KR101009427B1 - Method for the extract of volcanic ash used as cosmetic component and cosmetic composition thereof - Google Patents

Abstract

The present invention comprises the steps of pulverizing the ash to obtain a ash powder; And any extractant selected from purified water having a pH of 7 to 11, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, dichloromethane, hexane and mixtures thereof to the obtained volcanic ash powder, followed by ultrasonic extraction. Obtaining an extract; The present invention relates to a method of preparing a volcanic water for cosmetics and a cosmetic composition prepared therefrom, wherein the inhibitory effect of tyrosinase helps to produce melanin, a substance causing blemishes, freckles and skin pigmentation, and melanin synthesis. Inhibitory effect, inhibitory activity and expression of metalloproteinases affecting wrinkle formation and elasticity of skin, inhibitory effect of hyaluronidase, an inflammation-related enzyme, inhibitory effect on inflammatory cytokine expression, acne bacteria It is possible to produce volcanic water exhibiting an inhibitory effect of 5 alpha-reductase activity, which is the cause of antibacterial effect and acne.

Aging, whitening, solvent, manufacturing method, wrinkles, elasticity, anti-acne, anti-inflammatory, volcanic water, volcanic ash, cosmetics

Description

Method for preparing the volcanic water for cosmetics and cosmetic composition prepared therefrom {Method for the extract of volcanic ash used as cosmetic component and cosmetic composition

The present invention relates to a method for producing a cosmetic volcanic water and to a cosmetic composition prepared therefrom.

Human skin is constantly changing with age and skin aging. Skin aging is largely divided into natural aging (or endogenous aging) and external aging (EB Doris, Cosmetics & Toiletories , 111 : 31-37, 1996). While natural aging is influenced by genetic factors, it is difficult to control artificially. However, external aging is relatively easy to control because it is influenced by environmental factors. Representative external aging factors include heavy metals, yellow sand, ozone, ultraviolet rays, and chemicals. The most prominent external aging phenomenon is wrinkle formation (HW Daniel, Ann Intern Med , 75 (6): 873-880, 1971). GL Grove, et al ., J Am Acad Dermatol , 21: 631-637, 1989; CE Griffiths, et al ., Arch Dermatol , 128: 347-351, 1992).

In vivo, the synthesis and degradation of extracellular matrix such as collagen is properly regulated, but as aging progresses, its synthesis decreases and matrix metalloproteinase (MMP), an enzyme that breaks down collagen, In particular, the expression of collagenase (hereinafter referred to as 'MMP-1') and gelatinase (gelatinase (hereinafter referred to as 'MMP-2')) is promoted, thereby reducing the elasticity of the skin and forming wrinkles.

MMPs are classified into MMP-1, MMP-2, and MMP-9 according to their substrate specificity. MMP-1 is a fibroblast type collagenase, an enzyme of epilepsy, and collagen types I, II, III, Ⅶ, Ⅷ, Ⅹ and gelatin, originally cut into 3/4 or 1/4 length peptides to facilitate the action of nonspecific enzymes (gelatinase). MMP-2 belongs to 72 KD of gelatinase (A) and degrades substrates such as gelatin, collagen types IV, V, VIII, VIII, elastin and fibronectin. MMPs of neutrophils and polymorphonuclear leukocytes are primarily activated by oxidation, which first generates hydrogen peroxide in the cell and is converted to hypochlorous acid by myeloperoxidase in the presence of chlorine ions. Activate it.

In order to prevent aging due to external stimulation, the method of applying sunscreen products directly to the skin is the most common method.However, in 1988, retinoic acid was reported to be effective in reducing skin roughness and fine wrinkles of aged skin (KS). Weiss, et al ., JAMA , 259 : 527-532, 1988), research is being actively carried out around the world to develop substances that inhibit or improve skin aging. Among them, vitamins A, C, E, derivatives of vitamins and AHA (alpha-hydroxy acid) are representative substances for improving skin aging known to date (Hermitte, Cosmetics & Toiletries , 107 : 63-67, 1992; DR Rosenthal, et al ., J Invest Dermatol , 95 : 510-515, 1990; TD Ditre, et al ., J Invest Dermatol , 34 : 187-195, 1996). However, they are very unstable in the natural environment, so there is a problem in use, or somewhat insufficient to show a visible effect.

On the other hand, volcanic water is known to be rich in various minerals, and its form and composition are suitable for skin sensitivity and thus for the long-term sustainable mineral supply of skin. Mineral ions, such as iron, selenium, zinc, calcium, magnesium, sodium, potassium, and phosphorus, dissolved in mineral rocks over time, are cofactors of biochemical enzyme reactions that bring the skin's natural structure closer to old skin. Works. Sodium and potassium components are important for controlling moisture content, and calcium, magnesium and phosphorus contents play a crucial role in cell growth and aging. However, the use of volcanic water has been limited due to safety problems on the skin, formulation and stability problems when formulated in cosmetics, and research on the production method of volcanic water for cosmetics that can exhibit practically useful functionality is still insignificant. to be.

Accordingly, an object of the present invention is to provide a method for producing a volcanic water for cosmetics and a cosmetic composition prepared therefrom that exhibits skin improvement effects without causing formulation and stability problems when formulated in cosmetics.

In order to achieve the above object, the present invention comprises the steps of obtaining a ash powder by grinding the ash; And any extractant selected from purified water having a pH of 7 to 11, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, dichloromethane, hexane and mixtures thereof to the obtained volcanic ash powder, followed by ultrasonic extraction. Obtaining an extract; It provides a method for producing a cosmetic volcanic water comprising a.

The present invention also provides a cosmetic composition comprising the volcanic water obtained from the method for producing a volcanic water for cosmetics as an active ingredient.

Hereinafter, the problem solving means of the present invention will be described in detail.

Volatile water production method of the present invention is characterized by using an extraction solvent having a pH of 7 to 11, by using such an alkaline extraction solvent, it is possible to efficiently extract the alkaline material from the volcanic ash, such conditions Metalloproteinases affecting the activity of tyrosinase, which inhibits melanin formation, melanin synthesis, melanin synthesis, and wrinkle formation and decreased elasticity Inhibition effect of the activity and expression, inhibitory effect of hyaluronidiase, an inflammation-related enzyme, inhibitory effect of inflammatory cytokine expression, antibacterial effect against acne bacteria and inhibitory effect of 5 alpha-reductase activity Excellent skin improvement effect confirmed by exercise was confirmed from the following experiments.

On the other hand, if the pH is less than 7, the extraction yield of the useful material showing alkalinity from the volcanic ash is not high, the expression of various physiological effects confirmed in the present invention is insignificant, and if the pH exceeds 11, the alkalinity of the solvent is too high for the skin. It is not desirable because it gives a big stimulus.

On the other hand, the manufacturing method of the volcanic water for cosmetics of the present invention is the step of sterilizing after filtering the extract obtained from the ultrasonic extraction; It is better to include, because filtration and sterilization can remove impurities and harmful microorganisms to prevent irritation and infection on the skin.

On the other hand, volcanic ash powder in the manufacturing method of the volcanic water for cosmetics of the present invention is not necessarily limited to the size of the particular particles, preferably 50 to 200 mesh, because the extraction yield is the highest in the mesh degree of this range. to be.

On the other hand, in the manufacturing method of the volcanic water for cosmetics of the present invention, the ultrasonic extraction is not limited to specific conditions, but preferably 1 to 3 times for 1 to 5 hours, the ultrasonic extraction in the above range is most economically Because it can be done.

On the other hand, the present invention provides a cosmetic composition comprising the volcanic water obtained from the method for producing a cosmetic volcanic water as an active ingredient.

The volcanic water contained in the cosmetic composition of the present invention is not limited to a specific range, and may be any range within which the effect of the volcanic water described above is exerted without impairing the quality aptitude of the cosmetic composition. It is good to include 0.001 to 100% by weight.

In addition, the cosmetic composition according to the present invention preferably contains, in addition to volcanic water, other components that can give a synergistic effect to the main effect within the range of not impairing the main effect of the present invention.

On the other hand, the cosmetic composition of the present invention can be used as an example for skin whitening, anti-inflammatory, wrinkle improvement, anti-acne, elasticity, the formulation is most preferably a lotion, gel, water-soluble liquid, cream, essence, Basic cosmetic formulation consisting of oil-in-water (O / W) and water-in-oil (W / O) type, ointment oil-in-water and oil-in-water makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye bird In addition, it is preferred that any one selected from the color cosmetic formulation consisting of a cheek color and eyebrow pencils.

It may also be applied to the skin, optionally in the form of an aerosol, or may be in the form of a solid such as a stick. It may be used as a skin care product and / or makeup product.

The compositions according to the invention also contain conventional auxiliaries in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants and dyes. can do. The amounts of these various adjuvants are amounts conventionally used in the art, such as from 0.01 to 20% by weight relative to the total weight of the composition. In any case, the adjuvant and its proportions will be chosen so as not to adversely affect the desirable properties of the cosmetic composition according to the invention.

As described above, the volcanic water obtained from the method for preparing cosmetic volcanic water of the present invention is an inhibitory effect of tyrosinase, which inhibits melanin production, a melanin synthesis inhibitory effect, which helps to produce melanin, a substance causing blemishes, freckles and skin pigmentation. , Inhibitory effect of metalloproteinases on the formation of wrinkles on skin and decrease in elasticity, inhibitory effect of inflammatory enzymes, hyaluronidase, inhibitory effect on inflammatory cytokine expression, antibacterial effect against acne And it exhibits an excellent skin improvement effect by exerting an effect of inhibiting 5 alpha-reductase activity which is the cause of acne.

In addition, the cosmetic composition containing the volcanic water produced therefrom as an active ingredient also exhibits skin improvement effects such as excellent skin whitening, wrinkle and elasticity improvement.

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

Example  One: Volcanic water  Produce

After the ash was pulverized to about 130 mesh, 1 kg of purified water having a pH of 9 as an extraction solvent was mixed with 100 g of the obtained powder, and ultrasonically extracted three times at about 60 ° C. for 2 hours. The extract obtained above was put into a filtration tank and filtered, and the obtained filtrate was sterilized to obtain volcanic water (Example 1).

Experimental Example  One: Example  By 1 Mushroom Tyrosinase  Used Tyrosinase  Determination of activity inhibitory effect

In order to confirm the whitening effect of volcanic water obtained in Example 1, the degree of activity inhibition of tyrosinase was measured.

Tyrosinase is an enzyme that accelerates the oxidation of tyrosine in vivo and helps melanin production. In Experimental Example 1, a method of measuring the extent to which tyrosine is oxidized to inhibit the formation of a black polymer called melanin by inhibiting tyrosinase (SH Pomerantz, J Biol chem , 241: 161-168, 1966) was applied to measure the whitening effect.

In order to confirm the inhibitory effect of tyrosinase activity by volcanic water, koji acid, arbutin, lettuce extract and oil-soluble licorice extracts, which were known as whitening agents, were compared and used.

Inhibitory activity of tyrosinase of each sample was placed in a 96-well plate in a 96-well plate, 150 μl of 50 mM phosphate buffer (pH 6.5) and 25 μl of 1.5 mM L-tyrosine solution, followed by After 10 μl of tyrosinase (1,500 units / ml, Sigma) was added and reacted at 37 ° C. for 20 minutes, the absorbance was measured at 490 nm using a microplate reader (ELx800, USA) to measure the inhibition rate of tyrosinase activity. . The activity inhibition rate (%) for tyrosinase was calculated by Equation 1, and the IC ₅₀ value is the concentration of a substance that inhibits tyrosinase enzyme activity by 50%.

% Inhibition of tyrosinase = [(D-C)-(B-A) / (D-C)] X 100

A: Absorbance before reaction of the well containing the sample

B: Absorbance after reaction of the well in which the sample was placed

C: Absorbance before reaction of the well without sample

D: Absorbance after reaction of well without sample

 Sample Name Inhibitory effect of tyrosinase (IC ₅₀ )  Volcanic Water (Example 1)  0.110%  Arbutin  0.350%  Lettuce extract  0.900%  Soluble Licorice Extract  0.040%

As a result of measuring the inhibitory effect of tyrosinase activity by volcanic water (Table 1), the IC ₅₀ value of volcanic water was 0.110%, and it was confirmed that the inhibitory effect of tyrosinase was superior to that of the arbutin and lettuce extract used as the whitening agent.

From the above results, it can be seen that the manufacturing method of the volcanic water for cosmetics of the present invention can produce volcanic water having an excellent whitening effect.

Experimental Example  2: B16F1 Melanosite  Used Example  Melanin production inhibitory effect

In Experimental Example 2, to confirm the whitening effect of the volcanic water obtained in Example 1, the degree of inhibition of melanin production was measured using B16F1 melanosite.

B16F1 melanocytes used in Experimental Example 2 are cell strains derived from mice, and cells that secrete a black pigment called melanin. During the artificial culture of these cells, each sample (volcanic water (Example 1), hydroquinone, arbutin, lettuce extract, oil soluble extract) was treated to compare the degree of melanin black pigment reduction. The used B16F1 melanosite was used from the ATCC (American Type Culture Collection, Accession No .: 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocytes was measured as follows. B16F1 melanocytes were dispensed at a concentration of 2 X 10 ⁶ per well in a 6-hole plate incubator, and the cells were attached and incubated for 72 hours by treating the sample at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, and the cells were counted and centrifuged to recover the cells. Quantification of intracellular melanin was performed by slightly modifying the method of Rotan (R Lotan and D Lotan, Cancer Res , 40 : 3345-3350, 1980). The cell pellet was washed once with PBS, and then 1 ml of homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt the cells. Melt the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), and measure the absorbance of melanin at 405 nm with an absorbance spectrometer. Melanin biosynthesis inhibition rate (%) was measured. Inhibition rate (%) of melanin biosynthesis of B16F1 melanocytes was calculated by the equation (2), IC ₅₀ value is the concentration of a substance that inhibits melanin production by 50%.

% Melanin biosynthesis inhibition = [(A-B) / A] X 100

A: Melanin amount in wells without sample

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

Sample Name Melanin biosynthesis inhibitory effect ((IC ₅₀ ) Volcanic Water (Example 1) 0.10% Hydroquinone 0.05% Arbutin 0.20% Lettuce extract 0.05% Soluble Licorice Extract 0.03%

As a result of measuring melanin inhibitory effect of B16F1 melanocytes (Table 2), the IC ₅₀ value of volcanic water is 0.10%, which is similar to that of hydroquinone, arbutin, oil-soluble licorice extract and lettuce extract. It was confirmed that or excellent.

From the above results, it can be seen that the manufacturing method of the volcanic water for cosmetics of the present invention can produce volcanic water having an excellent whitening effect.

Experimental Example  3: Example  By 1 Measurement of collagen biosynthesis promoting effect

Experimental Example 3 measured the collagen biosynthesis promoting effect by the volcanic water obtained in Example 1.

In the test method, human skin fibroblasts were cultured in a 24-well plate until they grew to about 90% by seeding 3 × 10 ⁴ per well in a 24-well plate. After taking them once washed with PBS and the number of processing volcano and incubated 48 hours jongan CO ₂ incubator. These supernatants were removed and the amount of procollagen (procollagen) type (1) C-peptide (PICP) was tested using ELISA kits to promote procollagen biosynthesis. Synthetic capacity is prepared by the control (non-treated group) 100. Collagen biosynthesis rate was calculated by the equation (3).

Collagen biosynthesis rate (%) = (reaction absorbance of extract cell culture / response absorbance of control) × 100

 Sample Name Treatment concentration (%)  Collagen Biosynthesis Rate (%)  Volcanic Water (Volcanic Ash Beauty Water) 5  107.5% 10  120.0% 20  138.0%  Control -  100.0%

As a result of measuring collagen biosynthesis promoting effect by volcanic water obtained in Example 1 (Table 3), it was confirmed that the excellent collagen biosynthesis promoting effect of volcanic water.

From the above results, it can be seen that the manufacturing method of the volcanic water for cosmetics of the present invention can produce volcanic water having an excellent elasticity improvement effect.

Experimental Example  4: in in vitro in Example  By 1 Of metalloproteinases (MMP-1)  Activity inhibition rate measurement

As aging progresses, the synthesis of extracellular matrix, such as collagen, is reduced in vivo, and the expression of metalloproteinase (MMP-1), an enzyme that degrades collagen, is promoted, which not only reduces skin elasticity but also wrinkles. Is formed. In Experimental Example 4, in Comparative sample retinol and green tea extract were used to determine the inhibition rate of metalloproteinase (MMP-1) by volcanic water obtained in Example 1 in vitro .

Inhibition rates of substrate metalloproteinases were determined in a biochemical model, based on the use of collagen and gelatin conjugated to purified collagenase and its substrate, fluorescein (EnzChek ™ gelatinase / cola). Genase kit, molarula probe). Collagenase purified from Clostridium historiescom was supplied in the EnzChek ™ gelatinase / collagenase kit.

Collagen was purified from porcine skin and the reaction buffer consisting of DQ-collagen conjugated to fluorescein and 0.05M Tris-HCl, 0.15M NaCl, 5mM CaCl ₂ and 0.2mM sodium azide (pH 7.6) was EnzChek ™. Gelatinase / collagenase kit (Molcula Probes) was used. The volcanic water obtained in Example 1 was tested by dissolving in the reaction buffer. Dilutions of the test extracts were incubated with 25 ug / ml of DQ-collagen and 0.1 U / ml of collagenase for 15 minutes, 45 minutes, and 120 minutes at room temperature.

The final sample concentration was 0.1% (v / v) and 0.2% (v / v) and the inhibition rate of MMP-1 was measured.

In each experimental condition, the control mixture corresponding to the collagenase and DQ-collagen mixture (control; the same composition as the above experiment but no sample added) was also incubated in the same manner, and blank (Blank; Containing blank) was incubated in the presence of DQ-collagen and in the absence of collagenase, and each experiment was performed three times. After 15 minutes, 45 minutes and 120 minutes, the signal corresponding to the degradation of DQ-collagen was measured by a fluorimeter (excitation: 485 nm, emission 505 nm), and based on the 'enzyme-free blank' fluorescence value of each sample, Fluorescence values were measured. Results are expressed as percent variance for fluorescence units per sample and control.

Sample Name % Inhibition of MMP-1 Treatment Concentration 0.1% Treatment concentration 0.2% Volcanic Water (Example 1) 42 70 Retinol 0 9 Green Tea Extract 57 75

in As a result of measuring the inhibition rate of metalloproteinase (MMP-1) in vitro (Table 4), the volcanic water obtained in Example 1 retained anticollagenase / gelatinase activity in a dose dependent manner.

Volcanic water (Example 1) inhibited the collagen degrading activity of Clostridium collagenase by 42% and 70% at 0.1% and 0.2%, respectively, which was superior to or similar to that of retinol and green tea extract at each treatment concentration. -1 shows the inhibitory effect.

From the above results, the production method of the volcanic water for cosmetics of the present invention was confirmed that by effectively inhibiting metalloproteinases can produce volcanic water that can prevent the decrease in elasticity of the skin and the formation of wrinkles.

Experimental Example  5: after UV irradiation Volcanic water  by Metalloproteinases ( MMP -1) expression inhibition measurement

Metalloproteinase (MMP-1), which breaks down collagen and affects skin elasticity and wrinkle formation, is also activated by UV irradiation. In Experimental Example 5, the volcanic water obtained in Example 1 after UV irradiation The degree of inhibition of the expression of metalloproteinase (MMP-1) was measured.

After UV irradiation and sample addition (volcanic water (Example 1), retinol), ELISA was performed to measure metalloproteinase (MMP-1) concentration.

Human dermal fibroblasts are irradiated with UVA at an energy of 5 J / cm 2 using a UV chamber. UV irradiation dose and culture time were established through preliminary experiments to maximize the expression of MMP in fibroblasts. The negative control was wrapped in tinfoil and kept at the same time in the environment of UVA, UVA emission was measured using a UV radiometer. The cells during the UVA irradiation were intact with the previously dispensed medium and irradiated with UVA and then exchanged with the medium containing the sample for 24 hours of incubation, and then the medium was recovered and coated on a 96-well plate. The primary antibody (MMP-1 (Ab-5) monoclonal antibody and MMP-2 (Ab-3) monoclonal antibody) was treated and reacted for 60 minutes at 37 ℃. After reacting the secondary antibody anti-mouse IgG (whole mouse, alkaline phosphatase conjugated) again for about 60 minutes, the alkaline phosphatase substrate solution (1mg / mlρ-nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes, and then Absorbance was measured at 405 nm with a plate reader. As a control, one without addition of a sample was used.

Sample Name Treatment concentration (%) % Inhibition of MMP-1 expression Control - - Volcanic Water (Example 1) 0.1 23 Retinol 0.1 22

As a result of measuring the degree of inhibition of the expression of metalloproteinase (MMP-1) for each sample after irradiation with ultraviolet rays (Table 5), Example 1 showed more than 20% inhibition rate compared to the control group not treated with the sample. In particular, it showed better inhibition than retinol.

From the above results, the production method of the volcanic water for cosmetics of the present invention was confirmed that it is possible to produce volcanic water which can prevent the decrease in elasticity of the skin and the formation of wrinkles by inhibiting the expression of metalloproteinases even after UV irradiation.

Experimental Example  6: Example  By 1 Inflammation-related enzymes ( hyaluronidase ) Determination of activity inhibitory effect

In Experimental Example 6, in order to confirm the anti-inflammatory effect of the volcanic water obtained in Example 1, the activity inhibitory effect of hyaluroniadiaze, an inflammation-associated enzyme, was measured.

Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid and has a function of causing inflammation. In Experimental Example 6, a method of measuring anti-inflammatory effect by inhibiting the activity of hyaluronidia agent (Y Kakegawa, et. al . , Japanese J of Inflammation , 4 : 437-438, 1984) was used to determine the anti-inflammatory effect.

The effect of inhibiting hyaluronia diase activity was investigated using volcanic water (Example 1), Compri, Seesaw, and Triticale extract as samples. Inhibitory activity of hyaluronidiases of each sample was measured as follows.

100 μl of sample and 50 μl of hyaluronidiase solution (type IV-S, Sigma, 400U / mL) were added and reacted at 37 ° C. for 20 minutes, followed by enzyme activation solution (compound 48/80 CaCl ₂ ㆍ 2H ₂ O, Sigma). , 0.1 mg / ml) was added to 100 µl and reacted again at 37 ° C. for 20 minutes. 250 µl of a hyaluronic acid solution (0.4 mg / ml) was added thereto and reacted at 37 ° C. for 40 minutes, and 100 µl of 0.4N NaOH was added to terminate the reaction. 100 μl of potassium borate solution was added thereto, reacted at 95 ° C. for 3 minutes, cooled, and then, 3 ml of ρ -dimethylaminobenzaldehyde solution was added and reacted at 37 ° C. for 20 minutes, followed by color development. Absorbance was measured at 565 nm to determine the inhibition rate for hyaluronidiases. Inhibition rate (%) for hyaluronidiases was calculated by the equation (4), IC ₅₀ value is the concentration of a substance that inhibits the hyaluronidiase enzyme activity by 50%.

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

A: Enzyme activity of wells without sample

B: Enzyme Activity of Well Added Samples

 Sample Name Inhibitory effect of hyaluronidiases (IC ₅₀ )  Volcanic Water (Example 1)  0.1%  Comfrey Extract  0.3%  Seesaw extract  0.5%  Triticale extract  0.2%

As a result of measuring the inhibitory effect of hyaluronidiasis by volcanic water (Table 6), the IC ₅₀ value of volcanic water (Example 1) was 0.1%, and the existing anti-inflammatory agent known to be excellent in inhibiting hyaluronidase. Compared to the phosphorus extract, trichoola extract, seesaw extract and the like showed an excellent effect.

From the above results, it was confirmed that the production method of the volcanic water for cosmetics of the present invention can produce volcanic water exhibiting an anti-inflammatory effect by inhibiting the activity of hyaluronidia.

Experimental Example  7: Example  1, when processing Inhibition of Inflammatory Cytokine Expression by UV Irradiation

In Experimental Example 7, to determine the anti-inflammatory effect of the volcanic water obtained in Example 1 was measured the degree of inhibition of the expression of inflammatory cytokines (hereinafter referred to as 'IL-1α') expressed by ultraviolet irradiation.

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

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

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

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

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

Sample Name Treatment concentration (%) Inhibitory rate of inflammatory cytokine expression (%) Volcanic Water (Example 1) One 32 2 58

As a result of measuring the inhibitory effect of inflammatory cytokine expression by ultraviolet irradiation when treating volcanic water (Table 7), it was confirmed that volcanic water inhibited the expression of inflammatory cytokines by 32% and 58% at 1% and 2% concentrations, respectively. It can be seen that it effectively protects the occurrence of inflammation caused by ultraviolet rays at low concentrations.

From the above results, it was confirmed that the manufacturing method of the volcanic water for cosmetics of the present invention can produce volcanic water having an anti-inflammatory effect.

Experimental Example  8: Example  Antibacterial ability test (1 of acne bacteria by 1 Paper Disc Test )

In Experimental Example 8, a paper disc test was conducted to confirm the antibacterial activity of acne bacteria caused by volcanic water obtained in Example 1.

First, in order to activate propionibacterium acnes, a skin acne causing cause of acne, pre-cultivation was performed in BHI liquid medium (Brain-Heart Infusion Broth; 3.7%) for 48 hours. The culture medium thus prepared was smeared in 0.1 ml each of BHI solid medium (Brain-Heart Infusion Broth; 3.7%; agar 1.5%) and dried. The volcanic water obtained in Example 1 was diluted to 20% (w / v) in a 95% ethanol aqueous solution, and 50 µl each was added to a 8 mm diameter paper disc and placed on the solid medium prepared above. This was incubated for 3 days in a 35 ° C. anaerobic culture tank.

The antimicrobial activity was evaluated by observing the bacterial growth inhibition area around the paper disc and measuring the size of the ring.

As a result of testing the antibacterial activity of acne bacteria by volcanic water, the size of the bacterial growth inhibitory ring of volcanic water (Example 1) was 17 mm, and the antibacterial activity against acne bacteria could be confirmed.

From the above results, it was confirmed that the manufacturing method of the volcanic water for cosmetics of the present invention can produce volcanic water having an anti-acne effect.

Experimental Example  9: Example  5 alpha-by 1 Reductase  Activity inhibition effect measurement

This Example measured the inhibitory effect of 5 alpha -reductase activity to confirm the anti-acne effect of volcanic water obtained in Example 1.

As the 5 alpha-reductase used in this experimental example, an enzyme produced by epidermal fibroblasts was used.

Fibroblasts were inoculated to inoculate 10000 cells per microplate hole and then cultured. Each hole was cultured after adding 0.1.mμ Ci of radiolabeled testosterone with 3H (tritium), and then fibroblasts were used. No volcanic water (Example 1) was used as a control. After 24 hours of incubation, the supernatant was obtained, steroids were obtained with 1 ml of ethylacetate-cyclohexane (1: 1) extractant, and the resulting steroids were placed on a thin layer chromatography plate, followed by chloroform / methanol. It developed with the mixed liquid (98/2 (v / v)). The radioactivity of the points corresponding to testosterone and dihydrotestosterone was measured using a densitometer to calculate the conversion, and the results were compared with the control group (conversion rate when no volcanic water (Example 1) was added). 5 alpha-reductase inhibitory ability was evaluated by the equation (6).

5 Alpha-reductase inhibitory activity (%) = [A-B] / [A] × 100

A = conversion of testosterone to dihydrotestosterone (if no volcanic water (Example 1) is added)

B = conversion of testosterone to dihydrotestosterone (with addition of volcanic water (Example 1))

Sample Name Treatment concentration (% 0 Inhibitory Activity of 5 Alpha-Reductase (%) Volcanic Water (Example 1) 0.1 32 0.5 51 One 64 2 83

As a result of measuring the activity inhibitory effect of 5 alpha-reductase by volcanic water (Table 8), it was confirmed that volcanic water (Example 1) inhibited 5 alpha-reductase in a concentration-dependent manner.

From the above results, it was confirmed that the manufacturing method of the volcanic water for cosmetics of the present invention can produce volcanic water having an anti-acne effect.

Example  2 to Example  4 and Comparative example  One: Cosmetics  Produce

In Examples 2 to 4 and Comparative Example 1, a cosmetic was prepared using the volcanic water obtained in Example 1 above.

The prepared cosmetics were in the form of a cream, the composition of which is shown in Table 9.

The 'Na' phase recorded in Table 9 was heated and stored at 70 ° C. The preliminary emulsification was performed by adding the 'ga' phase to the preserved 'na' phase, and then uniformly emulsified with a homomixer, and then gradually cooled. Creams (Examples 2 to 4 and Comparative Example 1) were prepared.

Unit: weight%  Raw material Example 2 Example 3 Example 4  Comparative Example 1


end


Stearyl alcohol 8 8 8 8 Stearic acid 2 2 2 2 Stearic Acid Cholesterol 2 2 2 2 Squalane 4 4 4 4 2-octyldodecyl alcohol 6 6 6 6 Polyoxyethylene (25 mol addition) alcohol ester 3 3 3 3 Glyceryl Monostearic Acid Ester 2 2 2 2
I
Volcanic Water (Example 1) 10 5 2 - Propylene glycol 5 5 5 5 Purified water Rest about 100 100 to
One rest 100 to
One rest 100 to
One rest

Experimental Example  10: Skin whitening effect measurement

In Experimental Example 10, the whitening effect of the cosmetics prepared in Examples 2 to 4 and Comparative Example 1 was measured.

For 15 experimenters (women aged 20 to 35 years), Example 2 was applied to the right side of the face and Comparative Example 1 was applied to the left side of the face twice a day for 2 consecutive months. After completion of the test, the skin of the applied areas on both sides of the face was compared with the face color using an image analyzer and a color difference meter, and the darkest color was set to 5, the middle color to 3, and the brightest color to 1, and the intermediate degree was estimated and evaluated in Table 10 below. The facial skin color of the experimenter using Example 2 is shown in comparison with the facial skin color of the experimenter using Comparative Example 1.

Product used Example 2 Comparative Example 1 Experiment number Right skin color Left skin color One 2.0 3.0 2 2.5 3.5 3 1.5 2.5 4 2.0 2.5 5 3.0 3.5 6 1.5 2.0 7 2.5 3.5 8 2.0 2.5 9 2.5 3.5 10 1.5 2.5 11 2.0 3.5 12 1.5 2.5 13 1.5 2.5 14 2.0 2.5 15 2.5 3.5

As a result of measuring the skin whitening effect (Table 10), the facial skin of the experimenter who applied Example 2 containing volcanic water (Example 1) was coated with Comparative Example 1, which did not contain volcanic water (Example 1). It was found to be brighter than the facial skin of the experimenter.

The excellent whitening effect of the cosmetic composition of this invention was confirmed from the said result.

Experimental Example  11: Skin elasticity improvement effect measurement

In Experimental Example 11, in order to measure the skin elasticity improvement effect of the cosmetics prepared in Examples 2 to 4 and Comparative Example 1 by applying Example 2 and Comparative Example 1 to the human skin elasticity improvement effect A comparative experiment was conducted and measured.

15 experimenters (women aged 20 to 35 years) were applied to Example 2 on the right side of the face and Comparative Example 1 on the left side of the face for 2 consecutive months, 2 times a day.

The skin elasticity improvement effect after the completion of the experiment was measured using a skin elasticity measuring instrument (cutometer SEM 575, C + K Electronic Co., Germany) before and after using the product for 2 months. The experimental results are described as ΔR8 value of 'Cutometer SEM 575' in Table 11 below, where R8 value represents the nature of viscoelasticity of the skin.

Product used Skin elasticity effect (ΔR8) Example 2 0.26 Comparative Example 1 0.12

 n = 15, p <0.05

As a result of measuring the skin elasticity improving effect (Table 11), it can be seen that the skin elasticity improving effect of the experimenter who applied Example 2 containing volcanic water (Example 1) as compared to Comparative Example 1.

The skin elasticity improvement effect of the cosmetic composition of this invention was confirmed from the said result.

Experimental Example  12: Measured to improve skin wrinkles

In Experimental Example 12, the cosmetics prepared in Examples 2 to 4 and Comparative Example 1 were applied to humans to measure skin wrinkle improvement.

15 experimenters (women aged 20 to 35 years) were applied to Example 2 on the right side of the face and Comparative Example 1 on the left side of the face for 2 consecutive months, 2 times a day.

After the experiment was completed, wrinkles around the face of the eyes were collected with a silicone resin copy (replica) before and after using the product for two months, and the skin fine wrinkle device and the skin image analyzer were used to compare the condition of the eye wrinkles.

Table 12 compares the average eye wrinkle depth and wrinkle reduction rate of the experimenter using Example 2 with the experimenter using Comparative Example 1.

Example 2 Comparative Example 1 Before use 2 months later Before use 2 months later Average fine wrinkle depth (AU) 0.203 0.172 0.202 0.196 Wrinkle Reduction (%) 15.3% 3.0%

 n = 15, p <0.05

As a result of measuring the skin wrinkle improvement effect of the cosmetic (Table 12), compared to Comparative Example 1, it was found that the effect of improving the skin wrinkles was excellent in the facial periphery skin of the experimenter who applied Example 2 containing volcanic water (Example 1). Able to know.

From the above results, it was possible to confirm an excellent skin wrinkle improvement effect of the cosmetic composition of the present invention.

Claims (12)

As a method of manufacturing a cosmetic composition for improving wrinkles and elasticity, The volcanic ash was pulverized to obtain a volcanic ash powder having a size of 50 to 200 mesh, and then the volcanic ash powder obtained was purified water having a pH of 7-11, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, dichloromethane, hexane and mixtures thereof. After the addition of any one of the extraction solvent selected from, by ultrasonic extraction 1 to 3 times at 60 ℃ for 1 to 3 times to obtain an extract, the obtained extract is filtered and sterilized to prepare a cosmetic volcanic water ; And Method for producing a cosmetic composition for improving wrinkles and elasticity, comprising the step of including the cosmetic volcanic water 0.001 ~ 100% by weight based on the total cosmetic composition. delete delete delete delete delete delete delete delete delete delete delete