KR101463012B1 - External Compositions for Skin Using Mineral Obtained from Magma Seawater or the Fermentation Product Thereof - Google Patents

Abstract

The present invention provides a composition for external application for skin using lava sea water minerals. Specifically, the present invention provides a composition for external application for skin based on skin whitening activity, wrinkle improving activity and ultraviolet blocking activity of lava sea water minerals. The composition for external application for skin of the present invention can be made into a cosmetic product, a soap, or the like.

Description

[0001] The present invention relates to a composition for external application for skin using lava sea water minerals,

The present invention relates to a composition for external application for skin using lava sea water minerals or fermented products thereof.

Lava seawater, which is distributed only in the eastern part of Jeju Island (Gucheong-eup, Seongsan-eup, and Seokseon-myeon), is clean and has a constant water temperature throughout the year and contains a large amount of various minerals not found in ordinary saltwater or seawater. , A Study on the Hydrological Geology in the Eastern Part of Jeju Island (Ⅱ), 2003, Kyungwon University, "A Study on the Underground Geological Structure and Groundwater Variation and Water Quality in the Eastern Part of Jeju Island, , Vol. 3, pp. 15-43 "," Meiwon et al., Underground Distribution of Seogwipo Formation in Jeju Island and Its Relation to Groundwater, 1991, Geology Journal, Vol. 27, No. 5, The production of lava seawater from the existing data, including, can be summarized as follows. In other words, seawater infiltrated into the underground lava flows from the outer ocean and huge seawater mass was formed on the Seoguipo (or Seifuri) layer, which is an impervious sedimentary layer that is difficult to move water. This is supported by the fact that lava seawater is not found in the western part of Cheju island and northern part of Cheju island where the Seoguipo layer is thin and only Seogwipo is found only in eastern part of Cheju island.

The minerals content of the depths obtained from 150m of underground (based on average sea level), 44.35mm, 86.35m and 126.35m were collected from Handong-ri, Gyoung-eup, Jeju-do, As shown in Table 1.

Concentration of minerals by water depth in Jeju Lava Seawater (ppm) Test Items Fresh water 1m Lava seawater 44.35m Lava Seawater 86.35m Lava seawater 126.35m Na 146 11400 11400 10700 Mg 0.49 1510 1520 1390 Ca 9.20 460 490 431 K 38.5 643 540 621 Cu 0.00069 0.00157 0.00113 0.00080 Co - - - - Sn - - - - Mo 0.0014 0.011 0.012 0.011 V 0.013 3.28 3.53 0.01 Ge 0.00019 0.0032 0.003 0.0025 Br - - - - Sr 0.05 1.18 1.31 1.30 Ti - - - - Ni - - - - Si (SiO ₂₎ 1.21 7.23 6.19 1.21 Zn 0.001 0.0045 0.0039 0.0019 Fe 0.082 0.21 0.26 0.04 Mn 0.01 19.51 20.7 18.9 Cl ^- 262 16629 18500 19900 B 0.15 3.35 3.73 0.15 Li 0.09 0.37 0.41 0.09 Ba 0.015 0.032 0.019 0.015  Pb 0.00091 0.004 0.0013 0.00065 F - - - - As - 0.01 - - Se 0.000049 0.00005 0.00124 0.00086 Hg - - - - CN ^- - - - - Cr - - - - CD - - - - Al 0.14 0.63 0.49 0.47  * The above-mentioned freshwater is the result of analysis of groundwater at 1 m below sea level with an electric conductivity of 1186.58 μs / cm and a salt concentration of 0.3 millimeter.

From Table 1, lava seawater shows similarities to general salt groundwater in items such as salt, calcium, and magnesium, but there is a clear difference in trace element items such as vanadium, germanium, and selenium.

The inventors of the present invention have completed the present invention by confirming that the powdered lava sea water minerals obtained by desalinating lava sea water and evaporating water and the fermented products thereof such as lactic acid bacteria have skin whitening activity, wrinkle improving activity and ultraviolet blocking activity.

An object of the present invention is to provide a skin external composition for skin whitening using lava sea water minerals or fermented products thereof.

Another object of the present invention is to provide a composition for external application for skin using wrinkle improvement using lava sea water minerals or fermented products thereof.

Another object of the present invention is to provide a composition for external application for skin using ultraviolet rays using lava sea water minerals or fermented products thereof.

Other objects and specific objects of the present invention will be described below.

In one aspect, the present invention relates to a whitening dermatological composition for external use comprising a mineral or a fermented product thereof as an active ingredient.

The inventors of the present invention have found that, as shown in the following Examples and Experimental Examples, powdery minerals obtained by draining lava seawater at 86.35 m underground in the eastern part of Jeju Island, desalting lava seawater and evaporating the water, and lactic acid bacteria or yeast When the fermented product was treated at a concentration of 100 μg / ml in the melanoma cell line derived from rat, the production of melanin was inhibited by 36% in the case of the mineral treatment group, and when the fermented product was treated, And yeast fermented products were inhibited by 49% and 54%, respectively. Melanin protects skin organs below the dermis by blocking ultraviolet rays and protects the damage of proteins and genes in the skin caused by free radicals generated in the skin of the skin. However, when produced more than necessary, melanin causes aging of the skin, It is a substance that causes hyperpigmentation such as freckles and freckles.

Herein, "lava sea water" is defined and described in Korean Patent No. 0853244 (Application No.: 10-2008-0027861), entitled " Method for producing mineral composition and mineral composition obtained by the method & It is the same meaning as "salt groundwater which exists in the eastern part of Jeju Island" Specifically, "eastern part of Jeju Island" means Jeju-eup, Seongsan-eup and Seokseon-myeon in Jeju-do in administrative district, and "saltwater groundwater" means groundwater containing salinity of a certain amount or more. Specifically, According to the criteria for classification of groundwater in Jeju Island based on the salinity concentration used in the study of the hydrological geology in the eastern part of Cheju Island (Ⅱ), 2003, Kyungwon University, considered as part of this specification), low salt groundwater (electric conductivity of 1,700 ㎲ / cm (17,350 / / cm) and saltwater groundwater (electrical conductivity of 17,350 / / cm or more), but preferably the electrical conductivity is 17,350 / / cm or more or the salt concentration is 30 ‰ ) Or more (usually the saline concentration of the seawater is 32 to 35 ‰).

In this specification, the term "lava sea water minerals" is a result obtained by desalting lava seawater. The resultant may be a concentrated liquid phase state in which water is partially removed by evaporation, or a completely water-evaporated powder phase state. This lava sea water mineral can also be understood as a composition of mineral components, whether liquid or powder. Methods for desalinating lava seawater to obtain lava sea water minerals can be performed by applying any technique known in the art, such as reverse osmosis, ion exchange resin, electrodialysis, and the like.

In the present specification, the term "lava sea water mineral fermentation product" means a culture obtained by adding a carbon source and an energy source to a liquid or powdery lava sea water minerals to be fermented and mixing and inoculating lactic acid bacteria or yeast. The culture may be used as it is, or may be used in a purified form through filtration or in a liquid or powder form by reduced pressure concentration and / or lyophilization. When the lava seaweed mineral fermented product is to be fermented, it may be obtained by adding a carbon source and an energy source thereto and inoculating and fermenting the lactic acid bacteria or yeast when liquid lava seawater minerals are used as a fermentation target. When minerals are used, they can be obtained by adding and mixing a carbon source and an energy source, adding and mixing purified water, and then inoculating and fermenting lactic acid bacteria or yeast.

In the present specification, the term "lactic acid bacteria" means bacteria that decompose saccharides such as glucose to produce lactic acid, such as Streptococcus sp . ), Pediococcus lactic acid bacteria sp . ), Leuconostoc sp. Lactic acid bacteria ( Leuconostoc sp . ), Lactobacillus ( Lactobacillus sp . And Bifidobacterium sp. ) . Specific examples thereof include Lactobacillus delbrueckii , Lactobacillus spp., And Lactobacillus spp. bulgaricus , Lactobacillus < RTI ID = 0.0 > casei), Lactobacillus brevis (Lactobacillus brevis , Lactobacillus < RTI ID = 0.0 > acidophilus , Pediococcus pentosaceus , Pediococcus S. cerevisiae , Lactococcus lactis), Les Kono Stock reum sheet (Leuconostoc citreum), Les Kono Stock mesen steroid (Leuconostoc mesenteroides), Bifidobacterium Bifidobacterium Doom (Bifidobacterium bifidum , Lactobacillus rhamnosus and the like. Preferably Lactobacillus genus of lactic acid bacteria (Lactobacillus spp.), And a means, and particularly preferably is a filler's (Lactobacillus know the Lactobacillus used in the examples below, acidophilus , Lactobacillus plantarum , Lactobacillus < RTI ID = 0.0 > casei ) and / or Lactobacillus rhamnosus .

As used herein, "yeast" refers to the genus Saccharomyces As yeast, for example, Saccharomyces rouxii), in my process to the three Levy cyano Saccharomyces (Saccharomyces cereviciae), access to the MY Ob pole miss Saccharomyces (Saccharomyces oviformis), a saccharide My process stay Neri (Saccharomyces steineri ), and it is particularly preferable to use Saccharomyces cerevisiae.

In particular, even if lactic acid bacteria and yeast are used as inoculum microorganisms in the following examples, it is expected that similar results will be obtained even if useful microorganisms known in the art are used. Examples of useful microorganisms include Mycobacterium tuberculosis, Bacillus subtilis, and Mycobacterium tuberculosis. These microorganisms may be used in combination of two or more species. The usable strains of Aspergillus are Aspergillus oryzae , Aspergillus and the like sojae), the Bacillus subtilis may be used if both Bacillus microorganisms (Bacillus sp.) are involved in the fermentation of soybean, soybean paste, cheese, food, specifically Bacillus subtilis (B. subtilis), Bacillus leak B. lichnerformis , B. megaterium , B. amyloliquefaciens , B. natto , B. antharcis , Bacillus amyloliquefaciens , Bacillus amyloliquefaciens , B. lentus , B. pumilus , B. thuringiensis , B. alvei , B. azotofixans , Bacillus macerans , Bacillus subtilis, (B. macerans), Bacillus Poly miksa (B. polymyxa), Bacillus papil Rie (B. popilliae), Bacillus core posts lance (B. coagulans), Bacillus stearate loader Russ a brush (B. stearothermophilus), Bacillus Pas toeri (B . pasteurii ), Basil B. sphaericus , B. fastidiosus , and the like. Also as honggukgyun (Monascus sp.) Available from the Pseudomonas kusu louver (Monascus ruber , Monascus purpureus , Monascus rubervan , Monascus fuliginosus , Monascus pilosus , Monascus anka ), Monasukusubarikerry barykery , Monascus purpurens , Monascus albidus Sato).

Meanwhile, carbon sources and energy sources of useful microorganisms such as lactic acid bacteria are well known in the art in the production of fermented lava seawater minerals, and thus any of those known in the art can be used. Generally, oligosaccharides, lactose, glucose, Sugar, mixtures of these, and the like. These carbon sources and energy sources can be added in an arbitrary range in consideration of the intended fermentation time and degree of fermentation. Usually from 3 parts by weight to 30 parts by weight based on 100 parts by weight of citrus pulverized product.

In the production of the fermentation product of lava sea water, the fermentation time will be determined according to the fermentation temperature, the amount of the fermentation raw material (which is a mixture of the carbon source and the energy source of the lava seawater minerals and useful microorganisms), the amount of useful microorganisms to be inoculated and the like. A person skilled in the art can determine an appropriate fermentation time in consideration of the fermentation temperature, the amount of the fermentation raw material, the amount of the useful microorganism to be inoculated and the like within the ordinary skill of the person skilled in the art.

In the production of fermented lava seawater minerals, the fermentation temperature may be in the range of 15 ° C to 45 ° C. Preferably in the range of 20 [deg.] C to 40 [deg.] C. If the fermentation temperature is lower than the above range, the fermentation rate may be slowed down and a desired fermentation product may be produced. Even if the fermentation temperature is higher than the above range, the fermentation rate may be slowed down or the desired fermentation product may be produced have.

In the production of fermented lava sea water mineral fermentation, an aging step can be carried out after fermentation. This aging step is intended to stabilize the fermentation product by the microorganism even after the inoculated microorganism stops growing, and this aging step may be performed for about 10 to 12 months.

In another aspect, the present invention relates to a composition for external application for skin for improving wrinkles of lava sea water minerals or fermented products thereof as active ingredients.

The following Examples and Experimental Examples demonstrate that both the lava seawater minerals and fermented products thereof inhibit the activity of elastase degrading elastin and inhibit the production of collagenase that degrades collagen, Treatment of human fibroblasts at a concentration of 100 μg / mL improved the production of Type I collagen by about 60% as compared to the untreated group.

Skin is composed of epidermis and dermis. Dermis is connective tissue under the epidermis. It consists mostly of macromolecular network called extracellular matrix. This extracellular epilepsy is made in fibroblasts in the dermis. , Collagen, and elastin, and polysaccharides called acidic mucopolysaccharides such as hyaluronic acid. Collagen, elastin, and hyaluronic acid in these dermal tissues are important fiber tissues that maintain the flexibility and elasticity of the skin. Therefore, when collagen and elastin of the dermis tissue are degraded or the production is reduced by the influence of aging, ultraviolet rays, or the like, skin wrinkles occur. Collagenase and elastase are involved in degradation of collagen and elastin, respectively. Therefore, when collagenase and elastase activity are inhibited, wrinkle-reducing effects can be expected.

In the present specification, "wrinkle improvement" is meant to include treatment of wrinkles, suppression of wrinkles, and / or delay of wrinkles.

The meaning of lava sea water, meaning of lava sea water minerals, fermented product thereof, meaning of effective ingredient in the composition for improving wrinkles of the present invention and the like are as described in relation to the above-mentioned skin whitening composition of the present invention.

In another aspect, the present invention relates to a composition for external application to skin for ultraviolet shielding with an active ingredient of lava sea water minerals.

The inventors of the present invention prepared a cosmetic composition containing powdery lava sea water minerals and a cosmetic composition not containing lava sea water minerals according to the 'functional test method for ultraviolet blocking functionality' of 'functional cosmetics standards and test methods' published by Korea Food and Drug Administration SPF for UVB, and PFA, a blocking index for UVA, were measured to compare UV blocking effects of cosmetic composition containing lava sea water minerals and cosmetic composition not containing lava sea water minerals. It was confirmed that the SPF and PFA of the cosmetic composition were significantly higher than those of the cosmetic composition containing no lava sea water minerals. Here, the higher the SPF / PFA, the better the UVB / UVA blocking effect.

In the present specification, "ultraviolet ray" means a long wavelength ultraviolet ray (320 to 400 nm, hereinafter referred to as "UVA") and / or a medium to high ultraviolet ray (280 to 320 nm; hereinafter referred to as "UVB"). Ultraviolet rays are classified into long wavelength ultraviolet (UVA), medium wavelength ultraviolet (UVB), and short wavelength ultraviolet rays (200-280 nm, hereinafter referred to as UVC). Ultraviolet rays reaching the ground are 290-400 nm, Of UVA, and UVA is about 0.5% and UVA is about 5.5%. UVA and UVB are known to cause erythema, blisters, and skin wrinkles.

The significance of the meaning of lava sea water, the meaning of lava sea water minerals, the meaning of active ingredients, etc. in the ultraviolet screening composition of the present invention is still valid as described in connection with the skin whitening composition of the present invention.

The whitening dermatologic external composition for skin, the composition for external application for wrinkle improvement and the composition for external application for ultraviolet ray shielding (hereinafter collectively referred to as "composition for external application for skin of the present invention") of the present invention, (Effective amount), as long as it can exhibit ultraviolet ray activity. Typical effective amount is in the range of 0.001 wt% to 40.000 wt% based on the total weight of the composition. Will be determined. Herein, "effective amount" refers to an amount of an effective ingredient capable of exhibiting skin whitening, wrinkle improvement or ultraviolet activity. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

The composition for external application for skin of the present invention can be identified as a cosmetic composition in a specific embodiment.

When the composition for external application for skin of the present invention is recognized as a cosmetic composition, the cosmetic composition may be prepared in various forms such as an emulsion, a lotion, a cream (an oil-in-water type, Anhydrous and aqueous), anhydrous products (oil and glycol), gels, masks, packs, powders, and the like.

The composition for external application for skin of the present invention may contain an acceptable carrier in a cosmetic preparation besides its effective ingredient.

As used herein, the term " acceptable carrier for cosmetic preparation "refers to a compound or composition which is already known and used in cosmetics, or which is a compound or composition to be developed in the future, and which has no toxicity that the human body can adapt to when contacted with skin.

The carrier may be included in the skin topical composition of the present invention in an amount of from about 1% by weight to about 99.99% by weight, preferably from about 50% by weight to about 99% by weight of the composition, based on the total weight thereof.

However, since the ratio depends on the above-mentioned formulation of the cosmetic product and its specific application site (face or hands) or the desired amount of application thereof, the ratio is to limit the scope of the present invention in any aspect It should not be.

Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosifiers, emulsifiers, stabilizers, sunscreens, coloring agents and perfumes.

The compounds / compositions which can be used as the carrier and which can be used as alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosifiers, emulsions, stabilizers, sunscreens, A person skilled in the art can select and use appropriate substances / compositions.

The external preparation for skin of the present invention can be identified as a soap composition in another specific embodiment.

When the composition for external application for skin of the present invention is recognized as a soap composition, the soap composition of the present invention can be prepared by incorporating an active ingredient into a soap base material and can be prepared as an additive including a skin moisturizer, an emulsifier, have.

Examples of the soap base material include vegetable oils such as palm oil, palm oil, soybean oil, perm free oil, olive oil and palm kernel oil or animal fats such as tallow, lard, sunshine and fish oil. Examples of the skin moisturizers include glycerin, erythritol, polyethylene glycol , Propylene glycol, butylene glycol, pentylene glycol, hexyl glycol, isopropyl myristate, silicone derivatives, aloe vera, sorbitol and the like. Examples of the emulsifier include natural oils, wax fatty alcohols, hydrocarbons, May be used. As the water softening agent, tetrasodium ethylenediate and the like may be used.

The soap composition of the present invention may further contain, as an additive, an antibacterial agent, a dispersant, a foam inhibitor, a solvent, a scouring inhibitor, a corrosion inhibitor, a fragrance, a dye, a sequestering agent, an antioxidant and an antiseptic.

In the soap composition of the present invention, the soap base or additive may be included in a content commonly used in the art, wherein the soap base generally comprises from 99.999% to 50% by weight, based on the total weight of the soap composition And the additive may be added in an amount of 1 wt% to 20 wt%.

The external preparation for skin of the present invention may be identified as a pharmaceutical composition in another specific embodiment.

The pharmaceutical composition of the present invention may be formulated into a wide variety of dosage forms, including pharmaceutically acceptable carriers, excipients and the like, in addition to the active ingredient, and may be formulated into a wide variety of dosage forms such as a localized preparation such as cream, lotion, ointment (semi-solid external drug), micro loo emulsion, (TTS) (e.g., patches, bandages, etc.), and the like.

The term "pharmaceutically acceptable" as used herein means that the application (prescribing) subject does not have the above-mentioned toxicity that is adaptable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (e.g., corn starch, potato starch and the like), cellulose, derivatives thereof (e.g. sodium carboxymethylcellulose, ethylcellulose, etc.), malt, gelatin, talc, (E.g., peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g., TWEENS), wetting agents (For example, sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, water, a saline solution, and a phosphate buffer solution. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

The excipient may be selected according to the formulation of the pharmaceutical composition of the present invention. For example, suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydropropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, .

The pharmaceutical compositions of the present invention may be administered orally or parenterally, and preferably topically.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

INDUSTRIAL APPLICABILITY As described above, according to the present invention, it is possible to provide a composition for external application for skin having a whitening activity, a wrinkle-reducing activity or a UV-blocking activity using lava sea water minerals and fermented products thereof. The composition for external application for skin of the present invention can be made into a cosmetic product, a soap, a medicine, and the like.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Lava seawater  Used Powdery  Mineral compositions and their Fermented  Produce

Example 1 Preparation of Powdered Mineral Composition Using Lava Seawater

It was drilled 150m underground in Handong-ri, Gucheong-eup, Jeju-do, eastern part of Jeju Island, and lava seawater was collected underground (average of sea level), 44.35mm, 86.35m and 126.35m. The collected lava sea water was analyzed by the Korea Research Institute of Chemical Technology (Seoul, Korea). The results are shown in Table 1 above. As a result of measuring the electrical conductivity and salinity of the collected lava water, the electrical conductivity of the lava water collected at 44.35m underground was 48516.41㎲ / cm, the salinity was 14.4 ‰ (permilliage), and the lava water collected at 86.35m The conductivity was 50618.4 ㎲ / cm and the salinity concentration was 34.1 ‰ (permilliage). The lava seawater collected at 126.35 m underground had an electrical conductivity of 50474.3 ㎲ / cm and a salinity concentration of 34.1 ‰ (permilliage).

The desalted water was removed by filtering the impurities with a 0.2 μm filter, and then removing the monovalent ion using an ion exchange membrane by an electrodialyzer (ASTOM CO. LTD., Japan). The moisture of the obtained desalted lava waters was evaporated to obtain a powdery mineral composition.

Example 2: Preparation of fermentation product of lava sea water mineral composition

<Example 2-1> Production of Fermented Lactic Acid Bacteria from Lava Seawater Mineral Composition

The <Example 1> The molasses in sea water desalination lava 2ℓ obtained was mixed with 150g added, here in MRS (DeMan, Rogosa, Sharpe and) Lactic Acid Bacteria (Lactobacillus broth prepared from plantarum ) was inoculated at 5% (v / v) and fermented at 38 ℃ for 2 weeks to produce a fermented product of lactic acid bacteria of lava seawater. The fermented product was sterilized at 120 ° C for 10 minutes, concentrated under reduced pressure, and lyophilized to be used in the experiment.

Example 2-2 Preparation of Fermented Product of Yeast Lava Seawater Mineral Composition

150 g of molasses was added to 2 liters of the desalted lava water obtained in Example 1 and mixed. A 5% (v / v) culture of yeast ( Saccharomyces cerevisiae ) prepared in a PDB (Potato Dextrose Broth) For 2 weeks to prepare fermented lava seawater mineral yeast. The fermented product was sterilized at 120 ° C for 10 minutes, concentrated under reduced pressure, and lyophilized to be used in the experiment.

< Experimental Example > Whitening activity test, wrinkle improving activity test and UV blocking activity test

< Experimental Example  1> Whitening activity experiment

<Experimental Example 1-1> Cell culture

B16 / F10 cells were purchased from the Korean Cell Line Bank (KCLB) as a melanoma cell line in rats, and 100 units / ml penicillin-streptomycin (GIBCO, Grand Island, NY, USA) and 10% fetal bovine serum (FBS; GIBCO, (GIBCO, Grand Island, NY, USA) in RPMI 1640 or DMEM medium (Grand Island, NY, USA) at 37 ° C in a 5% CO ₂ incubator. Subculture was performed every 3-4 days Respectively.

<Experimental Example 1-2> Cytotoxicity evaluation

The cell survival rate of B16 / F10 cells of the samples was evaluated using the MTT assay method (Carmichael et al., 1987).

Each of the cells (2.0 to 5.0 × 10 ⁴ / ml) was placed in each well of a 96-well plate, and a sample (100 μg / ml) -2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, Mo., USA) was added and further incubated for 4 hours. Plates were centrifuged at 1,000 rpm for 10 min. After carefully removing the medium, 150 μl of dimethylsulfoxide (DMSO; Sigma, St. Louis, Mo., USA) was added to dissolve the formazan precipitate produced by the reduction of MTT. (Bio-TEK instrumenis, Inc., Winooski Vermont, Wis., USA) was used to measure the absorbance at 540 nm.

The results are shown in Table 2 below as a percentage of the sample-untreated group (negative control group).

Cell survival rate sample Cell viability (%) Negative control group 100% The mineral composition of Example 1 102% The mineral fermentation product of <Example 2-1> 97% The mineral fermentation product of <Example 2-2> 98%

The results of the above Table 2 show that neither the mineral composition of the examples nor the fermented product show any particular cytotoxicity.

&Lt; Experimental Example 1-3 > Evaluation of inhibitory activity on melanin formation

Melanin quantitation was performed by Yasunobu et al. (Yasunobu et al., 2003). 6 well plate in 3 × 10 ⁵ cell / well and then dispensed by culturing the cells in the embodiment sample (100㎍ / ㎖) the treatment with α-MSH (50nM) and the melanin formation stimulant, 37 ℃ for 48 hours, CO ₂ incubator. The cultured cells were collected, and the number of cells was measured. The cells were centrifuged at 1,200 rpm for 5 minutes to precipitate, and the cells were dissolved in 1 ml of homogenization buffer (50 mM sodium phosphate pH 6.5, 1% Triton X-100, 2 mM PMSF). To the pellet obtained, 200 1 of 1 N NaOH (in 10% DMSO) was added and the absorbance was measured at 405 nm after vortexing. The amount of melanin produced in each well was calculated using a standard calibration curve obtained from the melanin standard. Melanin compares melanin production in unit cells (1 × 10 ⁴ cells).

The results are shown in Table 3 in terms of percentage of melanin production relative to the sample-untreated group (negative control group).

Melanin formation inhibitory activity sample Melanin production (%) Negative control group 100% The mineral composition of Example 1 64% The mineral fermentation product of <Example 2-1> 46% The mineral fermentation product of <Example 2-2> 51%

The results of the above Table 3 show that both the mineral composition of the examples and the fermented product thereof have an activity of inhibiting melanin production. When combined with the cytotoxicity test results, the melanin production inhibitory activity is not a result of cytotoxicity Tell me. In particular, it shows that the activity of inhibiting the melanin production of the fermented product of minerals is high.

< Experimental Example  2> Wrinkle improvement activity test

<Experimental Example 2-1> elastase dehydratase (Elastase) Assay

An elastase assay was conducted to confirm that the activity of elastase degrading elastin involved in skin elasticity was inhibited by the treatment of the sample of the example.

First, to 170 mu l of 0.2 M Tris-Cl buffer (pH 8.0), 20 mu l of a sample of the concentration of 100 mu g / mL of the example was added. 30 μl of substrate 1 mM N-succinyl- (Ala) _3- p-nitroanilide was added to the mixed solution, followed by reaction at 25 ° C for 10 minutes and 30 μl of 2.5 U / ml porcine pancreas elastase (PPE) was added. After mixing well, the reaction was carried out at 25 ° C for 20 minutes and absorbance was measured at 405 nm using an ELISA reader.

Elastase activity is shown in Table 4 below as a percentage of negative control.

Elastase inhibitory activity division  Elastase activity (%) Negative control group 100% The mineral composition of Example 1 72% The mineral fermentation product of <Example 2-1> 58% The mineral fermentation product of <Example 2-2> 62%

The results of the above Table 4 show that both the mineral composition of the examples and the fermented product thereof have elastase inhibitory activity, and that the fermented product of mineral fermentation has a high activity of inhibiting elastase.

<Experimental Example 2-2> collagenase (collagenase I, MMP -1) inhibitory effect

Collagen degradation activity of collagenase which promotes wrinkle formation by decomposing collagen was examined to confirm wrinkle improving effect.

In order to measure the inhibition of collagenase activity, an antibody of collagenase was used. In this test, the absorbance was measured by an ELISA method using a type I collagenase assay kit (Type I collagenase assay kit).

First, human fibroblast cells (CCD-986SK cells) were cultured in a suitable medium at 37 ° C under 5% carbon dioxide. After subsequent to the test on the 48-well plate for cell division of 5X10 ⁴ and to a certain incubation time, make the attachment of the cells, and cultured 16 hours in serum-free culture medium. The sample (100 μg / mL) of the example was treated with the cell culture medium and TNF-α was added to the collagenase-activating substance for 24 hours. The cell culture was collected, and 100 를 was added to the assay well. The enzyme was added to each well as a detection reagent, and the mixture was incubated at 37 캜 for 90 minutes and then measured at 450 nm using an ELISA reader.

The results are shown in Table 5 below as the percentage of collagenase inhibition relative to the TNF-a treated group as a percentage.

Collagenase inhibitory activity division Collagenase activity (%) Negative control group 37% TNF-alpha treated group 100% The mineral composition of Example 1 73% The mineral fermentation product of <Example 2-1> 59% The mineral fermentation product of <Example 2-2> 62%

The results of the above Table 5 show that both the mineral composition of the examples and the fermented product thereof have a collagenase inhibitory activity, and in particular the collagenase inhibitory activity of the mineral fermented product is high.

<Experimental Example 2-3> Collagen biosynthesis experiment ( Collagen synthesis assay )

<1> Cell culture

Fibroblasts (CCD-986SK, human fibroblasts) were purchased from the American Type Culture Collection (ATCC , Rockville, USA). FBS (fetal bovine serum, Hyclone) and 1% Antibiotic-Antimycotic Gibco) supplemented with DMEM (Dulbecco's Modified Eagle Medium.Hyclone) at 37 ° C and 5% CO ₂ .

&Lt; 2 > Evaluation of cell survival rate

Human fibroblasts are divided into 96 wells at a concentration of 2 × 10 ⁴ cells / well and cultured for 24 hours under cell culture conditions. The medium was discarded and the sample of the example was diluted with serum-free medium, treated at a concentration of 200 μg / mL, and cultured for 24 hours. MTT solution (50 μl of 2 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5 diphenyl-2H-tetrazolium bromide solution) is added to each well and incubated for 4 hours. After the culture medium was removed, dimethylsulfoxide (DMSO) solution was added, and the mixture was shaken for 10 minutes. Absorbance was measured at 540 nm using an ELISA reader.

The results are shown in Table 6 below as a percentage of the sample-untreated group (negative control group).

Cell survival rate sample Cell viability (%) Negative control group 100% The mineral composition of Example 1 109% The mineral fermentation product of <Example 2-1> 101% The mineral fermentation product of <Example 2-2> 99%

The results of the above Table 6 show that neither the mineral composition of the examples nor the fermented product showed any cytotoxicity.

<2> Effect of Type I collagen synthesis promotion

Human fibroblasts were divided into 96 wells at a concentration of 2 × 10 ⁴ cells / well and cultured for 24 hours. The medium was discarded and the sample of the example was diluted with serum-free medium and incubated at a concentration of 200 / / mL for 24 hours. The amount of collagen synthesis in the cell culture medium was measured using procollagen type I peptide EIA kit (Takara Biomedical Co.). 100 μl of anticody-POD conjugate solution was added to each well, and 20 μl of sample and standard were added and incubated at 37 ° C. for 3 hours. After the reaction was completed, the culture medium was removed from each well and washed four times with 400 μl of PBS (phosphate buffered saline). 100 μl of the substrate solution was added and reacted at room temperature for 15 minutes. After stopping the reaction, 100 μl of stop solution (1N H ₂ SO ₄ ) was added and the absorbance at 450 nm was measured with an ELISA reader. A standard concentration curve was drawn to confirm the amount of collagen.

Table 7 below shows the percentage of the sample-treated group (negative control group).

Promotion rate of collagen biosynthesis sample Collagen biosynthesis rate (%) Negative control group 100% The mineral composition of Example 1 137% The mineral fermentation product of <Example 2-1> 167% The mineral fermentation product of <Example 2-2> 159%

The results of the above Table 7 show that both the mineral composition of the examples and the fermented product thereof have an activity of promoting collagen biosynthesis, and when combined with the results of the cytotoxicity experiment, the collagen biosynthesis promoting activity is not a result of cytotoxicity Tell me. Here too, the mineral fermented product has high collagen biosynthesis promoting activity.

< Experimental Example  3> Experiment of ultraviolet blocking effect

For the UV blocking activity test, the cosmetic composition of [Table 8] was prepared with the following components and contents (wt%). For the mineral fermented product, only the mineral composition of Example 2-1 was used, and the cosmetic composition of Comparative Example was prepared by further containing talc by the amount of the mineral composition and mineral fermented product of the examples.

Cosmetic composition division Production Example 1 Production Example 2 Comparative Example Mica 32.0 32.0 32.0 Talc 42.0 42.0 54.0 Octylmethoxy cinnamate 6.0 6.0 6.0 Titanium dioxide 4.0 4.0 4.0 Zinc oxide 4.0 4.0 4.0 The mineral composition of Example 1 12.0 - The mineral fermentation product of <Example 2-1> - 12.0 -

The SPF (Sun Protection Factor) and PFA (Protection Factor of UVA) of the cosmetic composition prepared above were measured according to the 'UV Protection Function Test Method' of the 'Standards and Test Methods for Functional Cosmetics' notified by the Korean Food and Drug Administration. Here, the higher the SPF / PFA, the better the UVB / UVA blocking effect.

Specifically, the SPF was applied to a transpore tape so that the sample was 2 mg / cm ² , dried for 15 minutes, and then measured with an SPF 290 measuring apparatus. PFA measurement was performed in 10 male and female subjects. ² and applied with a Xenon lamp. Using a multi-port solar simulator 601-300W (Solar Light, USA) as a light source, after a lapse of 2 to 4 hours from ultraviolet A irradiation, (MPPD) was calculated and PFA was calculated according to the following equation.

PFA = [MPPD of the sample application site] / [MPPD of the site where the sample is not toppled]

The results are shown in Table 9 below as MEAN ± SD, which is repeated three times.

SPF and PFA measurement results division SPF PFA The cosmetic composition of Preparation Example 1 31.27 ± 0.37 6.87 + - 0.46 The cosmetic composition of Preparation Example 2 35.31 + - 0.52 7.21 + - 0.37 The cosmetic composition of Comparative Example 18.63 + - 0.31 5.64 + - 0.41

The results of the above Table 9 show that the mineral composition and the fermented product exhibit ultraviolet shielding effect against UVA and UVB, and that the mineral fermentation product has a higher ultraviolet shielding effect.

Claims (9)

A carbon source and an energy source are mixed with minerals obtained by excavating a lava layer and desalting lava seawater having an electric conductivity of 17,350 μs / cm or more and having a salt concentration of 30 ‰ or more, taken from a water depth of 44.35 to 126.35 meters, A composition for external application for skin for whitening whitening comprising a fermented product obtained by inoculating and culturing Lactobacillus plantarum or Saccharomyces cerevisiae as an active ingredient.
A carbon source and an energy source are mixed with minerals obtained by excavating a lava layer and desalting lava seawater having an electric conductivity of 17,350 μs / cm or more and having a salt concentration of 30 ‰ or more, taken from a water depth of 44.35 to 126.35 meters, A composition for external application for skin for improving wrinkles comprising, as an active ingredient, a fermented product obtained by inoculating and culturing Lactobacillus plantarum or Saccharomyces cerevisiae .
A carbon source and an energy source are mixed with minerals obtained by excavating a lava layer and desalting lava seawater having an electric conductivity of 17,350 μs / cm or more and having a salt concentration of 30 ‰ or more, taken from a water depth of 44.35 to 126.35 meters, A skin external composition for ultraviolet screening comprising an effective ingredient of a fermentation product obtained by inoculating and culturing Lactobacillus plantarum or Saccharomyces cerevisiae .
delete delete delete 4. The method according to any one of claims 1 to 3,
Wherein the composition for external application for skin is a cosmetic composition.
4. The method according to any one of claims 1 to 3,
Wherein the composition for external application for skin is a soap composition.
delete