepared in Example 14 was applied to the right side of the subject and the cream prepared in Comparative Example 1 was applied to the left side of the face twice, twice a day for 20 consecutive months for 20 subjects (20 to 35 year old female).

The skin elasticity was measured by using a skin elasticity meter (SEM 575, C + K Electronic Co., Germany) before and after using the product for 2 months. The experimental results are shown in Table 12 below as ΔR7 values of Cutometer SEM 575, where R7 values indicate the viscoelasticity properties of the skin. As shown in Table 12, it can be seen that the skin elasticity improvement effect of the experimenter who applied the cream containing the persimmon tree fermented product is excellent.

Raw material Example 13 Comparative Example 1 Stearyl alcohol 8 8 Stearic acid 2 2 Stearic acid cholesterol 2 2 Squalane 4 4 2-octyldodecyl alcohol 6 6 Polyoxyethylene (25 molar parts
A) Alcohol ester 3 3 Glyceryl monostearate
Aster 2 2 In Example 2, One - Propylene glycol 5 5 Suitable amount Suitable amount Suitable amount

Note) Unit: wt%

Experimental product Skin elasticity effect (△ R7) Example 12 0.35 Comparative Example 1 0.10

n = 20, p < 0.05

The cream prepared in Example 12 was applied to the right side of the subject and the cream prepared in Comparative Example 1 was applied to the left side of the face twice for 2 consecutive months for 20 consecutive months.

To evaluate the wrinkle improvement effect of the skin before and after the use of the product after the completion of the experiment, a silicone replica was made and the wrinkle state of the designated area was measured with a visiometer SV60, C + K Electronic Co., Germany). The results are shown in Table 12, which shows the average of two parameter values after two months minus the parameter values two months ago. That is, the negative value indicates that the wrinkle improving effect is higher. Therefore, as shown in Table 13, it was confirmed that the effect of improving the wrinkles of the skin of Example 12 containing the ginkgo tree leaf culture of Example 2 was greatly improved.

Experimental product R1 R2 R3 R4 R5 Example 13 -0.21 -0.17 -0.11 -0.08 -0.09 Comparative Example 1 -0.12 -0.06 -0.05 -0.04 -0.02 R1: Difference between the maximum value and the minimum value of the wrinkle contour line
R2: The wrinkle contour line is arbitrarily divided into 5 squares, and the average of R1 values
R3: Maximum value of R1 divided by 5
R4: Mean value of the baseline of the wrinkle contour minus the value of each apex and valley
R5: Mean value of the value obtained by subtracting the outline of each wrinkle from the base line of the wrinkle contour line

The present example was prepared by preparing a cosmetic preparation containing the ginkgo tree leaf culture obtained in Example 2 and evaluating skin whitening effect in humans compared with Comparative Example 2.

The cosmetics used in the comparative experiments are in the form of a cream, and the composition thereof is shown in Table 14. First, the phase (b) shown in Table 14 is heated and stored at 70 ° C. To this was added a phase, followed by preliminary emulsification, uniformly emulsified with a homomixer, and then gradually cooled to prepare a cream (Example 14, Comparative Example 2). The cream prepared in Example 13 was applied to the right side of the face and the cream prepared in Comparative Example 2 was applied to the left side of the face for two consecutive months for 20 consecutive months for 20 examinees (20 to 35 year-old female) . After completion of the test, the skin of the applied area on both right and left sides of the face was analyzed by image analyzer and color change (ΔL) of the color was measured using a Minolta CR300, and an objective visual observation by a plurality of expert Subjective visual observation was performed and the effect was measured according to the following classification. The results are shown in Table 15 below. At this time, the degree of whitening efficacy was classified into the following seven grades and evaluated.

· Whitening efficacy evaluation criteria:

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

1: Slight improvement 2: Slight improvement 3: Slight improvement

As shown in Table 14, it can be seen that the whitening effect is excellent in the facial skin of the experimenter who applied the cream containing the ginkgo tree leaf culture.

Raw material Example 14 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 molar parts
A) Alcohol ester 3 3 Glyceryl monostearate
Aster 2 2 I

Fermented tree One - Propylene glycol 5 5 Suitable amount Suitable amount Suitable amount

Note) Unit: wt%

Change in skin color brightness (? L) Objective evaluation of expert Subjective evaluation of the subject Example 13 Comparative Example 2 Example 13 Comparative Example 2 Example 13 Comparative Example 2 3.98 1.44 2.77 1.55 3.07 1.7

Effect of skin irritation mitigation by SLS

In this experiment, the effect of stimuli relaxation of the cosmetic composition containing the cultivar of gravel tree obtained in Example 2 was evaluated by a human skin test.

1% of SLS (sodium lauryl sulfate) which stimulates general cosmetic prescription (cream, lotion, skin, essence) and the product manufactured in Example 13 are mixed and applied for 24 hours, 48 hours and 72 hours, And the effect of stimulation relaxation was evaluated.

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

As a result of the test, the area where the SLS was applied alone showed irritation on the skin after 24 hours, but when the product containing the fermented product of the field tree was applied, it caused no skin attack even after 24 hours, 48 hours and 72 hours I did.

The results of this evaluation indicate that there is a significant effect of reducing the skin irritation caused by stimulants (surfactants, fragrances, alcohols) when the ginkgo leaf cultures are mixed with cosmetics.

Other examples are shown below. Namely, lotion, milky lotion and essence solution containing the barnyard leaf cultures obtained in Example 2 were prepared as in Examples 16 to 18. The lotion, essence, and essence containing these cultivars have excellent antioxidative effect, promoting collagen synthesis, improving skin wrinkles, whitening effect, moisturizing effect, skin irritation alleviation effect, atopy improvement effect and anti-inflammatory effect Effect.

Manufacture of cosmetic lotions containing ginkgo leaf cultures

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 p-hydroxybenzoic acid methyl ester, a small amount of antioxidant and a small amount of coloring matters were mixed and dissolved in 8 g of 95% ethanol . 10 g of the gravel tree leaf culture obtained in Example 2 and 5 g of glycerin were dissolved in 85.33 g of purified water. The mixture was added to the mixture, followed by stirring to obtain a skin lotion having skin-improving effect.

Manufacture of emulsions containing gravel tree leaf cultures

0.2 g of cetyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of p-hydroxybenzoic acid ethyl ester, 1 g of glycerin monoestearaide, 1 g of polyoxyethylene (20 mols) monooleate and 0.1 g of coryne 0.5 g of the ginkgo biloba cultivar obtained in Example 2, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1500, 0.2 g of triethanolamine and 76.2 g of purified water were dissolved by heating at 75 캜. The two were mixed and emulsified and then cooled to obtain an oil-in-water (O / W) type milky lotion.

Production of essence containing gravel tree leaf culture

To 5 g of 95% ethyl alcohol, 1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of chitoolose, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E-acetate, 0.2 g of sodium permanganate, 0.1 g of p-hydroxybenzoic acid ethyl ester 1 g of the ginkgo tree leaf culture obtained in Example 2 and an appropriate amount of pigment were mixed to obtain a serum having skin-improving effect.

Claims (11)

Obtaining a barnyard extract;
Fermenting Jeju Nuruk to the barnyardgrass extract, mixing the culture solution, and then culturing it; After the main culturing, removing the Jeju yeast from the present culture liquid, the cosmetic composition containing 0.001-30 wt% of the filtrate obtained through the above step delete delete The method according to claim 1,
Wherein the Jeju Nuruk is at least one selected from the group consisting of Pseudomonas aeruginosa, Bacillus subtilis, and Bacillus subtilis. The method according to claim 1,
Wherein the composition is for skin whitening. The method according to claim 1,
Wherein the composition is for improving wrinkles. delete delete delete The method according to claim 1,
The composition may be a cosmetic preparation, a cosmetic preparation, a gel, a water-soluble liquid, a cream, an essence, an oil-in-water type or a w / o type; Wherein the cosmetic composition is selected from the group consisting of a makeup base, a foundation, a skin cover, a lipstick, a lip gloss, a face powder, a two-way cake, an eye shadow, a cheek color and an eyebrow pencil. delete