KR20160096272A - A skin-care agent containing Mesembryanthemum crystallinum L. fermentation extract using yeast - Google Patents

Abstract

The present invention relates to a cosmetic composition containing a fermented product of an ice plant, and more particularly, to a cosmetic composition containing an extract of a plant of the ice plant, which is fermented using Jeju Nuruk, and which contains a filtrate as an active ingredient And exhibits an excellent antioxidative effect, an effect of promoting collagen synthesis, an effect of improving wrinkles, a whitening effect, a moisturizing effect, a skin irritation alleviating effect, an atopy improving effect and an antiinflammatory effect.

Description

[0001] The present invention relates to a skin-care agent containing mesenteric hemp crystallinum L. fermentation extract using yeast,

The present invention relates to a composition for external application for skin, and more particularly, to a composition for external application for skin containing, as an active ingredient, a filtrate obtained by fermenting an outpost extract of an ice plant belonging to the genus Chrysanthemum.

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 healing 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 dysentery, 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).

Ice plant (Mesembryanthemum crystallinum L.) is a succulent plant native to the Namib Desert of South Africa. Its main distribution areas are South Africa, Eastern and Western, Northwest, Southwest Australia, and Western Drylands of the Americas. Transparent crystals (bladder cells) on the surface contain a variety of minerals and components useful for the human body, such as inositols and beta-carotene. By transforming photosynthetic pathway into C3 → CAM through environmental stress, the functional material is sent to the surface cells together with the saline contained in it and stored. The stem and leaf bladder cells have the shape like ice crystals, (Ice plant). Especially, since the ice plant contains a lot of pinitol which lowers the blood sugar level and myoinositol which suppresses the neutral fat, it is suitable for the diabetic patients when the leaves and the stem are reproduced or used as a juice.

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 Luchuensis mut. Kawachii, Asp awamori) are predominantly 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 present inventors completed the present invention by establishing an increase in the content of the active ingredient in the fermented product of the ice plant cultured by using the Jeju Nuruk and confirming that it has an excellent effect as a cosmetic composition without inducing skin irritation.

The present invention provides a composition for external application for skin, which comprises, as an active ingredient, a filtrate obtained by fermenting an ice plant extract with Jeju Nuruk.

The present invention also relates to a method for confirming the efficacy of an ice plant fermented product as a cosmetic raw material and exhibiting effects such as antioxidative effect, collagen synthesis promoting effect, wrinkle improving effect, whitening effect, moisturizing effect, It is an object of the present invention to provide a skin external application composition containing an ice plant fermentation product.

Accordingly, the present inventors have recognized the excellent efficacy of the ice plant extract, and have made it possible to use the filtrate obtained by fermentation and cultivation using Jeju Nuruk as an effective ingredient, .

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.

The present invention also relates to a composition for external application for skin containing an ice plant fermentation product characterized in that the content of the filtrate is 0.001 to 30.0% by weight with respect to the whole composition for external application for skin. 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,

The present invention also relates to a composition for external application for skin, wherein an aqueous solution containing distilled water or ethanol in an amount of 85 to 99 (v / v) is used as an extraction solvent.

The present invention also relates to a composition for external application for skin, characterized in that the extract is subjected to a vacuum concentration or lyophilization process.

In addition, the present invention relates to a composition for external application for skin, wherein 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 preparation, wherein the composition for external application for skin is a lotion, 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 a skin whitening effect containing an ice plant fermented product.

The present invention also relates to a composition for external application for skin, which exhibits an anti-aging effect containing an ice plant fermented product.

The present invention also relates to a composition for external application for skin, which exhibits a skin irritation mitigating effect containing an ice plant fermented product.

Further, the present invention relates to a composition for external application for skin showing an atopic skin improving effect containing an ice plant fermented product.

The present invention also relates to a composition for external application for skin, which contains an ice plant fermented product and exhibits an acne-preventing effect.

The present invention also relates to a composition for external application for skin wherein the composition for external application for skin contains PLASTERS, LPTIONS, LINIMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, A pharmaceutical composition selected from the group consisting of OINTMENTS, FLUIDEXTRACTS, EMULSIONS, SUSPESIONS, CAPSULES, CREAMS, soft or hard gelatin capsules, patches, Is used.

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 composition for external application for skin of the present invention may vary depending on individual characteristics of a patient, and a practitioner skilled in the art will, for the patient, , For example, the most appropriate treatment strategy in terms of the 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, a suitable dose of the inventive skin topical composition may be generally determined in vitro or in an animal model. For example, various concentrations of the inventive skin topical composition may be added to the target cells in vitro to determine their proper dosage.

The fermented product of the ice plant of the present invention exhibited a whitening effect, such as melanin production 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 ice plant fermented product of the present invention showed excellent anti-aging effects such as active oxygen scavenging effect, MMP inhibitory effect and MMP expression control effect by ultraviolet irradiation, and relieving skin irritation caused by ultraviolet irradiation.

In addition, the ice plant fermented product of the present invention showed an effect of alleviating skin irritation and improving atopy by an inflammatory substance.

Therefore, cosmetic compositions such as lotion, cream, emulsion, pack, powder and the like containing such an ice plant fermented product can be used as a cosmetic composition containing active substance, such as active oxygen scavenging effect and collagenase enzyme activity regulating effect, skin wrinkle improving effect, whitening, moisturizing, And exhibits excellent skin external agent effect such as stimulation relaxation effect.

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) from ice plant

10 kg of the dried and frozen ice plant was refluxed for 3 hours with 95% (v / v) distilled water or aqueous ethanol solution for 5 hours, cooled, and filtered through Whatman # 5 filter paper. The filtered extract was concentrated under reduced pressure and freeze-dried at a temperature of 50 ° C or less, and the extract was prepared from the ice plant by dissolving the lyophilized product in a mixed solvent of purified water, ethanol, butylene glycol and propylene glycol so as to contain 15% .

Ice plant fermentation production

The ice plant extract obtained in Example 1 was added with 5% (v / v) of Jeju Nuruk prepared from PDB (Potato Dexrtose Broth) medium and fermented at 26 캜 for 7 days to prepare an ice plant fermented product. The ice plant fermented product was 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 in the ice plant fermentation

Qualitative analysis of the phenolic materials, flavonoids, flavonoid glycosides and phenol contents and total flavonoid contents of the major components of the ice plant extract prepared in Example 1 and the ice plant fermented product 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 to 2 drops of 2.5% FeCl3 ethanol solution was left in each of Example 1 (ice plant extract by simple extraction) and Example 2 (fermented ice plant), it was color-changed to dark green or whether precipitation was observed or not 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
(Ice plant extract by simple extraction) 340.5 21.2 Example 2 (fermentation of ice plant) 585.1 68.1

The total phenol content and the total flavonoid content were measured (Table 1), and it was confirmed that the phenol content and the total flavonoid content of Example 2 were higher than that of Example 1, and the content of flavonoid was increased by about 3 times .

From the above results, it was confirmed that the filtrate obtained by fermenting the ice plant extract prepared by simple extraction with Jeju Neruk can increase the total phenol content and total flavonoid content. Based on this, it was confirmed that the filtrate As an active ingredient. The composition for external application for skin of the present invention, which comprises the fermented product of the ice plant, is prepared.

Experiment to measure free radical scavenging activity

To determine the free radical scavenging activity of the ice plant fermentation obtained in Example 2, free radical scavenging activity was measured using a DPPH method with extracts having excellent antioxidative 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: The absorbance of the control well without the ice plant fermentation of the present invention

B: Absorbance of the experimental group well treated with the ice plant fermentation of the present invention

As can be seen in Table 2, the ice plant culture showed better antioxidative effect than the green tea extract having the superior antioxidant activity.

Name of sample Antioxidative effect(%) 0.25 wt% of ice plant fermentation product 97% 0.1% by weight of the ice plant fermentation product 95% Ice plant extract 0.25 wt% 91% Green tea extract 92%

Cytotoxicity of the sample

The cytotoxicity of the ice plant fermentation product 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 the cells were treated with a solution containing 2.5 mg / ml of MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl tetrazolium bromide)

200 μl of the culture medium was added thereto, followed by culturing 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 0.25 wt% of ice plant fermentation product 2% 0.1% by weight of the ice plant fermentation product 2% Ice plant fermentation water 0.05 wt% 2% Ice plant fermented water 0.025 wt% 2%

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

in 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. The fluorescence value was measured by adding the reaction buffer instead of the enzyme solution as a control 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 0.25% of the ice plant fermented product of Example 2 was treated, and the inhibitory activity was better than that of the ice plant extract.

From the above results, it was confirmed that the effect of inhibiting the metalloproteinase (MMP-1) activity of the fermented product of the ice plant is excellent. Based on this fact, the skin external composition composition of the present invention effectively inhibits the metalloproteinase, And wrinkle formation can be prevented.

Name of sample MMP-1 activity inhibition rate (%) 0.25 wt% of ice plant fermentation product 91 Ice plant extract 0.25 wt% 58 Green tea extract 0.2 wt% 69

Assessment of Inhibition of MMP-1 Expression by Fermentation of Ice Plant after UV Irradiation

In this experiment, enzyme immunoassay (ELISA) was performed to measure the concentration of MMP-1 after UV irradiation and sample addition of the ice plant fermentation product obtained in 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, the ice plant fermentation of Example 2 showed an increase in the MMP-1 inhibition rate of MMP-1 induced by UV irradiation, and the inhibition rate of MMP-1 was 76% 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 - 0.25 wt% of ice plant fermentation product 76 Ice plant extract 0.25 wt% 44 Retinol 41

Tyrosinase activity inhibition experiment

As a comparative sample, arbutin, well known as a whitening agent, was used as a comparative sample to confirm the inhibitory effect of the tyrosinase activity of the ice plant fermentation product 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 ice plant extract of Example 1 and the ice plant fermentation of Example 2 were dissolved in a 0.05 M sodium phosphate buffer solution (pH 6.8) adjusted to an appropriate concentration and used. 0.5 ml of L-tyrosine solution was placed in a test tube, and 0.5 ml of the ice plant extract and the ice plant fermentation sample 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 inhibition rate of tyrosinase in treating the fermented product of the ice plant of Example 2 is similar to arbutin.

Name of sample Inhibition rate of tyrosinase activity (%) Control group - 0.25 wt% of ice plant fermentation product 74 Ice plant extract 0.25 wt% 45 Arbutin 0.2 wt% 72

Melanin synthesis inhibition experiment using B16F1 melanocytes

In order to confirm the whitening effect of the ice plant extract obtained in Example 1 and the ice plant fermentation product of Example 2 in this experiment, the effect of inhibiting melanin synthesis on B16F1 melanocytes was measured.

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 carried out 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 effect of inhibiting melanin synthesis of B16F1 melanocytes was tested. As a result, the ice plant fermented product of Example 2 showed a melanin production inhibitory effect of 0.25% to 47%. In particular, the melanin production inhibitory effect was superior to that of the ice plant extract of Example 1, which showed a similar inhibitory effect to arbutin.

Name of sample Melanin synthesis inhibition (%) Negative control group - 0.25 wt% of ice plant fermentation product 47 Ice plant extract 0.25 wt% 24 Arbutin 0.2 wt% 39

Cytotoxicity mitigation effect by ultraviolet irradiation

This example was conducted in order to evaluate the effect of mitigating cytotoxicity by ultraviolet irradiation of the ice plant extract obtained in Example 1 and the ice plant fermentation product 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. Here, the ice plant fermentation product to be evaluated was 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 shown in Table 8, the ice plant fermented product of Example 2 exhibited cytotoxicity by 34% ultraviolet light at a concentration of 0.25%. It was confirmed that cytotoxic relaxation was increased as compared with the ice plant extract of Example 1, and it was found that cytotoxicity by ultraviolet rays was effectively prevented even at low concentrations.

Name of sample Cytotoxic Relaxation Rate (%) 0.25 wt% of ice plant fermentation product 34 Ice plant extract 0.25 wt% 17

Inhibitory Effect on Inflammatory Cytokine Expression by UV Irradiation

To evaluate the effect of suppressing the expression of inflammatory cytokines expressed by ultraviolet irradiation of the ice plant extract obtained in Example 1 and the ice plant 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 suppressing the inflammatory cytokine expression of the fermented product of the ice plant 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 showed that the ice plant fermented product inhibited the production of IL-1α, which is an inflammatory cytokine caused by ultraviolet light, by 47% at a concentration of 0.25%, and effectively prevented the development of inflammation caused by ultraviolet rays at low concentrations (Table 9).

Name of sample Inhibitory rate of inflammatory cytokine expression (%) 0.25 wt% of ice plant fermentation product 47 Ice plant extract 0.25 wt% 21

Promoting effect of type 1 procollagen biosynthesis

Procollagen assay was performed as follows to examine the effect of accelerating the biosynthesis of type 1 procollagen, which is a skin substrate component, after adding the fermented product of the ice plant 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 experiment of measuring the amount of procollagen, the fibroblasts were cultured in a 48-well plate at 90% or more, and then the ice plant extract of Example 1 and the ice plant fermentation of Example 2 were 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 0.25% treatment of the ice plant fermentation of Example 2 promoted 64% of the biosynthesis of procollagen, and the promoting of the biosynthesis of procollagen was increased compared with that of the ice plant extract of Example 1, Signal transduction was similar to TGF-β.

Name of sample Promoting effect of procollagen biosynthesis 0.25 wt% of ice plant fermentation product 64 Ice plant extract 0.25 wt% 36 TGF-beta 52

The present example was prepared by preparing a cosmetic preparation containing the fermented product of the ice plant obtained in Example 2 and evaluating the skin elasticity improvement effect and the wrinkle reduction 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 coated with the cream containing the ice plant 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 The ice plant fermentation product of Example 2 One - Propylene glycol 5 5 Suitable amount Suitable amount Suitable amount

Note) Unit: wt%

Experimental product Skin elasticity effect (△ R7) Example 12 0.31 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 skin wrinkles of Example 12 containing the ice plant fermentation product of Example 2 was greatly improved.

Experimental product R1 R2 R3 R4 R5 Example 13 -0.19 -0.18 -0.10 -0.08 -0.10 Comparative Example 1 -0.11 -0.07 -0.05 -0.04 -0.03 R1: Difference between the maximum value and the minimum value of the wrinkle contour line
R2: The wrinkle contour line is arbitrarily divided into 5 squares, and the average of R1 values
R3: Maximum value of R1 divided by 5
R4: Mean value of the baseline of the wrinkle contour minus the value of each apex and valley
R5: Mean value of the value obtained by subtracting the outline of each wrinkle from the base line of the wrinkle contour line

The present example was prepared by preparing a cosmetic preparation containing the fermented product of the ice plant obtained in Example 2 and evaluating the effect of skin whitening on humans in comparison 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 user who applied the cream containing the high-temperature and high-pressure extract of the pyrethroid gauge.

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

Ice plant fermentation product 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 4.05 1.39 2.87 1.45 3.57 1.5

Effect of skin irritation mitigation by SLS

In the present experiment, the effect of relaxing the irritation of the cosmetic preparation containing the ice plant fermentation product obtained in Example 2 was evaluated by a human skin patch 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 ice plant fermentation product was applied, no skin episode occurred after 24 hours, 48 hours and 72 hours I did.

The results of this evaluation indicate that there is a significant effect of reducing skin irritation caused by stimulants (surfactants, flavors, alcohols) when the ice plant fermentation product is mixed with cosmetics.

Other examples are shown below. In other words, lotion, milky lotion and essence solution containing the ice plant fermentation product obtained in Example 2 were prepared as in Examples 16 to 18. Lotions and essences containing these high-temperature and high-pressure extracts have excellent antioxidant effects, promoting collagen synthesis, improving skin wrinkles, whitening effect, moisturizing effect, skin irritation alleviation effect, atopic improvement effect and anti-inflammatory effect Respectively.

Production of lotion containing ice plant fermented product

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 are mixed with 8 g of 95% ethanol . 10 g of the pyrethroid high-temperature high-pressure extract 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 lotion having skin-improving effect.

Manufacture of emulsion containing ice plant fermented product

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 monoestearylate, 1 g of polyoxyethylene (20 molar part) monooleate and 0.1 g of coryne 0.5 g of the pyrethroid high-temperature high-pressure extract obtained in Example 2, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1,500, 0.2 g of triethanolamine and 76.2 g of purified water were dissolved by heating at 75 캜. The two were mixed and emulsified and then cooled to obtain an oil-in-water (O / W) type milky lotion.

Production of Serum Containing Fermented Ice Plant

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 high-temperature and high-pressure extract obtained in Example 2 and an appropriate amount of pigment were mixed to obtain a serum having skin-improving effect.

Claims (11)

Obtaining an ice plant culture;
Fermenting Jeju Nuruk to the ice plant extract, mixing the culture solution, and then culturing the mixture; [Claim 7] The composition for external application for skin according to claim 1, wherein the filtrate obtained through the step of removing Jeju yeast from the present culture solution after the present culture is used as an effective ingredient. The method according to claim 1,
Wherein the content of the filtrate is 0.001 to 30.0% by weight based on the total amount of the composition for external application for skin. The method according to claim 1,
Wherein an aqueous solution containing 85 to 99% (v / v) of distilled water, purified water or ethanol is used as an extraction solvent at the time of extraction. The method according to claim 1,
Wherein the main culturing step is carried out at a temperature of 25 to 37 DEG C for 5 to 7 days after the pH of the culture medium is adjusted to 5.0 to 6.0, inoculated with 4 to 6% (v / v) &Lt; / RTI &gt; The method according to claim 1,
Wherein the composition for external application for skin is for skin whitening. The method according to claim 1,
Wherein the composition for external application for skin is an anti-aging composition. The method according to claim 1,
Wherein the composition for external application for skin is a composition for relieving skin irritation. The method according to claim 1,
Wherein the composition for external application for skin is for improving atopic skin. The method according to claim 1,
Wherein the composition for external application for skin is for the prevention of acne. The method according to claim 1,
The composition for external application for skin may be a cosmetic formulation, a gel, a water-soluble liquid, a cream, an essence, an oil-in-water (O / W) type or a water-in-oil (W / O) type; Wherein the 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, . The method according to claim 1,
The composition for external application for skin may be used as a skin irritant such as PLASTERS, LPTIONS, LINIMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, OINTMENTS, Characterized in that it is selected from among FLUIDEXTRACTS, EMULSIONS, SUSPENSIONS, CAPSULES, CREAMS, soft or hard gelatine capsules, patches, and sustained release agents and is used as a pharmaceutical composition Skin external composition.