KR100985143B1 - Cosmetic composition comprising minerals complex components for improving wrinkle - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving wrinkles comprising a mineral complex, in detail, a cosmetic composition for improving wrinkles comprising a mineral complex containing jade, talc, dolomite, biotite, kaolin, anatomical and organic germanium It is about. The mineral complex of the present invention has excellent antioxidant activity, no cytotoxicity, and increased collagenase (MMP-1) and gelatinase (MMP-2) after irradiation with ultraviolet (UVA) to HDF, a fibroblast cell line of human dermis. ) And the collagenase (MMP-1) mRNA and gelatinase (MMP-2) mRNA expression inhibitory effect, collagen synthesis promoting effect and elastase activity inhibitory effect, hepatocytes It is excellent in the regeneration promoting effect, safe and irritating to human skin, it can be usefully used in cosmetic compositions for improving skin wrinkles.

Description

Cosmetic composition for improving wrinkles including mineral complex components

1 is a view of a particle of the mineral composite component of the present invention observed under a scanning electron microscope (magnification: x1000).

Figure 2 is a diagram showing the emissivity and radiation energy of the mineral composite component of the present invention.

3 is a diagram showing the antioxidant activity of the mineral complex component of the present invention.

Figure 4 is a diagram showing the cytotoxicity results of the mineral complex component of the present invention.

5 is a diagram showing the collagenase (MMP-1) activity inhibitory effect of the mineral complex of the present invention after ultraviolet (UVA) irradiation to the fibroblast cell line HDF of human dermis.

Figure 6 is a diagram showing the inhibitory effect of gelatinase (MMP-2) activity of the mineral complex of the present invention after ultraviolet (UVA) irradiation to the fibroblast cell line HDF of human dermis.

7 is a diagram showing the mRNA expression inhibitory effect of collagenase (MMP-1) of the mineral complex of the present invention after ultraviolet (UVA) irradiation to the fibroblast cell line HDF of human dermis.

8 is a diagram showing the mRNA expression inhibitory effect of gelatinase (MMP-2) of the mineral complex of the present invention after ultraviolet (UVA) irradiation to the fibroblast cell line HDF of human dermis.

9 is a diagram showing the collagen synthesis promoting effect of the mineral complex of the present invention compared with the effect of the mineral complex according to vitamin C, Comparative Example 1 and Comparative Example 2.

10 is a diagram showing the effect of inhibiting the elastase activity of the mineral complex of the present invention compared with the effect of the mineral complex according to polyphenols, Comparative Examples 1 and 2.

11 is a view showing the regeneration promoting effect on the hepatocytes of the mineral complex component of the present invention.

The present invention relates to a cosmetic composition for improving wrinkles comprising a mineral complex, in detail, a cosmetic composition for improving wrinkles comprising a mineral complex containing jade, talc, dolomite, biotite, kaolin, anatomical and organic germanium It is about.

In general, skin aging is divided into chronologic aging and photo aging. Chronologic aging is the generation of wrinkles due to the natural degeneration of age, and photoaging is the aging observed in sunlight-exposed skin. Severe and early observed. Photoaging is a relatively large part of skin aging and is the aging of skin caused by ultraviolet (UVA, UVB). Skin exposed to ultraviolet light causes irregular pigmentation, roughness and dryness of the skin, reduced elasticity and many wrinkles. In addition, UV-exposed skin produces large amounts of reactive oxygen species, which cause nucleic acid and protein modifications and genetic mutations. And alters the expression of genes involved in signaling systems, cell cycles, and cancer induction. In particular, the dermis has been reported to increase matrix metalloproteinases (MMPs) activity by ultraviolet rays and to reduce the synthesis of collagen and elastin to cause wrinkles.

In general, the dermis is composed of the majority of Type I collagen and some Type III collagen, elastin, proteoglycan, fibronectin, and the like, and these fibrous tissues are involved in the formation of skin wrinkles. Collagen and elastin in the dermal tissue are important fibrous tissues that work closely together to maintain skin flexibility and elasticity.

Collagen is a fibrous tissue that gives skin flexibility. It is a major component that accounts for 90% of the dermal layer. It gives strength and tension to the skin and protects the skin from external irritation. Polypeptide chains of collagen fibers are synthesized in a state in which fibroblast granules have extended peptides (telepeptides) at both ends, and then secreted extracellularly in the form of procollagen combined in a triple helix structure. The secreted procollagen is digested with an extended peptide by a specific enzyme to produce tropocollagen, which is combined with each other in the extracellular matrix to synthesize collagen fibers. Synthesis of such collagen fibers is rapidly reduced as the skin ages, collagen fibers are deformed by the external environment, and wrinkles are generated and deepened on the skin. The collagen is involved in collagenase, which is a collagen degrading enzyme.

In addition, elastin, like collagen, is a major fibrous tissue that influences the elasticity of the skin. The amount of elastin is about 2%, which is a small amount compared to collagen, but plays an important role in skin elasticity because it forms a network tissue supporting the epidermis. Elastin is degraded by a degrading enzyme called elastase, and it continues to be produced by fibroblasts until 18-19 years, but after that, elastin is weakened once it stops producing and must take care of skin elasticity with only elastin already produced. It is very difficult to recover its elasticity. That is, the deficiency and aggregation of elastin fibers and the dramatic increase in the activity of elastase are the main factors in the production of skin wrinkles. Therefore, by inhibiting the activity of elastase, the improvement of skin wrinkles can be fundamentally reduced.

In addition, matrix metalloproteinase (MMP) is a group of enzymes involved in the decomposition of extracellular matrix (ECM) and basement membrane, and interstitial collagenase, stromelysin, and gela, depending on their structural and functional properties. It is divided into four subfamily (gelatinase) and membrane-type MMP (MT-MMP). More than 20 MMPs are known to date in various species including mammals, birds, amphibians, plants and nematodes. MMPs also differ in substrate specificity but have structural and functional similarities. Thus, the names of all MMPs are generally numbered. Collagenase-1, 2, 3 is MMP-1, 8, 13, gelatinase-A, B is MMP-2, 9, stromelysin-1, 2, 3 is MMP-3, 10, 11, metalloelastase is MMP-12, matlylysin is MMP-7, membrane-type MMPs (MT-MMPs) are MMP-14, 15, 16, 17 , 24, ennamelysin (enamelysin) is called MMP-20, MMP-19, MMP-23 and the like. These enzymes can decompose the corresponding substrates, such as collagen, fibronectin, laminin, proteoglycans, entaxin, elastin, and the like. MMP is a metalloproteinase having zinc in its active center and is secreted in the form of an inactive precursor (zymogen) in vivo. In order to have enzymatic activity, structural modifications occur and the amino terminus must be cleaved, and activated MMPs are controlled by inhibitors such as 2-macroglobulin or TIMPs (Tissue inhibitors of metalloproteinase). The vast majority of cells, including keratinocytes and fibroblasts, secrete MMPs. MMP has been reported as a major factor in photoaging, since even after a single UV irradiation, MMP activity in the skin can be increased to significantly reduce collagen.

Currently, cosmetics for improving wrinkles are known, such as retinoids, adenosine, animal placental-derived proteins, chlorella extracts, and the like. The most well known retinol is not only to promote collagen synthesis but also to inhibit wrinkle activity by inhibiting elastase enzyme activity, but it is unstable and has limited use due to safety problems such as irritation and seizures when applying skin. Chlorella extract is known to be difficult to expect the effect of improving the skin wrinkles because the effect is insignificant.

Therefore, there is a need for a cosmetic composition that is safe and irritating to human skin, reduces the activity of MMP, increases the synthesis of collagen and elastin, and is useful for inhibiting skin aging.

Therefore, while the present inventors are studying a cosmetic composition that is safe and non-irritating to human skin and is useful for inhibiting skin aging, after mixing various mineral components in a specific ratio, the present inventors apply the mineral complex component to human skin cells irradiated with ultraviolet rays. As a result of application, the mineral complex has excellent antioxidant activity, no cytotoxicity, the inhibitory effect of collagenase (MMP-1) and gelatinase (MMP-2) and collagenase (MMP-1) It is excellent in inhibiting mRNA expression of mRNA and gelatinase (MMP-2), promoting collagen synthesis and elastase inhibitory effect, reproducing effect on hepatocytes, and not irritating to human skin. It confirmed that it was safe and completed this invention.

The present invention is to provide a cosmetic composition for improving wrinkles comprising a mineral complex component.

The present invention provides a cosmetic composition for improving wrinkles comprising a mineral complex containing jadeite, talc, dolomite, biotite, kaolin, anatomical and organic germanium.

Hereinafter, the present invention will be described in detail.

Cosmetic composition for improving wrinkles of the present invention is 1 to 10% by weight of jade, 10 to 20% by weight of talc, 20 to 35% by weight of mica, 10 to 20% by weight of mica, 35 to 45% by weight of kaolin, 1 to 3 weight of dissected stone It characterized in that it comprises a mineral composite component containing 0.1 to 0.3% by weight and organic germanium.

The characteristics of each component in the mineral complex is as follows.

Nephrite (CaO ₃ MgO ₄ SiO ₂ ) refers to a two-sumstone or tung-xanthite, which is a dense mass of entangled with fine fibrous crystals, and corresponds to jadeite. The quality is strong, so it does not break well, and when polished, it gives a mild luster. Hardness is 5.5 ~ 6.0, specific gravity is 2.9 ~ 3.0, and color is white and dark green. In Dongbobogam, "Okgar is sweet and has no poison, heats up the stomach and intestines, relaxes the five intestines, and releases waste products from the body. Agate (玉 水) promotes blood circulation and brightens the eyes." Modern clinical trials on purgatory have shown that it has excellent therapeutic effects such as headache, insomnia, blasting, and hand-picking, and the adsorption of anion by Ca ²⁺ is excellent in sterilization and waste adsorption, and can prevent acidification of body fluids. . In the cosmetic composition of the present invention, the content of the nephrite is preferably 1 to 10 wt% based on the total weight of the composition. If it is less than 1% by weight, the adsorption force by Ca ²⁺ is weakened, and if it is more than 10% by weight, the absorption of zinc, magnesium and iron components is hindered.

Talc (talc, Mg ₃ Si ₄ O ₁₀ (OH) ₂ ) is a rock belonging to the monoclinic system with a mica-like crystal structure. It can be cut with a knife having a hardness of 1 and specific gravity of 2.7 to 2.8. And pale green. Magnesium in talc is known to promote skin metabolism, contribute to structural stabilization of ATP energy, and promote protein synthesis. Magnesium also has the ability to regulate muscle contraction and relaxation as opposed to calcium. Talc is mainly used as a main ingredient in infant sweat bands, which removes the dust and lowers the heat, and is used to treat eczema. It is used as a base in cosmetics and as an additive in food or pharmaceuticals. The content of talc in the cosmetic composition of the present invention is preferably included 10 to 20% by weight relative to the total weight of the composition. If less than 10% by weight of the skin metabolism and muscles are too constricted, if more than 20% by weight the skin metabolism and muscles are too relaxed.

Muscovite (K (OHF ₂ ) ₂ A1 ₃ Si ₃ O ₁₀ ) is a monoclinic mineral containing small amounts of iron and magnesium. Hardness is 2 ~ 2.4, specific gravity is 2.7 ~ 3, colorless, white, yellow, gray, green, brown, and chloroform when chromium is contained. The fluorine component of the mica is known to inhibit the activation of melanin pigment, which has a whitening effect and moisturizes the skin. The content of the mica in the cosmetic composition of the present invention is preferably included 20 to 35% by weight based on the total weight of the composition. If it is less than 20% by weight, the skin whitening and moisturizing effect is hardly exhibited, and when it exceeds 35% by weight, the skin whitening and moisturizing effect is excessive.

Sericite, KAl ₂ (OH) ₂ (AlSi ₃ O ₁₀ )), belongs to the monoclinic system and has a white or off-white pearl luster. Originally referred to as the dominant mineral of crystalline schist rock, especially chorionic schist rock, today it refers to clay-like fine muscovite formed by hydrothermal action. The chemical composition is almost the same as that of the mica, but generally potassium (K) is less than that of the mica and somewhat more water (H ₂ O). In addition, the mica contains selenium (Se), zinc (Zn), magnesium (Mg), calcium (Ca) and various minerals essential to the human body, and heavy metal detoxification and antioxidant activity. In addition, Cicada is used as a mixed material of ceramics or refractory bricks, and various other uses such as paints, electrical insulators, active materials and cosmetics. The content of the mica in the cosmetic composition of the present invention is preferably included 10 to 20% by weight based on the total weight of the composition. If it is less than 10% by weight, heavy metal detoxification and antioxidant effects are weakened. If it exceeds 20% by weight, the regulation of the entire antioxidant system in the cell is destroyed, and the heavy metal detoxification and antioxidant effects tend to be weaker.

Kaolin (Kaolin, Al ₂ Si ₂ O ₅ (OH) ₄ ) is composed mainly of kaolinite and halosite, and feldspars such as feldspar, feldspar, soda feldspar, and feldspar in rock are chemically reacted by carbonic acid or water. Produced by weathering which decomposes and is white or slightly gray. Kaolin is good for oily skin and acne skin because it cleans the skin and removes dirt, toxins, pores, and dead skin cells from the skin surface. In oriental terminology, it is called white stone or yellow stone depending on the color and it is used for skin troubles. The content of kaolin in the cosmetic composition of the present invention preferably contains 35 to 45% by weight relative to the total weight of the composition. If it is less than 35% by weight, the affinity between components is weak and the adsorption power is weakened. If it exceeds 45% by weight, the affinity is too high and the adsorption power is too strong, causing problems on the skin.

Pumice is a highly porous mineral formed by the solidification of rocks ejected from volcanoes. Anatomical stone is a silicate mineral ([SiO ₂ ] x) including rhyolite, trachyte and phonolite. Its essential minerals are calcium, iron, zinc, copper, nickel, chromium, cobalt, vanadium and selenium. It contains. According to the herbaceous tree, anatomical stone is known to have a lump or bruise on the skin, to treat extreme lower abdominal pain, and to lower the swelling. It is also known to treat the symptoms of obstruction of the eye and obstruction. The content of anatomical stone in the cosmetic composition of the present invention is preferably included 1 to 3% by weight based on the total weight of the composition. If less than 1% by weight has little effect on the skin, if it exceeds 3% by weight heavy metals are harmful to the skin.

Organic germanium (Ge) is present in trace amounts, and oxygenation, semiconductor action (electron flow regulation, anion effect), immunity strengthening, interferon production, endorphin production promotion, pain elimination, acid constitution Efficacy of detoxification, heavy metal excretion, and natural healing power is known. Therefore, cosmetics using high purity germanium; Health tools such as bracelets, necklaces and patches; Soap, etc. has been commercialized, and is a material that is showing a strong increase in demand worldwide. In the cosmetic composition of the present invention, the content of anatomical stone is preferably 0.1 to 0.3% by weight based on the total weight of the composition. If it is less than 0.1% by weight, germanium does not show the efficacy, and if it exceeds 0.3% by weight, it is toxic to the skin.

Among the mineral components, mineral components other than organic germanium are essential macro-elements such as Ca, Mg, P, and K, Fe, Zn, Se, Mn, Cu, Mo, Co, Cr, It contains essential micro-elements such as F, Ni, V, B, and Se, which can be helpful for the life of cells. In particular, the selenium of the elements is a representative bio-defense enzyme that acts to remove hydrogen peroxide generated in the body as a coenzyme of glutathione peroxidase enzyme. Therefore, elements such as selenium contained in the mineral complex component are known to promote the action of glutathione peroxidase to remove the by-products of intracellular metabolic processes and to participate in cell division and differentiation.

In addition, silicon (Si), which is commonly contained in the mineral components, is a natural substance contained in grains that have not been milled, and plays an important role in the synthesis of proteins forming connective tissues as an important element that accounts for 0.5% of body weight. Because the connective tissue is an essential component to maintain elasticity. There is also research on silicones to boost immunity. In order for the cells to consume the silicon usefully, several essential minerals such as boron, calcium, magnesium, manganese and potassium are needed at the same time.

Mineral composite component of the active ingredient in the cosmetic composition for improving wrinkles of the present invention is prepared as follows. First, the nephrite, talc, dolomite, biotite, kaolin, anatomical and organic germanium is washed with water and then dried to remove moisture. Each dried mineral is placed in a conventional rotary powder and subjected to a high speed powder to be ground into an ultrafine powder having a particle size of 1 to 10 microns. The pulverized ultra-fine powder is transmitted through a strong magnetic force line to the roller-type electromagnet drum and the conveyor belt, respectively, to form a strong magnetic field to remove unnecessary iron components as much as possible. After the iron component is removed, the powder is placed in 20 times the weight of water, and the stirrer is operated. The supernatant is discarded and the precipitate is recovered, or the suspended solid is recovered and the specific gravity is discarded. Among the minerals, mineral components other than purgatory and organic germanium, that is, talc, dolomite, biotite, kaolin, and anatomical stones are mixed according to the mixing ratio. The mixture is evenly divided into a thin layer on a porcelain plate and then placed in an electric furnace and heated at a constant temperature of 700 ° C. When the mixture is heated red, this state is maintained for 15 to 25 minutes and then the porcelain plate is taken out. Purgatory and organic germanium are added to obtain a mineral complex.

The mineral complex of the present invention has excellent antioxidant activity and shows almost no cytotoxicity at a concentration of 0.244 µg / ml, resulting in almost 100% cell viability. In addition, the mineral complex of the present invention is the collagenase (MMP-1) and gelatinase (MMP-2) activity inhibitory effect and collagenase (HMP) increased after ultraviolet (UVA) irradiation to the fibroblast cell line HDF of human dermis MMP-1) mRNA and gelatinase (MMP-2) mRNA expression inhibitory effect is excellent, collagen synthesis promoting effect and elastase inhibitory effect is also excellent. In addition, the mineral complex component of the present invention is excellent in the regeneration promoting effect on hepatocytes can predict the regeneration promoting effect of the skin cells, it is safe and irritating to human skin. Therefore, the mineral complex component of the present invention can be usefully used in cosmetic compositions for improving skin wrinkles.

In the cosmetic composition of the present invention, the content of the mineral complex component is included in an amount of about 0.0001 to 10.0% by weight, preferably 0.001 to 5.0% by weight, more preferably 0.01 to 1% by weight based on the total weight of the cosmetic composition. Can be. If the content of the mineral complex is less than 0.0001% by weight, it is difficult to expect a substantial effect. If the content of the mineral complex is greater than 10.0% by weight, there is no apparent increase in effect due to the increase in content, causing skin irritation and instability of the formulation. .

The cosmetic composition for improving wrinkles of the present invention can be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, interfaces It may be formulated as an active agent-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, and the like.

The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the dosage form. When the formulation of the present invention is an ointment, paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures of these may be used.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or mixtures thereof may be used, especially in the case of sprays, additionally chlorofluoro Propellants such as rohydrocarbon, propane / butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, solvents, solubilizers or emulsions are used as carrier components, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, There are 1,3-butylglycol oils, in particular cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycols or fatty acid esters of sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, micro Crystalline cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars and the like may be used as carrier components. Can be.

In addition, the cosmetic composition of the present invention may include not only a carrier but also an adjuvant, and may include, for example, a preservative, an antioxidant, a stabilizer, a solubilizer, a vitamin, a pigment, a perfume, and a mixture thereof, but is not limited thereto. .

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  1-3 and Comparative example  1-2  : Preparation of Mineral Complex

Purity, talc, dolomite, biotite, kaolin, anatomical and organic germanium were washed clean with water, and then dried in a drier at 100 ° C. for at least 3 hours to remove moisture. Each dried mineral was placed in a conventional rotary powder and subjected to a high-speed powder, which was ground to an ultrafine powder having a particle size of 1 to 10 microns. The pulverized ultrafine powders respectively penetrated the strong magnetic lines through the roller-type electromagnet drum and the conveyor belt to form a strong magnetic field, thereby removing unnecessary iron components as much as possible. The iron component was removed, and the stirrer was put in a 20-fold weight of water, and the supernatant was discarded to recover the precipitate. The process may recover the suspended matter and discard the precipitate in the stirrer operating state.

Mineral components other than purgatory and organic germanium of the minerals were mixed according to the blending ratio shown in Table 1 to prepare Examples 1 to 3. In addition, Comparative Examples 1 and 2 were prepared according to the blending ratios shown in Table 1 below to compare the effects of purgatory and organic germanium contents. The mixture was evenly divided into a thin layer on a porcelain plate and then placed in an electric furnace and heated at a constant temperature of 700 ° C. When the mixture was heated red, this state was maintained for 20 minutes and then the porcelain plate was taken out. Purgatory and organic germanium were added to the mixture according to the blending ratios shown in Table 1 below.

Example 1 (% by weight) Example 2 (% by weight) Example 3 (% by weight) Comparative Example 1 (% by weight) Comparative Example 2
(weight%) purgatory 9 7 4 0 15 talc 11.9 18.8 13.7 22 7 muscovite 25 20 31 35 20 Cicada 15 13 16 18 10 china clay 38 38 34 25 43.5 Anatomy stone One 3 One 0 4 Organic germanium 0.1 0.2 0.3 0 0.5

Experimental Example 1  : Physicochemical Properties of Mineral Complex Components of the Present Invention

In order to find out the physicochemical properties of the mineral composite component of the present invention, the following experiment was performed.

1. Observation of particle shape of mineral complex

After adhering the mineral composite components prepared in Examples 1 to 3 with a conductive adhesive to a scanning electron microscope (SEM) sample bed, an ion sputter coater (SEM auto coating E 5200) was used. The surface was coated at 18 mA for about 3 minutes and observed under an accelerating voltage of 20 kV with a scanning electron microscope.

The results are shown in FIG.

As shown in Figure 1, the mineral composite component of the present invention is evenly distributed, it was confirmed that the particle size of the mineral composite component is 3.26㎛.

2. Far Infrared Spectroradiance Analysis of Mineral Complex Components

The far-infrared emissometer (FT-IR Spectrometer) equipped with the MCT detector used to measure the far infrared spectral emissivity of the mineral composite components prepared in Examples 1 to 3 is MIDAC M2400-C (USA), and the measurement temperature range is 37 ℃, the measurement wavelength is 5 ~ 20㎛, and measured results compared to the black body (Black body).

The results are shown in Fig.

As shown in Figure 2, the emissivity of the mineral composite component of the present invention was 0.916, the emission energy was 3.53 × 10 ² W / ㎡. Therefore, it can be seen that the far-infrared radiation energy efficiency of the mineral composite component of the present invention is excellent.

Experimental Example 2  : Determination of Antioxidant Activity of Mineral Complex Components of the Present Invention (DPPH Method)

In order to determine the antioxidant activity of the mineral complex component of the present invention, the following experiment was performed.

Antioxidant activity of the sample was indirectly measured by measuring the extent of DPPH radical decrease by reaction with a certain amount of sample solution using DPPH · (1,1-diphenyl-2-picryl hydrazyl), which is a stable free radical. Activity was measured. The mineral composite component prepared in Example 1 of 1.25 µg / ml dissolved in methanol was added to 100 µl of 0.15 mM DPPH solution dissolved in 100 µl of methanol and reacted at 37 ° C for 10 minutes. As a positive control group, 0.97 ㎍ / ㎖ polyphenol was used, the antioxidant power was expressed by the reducing power by the electron donating ability (EDA%) of the formula (1) to measure the antioxidant effect of the mineral complex component. Absorbance reduction at 570 nm was measured using an ELISA reader. The results are shown in FIG.

EDA (%) = [(Control Absorbance-Sample Absorbance) / Control Absorbance] × 100

※ control absorbance: absorbance of test solution

   Sample absorbance: absorbance of the test solution to which the mineral complex component of the present invention was added

As shown in FIG. 3, the mineral complex component of the present invention exhibits an active oxygen scavenging ability similar to that of the positive control group, and thus has excellent antioxidant activity.

Experimental Example 3  : Cell viability measurement of human skin cells of the mineral complex component of the present invention (MTT method)

In order to determine the cell viability of the human skin cells of the mineral complex component of the present invention, the following experiment was performed.

1. Cell Culture

Human dermal fibroblast (HDF), a fibroblast cell line of human dermis used in this experiment, was purchased from Young Cell (MCTT, Modern Cell & Tissue Technologies). For cell culture, put HDF into a T-75 cell culture flask and incubate the medium and cells in a 5% CO ₂ , 37 ° C incubator, and measure the cell culture state and the number of cells using a microscope and a hematocytometer. It was. Cells that supply nutrients to the cells were prepared by adding DMEM (dulbecco's modified eagles's medium) with 10% heat inactivated fetal bovine serum (FBS) (Hyclone) and 1% antibiotic (penicillin-streptomycin) (Hyclone). Used as a culture. The passage of HDF used in all experiments did not exceed 6-10.

2. Cell viability measurement

Cell viability was measured using the MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] method and% relative to the control group was measured. HDF was transferred to a 96-well plate so that the cell number was 2 × 10 ^{4 per} well, and then incubated for 24 hours in a 5% CO ₂ , 37 ° C. incubator. The sample solution was prepared by adding 500 μg per ml of the mineral composite component prepared in Example 1 to DMEM containing 10% FBS and 1% antibiotics and treating the mixture by serial dilution until (1/2) ¹⁷ . After 24 hours, the culture medium was removed, 200 μl of the sample solution was added thereto, and then cultured for 24 hours in a 5% CO ₂ , 37 ° C. incubator for 24 hours. After 24 hours of sample solution treatment, MTT solution (5 mg / ml) was added to the cells and incubated for 4 hours in a 5% CO ₂ , 37 ° C. incubator. After incubation, the MTT solution was removed, 200 μl of DMSO (dimethyl sulfoxide) solution was added to dissolve blue formazan, and the absorbance values of the control group and each sample were measured at 570 nm using an ELISA reader.

The results are shown in FIG.

As shown in FIG. 4, the mineral complex of the present invention showed almost no cytotoxicity at a concentration of 0.244 μg / ml, resulting in almost 100% cell viability.

Experimental Example 4  : Inhibitory effect of collagenase (MMP-1) and gelatinase (MMP-2) of the mineral complex component of the present invention

In order to determine the inhibitory effect of collagenase (MMP-1) and gelatinase (MMP-2) of the mineral complex of the present invention, the following experiment was performed.

1. UV irradiation

HDF, a fibroblast cell line of human dermis, was transferred to 1.5 × 10 ⁵ cells in a 100Ф plate, and the medium was removed when the confluence of HDF was 70-80%. Then, after washing once with PBS (phosphate buffered saline), UVA (320-400 nm) of 300mJ / cm 2 was irradiated for 172 seconds at a height of 5 cm away from a 100Ф plate without removing PBS. After UV irradiation, PBS was removed and again washed with PBS. The control group is not exposed to UV, and after UV exposure of HDF, a serum-free media containing the mineral complex prepared in Example 1 was added thereto, and then 48 hours in a 5% CO ₂ , 37 ° C. incubator. Incubated for

2. Inhibitory effect of collagenase (MMP-1) and gelatinase (MMP-2): protease assay (zymography)

When HDF, a fibroblast cell line of human dermis, filled about 70-80% of the bottom of the culture dish, was washed once with PBS (phosphate buffered saline), exposed to 300mJ / cm 2 UVA, and washed again with PBS. PBS was removed, and serum-free media containing the mineral complex (0.244 μg / ml) prepared in Example 1 was added thereto, and cultured in a 5% CO ₂ , 37 ° C. incubator for 48 hours. Then, the supernatant was collected separately and 8% SDS-PAGE (Sodium dodecyl slufate-polyacrylamide gel electrophoresis) containing 0.1% gelatin was performed. After the electrophoresis gel was briefly washed in distilled water and soaked in 100ml of 2.5% Triton X-100 solution and washed twice in a shaker incubator for 30 minutes. 100 ml of culture buffer (50 mM Tris-HCl, 10 mM CaCl ₂ , 50 mM NaCl, 0.05% Brij35, pH 7.6) was added thereto and reacted at 37 ° C. for 20 hours. After the reaction, the solution was stained for about 40 minutes in coomassie brilliant blue staining solution, and then decolorized with a solution containing acetic acid, methanol, and distilled water 1: 5: 4 until a white band having proteolytic activity was observed. The decolorized gel was scanned with a scanner to quantify the white band.

Polyphenols were used as a positive control group, and polyphenols were also treated at the highest concentration (0.122 ㎍ / mL) without cytotoxicity.

The inhibitory effect of collagenase (MMP-1) activity of the mineral complex of the present invention is shown in Figure 5, the inhibitory effect of gelatinase (MMP-2) is shown in Figure 6.

As shown in Figure 5, the mineral complex of the present invention effectively reduced the activity of collagenase (MMP-1) increased after ultraviolet (UVA) irradiation to the fibroblast cell line HDF of human dermis.

In addition, as shown in Figure 6, the mineral complex of the present invention effectively reduced the activity of gelatinase (MMP-2) increased after ultraviolet (UVA) irradiation to HDF, a fibroblast cell line of human dermis.

Experimental Example 5  : Inhibitory Effect of Collagenase (MMP-1) and Gelatinase (MMP-2) of the Mineral Complex of the Present Invention

In order to determine the mRNA expression inhibitory effect of collagenase (MMP-1) and gelatinase (MMP-2) of the mineral complex of the present invention, the following experiment was performed.

1. Isolation of Total RNA from Fibroblast Line (HDF) of Human Dermis

TRI reagent in HDF, fibroblast cell line of human dermis grown on plate 1 ml was added, transferred to an eppendorf tube and allowed to react at room temperature for 5 minutes. Then, 200 μl of chloroform was added and mixed vigorously for 15 seconds, and then reacted for 3 minutes at room temperature, followed by centrifugation at 4 ° C. and 12,000 rpm for 15 minutes. The supernatant was removed, transferred to a new tube, and the RNA was purified by repeating the above procedure once. The supernatant in which the purified RNA was dissolved was taken, 500 μl of isopropanol was added thereto, mixed lightly, and then reacted at room temperature for 10 minutes, followed by centrifugation at 4 ° C. and 12,000 rpm for 15 minutes. 1 ml of 75% ethanol was added to the resulting precipitate and washed. After centrifugation again to remove the supernatant, the precipitate was dried for 5 minutes, dissolved in TE (RNase-free) buffer, and the absorbance (A ₂₆₀ / A ₂₈₀ ) was measured to determine the amount and purity. The measured absorbance value is appropriate between 1.7 and 2.0, and the total RNA isolated was stored in a cryogenic freezer at -70 ° C until used in the experiment.

2.RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction)

2-1. Synthesis of First Strand cDNA

Using the total RNA (2 μg / μl) isolated in 1 as a template, cDNA was synthesized for performing reverse transcription polymerase chain reaction (RT-PCR) using Accuone RT Premix kit of Bioneer. The reaction for cDNA synthesis was mixed with 3 μl Template RNA, 2 μl Oligo-dT primers, reacted at 70 ° C. for 5 minutes, and then placed on ice to stop further reactions. The reaction mixture was placed in an RT Premix tube, and the final volume was adjusted to 50 μl with DEPC-water, mixed, and reacted at 42 ° C. for 60 minutes and 94 ° C. for 5 minutes. And stored at 4 ° C. for 30 minutes. The synthesized cDNA was stored in a cryogenic freezer at -70 ℃ until used in the experiment.

2-2. Preparation of the primer

In order to amplify the human β-actin gene, which is always expressed from the genes of the human dermal fibroblast cell line (HDF) and can be used as a control, a sense primer and an anti-sense primer were prepared. . A sense primer and an anti-sense primer were prepared to amplify the MMP-1 and MMP-2 genes. All primers were custom made by Genotech Co., Ltd. Primer sequences and fragment sizes of the genes tested in RT-PCR are shown in Table 2.

gene Primer Sequence Fragment size (bp) MMP-1 F 5'-AAAGGGAATAAGTACTGGGC-3 '
R 5'-AATTCCAGGAAAGTCATGTG-3 ' 237 MMP-2 F 5'-CACCCCTAAGAGATCCT-3 '
R 5'-GTGCATACAAAGCAAACTGC-3 ' 274 β-actin F 5'-TATGCCCTCCCCCATGCCAT-3 '
R 5'-GAAGCATTTGCGGTGGACGATGG-3 ' 622

2-3. PCR (Polymerase Chain Reaction)

The PCR reaction was carried out using 20 μl of the reaction solution using the synthesized 2 μl cDNA as a template and a corresponding primer combination and 2 × Dye MIX DNA Polymerase from Enzynomics ^™ . A total of 20 μl of PCR reaction solution contains nTaq ^tenuto 2.5 units, nTaq ^tenuto reaction buffer 4 mM Mg ²⁺ , dNTP mixture 0.4 mM, glycerol, and dyes include xylene cyanol and orange G. Each reaction solution was added with 10 pmol of primer, followed by denaturation at 94 ° C. for 3 minutes using Techne's PROGENE, followed by 30 minutes of reaction at 94 ° C., 55 ° C. and 72 ° C., respectively. The reaction was extended at 3 ° C. for 3 minutes and then stored at 4 ° C. for 30 minutes. After the reaction, the product was stored in a freezer at -20 ° C until use in the next experiment, and electrophoresed using a 1% agarose gel to check the band on UV.

2-4. Quantitative RT-PCR

CDNA was synthesized by quantifying the amount of template RNA (total RNA) at the concentration of 6 μg at the time of initial cDNA synthesis, and serially diluted cDNA was used for quantitative PCR. Continuous cDNA dilution ranged from 1/2 to 1/32. Treatment with control, UVA, mineral complex of Example 1 and polyphenol under dilution conditions amplified by the same amount as the control gene β-actin primer used as the internal standard in the control and treatment groups for quantitative analysis The amplified amounts of one cell were compared with each other.

The mRNA expression inhibitory effect of collagenase (MMP-1) of the mineral complex of the present invention is shown in Figure 7, the mRNA expression inhibitory effect of gelatinase (MMP-2) is shown in Figure 8.

As shown in Figure 7, the mineral complex of the present invention effectively reduced the expression of collagenase (MMP-1) increased after ultraviolet (UVA) irradiation to HDF, a fibroblast cell line of human dermis.

In addition, as shown in Figure 8, the mineral complex of the present invention effectively reduced the amount of gelatinase (MMP-2) expression increased after ultraviolet (UVA) irradiation to the fibroblast cell line HDF of human dermis.

Experimental Example 6  : Promoting Collagen Synthesis of Mineral Complex Components of the Present Invention: Measurement of Procollagen Type 1 C-peptide (PIP)

In order to determine the collagen synthesis promoting effect of the mineral complex of the present invention, the following experiment was performed.

100 μl of antibody-horseradish peroxidase (POD) conjugate solution was added to each well of a 96-well plate, and the same protein amount as that of the standard (PIP 640 ng / ml) previously diluted by 1/2 was prepared in Example 1. The mineral complex (0.244 ㎍ / ㎖) was added and mixed well, and reacted for 3 hours at 37 ℃. After 3 hours, 400 μl of PBS was added to the well from which the reaction product was removed, and washed about 4 times. Then, 100 μl of the substrate was added thereto, followed by reaction at 20˜30 ° C. for 15 minutes. Then, the absorbance of the reactant turned from blue green to yellow was measured at 450 nm by adding 100 µl each of a stop solution (1 N H ₂ SO ₄ ). Vitamin C was used as a positive control, and vitamin C was also treated at the highest concentration (35 μg / ml) without cytotoxicity.

The results are shown in FIG.

As shown in Figure 9, the mineral complex prepared in Example 1 of the present invention promoted procollagen biosynthesis by 60% compared to the control. In addition, the mineral complex prepared in Example 2 or Example 3 also promoted about 60% procollagen biosynthesis compared to the control. In comparison, vitamin C promoted procollagen biosynthesis by about 30%, and the mineral complex prepared in Comparative Example 1 or Comparative Example 2 hardly promoted procollagen biosynthesis. Therefore, it was confirmed that the mineral composite prepared with the content of purgatory and organic germanium according to the present invention effectively promotes collagen synthesis.

Experimental Example  7  : Of the mineral complex component of the present invention Elastase  Inhibitory effect

In order to determine the elastase inhibitory effect of the mineral complex of the present invention, the following experiment was performed.

80 µl of 0.2 M Tris-HCl (pH 8.0) was mixed with 120 µl of 3.2 mM Succ-Ala-Ala-Ala-p-nitroanilide and 100 µl of the mineral complex (0.244 µg / ml) prepared in Example 1. . Here, 1 U of human leukocyte elastase (Human Leukocytes Elastase) was dissolved in 1 ml of 0.2 M Tris-HCl (pH 8.0) and 50 μl (50 mU) was added thereto. The enzyme activation reaction was performed at 25 ° C. for 15 minutes. The control group was reacted under the same conditions by adding the same amount of distilled water instead of 100 μl of the mineral complex component. After the reaction, the absorbance values of the control and each sample were measured at 415 nm using an ELISA reader.

The results are shown in FIG.

As shown in Figure 10, the mineral complex prepared in Examples 1, 2 or 3 of the present invention reduced the elastase activity by about 40% compared to the control. On the other hand, the mineral complex prepared in Comparative Example 1 or Comparative Example 2 had little effect on the elastase activity. Therefore, it could be seen that the effect of inhibiting the elastase activity of the mineral composite prepared with the content of nephrite and organic germanium according to the present invention was excellent.

Experimental Example 8  : Promoting regenerative effect on hepatocytes of the mineral complex component of the present invention

In order to confirm the regeneration promoting effect on the hepatocytes of the mineral complex component of the present invention, the following experiment was performed.

1. Materials of Liver Regeneration Experiment

Experimental animals were rats that had better liver regeneration than other animals, and male rats of 5-week-old Sprague-Dowley strains weighing 140g at the base for 2 weeks were used as standard feed and water in the animal breeding room. . The experimental group was intubated by diluting the mineral complex prepared in Example 1 once every day from 3 days before the partial liver resection, and the control group was treated with the same saline solution. The reagents used in this experiment were purchased from Sigma and specialty products from commercial molecular biology reagents specialists.

2. Partial hepatectomy of rats

Partial ablation of rat liver was performed by improving Higgins and Anderson's method (Higgins G, M., Anderson, R.M., Experimental pathology of liver, Arch Pathol; 12, 186-202, 1936). In order to stimulate liver regeneration, male rats were anesthetized with weak ether and liver resection was performed for about 70%. That is, under mild ether anesthesia, the incision was sterilized with alcohol, the hair was removed, and then the abdomen was opened from the central part of the abdomen to the upper side of the apex. After removing the ligaments of the liver to be dissected, the livers were removed by lifting them vertically to bind the vessels with silk threads and cutting as close to the vessels as possible (medium and left lobes, about 70%). The muscles and skin were then sutured and disinfected. 6 hours, 3 days, and 7 days after the operation were sacrificed and used as experimental materials.

3. Optical microscope sample preparation

Liver fixed in 10% formalin was washed with running water to remove formalin remaining in the tissue. Then, dehydration was performed for 1 hour in order of increasing ethanol concentration up to 60%, 70%, 80%, 90%, 95%, and 100%. Substituted twice in xylene for 1 hour, and after the paraffin infiltration process was embedded in paraffin to prepare a block. The prepared paraffin block was cut to a thickness of 7 μm using a rotary cutting machine (microtome, Leica Co., Ltd.), attached to a slide glass, and paraffin was removed twice in 3 to 5 minutes in xylene. Double staining with hematoxylin and eosin, encapsulated with canada balsam, followed by microscopy and photographed.

The results are shown in FIG.

As shown in FIG. 11, in the experimental group treated with the mineral complex component of the liver of the partially excised rat liver, the remaining liver size was increased after 3 days after partial liver resection, and the liver size of the normal control group was 7 days. It was visually confirmed to increase to a similar degree. In addition, as a morphological change during liver regeneration, in the experimental group after 6 hours, hepatic cell plate around the central vein collapsed and clustered in several places, and cells with two nuclei increased and cytoplasm. The size of the nucleus for was greater than that of the normal control. This process was observed more clearly the cell division in the experimental group treated with the mineral complex of the present invention. It is believed that the mineral complex component of the present invention is an effect of promoting cell division and differentiation by regulating the action of the enzyme to remove the generator oxygen generated in the metabolism process.

Therefore, the mineral complex component of the present invention can predict the regeneration promoting effect of skin cells through the regeneration promoting effect on hepatocytes.

Experimental Example 9  : Accumulated skin irritation test

In order to confirm the safety of human skin of the mineral complex component of the present invention, the following experiment was performed.

Thirty healthy men and women in their 20s and 40s (man-15 -15, -15 -15) using nutrition creams (Formulations -A, B, C) containing the mineral complex components prepared in Examples 1 to 3 above. A total of nine 24-hour cumulative blisters were applied to the upper forearm every other day for 24 days, and the mineral complex components according to the present invention were measured to irritate the skin.

The patch method used the fin chamber (Finn chamber, Epitest Ltd, Finland). 15 µl of nourishing cream (Formulations -A, B, C) containing a mineral complex component was added dropwise to the chamber, and then patched. The degree of response that appeared on the skin each time was scored using Equation 2 below.

Average responsiveness = {[(response index × responsiveness) / (total number of subjects × highest score (4 points))] × 100} ÷ number of tests (9 times)

At this time, ± 1 point, + 2 points, + + 4 points in the reactivity, and the average response was less than 3 was determined as a safe substance.

The results are shown in Table 3.

Number of subjects with response Average responsiveness 1 week 2 weeks 3 weeks Primary Secondary 3rd 4th 5th 6th 7th 8th 9th ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ Formulation-A 2 - - --- --- --- --- --- --- --- --- 0.18 Formulation-B 2 - - --- --- --- --- --- --- --- --- 0.18 Formulation-C 2 - - --- --- --- --- --- --- --- --- 0.18 Inspector 30 30 30 30 30 30 30 30 30

As shown in Table 3, in the case of the nourishing cream containing the mineral complex component of the present invention, the number of persons corresponding to ±, +, and ++ was 2, 0, and 0, respectively, and the rest was not shown. Did. In addition, since the average reactivity was 0.18 and less than 3, it was determined to be a safe substance for human skin.

Examples of formulations for the composition of the present invention are illustrated below.

Formulation example  Cosmetic preparations

1. Flexible cosmetics (content: wt%)

Mineral Complex 0.01

Glycerin 3.0

Butylene Glycol 2.0

Propylene Glycol 2.0

Carboxy Vinyl Polymer 0.1

Ethanol 10.0

Triethanolamine 0.1

Preservatives, trace pigments, trace fragrances, trace amount of purified water

Total 100.0

2. Nutritional Cosmetics (Content: Weight%)

Mineral Complex 0.01

Beeswax 4.0

Polysorbate 60 1.5

Sorbitan Sesquioleate 0.5

Liquid Paraffin 5.0

Squalane 5.0

Caprylic / Capric Triglycerides 5.0

Glycerin 3.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Carboxy Vinyl Polymer 0.1

Triethanolamine 0.2

Preservatives, trace pigments, trace fragrances, trace amount of purified water

Total 100.0

3. Massage cream (content: wt%)

Mineral Complex 0.005

Beeswax 10.0

Polysorbate 60 1.5

Sorbitan Sesquioleate 0.8

Liquid Paraffin 40.0

Squalane 5.0

Caprylic / Capric Triglycerides 4.0

Glycerin 5.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Triethanolamine 0.2

Preservatives, trace pigments, trace fragrances, trace amount of purified water

Total 100.0

4. Pack (Content: Weight%)

Mineral Complex 0.005

Polyvinyl Alcohol 13.0

Sodium Carboxymethylcellulose 0.2

Allantoin 0.1

Ethanol 5.0

Nonylphenyl Ether 0.3

Preservatives, trace pigments, trace fragrances, trace amount of purified water

Total 100.0

The mineral complex of the present invention has excellent antioxidant activity, no cytotoxicity, and increased collagenase (MMP-1) and gelatinase (MMP-2) after irradiation with ultraviolet (UVA) to HDF, a fibroblast cell line of human dermis. Inhibitory activity of collagenase and mRNA expression of collagenase (MMP-1) and gelatinase (MMP-2) are excellent, as well as promoting collagen synthesis and elastase inhibitory effect, and hepatocytes It is excellent in the regeneration promoting effect, and because there is no irritation and safe on human skin, it can be usefully used in cosmetic compositions for improving skin wrinkles.

Claims (4)

Purgatory 1-10 wt%, Talc 10-20 wt%, Dolomite 20-35 wt%, Cicada 10-20 wt%, Kaolin 35-45 wt%, Anatomical 1-3 wt% and Organic germanium 0.1-0.3 wt% Wrinkle improvement cosmetic composition comprising a mineral composite component containing. delete The cosmetic composition for improving wrinkles according to claim 1, wherein the content of the mineral complex component is 0.0001 to 10.0 wt% based on the total weight of the cosmetic composition. The composition of claim 3, wherein the cosmetic composition is a group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray Cosmetic composition for improving wrinkles, characterized in that it is formulated in any one selected from.

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