WO2018151563A1 - Composition for preventing photoaging and alleviating skin wrinkles, containing cosmos bipinnatus extract - Google Patents

Abstract

The present invention relates to a composition for improving skin condition, containing a Cosmos bipinnatus extract. The Cosmos bipinnatus extract, which is used as an active ingredient in the composition of the present invention, has an excellent effect of alleviating wrinkles through a molecular mechanism such as collagen synthesis promotion and fibroblast protection action, prevents skin damage caused by ultraviolet irradiation so as to prevent photoaging, and is noncytotoxic and exhibits excellent skin safety, thereby being usable in a cosmetic or a pharmaceutical composition.

Description

Composition for preventing photoaging and improving skin wrinkles, including cosmos extract

The present invention relates to a composition for preventing photoaging and anti-wrinkle including Cosmos ( Cosmos bipinnatus ) extract, more specifically, to enhance collagen synthesis, inhibit collagen degrading enzymes, increase the inhibitory activity by increasing the amount of TIMP-1 expression, skin cell proliferation The present invention relates to a photo-aging and anti-wrinkle cosmetic or pharmaceutical composition comprising a cosmos extract having an effect, an anti-aging effect on skin cell damage by ultraviolet rays, and an anti-aging effect.

Aging is a series of processes that occur due to the loss of homeostasis in the body due to functional, structural, and biochemical changes that occur throughout cells and throughout the body. Human skin aging usually begins around 25 years of age and starts around 40 years of age. The cause of skin aging can be classified into two types: endogenous aging and exogenous aging. Endogenous aging is the aging that progresses by decreasing the secretion of various hormones that regulate metabolism internally over time, decreasing the physiological function of cells and reducing the biosynthesis of the elements necessary for living organisms. Exogenous aging is ultraviolet and pollution Aging is caused by external stimuli such as temperature, and various pollutants.

Meanwhile, collagen (Collagen), which accounts for 90% of the dermal layer of the skin, and elastin (elastic fiber), which account for 3-4%, provide elasticity and protect the skin. The number and activity of fibroblasts involved in the body is reduced and their amount of synthesis is also reduced. Accordingly, as the amount of water loss in the skin cells increases and the structure of the stratum corneum changes, the activity of collagenase (collagenase) increases, the crosslinking form of collagen increases, skin elasticity, moisturization, etc. decrease, and wrinkles occur. . Dermis atrophy that occurs after the age of 70 is a typical aging phenomenon, such as the separation of collagen bundles, reduced polysaccharide synthesis, decreased number and diameter of collagen and elastin, crushing collagen and elastic fibers, and swelling of blood vessels.

Photoaging and various contaminants caused by ultraviolet rays cause chain breakage of collagen and elastin, abnormal crosslinking and cleavage of hyaluronic acid to increase direct skin cell damage and pigmentation. This directly leads to a loss of function of the skin's major components, thereby aging the skin.

As the skin ages, sebum secretion decreases, the skin becomes dry, cell regeneration slows down, and aging keratin accumulates, skin roughness occurs. Collagen decreases and elastin degeneration causes wrinkles.

In general, the greatest influence on the formation of wrinkles is the activity of collagenase to reduce the amount of collagen production and collagen content. Therefore, research is focused on the development of materials that can induce collagen synthesis, search for substances that can inhibit the synthesis or activity of collagenase, and alleviate skin aging using substances that have secured safety. Development of natural herbal materials is required to find safer and more effective materials.

Accordingly, the present inventors have been researching natural materials to prevent photoaging, develop wrinkles, and have high safety and stability, and thus have no skin side effects, while cosmos extract ( Cosmos bipinnatus ) will become an excellent natural material. The present invention has been completed by discovering that it is possible and continuing the research on this.

One object of the present invention is to provide a cosmetic composition for preventing photoaging and / or anti-wrinkle comprising Cosmos extract ( Cosmos bipinnatus ) as an active ingredient.

It is another object of the present invention to provide a photoaging preventing and / or anti-wrinkle pharmaceutical composition comprising Cosmos extract ( Cosmos bipinnatus ) as an active ingredient.

In one embodiment, the present invention provides a composition for improving wrinkles comprising cosmos extract as an active ingredient.

In the present invention, Cosmos ( Cosmos bipinnatus ) is a perennial herb belonging to the Asteraceae family, grows up to 1 to 2 m in height and blooms in June-October, and the flowers have various colors such as red, white, and purple. Cosmos is characterized by branching at the top and hairless, with one headpiece at the end of branches and stems.

In the present invention, "improvement of skin wrinkles" is to relieve and remove wrinkles formed on the skin of animals including humans by endogenous skin aging due to hormonal secretion reduction or physiological function decrease or exogenous skin aging due to ultraviolet rays or pollution. Or to prevent.

Cosmos extract of the present invention refers to a substance having an activity of improving skin wrinkles extracted from or separated from cosmos outposts, leaves, roots, stems, flowers and the like.

In addition, the cosmos extract refers to all extracts, fractions, purified products, their dilutions, concentrates, dried products or all forms formulated using each extract, fraction, or each step of purification (separation, fraction).

Extraction method of the cosmos extract may include cold needle extraction, ultrasonic extraction, reflux cooling extraction method or supercritical extraction method, preferably ultrasonic extraction method.

The cosmos extract may be extracted using conventional solvents known in the art, and the extracted liquid may be used in liquid form or concentrated and / or dried. At this time, the solvent is water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), ethylene, acetone, hexane, ether, ethyl acetate, butyl acetate, chloroform, 1,3-butylene A solvent of any one of glycol, propylene glycol, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or a mixed solvent thereof can be used. Preferably, water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.) or a mixed solvent of water and the lower alcohol is used. More preferably, a mixed solvent of water and methanol is used. However, the present invention is not limited thereto, and the extraction degree and loss degree of the active ingredient of the extract may vary depending on the organic solvent to be extracted. Therefore, it is preferable to select and use an appropriate organic solvent.

As one specific example, the mixed solvent of water and anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.) is a mixture of water and the lower alcohol in a weight ratio of 1: 0.1 to 1:10. It is preferably mixed in a weight ratio of 1: 0.2 to 1: 9.

As one specific example, the cosmos extract may be obtained by immersion extraction after pulverizing the cosmos, and then filtering and recovering the extract. The filtration is a process of removing the suspended solid particles from the extract, it may be used to filter the particles using cotton, nylon, or the like, ultrafiltration, cryofiltration, centrifugal separation, etc., but is not limited thereto. It may also further comprise a separation process by various chromatography (chromatography according to size, charge, hydrophobicity or affinity).

As one specific example, the cosmos extract may be obtained by drying the filtrate. Freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying or infrared drying may be used as a method of drying the filtrate, but is not limited thereto.

The cosmos extract included in the composition of the present invention increases TI collagen synthesis in skin cells, inhibits matrix metaloproteinase-1 (MMP-1) collagen degrading enzyme, and inhibits the activity of MMP-1 collagen degrading enzyme. Tissue Inhibitors of Metalloproteinase-1) increases the expression level, thereby improving the skin wrinkles.

As another aspect, the present invention provides a composition for preventing photoaging comprising the cosmos extract as an active ingredient.

Cosmos extract contained in the composition for preventing photoaging of the present invention is as described above, the cosmos extract is a material having a photoaging prevention activity extracted from or separated from the outpost, leaves, roots, stems, flowers and the like of cosmos.

In the composition for preventing photoaging according to the present invention, "photoaging prevention" protects the skin from skin damage caused by external factors such as ultraviolet rays, and prevents skin aging caused by ultraviolet rays and skin aging such as sagging of the skin and wrinkle formation of the skin. It is to suppress.

In the present invention, the external factors such as ultraviolet rays include stress and external harmful chemicals, etc. in addition to ultraviolet rays, the factors may generate free radicals (eg, hydroxyl radicals, superoxide radicals, active oxygen and hydrogen peroxide) to the skin. Increasing oxidative stress causes inflammation, skin cell death and skin cell DNA damage, resulting in skin cancer or aging of the skin.

In the present invention, the "skin damage" means that the skin does not perform its normal function, including damage to the skin constituents (such as collagen and elastin), impaired function of skin cells and death of skin cells, etc. do.

In the present invention, the skin cells include all cells present in the skin, preferably keratinocytes and fibroblasts.

In the present invention, "skin protection" includes preventing damage to the skin and improving damaged skin.

Cosmos extract contained in the composition of the present invention inhibits the expression of MMP-1 in the skin cells damaged by ultraviolet light, increases the cell survival rate by protecting the skin cells damaged by ultraviolet light, collagen in the skin cells damaged by ultraviolet light And anti-aging effects such as preventing cell aging by inhibiting destruction of elastin fibers.

In one specific embodiment, the composition of the present invention is provided as a cosmetic composition for preventing photoaging or improving skin wrinkles.

The cosmetic composition of the present invention may include the cosmos extract as an active ingredient in an amount of 0.00001 to 15.0% by weight, preferably 0.0001 to 10% by weight, more preferably 0.0001 to 5% by weight, based on the total weight of the total cosmetic composition. . If the content is less than 0.0001% by weight can not be expected to improve the skin wrinkles and prevent photoaging, when the content exceeds 15.0% by weight the difference between the wrinkle improvement effect and photoaging prevention is insignificant or difficult to prepare a formulation.

The cosmetic composition may further include components commonly used in cosmetic compositions in addition to cosmos extract. For example, it may include conventional auxiliaries and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and agents.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art. For example, it may be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation or spray, and the like. More specifically, it may be prepared in a formulation such as nourishing cream, astringent lotion, flexible lotion, lotion, essence, nourishing gel or massage cream. However, it is not particularly limited to this.

When the formulation of the cosmetic composition is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components. Can be.

When the formulation of the cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, and in the case of a spray, additionally chlorofluorohydrocarbon, Propellant such as propane / butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, a solubilizer or an emulsifier may be used as a carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester can be used. have.

When the formulation of the cosmetic composition is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester as a carrier component, Crystalline cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the cosmetic composition is a surfactant-containing cleansing agent, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid. Amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention exhibits excellent skin wrinkle improvement effect through molecular mechanisms such as fibroblast protection and promotion of collagen synthesis while having low cytotoxicity.

In addition, the cosmetic composition of the present invention prevents and inhibits skin cell damage caused by ultraviolet rays, thereby exhibiting an anti-aging effect.

In one specific embodiment, the composition of the present invention is provided as a pharmaceutical composition for preventing photoaging or improving skin wrinkles.

The pharmaceutical composition of the present invention may include the cosmos extract as an active ingredient in an amount of 0.00001 to 15.0% by weight, preferably 0.0001 to 10% by weight, more preferably 0.0001 to 5% by weight based on the total weight of the total pharmaceutical composition. Can be. If the content is less than 0.0001% by weight can not be expected to improve the skin wrinkles and prevent photoaging, when the content exceeds 15.0% by weight the difference between the wrinkle improvement effect and photoaging prevention is insignificant or difficult to prepare a formulation.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form or in multidose form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which can be easily carried out by those skilled in the art. It may be prepared by incorporation into a container. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or an aqueous medium, or may be in the form of an exir, powder, powder, granule, tablet or capsule, and may further include a dispersing or stabilizing agent.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are commonly used in the preparation of pharmaceutical compositions, and include carbohydrate compounds (eg, lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch). , Cellulose, etc.), acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, salt solutions, alcohols, gum arabic, vegetable oils (e.g. corn Oils, cotton seed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, and the like.

In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention can be used in the form of injections or external preparations according to conventional methods.

The pharmaceutical composition of the present invention has an effect of improving skin wrinkles and preventing photoaging, and the method of administration includes administering the pharmaceutical composition in a pharmaceutically effective amount to mammals such as rats, mice, livestock, humans, and the like by various routes. .

The pharmaceutical composition may be administered orally or parenterally, and is preferably applied by topical application by parenteral administration, more preferably by application.

The dosage of the pharmaceutical composition may vary depending on factors such as the method of formulation, the mode of administration, the age, weight, sex, morbidity, etc. of the patient, and the dosage ranges from 0.001 to 300 mg / kg on an adult basis. However, the dosage does not limit the scope of the present invention.

Although the pharmaceutical composition of the present invention has low cytotoxicity, it has an excellent skin wrinkle improvement effect through molecular mechanisms such as fibroblast protection and promotion of collagen synthesis.

In addition, the cosmetic composition of the present invention prevents and inhibits skin cell damage caused by ultraviolet rays, thereby exhibiting an anti-aging effect.

The pharmaceutical composition comprising the cosmos extract of the present invention as an active ingredient has the advantage of achieving the purpose of improving the skin wrinkles and preventing photoaging of the individual by topical administration, such as administration in the subject, subcutaneous administration or application.

Cosmos extract of the present invention is a natural extract derived from the plant as a safe substance harmless to the human body without toxicity and side effects as a result of skin irritation experiments on the human body, as an active ingredient for improving skin wrinkles and preventing photoaging, and safely in cosmetics and pharmaceutical compositions. Can be applied.

In another specific embodiment, the present invention provides a method for preventing photoaging and / or improving wrinkles of the skin by administering to the individual a photoaging preventing and / or anti-wrinkle composition comprising the cosmos extract as an active ingredient.

The subject is a mammal, including a human, that is intended to prevent photoaging of the skin and / or to improve wrinkles of the skin.

The administration is oral or parenteral administration, preferably parenteral administration, more preferably topical administration by application, the dosage and the like are formulated method, mode of administration, age, weight, sex, morbidity, etc. It can be easily changed and used by those skilled in the art according to the factors of the.

By administering to the subject a composition for preventing photoaging and / or improving wrinkles according to the present invention, it is possible to prevent photoaging of the skin of the subject and to improve wrinkles.

In the composition for preventing photoaging and improving skin wrinkles of the present invention, the cosmos extract is included as an active ingredient, and the cosmos extract has an increase in collagen degradation inhibitory activity and fiber due to the promotion of collagen synthesis, inhibition of MMP-1, and increased expression of TIMP-1. Through detailed mechanisms such as promoting blast proliferation, it exhibits an excellent effect of improving skin wrinkles, and inhibits damage to skin cells caused by ultraviolet rays, thereby preventing photoaging. In addition, the extract of the present invention is derived from a natural material can be safely applied to cosmetics or pharmaceutical compositions as a safe substance harmless to the human body without toxicity and side effects as a result of skin irritation experiments on the human body.

1 is a graph showing the collagen (type I collagen) biosynthesis promoting effect of the cosmos extract as an embodiment of the present invention.

2 is a graph showing the inhibitory effect of MMP-1 collagenase of cosmos extract of one embodiment of the present invention.

3 is a graph showing the effect of increasing the amount of TIMP-1 (MMPs inhibitor) produced by the cosmos extract as an embodiment of the present invention.

4 is a graph showing the cell proliferation induction effect of the human-derived fibroblasts of the cosmos extract as an embodiment of the present invention.

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example  1: Cosmos Extract Manufacturer

The leaves, stems, and flowers of the cosmos were collected and washed to completely remove foreign substances and impurities, and then shaded at 20 to 35 ° C., respectively, and then pulverized to have a particle size of 1 mm or less. Then, 1 kg of the pulverized cosmos powder was immersed in 10 L of 70% ethanol and ultrasonically extracted for 24 hours. Thereafter, the obtained extract was filtered through filter paper (Advantes, No. 2), and concentrated under reduced pressure to prepare a cosmos extract.

Test Example  1: Measurement of Collagen Synthesis Effect of Cosmos Extract

Skin wrinkle improvement can be usually measured through the effect of enhancing collagen biosynthesis, and the amount of collagen synthesized can be determined by measuring the amount of propeptide isolated from procollagen, a precursor of collagen.

For collagen biosynthesis experiments, human-derived normal fibroblasts (Azo dermatology) were inoculated into 24-well microplates containing DMEM medium (2 × 10 ⁵ cells / well), 37 ° C. at 5% concentration, Incubated for 24 hours in a CO ₂ incubator. After removing the medium from each well, and treated with a positive control TGF-β1 10 ng / ml and cosmos extract by concentration and incubated again for 24 hours, a cell medium was collected to prepare a sample.

Collagen synthesis amount was measured by using a collagen measurement kit (Procollagen Type I C-peptide EIA kit MK101, Takara, Japan) to measure the amount of procollagen type I C-peptide (Type I C-peptide: PICP). Absorbance was measured and quantified at 450 nm, and the test was performed using human fibroblasts not treated with TGF-β1 and cosmos extract as a control. Detailed test method was performed according to the kit instructions of Takara Co., Ltd.

As shown in Figure 1, the test results showed that the cosmos extract in human-derived fibroblasts increases the degree of collagen biosynthesis in a concentration-dependent manner.

Test Example  2: of cosmos extract MMP -1 inhibition activity measurement

Inhibitory effects of cosmos extracts against matrix metalloproteinase-1 (MMP-1) collagen degrading enzymes and the anti-wrinkle effects were tested. MMP-1 is produced in fibroblasts, keratinocytes, macrophages, and the like, and is a collagen degrading enzyme that promotes secretion during tissue remodeling, wound healing, and inflammatory reactions. Inoculate human-derived fibroblasts into 24-well microplates, incubate for 24 hours in a CO ₂ incubator, remove the medium from each well, and treat the positive control TGF-β1 10 ng / ml and cosmos extract by concentration, Samples were prepared by collecting the cell medium after the reaction.

MMP-1 secretion was measured using an MMP-1 measurement kit (Human Pro-MMP-1 Quantikine ELISA kit, R & D systems,) and controls human-derived normal fibroblasts not treated with TGF-β1 and cosmos extracts. The test was carried out as described above. Detailed test methods were performed according to the R & D systems kit instructions.

As shown in FIG. 2, the cosmos extract in human-derived fibroblasts was found to decrease MMP-1 in a concentration-dependent manner.

Test Example  3: of cosmos extract TIMP -1 inhibition activity measurement

The activity of MMP-1 collagenase is regulated by inhibitors such as Tissue Inhibitors of Metalloproteinases-1 (TIMP-1). TIMP-1 is expressed in many human cells, including fibroblasts, and is secreted to maintain cell homeostasis. It is involved in the normal collagen synthesis process by inhibiting the action of MMPs in collagen synthesis and degradation in vivo.

The effects of TIMP-1 on inducing collagen synthesis and MMP-1 inhibitory activity of cosmos extracts were confirmed using a TIMP-1 measurement kit (Human TIMP-1 Quantikine ELISA kit, R & D systems). After culturing human-derived normal fibroblasts at 5% concentration, 37 ° C, CO ₂ incubator for 24 hours, the medium was replaced and the positive control TGF-β1 10 ng / ml and cosmos extract by concentration After treatment, and further incubated for 24 hours to prepare a sample was performed by the ELISA test method according to the R & D systems kit instructions, the absorbance was measured at 450nm wavelength to quantify the amount of TIMP-1 production. Human fibroblasts derived from humans without TGF-β1 and cosmos extracts were tested as controls.

As shown in FIG. 3, it was confirmed that the cosmos extract increased the concentration of TIMP-1 in human-derived fibroblasts and showed the same tendency as the collagen synthesis amount.

Test Example  4: Determination of the Protective Effect of Cosmos Extracts Against Collagen Damage in Ultraviolet Irradiated Cells

In order to investigate the cellular protective effect of cosmos extract in the aging of the cells by UV irradiation, the cosmos extract was treated on fibroblasts, irradiated with a certain amount of UVA, and then the collagen amount was examined.

Changes in the amount of collagen were inoculated into 24-well microplates containing DMEM medium (2 × 10 ⁵ cells / well), and 5% concentrations of 37 ° C., CO _The incubator was incubated for 24 hours. Medium was removed from each well and treated with positive control TGF-β1 10 ng / ml and cosmos extract for 4 hours by concentration. Samples were prepared by irradiating UVA at 5 J / cm 2 and then collecting the cell media after 48 hours.

Collagen synthesis amount was measured by using a collagen measurement kit (Procollagen Type I C-peptide EIA kit MK101, Takara, Japan) to measure the amount of procollagen type I C-peptide (Type I C-peptide: PICP). The absorbance was measured and quantified at 450 nm, and the test was performed by dividing the sample without UVA and medicinal herbs, the UVA-treated sample, the TGF-β1 and the cosmos extract, and the UVA-treated sample. Detailed test method was performed according to the kit instructions of Takara Co., Ltd.

As shown in FIG. 4, collagen reduced and damaged during UVA irradiation in human-derived fibroblasts was inhibited during pretreatment of TGF-β1 or cosmos extract, indicating a protective effect of restoring the reduced amount.

Test Example  5: in ultraviolet irradiation cells Increased MMP Of inhibitory effect of cosmos extract on -1

In order to examine the cell protective effect of the cosmos extract in the aging of the cell by ultraviolet irradiation, following Experimental Example 4, the fibroblasts were treated with cosmos extract and irradiated with a certain amount of UVA, and then the change of MMP-1 amount was investigated.

Changes in the amount of MMP-1 were inoculated into human well-derived normal fibroblasts (Azo dermatology) into 24-well microplates containing DMEM medium (2 × 10 ⁵ cells / well) and 37 ° C. at 5% concentration. Incubated for 24 hours in a CO ₂ incubator. Medium was removed from each well and treated with positive control TGF-β1 10 ng / ml and cosmos extract for 4 hours by concentration. Samples were prepared by irradiating UVA at 5 J / cm 2 and then collecting the cell media after 48 hours.

MMP-1 secretion was measured using an MMP-1 measurement kit (Human Pro-MMP-1 Quantikine ELISA kit, R & D systems,) and controls human-derived normal fibroblasts not treated with TGF-β1 and cosmos extracts. The test was carried out as described above. Detailed test methods were performed according to the R & D systems kit instructions.

As shown in Figure 5, it was confirmed that the increase in MMP-1 when pretreatment with TGF-β1 or cosmos extract amount of MMP-1 increased UVA irradiation in human-derived fibroblasts.

Test Example  6: Measurement of Cytoprotective Effect of Cosmos Extract on Human Fibroblast Damage by UV Irradiation

In order to determine the cellular protective effect of cosmos extract in human-derived fibroblasts damaged by UV irradiation, it was confirmed by MTT assay.

Human normal fibroblasts inoculated in 24 well-plates were incubated for 24 hours at 37 ° C. in a 5% CO ₂ incubator, followed by 10 ng / ml of the positive control TGF-β1 and The cosmos extract for each concentration was treated for 4 hours and irradiated with UVA at 5 J / cm 2 for 44 to 46 hours. After further incubation, MTT solution was added, and then reacted in a 37 ° C., CO ₂ incubator for 1-2 hours. The medium was removed from each well, DMSO (dimethyl sulfoxide) was added, and crystal violet crystals were dissolved. Thereafter, absorbance was measured at 540 nm using a Spectrophotometr.

As shown in Figure 6, it was confirmed that the reduced cell viability during UV irradiation was increased by the cosmos extract treatment.

Test Example  7: Determination of Anti-aging Effect of Cosmos Extracts on Aging of Human Fibroblasts by UV Irradiation

Prolonged exposure to the sun promotes skin aging by destroying the skin's collagen and elastin fibers, sagging skin and creating wrinkles. A senescence β-galactosidase assay was performed to determine whether the cosmos extracts have photoaging inhibitory activity that can inhibit skin aging caused by UV light.

The senescence β-galactosidase assay is a test method that utilizes high expression of lysosomal β-galactosidase in activated cells and is activated in the pH range of 5.8-6.0 compared to normal cells. Lysosomal β-galactosidase is a representative marker of senescent cells. It is widely used and is called senescence β-galactosidase.

Inoculate human-derived normal fibroblast (Ajou University dermatology) on 6-well plate and incubate for 24 hours at 37 ° C in 5% CO ₂ incubator. Were treated and UVA was irradiated at 5 J / cm 2 and further incubated for 24 hours. After further incubation, cells were fixed and stained with senescence β-galactosidase, and cell fixation and staining were performed using a senescence β-galactosidase staining kit (Cell signaling technology, # 9860). Removing the liquid medium and stained fixed to the two wash after the cells for 10 minutes at room temperature in 1X fixative solution in 1X PBS and, 18 hours or more in the β-galactosidase staining solution with CO senescence 37 ℃ incubator of no _second state. The cells stained blue were observed and photographed on a 200x optical microscope.

As shown in FIG. 7, the senescence β-galactosidase overexpressed senescent cells stained blue rapidly increased when human-derived fibroblasts were irradiated with a certain amount of UVA, but the number of the cosmos extracts was significantly reduced when pre-treated with cosmos extract.

Example  2: Preparation of Nutritional Cream Containing Cosmos Extract

The composition of the nourishing cream containing cosmos extract is shown in Table 1 below. The aqueous phase purified water, triethanol amine and propylene glycol were heated and dissolved at 70 占 폚, and the oily fatty acid, oil component, emulsifier, and preservative were heated to 70 占 폚 to dissolve the solution. After emulsification was completed the solution was cooled to 45 ° C., cosmos extract and flavor were added and dispersed and then cooled to 30 ° C.

ingredient	Content (% by weight)
Cosmos extract	0.01
Jojoba oil	5.0
Floating paraffin	7.0
Cetylaryl alcohol	2.0
Polyglyceryl-3 Methyl Glucose Disteare	2.0
Glyceryl Stearate	0.5
Squalane	3.0
Propylene glycol	4.0
glycerin	5.0
Triethanol amine	0.3
Carboxy Vinyl Polymer	0.3
Tocopheryl acetate	0.2
Preservative, incense	a very small amount
Purified water	Remaining amount
Sum	100

Example 3: Preparation of Nutritional Cream Containing Cosmos Extract

Nutritional creams were prepared in the same manner as in Example 2, with the content of the cosmos extract being 0.05% by weight instead of 0.01% by weight.

Example 4: Preparation of a Nourishing Cream Containing Cosmos Extract

A nourishing cream was prepared in the same manner as in Example 2, with the content of the cosmos extract being 1.00 wt% instead of 0.01 wt%.

Comparative example  1: Preparation of nourishing cream without cosmos extract

The composition of the nutrient cream not containing cosmos extract is shown in Table 2 below. The aqueous phase purified water, triethanol amine and propylene glycol were heated and dissolved at 70 占 폚, and the oily fatty acid, oil component, emulsifier, and preservative were heated to 70 占 폚 to dissolve the solution. After emulsification was completed, the solution was cooled to 45 ° C., purified water and flavor were added and dispersed, and then cooled to 30 ° C.

ingredient	Content (% by weight)
Cosmos extract	0.00
Jojoba oil	5.0
Floating paraffin	7.0
Cetylaryl alcohol	2.0
Polyglyceryl-3 Methyl Glucose Disteare	2.0
Glyceryl Stearate	0.5
Squalane	3.0
Propylene glycol	4.0
glycerin	5.0
Triethanol amine	0.3
Carboxy Vinyl Polymer	0.3
Tocopheryl acetate	0.2
Preservative, incense	a very small amount
Purified water	Remaining amount
Sum	100

Test Example 8: Measurement of the wrinkle improvement effect of the cosmetic containing the cosmos extract

Wrinkle improvement effect of the cosmetic containing the cosmos extract was measured through clinical trials. The anti-wrinkle effect was evaluated by measuring the change in skin elasticity. In Test Example 8, the nutrition creams of Example 2 to 4 (nutrition creams containing 0.01 wt%, 0.05 wt% and 1.00 wt% of cosmos extract) and comparative examples Nourishing cream of 1 was used.

Skin elasticity is measured in the nutritional creams of Examples 2 to 4 and Comparative Example 1 of 30 healthy women (25 to 35 years) in an environment of constant temperature 24 to 26 ℃, humidity 38 to 40% The nourishing cream was applied to the face of each subject twice a day for 3 months. Skin elasticity was measured before and after using the cream by using a skin measuring instrument (Cutometer SEM 474, Courage + Khazaka, Cologne, Germany). . The test results are shown in Table 1 below.

Test Items	Example 2 (0.01%)	Example 3 (0.05%)	Example 4 (1.00%)	Comparative Example 1 (0.00%)
Skin elasticity	2.38	2.64	2.9	2.31

As shown in Table 3, Examples 2 to 4 of the present invention was much better skin elasticity than Comparative Example 1, the skin elasticity was shown to increase with increasing the concentration of the cosmos extract concentration-dependent wrinkle improvement It was found that the effect was improved.

Test Example 9 Safety Confirmation Experiment of Cosmos Extract on Human Skin

Skin safety verification experiments were conducted to determine the harmful effects of cosmos extracts on human skin, and cumulative skin irritation tests were performed to confirm this. A formulation containing 0.1%, 0.5%, and 1% of cosmos extracts, respectively, was prepared based on squalene, and nine 30-hour cumulative patches were applied to the upper forearm every other day for a total of 30 healthy adults. Whether the extract irritated the skin was measured. It was patched using a fin chamber (Finn chamber, Epitest Ltd, Finland), and 15 µl of each skin external preparation was added dropwise to the chamber and then patched. The degree of response to the skin each time was scored using Equation 1 below, and the results are shown in Table 3 below.

In the reactivity, ± gives 1 point, + gives 2 points, and ++ gives 4 points, which are judged to be safe compositions when the average reactivity is less than 3.

Test substance	Number of subjects with response									Average reactivity
	1 week			2 weeks			3 weeks
	Primary	Secondary	3rd	4th	5th	6th	7th	8th	9th
	± + + +	± + + +	± + + +	± + + +	± + + +	± + + +	± + + +	± + + +	± + + +
Squalane [Control]	One - -	0 - -	---	---	---	---	---	---	---	0.09
Cosmos Extract (0.1%) [Test Group 1]	0 - -	0 - -	---	---	---	---	---	---	---	0.00
Cosmos Extract (0.5%) [Test Group 2]	0 - -	0 - -	---	---	---	---	---	---	---	0.00
Cosmos Extract (1%) [Test Group 3]	0 - -	0 - -	---	---	---	---	---	---	---	0.00

As shown in Table 4, the number of people corresponding to ±, + and ++ in test groups 1, 2 and 3 were 0, 1 and 0, respectively, and the average reactivity was 0.00, 0.00 and 0.00, respectively. Therefore, since the average reactivity was 3 or less, the cosmos extract was determined to be a safe substance for human skin that does not exhibit a distinct cumulative stimulus pattern.

The following exemplifies a formulation containing a composition of the present invention having an anti-wrinkle effect, and the preparation examples are as follows.

Formulation Example 1: Cosmetic Preparation

1-1. Flexible Cosmetics

ingredient	weight%
Cosmos extract	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, Micro Pigments, Micro-Fragrances and Micro-Purified Water	82.79
sum	100.0

1-2. Nutrition

ingredient	weight%
Cosmos extract	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, Micro Pigments, Micro-Fragrances and Micro-Purified Water	69.69
sum	100.0

1-3. Nutrition Cream

ingredient	weight%
Cosmos extract	0.01
Beeswax	10.0
Polysorbate 60	1.5
Sorbitan sesquioleate	0.5
Liquid paraffin	10.0
Squalane	5.0
Caprylic / Capric Triglycerides	5.0
glycerin	5.0
Butylene Glycol	3.0
Propylene glycol	3.0
Triethanolamine	0.2
Preservatives, Micro Pigments, Micro-Fragrances and Micro-Purified Water	56.79
sum	100.0

1-4. Massage cream

ingredient	weight%
Cosmos extract	0.01
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, Micro Pigments, Micro-Fragrances and Micro-Purified Water	27.49
sum	100.0

1-5. pack

ingredient	weight%
Cosmos extract	0.01
Polyvinyl alcohol	13.0
Sodium Carboxymethylcellulose	0.2
Allantoin	0.1
ethanol	5.0
Nonylphenyl Ether	0.3
Preservatives, Micro Pigments, Micro-Fragrances and Micro-Purified Water	81.39
sum	100.0

Formulation example  2: pharmaceutical preparation

2-1. Manufacture of powder

ingredient	Weight (g)
Cosmos extract	2
Lactose	One

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2-2. Manufacture of tablets

ingredient	Weight (mg)
Cosmos extract	100
Corn starch	100
Lactose	100
Magnesium Stearate	2

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

2-3. Preparation of Capsule

ingredient	Weight (mg)
Cosmos extract	100
Corn starch	100
Lactose	100
Magnesium Stearate	2

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

Claims (6)

1. A cosmetic composition for preventing photoaging or improving skin wrinkles, including cosmos extract (Cosmos bipinnatus) as an active ingredient.
2. The method of claim 1,

   The skin wrinkle improvement is to reduce, eliminate or prevent the wrinkles formed on the cosmetic composition.
3. The method of claim 1,

   The photoaging prevention is to prevent skin damage by ultraviolet rays and to protect the skin composition.
4. A pharmaceutical composition for preventing photoaging or improving skin wrinkles comprising Cosmos extract (Cosmos bipinnatus) as an active ingredient.
5. The method of claim 4, wherein

   The skin wrinkle improvement is a pharmaceutical composition that will alleviate, remove or prevent wrinkles formed on the skin.
6. The method of claim 4, wherein

   The photoaging prevention is to prevent skin damage caused by ultraviolet rays and to protect the skin.

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