KR102485135B1 - A multi-fuctional cosmetic composition comprising astaxanthin complex - Google Patents

Abstract

The present invention relates to a cosmetic composition for skin moisturizing and antioxidation. Specifically, the present invention provides a cosmetic composition containing astaxanthin and madecassoside as active ingredients. An objective of the present invention is to identify excellent synergistic activity through raw materials whose skin safety has been verified, and to provide the cosmetic composition with excellent biosafety along with functionality through the same.

Description

Multifunctional cosmetic composition containing astaxanthin complex component as an active ingredient

The present invention relates to a multifunctional cosmetic composition comprising an astaxanthin complex component as an active ingredient.

Inflammation is a phenomenon that occurs for a series of defense purposes to minimize the reaction when cells or tissues are damaged by any cause and to restore the damaged area to its original state.

Inflammation causes nerves, blood vessels, lymphatics, humoral reactions, and cellular reactions, resulting in pain, swelling, redness, fever, and the like, which can lead to functional disorders.

Causes of inflammation include physical factors such as trauma, frostbite, burns, and radiation, chemical factors such as acids, and immunological factors such as antibody reactions. may occur

Cells damaged by external stimuli secrete various biological mediators such as pro-inflammatory cytokine, chemokine, interleukine, and interferon, and their permeability increases by vasodilation, thereby increasing antibody production. , complement, plasma, and phagocytic cells are concentrated in the inflamed area, which can cause erythema.

On the other hand, nonsteroidal benzidamine, indomethacin, ibuprofen, etc. are used as anti-inflammatory agents, and steroidal dexamethasone and hydrocortisone are used.

However, the use of the anti-inflammatory agent is limited due to human safety and side effects, and the effect is insignificant, so there is a problem in that the anti-inflammatory effect cannot be expected.

Therefore, there is an urgent need for the development of multifunctional ingredients that are superior in biosafety, stable, and, above all, more effective than existing materials with skin elasticity, wrinkle improvement, moisturizing and anti-inflammatory effects.

The present invention is to solve the above problems, and an object of the present invention is to identify excellent synergistic activity through raw materials whose skin safety has been verified, and to provide a cosmetic composition with excellent biosafety as well as functionality through this.

According to one aspect of the present invention, a cosmetic composition for skin moisturizing and antioxidant containing astaxanthin and madecassoside as active ingredients is provided.

In one embodiment, the cosmetic composition may further include hyaluronic acid.

In one embodiment, the cosmetic composition may further include one or more of betaine and centella asiatica extract.

In one embodiment, the cosmetic composition may further include Haematococcus fluvialis extract.

In one embodiment, the Haematococcus fluvialis extract may be fermented by Candida bombicola .

In one embodiment, the cosmetic composition may further include fermented olive oil.

In one embodiment, the astaxanthin may be stabilized by liposomes.

In one embodiment, the cosmetic composition may include anti-inflammatory, acne improvement or skin regeneration applications.

In one embodiment, the cosmetic composition may further include an anti-wrinkle or anti-photoaging application.

According to the present invention, the cosmetic composition has excellent skin improvement effects such as skin soothing and regeneration, anti-inflammatory, and skin moisturizing, and has excellent human safety.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.

The terms used in this specification have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art, precedent, or the emergence of new technologies. In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't

Numerical ranges are inclusive of the values defined therein. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written. Every numerical limitation given throughout this specification will include every better numerical range within the broader numerical range, as if the narrower numerical limitations were expressly written.

Hereinafter, embodiments of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.

One aspect of the present invention provides a cosmetic composition for skin moisturizing and antioxidant containing astaxanthin and madecassoside as active ingredients.

The "moisturizing skin" may refer to all actions to maintain the homeostasis of skin tissue by appropriately controlling the loss of moisture (evaporation of moisture) of the skin. The skin moisturizing effect may be accompanied by additional skin improving effects in various aspects, such as an exfoliating effect and a skin irritation reducing effect.

The “anti-oxidant” refers to an action that inhibits oxidation. In the human body, oxidation promoters and oxidation inhibitors are in balance, but due to various factors, this balance is lost and oxidation is promoted. If it is tilted in the direction, oxidative stress is induced in the living body, which can cause cell damage and pathological diseases.

Reactive oxygen species (ROS), which is a direct cause of the oxidative stress, are chemically unstable and highly reactive, and can easily react with various biomaterials such as DNA, proteins, lipids, and carbohydrates. It attacks and causes irreversible damage to cells and tissues, or causes mutations, cytotoxicity and cancer, etc., and may also be a direct cause of aging.

By removing or reducing the reactive oxygen species, an antioxidant effect can be obtained to prevent aging and maintain health.

The cosmetic composition may exhibit excellent skin improvement effects through the synergistic activity of various complex components together with astaxanthin.

The “astaxanthin” is a carotenoid-based pigment that belongs to a class of phytochemicals known as terpenes, has a structural formula of C ₄₀ H ₅₂ O ₄ , and may include derivatives thereof.

The astaxanthin is a pigment widely distributed in nature obtained from many organisms such as algae, mushrooms, crustaceans, etc., and exhibits active oxygen and free radical scavenging ability, and has a very high antioxidant activity compared to other carotenoid pigments.

The astaxanthin may be derived from natural products such as plants, algae, crustaceans and bacteria, or may be synthesized. The astaxanthin may be used after being extracted from red yeast papia, green alga Hematococcus, marine bacteria, krill, or the like, or may be purchased and used commercially.

The “madecassoside” is a substance isolated from Centella asiatica , and the madecassoside can soothe the skin, promote collagen synthesis, and impart skin barrier strengthening and tissue recovery effects.

At this time, when the madecassoside acts simultaneously with the astaxanthin on the skin, more excellent skin improvement activity can be realized.

The cosmetic composition may further include hyaluronic acid.

The "hyaluronic acid" is a type of anionic non-sulfated glycosaminoglycan widely distributed in connective tissue, epithelial tissue, skin tissue and nerve tissue, and is a high molecular weight material having a molecular weight of 20 to 100.

The hyaluronic acid is known to be involved in the storage and diffusion of cell growth factors and nutrients as a major component of the cell matrix, as well as in cell differentiation, migration, water storage, and cell spacing maintenance.

The present inventors have found that the hyaluronic acid can exhibit excellent skin improvement activity through synergistic activity with the astaxanthin and madecassoside, compared to the case where the hyaluronic acid acts alone on the skin.

The cosmetic composition may further include betaine.

The “betaine” is an amino acid with three methyl groups and is widely present in the animal and plant world, and is particularly abundant in marine invertebrates, spinach, goji berries, and radishes, so it can be supplied through food or dehydrogenase in vivo. ) and betaine aldehyde dehydrogenase.

The betaine changes the metabolism of sulfur-containing amino acids to convert methionine and cysteine to taurine and glutathione through the transsulfuration pathway (TS). It has been reported to have a protective effect against hepatotoxicity, and there have been several previous research results, but it has not been clarified that it can exhibit skin improvement activity together with the astaxanthin and madecassoside.

The cosmetic composition may further include a Centella Asiatica extract.

The Centella asiatica is a creeping perennial herb belonging to the Umbel family and is native to the island of Madagascar in Africa, but is widely native to high-temperature and humid places such as coastal areas of the Indian Ocean, northern Australia and some Central and South America, and domestic In , it grows wild in Jeju Island belonging to the temperate zone and island areas in the southern region.

The Centella asiatica is excellent in wound treatment and is effective in anti-dementia and gastrointestinal diseases, so it is used in various ways as a raw material for treating diseases. It was confirmed that the skin regeneration effect could be maximized.

The astaxanthin may be stabilized by liposomes.

The "liposome" refers to a vesicle with a diameter of about 50 to 2000 nm surrounded by a fine spherical membrane, and may be a concept including all compartments surrounded by a lipid bilayer.

The liposome may exhibit more advantageous effects in carrying active ingredients and permeating active ingredients into the skin.

The average particle diameter of the liposome may be 50 to 250 nm. If the average particle diameter of the liposome is more than 250 nm, the skin penetration effect and formulation stability may be reduced, and if it is less than 50 nm, astaxanthin may not be properly contained in the liposome.

The liposome may be prepared by a mixture containing a polyol, an oil component, a surfactant, a phospholipid, a fatty acid, and water, and may be obtained through various methods known in the art.

The present inventors have confirmed that when the astaxanthin complex component acts on the skin, only the astaxanthin is collected through liposomes, thereby maximizing skin penetration and thereby significantly improving the skin improvement effect.

The cosmetic composition may further include Haematococcus fluvialis extract.

The Haematococcus fluvialis is a microalgae that lives in freshwater and accumulates secondary metabolites such as lipids and carotenoids to resist stress. The Haematococcus fluvialis extract is In addition to astaxanthin, skin functional ingredients may be included.

The Haematococcus fluvialis extract may be fermented by Candida bombicola , and the fermentation process may be performed by a method known in the art.

The Candida Bombicola is a yeast that produces a large amount of Sophorolipid, and the Haematococcus fluvialis extract can exhibit better skin functionality by fermentation of Candida Bombicola, especially synergistic with astaxanthin action can be shown.

The cosmetic composition may further include fermented olive oil, and specifically, may further include olive oil fermented by Candida bombicola.

The olive ( Olea europaea Linnaeus )” is a plant of the Oleaceae family, and oleuropein, which is abundantly contained in the olive leaf, is known to be effective in improving vascular health, anti-aging, anti-inflammation, and preventing constipation. Rosol (hydroxytyrosol) is known to be effective in inflammation.

The cosmetic composition may further include anti-inflammatory, acne improvement or skin regeneration purposes.

The "skin regeneration" refers to a process of recovering skin tissue from damage caused by external and internal skin causes.

The damage caused by the external cause may include ultraviolet rays, external contaminants, wounds, trauma, and the like, and the damage caused by the internal cause may include stress.

The cosmetic composition can restore the skin barrier, promote skin regeneration through this, increase moisture to protect the skin from ultraviolet rays or pollution, increase immunity, and inhibit skin aging.

The cosmetic composition can effectively improve skin damaged by acne through various activities such as skin moisturizing and regeneration by the astaxanthin complex component effectively acting on the skin.

The “anti-inflamatory” means an effect of improving inflammation.

The "inflammation" is a mechanism for restoring and regenerating the damaged area when an invasion that causes any organic change such as physical action, chemical substance, bacterial infection, etc. is applied to a living body or tissue. Once stimulation is applied, histamine, Inflammation may be induced as vascular active substances such as serotonin, bradykinin, prostaglandin, HETE (hydroxyeicosatetraenoic acid), and leukotrienes are liberated to increase vascular permeability.

The cosmetic composition may further include an anti-wrinkle or anti-photoaging application.

The "wrinkle improvement" refers to a phenomenon in which elastic fibers composed of elastin exist together with collagen, and skin elasticity is maintained or improved in a state in which the elastin and collagen are sufficiently present. In addition, the "wrinkle improvement" refers to suppressing or inhibiting the formation of wrinkles on the skin, or mitigating already generated wrinkles.

The "anti-photoaging" may be for preventing skin aging induced by ultraviolet rays or UVB.

The “photoaging” is an aging phenomenon caused by ultraviolet rays, which are external environmental factors. The UV light activates proteolytic enzymes and causes chain scission and abnormal cross-linking of matrix proteins, resulting in damage to biocomponents, and repetition of the mechanism may result in apparent skin aging.

The cosmetic composition can effectively improve skin aging induced by ultraviolet rays, unlike endogenous aging caused by genetic factors, regardless of the external environment.

The cosmetic composition is at least one selected from the group consisting of softening lotion, nutrient lotion, moisture cream, nutrient cream, massage cream, essence, ampoule, gel, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, and powder can be formulated.

The cosmetic composition may be formulated by conventional methods. In the formulation of external skin preparations, reference may be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, and in the formulation of cosmetic compositions, International Cosmetic Ingredient Dictionary, 6th ed (The cosmetic, Toiletry and Fragrance Association , Inc., Washington, 1995) may be referred to.

Specifically, the cosmetic composition may be prepared in a general emulsified formulation and solubilized formulation. For example, cosmetic lotion, such as softening lotion or nourishing lotion; emulsions such as facial lotion and body lotion; creams such as nourishing creams, moisture creams, and eye creams; essence; cosmetic ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Alternatively, it may be formulated as a cleanser such as a cleansing foam, soap, body wash, etc., but is not limited thereto. In addition, the skin external preparation may be formulated as an ointment, patch, gel, cream or spray, but is not limited thereto.

In addition to the essential components in each formulation, the cosmetic composition may be appropriately mixed with other components within a range that does not impair the purpose of the present invention depending on the type of formulation or purpose of use.

The cosmetic composition may include a conventionally acceptable carrier, and for example, oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, a chelating agent, a colorant, a preservative, a flavoring agent, and the like may be appropriately blended, but are not limited thereto. no.

The acceptable carrier may vary depending on the formulation. For example, animal oil, vegetable oil, wax, paraffin, starch, tracanthate, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or any of these as a carrier component when formulated into an ointment, paste, cream or gel extracts may be used.

When the cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or extracts thereof may be used as a carrier component, and in the case of a spray, chlorofluorohydro It may further contain a propellant such as carbon, propane, butane or dimethyl ether.

When the cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan may be used.

When the cosmetic composition is formulated as a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like can be used.

When the cosmetic composition is formulated as a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. can be used

The cosmetic composition includes fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, and fragrances commonly used in the industry depending on the quality or function of the final product. , surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives commonly used in cosmetics It may additionally contain adjuvants commonly used in cosmetology or dermatology, such as any other ingredient.

However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect desirable properties of the cosmetic composition according to the present invention.

Through the following examples, the present invention is further detailed, but it is obvious that the present invention is not limited by the following examples.

manufacturing example

For the preparation of astaxanthin liposomes, phase B was added to phase A according to the composition shown in Table 1 below and uniformly wetted, and then phase C was added and heated and mixed at 70 to 80 ° C.

The mixed solution was passed through a high-pressure homogenizer three times under a condition of 600 to 1000 bar and then cooled, and liposomes having an average particle diameter of about 90 nm were obtained.

division ingredient Content (% by weight) A propylene glycol 40 B lecithin 5 cholesterol 2.5 ceramide 2.5 Sodium Stearoyl Lactate 0.5 Polysorbate-80 0.5 stearic acid 0.5 C astaxanthin 20.0 potassium hydroxide a very small amount vitamin C 15.0 Purified water balance

Examples and Comparative Examples

In order to verify the stability and functionality of the astaxanthin complex component of the present invention, examples and comparative examples were set by mixing samples as shown in Tables 1 and 2 below.

For each sample, a product in a powder state was purchased from the market and used.

Olive Oil Hematococcus fluvialis fermented extract and fermented olive oil obtained by inoculating and fermenting Candida bombicola were used.

[Amount (parts by weight)] division Example One 2 3 4 5 6 7 8 9 10 11 12 astaxanthin 60 40 40 30 24 60 - - 20 20 20 Madecassoside 60 40 40 30 24 - 30 24 20 20 20 20 hyaluronic acid - 40 - 30 24 - 30 24 20 20 20 20 betaine - - 40 30 24 - 30 24 20 20 20 20 centella extract - - - - 24 - - 24 20 20 20 20 Haematococcus fluvialis extract - - - - - - - - 20 - - - Haematococcus fluvialis fermented extract - - - - - - - - - 20 10 10 fermented olive oil - - - - - - - - - - 10 10 astaxanthin liposome
(Manufacture example) - - - - - 60 30 24 - - - 20

[Amount (parts by weight)] division comparative example One 2 3 4 5 6 7 8 9 10 11 12 astaxanthin 120 - - - - - 60 60 - - - 40 Madecassoside - 120 - - - - - - 60 60 40 - hyaluronic acid - - 120 - - - 60 - 60 - 40 40 betaine - - - 120 - - - 60 - 60 40 40 centella extract - - - - 120 - - - - - - - astaxanthin liposome
(Manufacture example) - - - - - 120 - - - - - -

Experimental Example 1: Skin safety test

In order to verify the skin safety of the samples of Examples and Comparative Examples, MTT assay experiments were performed on fibroblasts (HDF).

Samples of Examples and Comparative Examples were suspended in purified water at concentrations of 25, 50, 100, 200, and 400 μg/mL, respectively, and cell viability was measured.

Cytotoxicity was performed by modifying Mosmann's method for measuring cell viability using MTT{3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide} reagent.

Fibroblasts (HDF) were each dispensed in a 96-well plate at a concentration of 1×10 ⁴ cells/well and cultured at 37° C., 5% CO ₂ for 48 hours.

The culture medium was removed, and the samples were cultured in the medium treated for each concentration for 48 hours, and then the medium was removed and washed repeatedly with PBS (Phosphate buffered saline).

50 μL of MTT was dissolved in PBS at 5 mg/mL, and cultured at 37° C. and 5% CO ₂ for 48 hours. 100 L of dimethyl sulfoxide (DMSO) was added per well, stirred for 10 minutes, and absorbance was measured at 540 nm.

As a result of the measurement, cytotoxicity was not confirmed at all concentrations of each sample. The results suggest that the sample is not only harmless to the human body but also has excellent human safety.

Experimental Example 2: Evaluation of free radical scavenging activity

Extracts of Examples and Comparative Examples were suspended in purified water to evaluate free radical scavenging acticity.

The DPPH measurement method is a method of measuring absorbance at 540 nm to determine the degree of decolorization by scavenging the stable radical DPPH (2,2-Diphenyl-1-picrylhydrazyl radical) by an inhibitor.

Vitamin C was used as a control group, and the same experiment was repeated three times for the accuracy of the experiment, and the results are shown in Table 4 below.

[Concentration that scavenges 50% of radical DPPH] division SC ₅₀ (μg/mL) Example 1 133.6 Example 2 122.8 Example 3 124.3 Example 4 118.3 Example 5 114.2 Example 6 127.1 Example 7 112.6 Example 8 108.7 Example 9 112.5 Example 10 107.4 Example 11 108.4 Example 12 105.8 Comparative Example 1 225.9 Comparative Example 2 181.8 Comparative Example 3 183.8 Comparative Example 4 182.5 Comparative Example 5 179.4 Comparative Example 6 183.8 Comparative Example 7 158.1 Comparative Example 8 163.0 Comparative Example 9 151.6 Comparative Example 10 156.1 Comparative Example 11 149.6 Comparative Example 12 154.0 control (vitamin C) 128.2

The lower the value of SC ₅₀ , the higher the DPPH free radical scavenging activity. Referring to Table 4, the samples of the examples were significantly superior in antioxidant effect compared to the comparative examples.

In particular, Example 1 containing astaxanthin and madecassoside at the same time was significantly superior in free radical scavenging activity compared to Comparative Examples 7 to 12 lacking some of them, and the result shows that the combination of astaxanthin and madecassoside suggests a synergistic effect.

Experimental Example 3: Evaluation of anti-inflammatory activity

Anti-inflammatory activity of the samples of Examples and Comparative Examples was evaluated by analyzing the ability of EAME to inhibit NO production in the mouse macrophage cell line RAW 264.7 cells stimulated with Lipopolysaccharide (LPS).

For the experiment, Raw 264.7 cells, a murine macrophage cell line, were purchased from the Korea Cell Line Bank.

Using DMEM medium containing 100 units/mL penicillin-streptomycin and 10% fetal bovine serum (FBS), they were cultured in a 37°C, 5%, CO ₂ incubator, and subcultured once every 2 to 3 days.

Raw 264.7 cells (3 x 10 ⁵ cells/mL) were cultured before 18 hours, and the samples (concentration: 1%) and LPS (1 μg/mL) of Examples and Comparative Examples were simultaneously treated and cultured for 24 hours.

Produced NO was measured in the form of NO ₂ ^- present in the cell culture medium using Griess reagent.

The negative control was not treated with LPS, and the positive control was treated with only LPS, and the amount of NO produced was measured.

100 μL of cell culture supernatant and Griess reagent (1% (w/v) sulfanilamide, 0.1% (w/v) naphtylehylenediamine in 2.5% (v/v) phosphoric acid) were mixed in equal amounts and reacted in the dark at room temperature for 10 minutes, followed by ELISA Absorbance (540 nm) was measured using a reader.

The standard concentration curve was calculated by serial dilution of sodium nitrite (NaNO ₂ ) (10-100 μM). The measurement results are shown in Table 5 below.

division NO production rate (%) Example 1 50.0 Example 2 47.5 Example 3 45.8 Example 4 43.9 Example 5 39.5 Example 6 48.0 Example 7 42.2 Example 8 37.9 Example 9 38.2 Example 10 36.2 Example 11 34.7 Example 12 32.5 Comparative Example 1 65.3 Comparative Example 2 68.3 Comparative Example 3 64.6 Comparative Example 4 70.5 Comparative Example 5 73.3 Comparative Example 6 69.6 Comparative Example 7 63.1 Comparative Example 8 69.8 Comparative Example 9 70.7 Comparative Example 10 65.1 Comparative Example 11 63.4 Comparative Example 12 61.1 control group 29.5 Negative control (untreated) 100.0

Referring to Table 5, the samples of Examples 1 to 12 were superior in nitric oxide (NO) production inhibitory activity compared to Comparative Examples 1 to 12.

In particular, Example 1 containing astaxanthin and madecassoside at the same time was significantly superior in anti-inflammatory activity compared to Comparative Examples 7 to 12 lacking some of them, and the above results suggest a synergistic effect according to the combination of each component.

Experimental Example 4: Cell protection effect test according to UV irradiation

The cell protection effect of the samples of Examples against UVB was measured.

Fibroblasts at a concentration of 1.0 x 10 ⁴ cells/mL were dispensed in a 96-well plate at a concentration of 1 x 10 ⁴ cells/well and cultured at 37°C, 5% CO ₂ for 48 hours.

The culture medium was removed and washed twice with PBS to remove the remaining medium. The sample of Example was replaced with PBS adjusted for each concentration, and UVB (30mJ/cm ² ) was irradiated for 5 minutes.

After removing the PBS and removing the remaining sample, the culture was further cultured for 24 hours.

50L of MTT was dissolved in PBS at 5 mg/mL, and cultured at 37°C and 5% CO ₂ for 4 hours.

After dissolving the stained cells by adding 100 μL of dimethyl sulfoxide solution to each well, the absorbance was measured at 570 nm using a microplate reader (Table 6).

division relative cell viability (% of control) Example 1 82.1 Example 2 84.2 Example 3 85.7 Example 4 86.3 Example 5 88.6 Example 6 86.3 Example 7 89.7 Example 8 91.4 Example 9 88.8 Example 10 90.1 Example 11 89.5 Example 12 91.9 Comparative Example 1 61.9 Comparative Example 2 52.3 Comparative Example 3 47.6 Comparative Example 4 51.4 Comparative Example 5 53.3 Comparative Example 6 54.7 Comparative Example 7 62.4 Comparative Example 8 63.5 Comparative Example 9 64.3 Comparative Example 10 64.7 Comparative Example 11 62.5 Comparative Example 12 67.2 Negative control group (not irradiated with UV rays) 100

Referring to Table 6, the samples of Examples containing astaxanthin and madecassoside at the same time effectively recovered cells damaged by ultraviolet rays compared to Comparative Examples 7 to 12, which lacked some of them, and the above results show the combination of each component. suggests a synergistic effect.

Experimental Example 5: Evaluation of cell senescence inhibition ability according to UV irradiation

SA-β staining assay was performed to analyze the cell protection mechanism against ultraviolet rays according to the samples of the examples.

The SA-βgalactosidase enzyme, which is specifically expressed in cells that have stopped growing due to aging, was stained using a Cellular senescence assay kit.

After pre-treatment of the sample to cells cultured for 38 hours, UVB (30 mJ/cm ² ) was irradiated and cultured for 24 hours, washed with 1× PBS, and 7 in 1× fixative (2% formaldehyde, 0.2% glutaraldehyde). fixed for min.

The cells from which the fixative was removed were washed again with 1x PBS three times, and then incubated in a dark room at 37°C for 17 hours in a state where the staining mixture (10x staining solution, Reagent B, Reagent C, X-gal solution) and carbon dioxide were removed. Beta-galactosidase activity was measured by reacting (Table 7).

division Numbers of SA-β-Gal (% of control) Example 1 165 Example 2 154 Example 3 152 Example 4 155 Example 5 145 Example 6 158 Example 7 152 Example 8 137 Example 9 141 Example 10 138 Example 11 136 Example 12 131 Comparative Example 1 228 Comparative Example 2 225 Comparative Example 3 195 Comparative Example 4 193 Comparative Example 5 204 Comparative Example 6 205 Comparative Example 7 197 Comparative Example 8 185 Comparative Example 9 192 Comparative Example 10 193 Comparative Example 11 184 Comparative Example 12 178 Negative control group (not irradiated with UV rays) 100 Positive control group (ultraviolet irradiation) 312

Referring to Table 7, the cells treated with only UV light increased the number of stained cells by more than 3 times compared to the control group, and the cells treated with the sample of Example after UV treatment were inhibited from aging, so that the sample treated with the comparative sample Compared to cells, the number of cells was significantly decreased.

Experimental Example 6: Skin moisturizing effect test

In the Example, the skin moisturizing effect was evaluated by suspending the sample in purified water.

Expression levels of HAS2 gene and AQP3 gene involved in skin moisturizing in human keratinocytes were measured at the mRNA level.

Human keratinocytes (HaCaT) were cultured in a 100 mm/60.1 cm culture dish with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS), and 1% Antibiotic-Antimycotic at 37°C and 5% CO ₂ . did

Keratinocytes were dispensed into a 96-well plate at a concentration of 1.0 x 10 ⁶ cells/mL and cultured for 24 hours.

After exchanging with DMEM free medium, the diluted samples of Examples and Comparative Examples were treated and cultured for 24 hours.

The cells were harvested, washed with chilled phosphate buffered saline (PBS), and 1 mL of Trizol reagent [TRIzol™reagent, Life Technologies, Inc.] was added and total RNA was extracted.

Changes in gene expression were measured by quantitative real time PCR (qRT-PCR), which binds a fluorescent substance to a DNA product amplified in a polymerase chain reaction (PCR) and continuously detects the fluorescent substance.

To quantify changes in HAS2 and AQP3 gene expression, fluorescence values emitted by the PCR products were measured using SYBR green I (Invitrogen).

A reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris/HCl pH 8.4, 0.8 mM dNTP, 0.5 U Extaq DNA polymerase, 3 mM MgCl2, and 1×SYBR green in a PCR tube.

After preliminary denaturation at 94°C for 3 minutes using Linegene K (BioER, China), denaturation, annealing, and polymerization were performed at 94°C for 30 seconds and 58°C for 30 seconds. A total of 40 cycles were performed at 72° C. for 30 seconds, and fluorescence intensity was measured after each cycle was completed.

PCR results were verified by melting curve for each result. After standardizing the threshold cycle (Ct) value of each gene with the Ct value of β, the amount of change in the Ct value was compared and analyzed.

The Ct value is the number of cycles when the amount of fluorescence generated by the PCR product (the number of amplified PCR products) reaches a certain standard value higher than the basic value, and the gene expression level can be confirmed through the Ct value.

The HAS2 and AQP3 mRNA expression level measurement results are shown in Table 8 below, and the mRNA expression level was measured after sample treatment at a concentration of 20 μg / mL in Example.

The higher the HAS2 and AQP3 expression promoting ability, the better the skin moisturizing effect.

[Fold of control] division HAS2 expression level AQP3 expression level Example 1 1.36 1.43 Example 2 1.41 1.52 Example 3 1.42 1.54 Example 4 1.47 1.58 Example 5 1.54 1.63 Example 6 1.38 1.45 Example 7 1.49 1.60 Example 8 1.56 1.65 Example 9 1.56 1.64 Example 10 1.59 1.67 Example 11 1.61 1.68 Example 12 1.64 1.71 Comparative Example 1 1.13 1.19 Comparative Example 2 1.08 1.12 Comparative Example 3 1.10 1.13 Comparative Example 4 1.06 1.15 Comparative Example 5 1.03 1.14 Comparative Example 6 1.15 1.18 Comparative Example 7 1.11 1.15 Comparative Example 8 1.09 1.17 Comparative Example 9 1.18 1.21 Comparative Example 10 1.21 1.24 Comparative Example 11 1.23 1.25 Comparative Example 12 1.25 1.28 negative control group 1.00 1.00

Referring to Table 8, the expression levels of the HAS2 gene and the AQP3 gene increased according to the sample treatment of the examples.

The above results suggest that the samples of Examples can effectively enhance skin moisturizing by promoting the expression of aquaporin protein and the synthesis of hyaluronic acid.

Experimental Example 7: Evaluation of skin wrinkle improvement activity

In order to evaluate the effect of improving skin wrinkles, an experiment was conducted as follows.

120 adult women aged 30 years or older were divided into 20 groups of 6 people each, and the test subjects applied the cosmetics formulated with the samples of Examples and Comparative Examples evenly to the face after washing their face every morning and evening for 4 weeks to be sufficiently absorbed. .

At this time, the samples of Examples and Comparative Examples were all formulated with the same components.

ANTERA 3D (Miravex, Ireland) was applied to evaluate wrinkle improvement, and instrument measurement was performed before, after 2 weeks of use, and after 4 weeks of use of the test substance.

The same tester measured the left corner of the eye of all test subjects, and for reproducibility of the measurement, the same area was measured by overlapping with the image measured before the test substance was used.

The captured images were matched using ANTERA pro software, a dedicated software for ANTERA 3D, and the matched measurement area was used for analysis.

As for the measurement value, the Wrinkles small value representing the wrinkles of the skin was analyzed using the indentation index, which is a measurement variable.

The amount of change in Wrinkles small value after 4 weeks of the test subject according to the use of cosmetics formulated with the samples of Examples and Comparative Examples was shown (Table 9). The greater the wrinkle improvement effect, the greater the change in Wrinkles small value.

division Wrinkles small average value Example 1 1.38 Example 2 1.41 Example 3 1.42 Example 4 1.47 Example 5 1.56 Example 6 1.44 Example 7 1.54 Example 8 1.62 Example 9 1.57 Example 10 1.61 Example 11 1.61 Example 12 1.64 Comparative Example 1 1.09 Comparative Example 2 1.18 Comparative Example 3 1.09 Comparative Example 4 1.09 Comparative Example 5 1.12 Comparative Example 6 1.10 Comparative Example 7 1.14 Comparative Example 8 1.18 Comparative Example 9 1.17 Comparative Example 10 1.21 Comparative Example 11 1.22 Comparative Example 12 1.24

Referring to Table 9, the cosmetic composition formulated with the sample of Example containing both astaxanthin and madecassoside had a remarkably excellent skin wrinkle improvement effect, particularly Example containing astaxanthin encapsulated by liposomes. The skin wrinkle improvement effect of 6 to 8 was further improved.

Experimental Example 8: Evaluation of cell migration promoting activity

Skin regeneration and wound healing effects of the samples of Examples and Comparative Examples were confirmed through a wound healing assay.

Specifically, HaCat cells were dispensed in a 96-well plate at a number of 1×10 ³ cells/well.

After 24 hours, the medium was replaced with DMEM medium supplemented with 5% FBS, and samples of Examples and Comparative Examples were treated at the same concentration (1%) in each well.

After 48 hours, a wound was applied using a yellow tip, and after 24 hours, a photograph was taken of the extent to which the wound was closed, and the area was measured using an image J program.

division Cell mobility (%) Example 1 57.2 Example 2 62.0 Example 3 64.6 Example 4 66.6 Example 5 70.4 Example 6 58.9 Example 7 68.2 Example 8 72.6 Example 9 71.3 Example 10 72.6 Example 11 73.7 Example 12 75.2 Comparative Example 1 29.1 Comparative Example 2 28.2 Comparative Example 3 27.6 Comparative Example 4 26.8 Comparative Example 5 28.8 Comparative Example 6 27.5 Comparative Example 7 28.6 Comparative Example 8 33.4 Comparative Example 9 31.3 Comparative Example 10 32.4 Comparative Example 11 33.0 Comparative Example 12 34.2

Referring to Table 10, the sample of Example significantly improved cell migration compared to Comparative Example. In particular, Example 1 containing astaxanthin and madecassoside at the same time compared to Comparative Examples 7 to 12 lacking a part of cell migration. The accelerating activity is significantly superior, and the above results suggest that skin regeneration and wound healing effects are improved by synergy according to the combination of each component.

Experimental Example 9: Evaluation of acne improvement effect

Samples of Examples and Comparative Examples 1 to 5 formulated for 80 women aged 19 to 28 with acne were applied to the face and the first half of the body twice daily for 1 month, and the degree of improvement was determined according to the user's opinion And the results are shown in Table 11 below.

As a result of the evaluation, it was confirmed that the sample of Example 1 (Example) was remarkably superior in the acne improvement effect compared to the samples of Comparative Examples 1 to 5.

division 2 weeks 4 weeks Example 1 + ++ Example 2 ++ ++ Example 3 ++ ++ Example 4 ++ +++ Example 5 ++ +++ Example 6 ++ ++ Example 7 ++ +++ Example 8 ++ +++ Example 9 ++ +++ Example 10 +++ +++ Example 11 ++ +++ Example 12 +++ +++ Comparative Example 1 ± ± Comparative Example 2 ± ± Comparative Example 3 ± + Comparative Example 4 ± ± Comparative Example 5 + + Comparative Example 6 ± ± Comparative Example 7 ± + Comparative Example 8 + + Comparative Example 9 ± + Comparative Example 10 + + Comparative Example 11 + ++ Comparative Example 12 + +

<Evaluation Criteria> +++: Very good improvement / ++: Significant improvement / +: Slight improvement / ±: No improvement, but no deterioration / -: No improvement, rather weakened

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (9)

Contains astaxanthin, madecassoside, hyaluronic acid and Haematococcus fluvialis extract;
The Haematococcus fluvialis extract is fermented by Candida bombicola ,
A cosmetic composition for skin moisturizing and antioxidant comprising at least one of betaine and centella asiatica extract as an active ingredient. delete delete delete delete According to claim 1,
A cosmetic composition further comprising fermented olive oil. According to claim 1,
The astaxanthin is a cosmetic composition stabilized by liposomes. According to any one of claims 1, 6 and 7
A cosmetic composition comprising anti-inflammatory, acne improvement or skin regeneration applications. The method of any one of claims 1, 6 and 7,
A cosmetic composition further comprising an anti-wrinkle or anti-photoaging application.