KR20110041103A - Skin external composition for antiaging containing caviar and ocean minerals - Google Patents

Abstract

PURPOSE: An external use skin composition containing mineral complex ingredients is provided to ensure antioxidation, gene protection, and collagen biosynthesis. CONSTITUTION: An external use skin composition for anti-aging contains 0.0001-10 weight% of mineral complex ingredients isolated from caviar, oyster shell, and surwater sediment. The mineral complex ingredients are obtained by primary extraction of caviar, oyster shell, and surwater sediment using anhydrous or hydrous low alcohol and acetone, and by reextraction of the extract using ethyl acetate, diethyl ether, benzene, chloform, and hexane.

Description

Skin external composition for antiaging containing caviar and ocean minerals containing mineral complex components extracted from caviar, oyster shell and sea persimmon

The present invention relates to a skin external preparation composition containing a mineral complex component extracted from caviar and oyster shell, seaweed (sea sediment), specifically, the invention is a mineral complex extracted from caviar, oyster shell, seaweed (sea sediment) The present invention relates to a skin external preparation composition for preventing skin aging, which contains components, has been found to be excellent in antioxidant, cell membrane protective effect, gene protective effect, collagen biosynthesis promoting effect, and is excellent in anti-aging skin and improving wrinkles.

Skin tissues have various antioxidant systems and protective factors, such as heat shock protein (HSP), SODs, catalases, etc. to protect the skin.However, skin cells may be damaged due to deactivation caused by endogenous or photoaging. Injury occurs and the biosynthesis of matrix metalloproteinases (MMPs), an enzyme that degrades skin tissues, increases collagen biosynthesis, reduces wrinkles, reduces elasticity, and induces skin aging. In addition, DNA damage and the structure of the nuclear membrane collapses with aging. Repairing damaged DNA and maintaining the structure of the nuclear membrane are important factors in preventing aging. Thus, antioxidants can protect skin cells, MMPs can inhibit biosynthesis, which breaks down collagen, a substrate that makes up skin, substances that prevent damage to skin cells, DNA damage repair proteins and nuclear membrane structures Substances that induce the production of oil and fat proteins have been found to mitigate skin aging.

Among the substances that alleviate the skin aging, research and development of functional natural materials have been steadily made to prevent or solve the skin aging phenomenon using natural raw materials. The greatest feature of natural skin care is not only to identify the cause of skin aging and improve systemic symptoms by looking at the human body from the whole point of view, but also to fundamental treatment to prevent the recurrence by fundamentally improving the cause of skin problems. The emphasis is on.

The present invention contains a mineral complex component extracted from caviar, oyster shell, and sea persimmon (sea sediment) sequentially by the polarity change as an active ingredient to protect and activate skin cells, the skin external agent for preventing skin aging excellent in promoting collagen production It is to provide a composition.

[Configuration of Invention]

In order to achieve the object as described above, the external preparation composition for skin according to the present invention is characterized in that the invention contains a mineral complex component extracted from caviar, oyster shell, seaweed (sea sediment). In the external preparation composition according to the present invention, the present invention is characterized in that the mineral composite component extracted from caviar, oyster shell, and sea persimmon (sea sediment) is contained in a total weight of 0.0001% by weight to 10% by weight of the total composition.

In the external preparation composition for skin according to the present invention, the external preparation composition has been found to be excellent in antioxidant effect, cell membrane protection effect, gene protection effect, collagen biosynthesis promoting effect, and has the effect of preventing skin aging and improving wrinkles.

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

In the present invention, caviar, oyster shell, and seaweed (sea sediment) are first extracted with anhydrous or lower alcohol, acetone, and then reextracted with low polar solvent such as ethyl acetate, ethyl ether, benzene, chloroform, and hexane. Maximizing the content of ingredients and removing impurities to improve stability and at the same time, extracts with maximum efficacy are added to external cosmetics such as supple cosmetics using solubilization technology to nutrient cosmetics, cream, and essence applying emulsification technology.

The present invention will be described in more detail as follows. Caviar, oyster shell and sea persimmon (sea sediment) are dried to dry weight with water, lower alcohol (methanol, ethanol), mixture of water and lower alcohol, acetone, 1,3-butylene glycol, normal propanol, Polar solvents such as isopropanol and normal butanol and mixed solvents thereof are added by 5-20 times by volume, followed by immersion extraction and filtration. The filtrate is concentrated under reduced pressure. Water is added to the concentrate to disperse the same, and then the same amount of low polar organic solvents such as ethyl acetate, chloroform, and ethyl ether are added and shaken. The organic layer is separated and concentrated under reduced pressure to obtain an excellent extract. Extraction method is about 12-96 hours for cold acupuncture and percolation or 3-20 days at 4-25 ° C at room temperature using anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms, ethyl acetate or dietyl ether as solvents. The active ingredient can also be extracted by aging. In the case of a warm needle, it is different depending on the type and temperature of the solvent and the like used, but it is preferable to perform about 5-24 hours at a temperature close to the reflux temperature of the solvent.

As described above, the external preparation composition for skin containing the mineral complex component extracted from caviar, oyster shell and sea persimmon (sea sediment) of the present invention was confirmed to be excellent in antioxidant, cell membrane protective effect, gene protective effect collagen biosynthesis promoting effect It prevents skin aging and improves wrinkles. Due to this effect, it can be usefully used as an external skin preparation for the purpose of anti-aging and wrinkle improvement.

Example 1

10 g of caviar, oyster shell, and sea persimmon (sea sediment) dried in 100 ml of 50% ethanol aqueous solution, boiled for 12 hours in an extractor with a cooling condenser, extracted with 300 mesh filter cloth, and the filtrate was dried at 4-15 ° C. After aging for one day, it is filtered with Whatman No. 2 filter paper. The extract was added to a 3-liter separatory funnel, and 1 liter of lactate was added thereto. After shaking, the mixture was rotted well, completely separated into two layers, and the upper layer (ethyl acetate layer) was taken. The lower layer (water layer) is again extracted twice with a separatory funnel. The combined upper layers were combined, concentrated under reduced pressure at 50 ° C. using a distillation apparatus equipped with a cooling condenser, and dried to obtain a dry weight of 22.1 g.

[Test Example 1] Wrinkle improvement effect

Wrinkle improvement effect of the mineral complex extracted from caviar, oyster shell and sea snail (sea sediment) is a 96-well microplate (96-well microplate) in which human normal fibroblasts are used to see the skin cell proliferation effect. Inoculate each well to 1 × 10 ⁴ cells and incubate for 24 hours in DMEM (dulbecco, Modified Eagle's Medium) medium. After culturing, the pine root extract of Example 1 from the pineal extract of Comparative Example 1 prepared above was replaced with a DM medium without serum adjusted to a final concentration of 500 g / ml, followed by further culturing for 24 hours. 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (MTT: (3- (4,5-dimdimeth-thiazol-2-yl) 2,5-diphenyl tetrazolium: 5 mg / ml) was added 10 l and left for 4 hours, and then the media section was discarded, and 100 l of dimethyl sulfoxide urea was added to each well, and the absorbance at 570 nm was measured with a microplate reader.

By performing the above procedure, the cell proliferation effect was obtained from Equation 1 below, and the results are shown in Table 1.

Equation 1

Cell proliferation effect (%) = [(absorbance-control absorbance at extract treatment) / absorbance of control] × 100

TABLE 1 Cell proliferation effect

As shown in Table 1, it was found that the normal fibroblast cell proliferation effect of the mineral complex extracted from caviar and oyster shell, sea persimmon (sea sediment).

Test Example 2 Collagen Biosynthesis Promoting Effect

To investigate the collagen biosynthesis-promoting effect of mineral complexes extracted from caviar, oyster shell and seaweed (sea sediment), human well fibroblasts were collected from each well of a 96-well microplate. Inoculate 1 × 10 ⁴ cells and incubate for 24 hours in DMEM (dulbecco, s Modified Eagle's Medium) medium. After culturing, Example 1 prepared above was replaced with cadmium, oyster shell, and mineral complex components extracted from sea persimmon (sea sediment) with serum-free DM medium adjusted to a final concentration of 500 g / ml, followed by further incubation for 48 hours. . Ascorbic acid 50 g / ml is added to promote collagen synthesis before the last 24 hours of culture. After incubation, each well is washed, replaced with DM medium without serum, and incubated for another 24 hours. After incubation, the supernatant of each well was collected, and the amount of newly synthesized collagen was measured by the amount of PICP using a kit (Kit, Takara, Kyoto, Japan) using procollagen type I-peptide (PICP). PICP amount is shown in Table 2 below to synthesize the collagen in terms of ng / 2 × 10 ⁴ cells.

[Table 2] Collagen biosynthesis promoting effect

As shown in Table 1, it was found that the collagen biosynthesis promoting effect of the mineral complex component extracted from caviar and oyster shell, sea persimmon (sea precipitate).

Test Example 3 Free Radical Scavenging Activity

The method used to measure the antioxidant power was 1,1-diphenyl-2-pycryl-hydrazyl (DPPH) method, caviar, oyster shell, seaweed ( Mineral complex extracted from marine sediment) diluted in ethanol by concentration (0, 50, 100, 200, 300, 400 ㎍ / mL) in 10-well 96-well plates, and DPPH prepared in 5 mM ethanol solution After the volume was left to 200 and left at 37 to 30 minutes, absorbance was measured using a 520 nm ELISA reader (DI biotech, Korea). At this time, vitamin C, which is widely known to have excellent antioxidant power, was used as a positive control group.

[Equation 1]

% Radical scavenging activity = [(absorbance of control-absorbance of sample) / absorbance of control] × 100

The radical scavenging activity of the mineral complex extracted from caviar, oyster shell, and sea persimmon (sea sediment) measured by the method of DPPH is shown in Table 3.

The results showed that the mineral complex extracted from caviar, oyster shell, and sea persimmon (sea sediment) is less potent than vitamin C, which is known as a powerful antioxidant, but has radical scavenging activity at concentrations of about 100 µg / mL or more.

Table 3

Test Example 4 Measurement of Cell Membrane Damage Protection

Human keratinocytes HaCaT cell line (cell line) in each well of a 96-well plate 100 cells of 1 × 10 ⁵ cells / mL were put in 100, 37 and 5% CO ₂ incubator for 24 hours to attach. After incubation, the concentration of the mineral complex extracted from the caviar, oyster shell, and sea persimmon (sea sediment) of the example was replaced with a culture solution containing 0.625, 1.25, 2.5, and 5 µg / mL, followed by incubation for 3 hours. The culture was removed and 50 μl of phosphate buffered salt was added to each well. After irradiating with UV 30mJ / cm ² using an ultraviolet B lamp, remove the phosphate buffered saline solution and 200µl of the cell culture medium containing the mineral complex extracted from the caviar, oyster shell, and seaweed (sea sediment) of the above concentration in each well. Addition was incubated for 24 hours. After 24 hours, an appropriate amount of the culture supernatant was taken and the amount of lactate dehydrogenase (LDH), an indicator of cell damage, was measured using the cytotox 96 non-radioactive cytotoxicity assay kit, and the average value was calculated after 6 repeated measurements. At this time, CTL is a control group that did not process the mineral complex extracted from caviar and oyster shell, sea persimmon (sea sediment).

The results of measuring the amount of lactate dehydrogenase (LDH), an indicator of cell damage, using 96 non-radioactive cytotoxicity assay kits are shown in Table 4 below.

Table 4

In the group treated with concentrations of mineral complexes extracted from caviar, oyster shell, and sea persimmon (sea sediment) and irradiated with ultraviolet B, the increase in activity in LDH cell culture medium by UVB irradiation showed a concentration-dependent decrease. . In particular, the treatment of mineral complexes extracted from 5 μg / mL caviar, oyster shell, and sea persimmon (sea sediment) showed that LDH was suppressed to the control level without irradiation of UVB. From these results, it can be judged that the mineral complex component extracted from caviar, oyster shell and sea persimmon (sea sediment) effectively inhibits cell membrane damage of HaCaT cells by ultraviolet B irradiation.

Test Example 5 Determination of Reactive Oxygen Species Production

In each well of a fluorescence 96-well black plate, 2.0 × 10 ⁵ cells / mL of human keratinocytes HaCaT cells were dispensed at 100 and incubated in 37, 5% CO ₂ incubator for 24 hours. After the caviar and oyster shell of the above example, was treated with a mineral composite component extracted from sea persimmon (sea sediment). Concentrations of the extract were 0.625, 1.25, 2.5, and 5 µg / mL. Incubate the test sample for 24 hours, wash with HEPES-buffered control salt solution (HCSS) to remove the remaining medium, and 100µl of 2 ', 7'-dichlorodihydro-fluorescein diacetate (DCFH-DA) prepared at 20μM in HCSS. Incubated for 20 minutes in a 37, 5% CO ₂ incubator and washed again with HCSS. Thereafter, 100 μl of HCSS treated for each concentration of the mineral complex extracted from caviar, oyster shell, and seaweed (sea sediment) was added, followed by irradiation with ultraviolet light B (30mJ / cm ² ), followed by fluorescence spectroscopy (fluorometer). ) (Ex = 485 nm, Em = 530 nm). The measured results showed the average value after 6 repeated measurements, and CTL was a control group that was not treated with mineral complex components extracted from caviar, oyster shell, and seaweed (sea sediment).

After treatment with the extract, the cells were irradiated with ultraviolet rays and measured for fluorescence after 3 hours. As a result, the active oxygen species increased by 182.1% in the group irradiated with ultraviolet B as compared to the control group not irradiated with ultraviolet rays. This increase was observed in the generation of reactive oxygen species in a concentration-dependent manner when the mineral complex components extracted from caviar, oyster shell and sea persimmon (sea sediment) by concentration (Table 5). From these results, it can be judged that the mineral complex component extracted from caviar, oyster shell and sea persimmon (sea sediment) has an excellent effect of inhibiting the generation of intracellular reactive oxygen species by UV irradiation.

[Table 5]

[Test Example 6] Measurement of recovery of reduced expression of superoxide dismutase (SOD) and catalase

2 mL of 1 × 10 ⁵ cell / mL human keratinocytes HaCaT cells were placed in a 6-well plate and incubated for 24 hours in a 37, 5% CO 2 incubator, followed by caviar, oyster shell, and seaweed (sea sediment). The extracted mineral composite component was treated at a concentration of 5 μg / mL. Next, after culturing for 24 hours, the remaining medium was removed by washing with phosphate buffered saline solution, 50 μl of phosphate buffered saline was added and irradiated with ultraviolet ray B. After removing the phosphate buffered saline solution, caviar, oyster shell, and seaweed (sea sediment) The mineral complex components extracted from the treatment were incubated for 48 hours. After 48 hours of incubation, the cells were collected and cell lysate (Lysis buffer: 250 mM NaCl, 25 mM Tris-HCl pH 7.5, 5 mM EDTA pH8.0, 1% NP-40, 0.1M PMSF, 1M DTT, protease inhibitor cocktail.DW) After crushing at 4, the amount of protein was quantified with BCA solution. Samples were prepared by mixing the same amount of protein and buffer solution, and then separated by sodium dodesyl sulfate (SDS) polyacrylamide gel electrophoresis. Acrylamide gel containing protein isolated for Western blot analysis was transferred to nitrocellulose membrane by electroblotting and washed twice with TBS-T (0.1% Tween 20 in TBS) containing 5% skim milk. . After washing, the SOD and the catalase primary antibody (1: 2000) were treated and left for 4 to 12 hours, and then reacted at room temperature for 1 hour using a secondary antibody (1: 2000) suitable for the primary antibody. After washing with TBS-T, an enhanced chemiluminescence (ECL) solution was applied, and the expression patterns of SOD and catalase proteins were compared by photosensitive X-ray film in the dark. In order to confirm that the protein has been transferred in the same amount, the above procedure was repeated using actin as the primary antibody.

After 48 hours of irradiation with UVB on HaCaT cells, the expression of SOD and catalase was significantly reduced, and the expression of SOD and catalase was remarkably decreased. From caviar, oyster shell, and seaweed (sea sediment) It was observed that the treatment of 5 ㎍ / mL of the extracted mineral complex component effectively inhibited the decrease of antioxidant enzyme expression by ultraviolet rays (Table 6). From these results, mineral complexes extracted from caviar, oyster shells and seaweeds (sea sediments) not only have antioxidant properties in themselves, but also damage the antioxidant enzyme system held in the cell against oxidative stress caused internally or externally. It can be judged that it has an excellent protection effect and ultimately protects skin cells.

Table 6

Test Example 7 Cell Protection Measurement

HSP70, a protein induced in response to stress, is known to protect cells and individuals against various stresses and to prevent cell death. In addition, HSP70 protein, which is overexpressed in advance, is known to protect cells from stress and various toxicity. To measure cell protection by promoting HSP70 production, 2 mL of 1 × 10 ⁵ cell / mL normal fibroblasts were added to a 6-well plate and cultured for 24 hours in a 37, 5% CO ₂ incubator to attach cells. After treatment, the mineral composite components extracted from caviar, oyster shell, and sea persimmon (sea sediment) were treated with 0.25, 0.5, and 1 ㎍ / mL. After the test sample was treated and cultured for 24 hours, the cells were collected, crushed at 4 with cell disruption solution, and the protein amount was quantified with BCA solution. Samples were prepared by mixing the same amount of protein and buffer solution, and then separated by sodium dodesyl sulfate (SDS) polyacrylamide gel electrophoresis. Acrylamide gel containing protein isolated for Western blot analysis was transferred to nitrocellulose membrane by electroblotting, and then stained with Ponceus S solution to confirm the same amount of transfer. The protein-transferred membrane was incubated for 1 hour at room temperature with TBS-T (0.1% Tween 20 in TBS) containing 5% skim milk to block nonspecific proteins and washed twice with TBS-T. It was. After washing, the HSP70 primary antibody (1: 1000) was treated and left at 4 ° C. overnight, and then reacted at room temperature for 1 hour using a secondary antibody (1: 2000) suitable for the primary antibody. After washing with TBS-T, an enhanced chemiluminescence (ECL) solution was applied to the X-ray film in the dark, and the expression patterns of HSP70 proteins were analyzed.

Fibroblast was treated with mineral complexes extracted from caviar, oyster shell, and sea persimmon (sea sediment) and western blot using HSP70 antibody. As a result, plant stem cell extract isolated from pine root growth point was treated more than 1㎍ / mL. HSP70 protein production was induced a lot, and HSP70 protein production was induced depending on treatment concentration. Therefore, the mineral complex extracted from caviar, oyster shell and sea persimmon (sea sediment) was able to determine the HSP70 protein to a certain level or more can be shown to protect the cells against the stress given later.

Table 7

Test Example 8 Measurement of MMP-2 Biosynthesis Inhibition

When UV rays are irradiated to cultured skin cells and human skin, the expression of various matrix metalloproteinases (MMPs) increases, and the increased MMPs break down collagen constituting the skin and form skin wrinkles. The mineral complex extracted from caviar, oyster shell and seaweed (sea sediment) for the inhibition of ECM damage by UV rays by increasing the amount of deficiency of collagen in aged skin by evaluating the degree of aging of aged cells. In order to test the effects of the ingredients, 2 mL of 1 × 10 ⁵ cell / mL normal fibroblasts were added to a 6-well plate and incubated in 37, 5% CO ₂ incubator for 24 hours to attach the cells, followed by caviar and Mineral complex components extracted from oyster shell and sea persimmon (sea sediment) were treated with 0.25, 0.5, 1 μg / mL. After irradiating with UV B 30mJ / cm ² and treated with caviar and oyster shell, mineral complex components extracted from sea persimmon (sea sediment) was incubated for 24 hours. After incubation, the cell culture solution was collected, a sample was prepared by mixing the cell culture solution and the buffer solution, and then introduced into a gelatin-containing zymogram gel and separated by electrophoresis. In accordance with the reagents, the gramogram gel containing the separated protein for MMP-2 identification was incubated for 30 minutes at room temperature with renaturing buffer (2.7% Triton X-100), and then developed (developing buffer) (50mM Tris Base, 40mM 6N HCl, 200mM NaCl, 5mM CaCl ₂ 2H ₂ O, BriJ 35 0.02%) was incubated at room temperature for 1 hour, and then changed to fresh developing buffer and reacted at 37 ° C overnight. After completion of the reaction, the gel was stained to compare the degree of biosynthesis. In order to confirm that there is no difference between samples, cells were collected and crushed at 4 ° C. with cell lysate, and then transferred to nitrocellulose membrane by electroblotting and stained with Ponceus S solution.

MMP-2 biosynthesis was measured in cell cultures treated with mineral complexes extracted from caviar, oyster shell, and sea bream before and after UV irradiation. It was confirmed that MMP-2 biosynthesis was inhibited by 1 μg / mL treatment of the composite component and was dependent on the treatment concentration [Table 8].

[Table 8]

Test Example 9 Expression Induction Effect of SIRT1 and LMNA1 Genes

HaCaT cells were injected into DMEM medium containing 10% (v / v) FBS, 100 U / ml penicillin and 100 μg / ml of streptomycin and cultured in an animal cell incubator with 37 ° C., 5% CO ₂ feed conditions. HaCaT cells prepared at a concentration of 1.5 × 10 ⁶ per well were acclimated with medium without FBS for 3 hours. Thereafter, the mineral complex components extracted from caviar, oyster shell, and sea persimmon (sea sediment) were each treated at 10 ppm and incubated for 24 hours. Total RNA was extracted using TRIzolTM (GIBCO BRL, MD, USA) and stored at -80 ° C. 1 g total RNA was placed in 25 l reverse transcription reaction buffer containing 50 mM Tris-HCl (Tris-HCl, pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , 0.1 M DTT, 10 mM dNTP and 40 units / l RNase inhibitor, 0.5 g / l Primer of oligo (dT) 16 and reverse transcriptase of 200 units SuperScript II (GiboBRL) were added and reacted at 42 ° C for 1 hour, and 2.5l of reverse transcript reaction solution was amplified (AmpliTaq). ) 50 ml of PCR reaction buffer containing DNA polymerase (0.04 U, Perkin Elmer, Shelton, CT), 50 mM Tris (pH 8.3), 0.25 mg / ml bovine serum albumin, 3 mM MgCl ₂ , 0.25 mM dNTPs, 10M 30 cycles of denaturation at 94 ° C for 30 seconds, annealing at 53 ° C for 30 seconds, and expansion at 72 ° C for 1 minute were performed. Images were acquired after electrophoresis on 1% agarose gel. Each primer was prepared based on SCI papers.

Cells were treated with caviar, oyster shell, and mineral complex components extracted from seaweed (sea sediment), and PCR was confirmed to express SIRT1 and LMNA1 genes (Table 9). By inducing the expression of the SIRT1 gene that repairs DNA damage and the LMNA1 gene that maintains the structure of the nuclear membrane, it has been shown to be effective in preventing skin aging.

TABLE 9

Claims (8)

Anti-aging skin external composition comprising caviar, oyster shell and mineral complex extracted from sea persimmon (sea sediment) The method of claim 1, The external skin composition is an external composition for skin, characterized in that it has a skin cell (fibroblast) proliferation effect The composition for external application for skin according to claim 1, wherein the composition for external application for skin has a function of promoting collagen biosynthesis. The external preparation composition for external skin, characterized in that it has a function of free radical scavenging and inhibiting the generation of reactive oxygen species. The external preparation composition for external skin, characterized in that it has a cell membrane damage protection function The external skin composition is a skin external composition, characterized in that it has a superoxide dismutase (SOD) and catalase expression inducing function The external preparation composition for external skin, characterized in that it has a function of inhibiting MMP-2 biosynthesis. The external skin composition is an external composition for skin, characterized in that it has a function of inducing the expression of SIRT1 and LMNA1 gene