KR20170076643A - Cosmetic composition comprising 7,8-dihydroxyflavone or salt thereof for prevention and treatment of skin aging and wrinkle - Google Patents

Abstract

The present invention relates to a cosmetic composition and a food composition for preventing skin aging and improving skin wrinkles containing 7,8-dihydroxyflavone or a salt thereof as an active ingredient, which is one of flavonoid compounds, More particularly, the present invention relates to a method for promoting collagen synthesis in human skin cells and suppressing the expression of collagenase, an enzyme that degrades collagen, And antioxidant enzymes (catalase, Mn-SOD, and HO-1), and to provide a cosmetic composition and a food composition excellent in skin aging prevention and skin wrinkle improving activity.

Description

Cosmetic composition comprising 7,8-dihydroxyflavone or salt thereof for prevention and treatment of skin aging and wrinkle}

The present invention relates to a cosmetic composition and a food composition for preventing skin aging and improving skin wrinkles, comprising 7,8-dihydroxyflavone, one of the flavonoid compounds, or a salt thereof as an active ingredient, More specifically, it has excellent anti-wrinkle effect by inhibiting the expression of collagenase, an enzyme that promotes collagen synthesis and decomposes collagen in human skin cells, and has a reactive oxygen species (ROS) and associated inflammatory signals (MAPKs). And Akt) and antioxidant enzymes (catalase, Mn-SOD, and HO-1), thereby preventing skin aging and improving skin wrinkles.

The skin, the primary defense system of our body, not only protects our body from external stimuli and invasion of external bacteria, but also maintains body temperature and moisture, and performs a wide range of functions. The skin is composed of the dermis, subcutaneous tissue, and appendages (sweat glands, hair holes). The epidermis is composed of keratinocytes, which are made of keratin, and melanocytes that form and secrete melanin, and the dermis is composed of fibrous and matrix components. As a fibrous component in the dermis, collagen gives strength and tension to the skin, occupies 90% of the dermal layer, and affects the elasticity of the skin. Collagen is synthesized by fibroblasts and decomposed by collagenase, and type 1 collagen is mostly.

Skin aging is generally caused by dividing into intrinsic and extrinsic factors. Intrinsic factors are caused by inflammatory mediators, and external factors are mainly caused by UV exposure. UV exposure increases the release of ROS and inflammatory cytokines in the skin, thereby further promoting skin aging, thereby reducing the collagen content in the skin tissue, causing dryness and wrinkles. In particular, TNF-α (tumor necrosis factor-α), an inflammatory cytokine, induces an inflammatory reaction in cells and activates the collagen-degrading enzyme MMP (matrix metalloproteinase) to degrade collagen and reduce skin moisturization and tightness.

In general, among the factors that cause skin wrinkles, such as immunity against the external environment and skin moisture content, the biggest influence is the collagen-degrading enzyme that reduces the collagen production and collagen content of fibroblasts in the dermis. It is the expression level and activity of MMPs (matrix metalloproteinases). MMPs (matrix metalloproteinases) are classified according to the type of substrate, and MMPs expressed by UV exposure and inflammatory cytokines include MMP-1 (collagenase), MMP-3 (stromelysin), and MMP-9 (gelatinase). In particular, MMP-1 expressed in fibroblasts fragments collagen and causes skin aging by changing its function.

External factors that cause skin wrinkles are environmental pollution including the destruction of the ozone layer, which induces physical and chemical stress due to the increase in ultraviolet rays and free radicals, thereby deteriorating skin function and promoting skin aging. When the skin is exposed to UV rays, reactive oxygen species (ROS) accumulate in the skin due to photochemical reactions, causing oxidative stress, and continued oxidative stress activates collagenase enzymes and affects the moisture content of the skin. It causes damage to skin components such as cleavage of the hyaluronic acid chain and promoting DNA mutation. In addition, oxidative stress may induce skin inflammation through mitogen-activated protein kinases (MAPKs) and Akt signaling. MAPKs and Akt signaling systems are important mediators involved in proliferation, death and differentiation, including inflammation, within cells. Particularly in aging skin cells, it is known that ROS accumulates in cells by decreasing the activity of catalase, an enzyme that destroys ROS due to changes in the MAPKs and Akt signaling systems, and thereby further progresses skin aging and inflammation. Yes (Fisher et al. , 2002). As a result, due to oxidative stress, the thickness of the dermis becomes thinner and collagen is decomposed, causing changes in the extracellular matrix components of the dermal tissue. Therefore, antioxidants that inactivate reactive oxygen species can delay the skin aging process and improve wrinkles.

Flavonoids, a natural product derived from plants, are widely distributed in fruits (apples, oranges, etc.), vegetables (tomatoes, onions, etc.), beverages (wine, etc.), and the skins of plant stems and roots, especially in the epidermis. Flavonoids not only affect color and scent, but also play an important role in protecting themselves from UV rays. Flavonoids, based on their useful physiological activity, have already developed specialized medicines using antithrombotic agents, anti-gastric ulcers, anti-gastric cancer specialty drugs, dementia prevention and treatment, etc., and the demand for flavonoid compounds in the pharmaceutical and health functional food market is It continues to increase (Bradley et al. 1998; Bradley et al. 1999; Dixon and Steele 1999).

In particular, flavonoids have a wide range of antioxidant properties and thus inhibit the action of oxides that cause aging or disease in the human body or skin, so their research value is highly evaluated in the cosmetics market as well as the pharmaceutical and health functional food market. Among them, EGCG (Epigallogatechin gallate), a representative flavonoid component obtained from green tea, protects skin cells from strong ultraviolet rays with a strong anticancer effect, promotes the creation of new skin cells, and inhibits the breakdown of collagen to prevent and improve skin aging. It has been reported to be able to do so (katiyar 2003). In addition to EGCG, well-known skin anti-aging flavonoids include proanthocyanidins (grape flavonoids), quercetin (apple and onion flavonoids), and hespyridine (mandarin and orange flavonoids).

Among various kinds of flavonoids, 7,8-dihydroxyflavone has been reported for anti-inflammatory, vasodilating, antihypertensive, and antioxidant functions. In particular, it protects DNA, nerve cells and keratinocytes against oxidative stress. It is known to be outstanding. However, despite the close relationship between the antioxidant ability of substances in skin aging and wrinkle formation, no studies or reports have been made on the direct antioxidant, anti-aging, and anti-wrinkle of 7,8-dihydroxyflavones against skin cells.

In order to develop the domestic cosmetics industry and to cultivate competitiveness with foreign cosmetics, a separate cosmetics law was enacted (July 2000) and functional cosmetics were introduced, which spurred the development of cosmetics research and development and manufacturing technology. In addition, the cosmetics market, which was considered exclusively for adult women due to lifestyle changes, is expanding to include male consumers, and as income levels increase, interest in appearance management has increased, and the cosmetics market is growing. In particular, about 75% of the anti-aging market, which currently stands at KRW 11.9 trillion, is cosmetics, and according to a survey on considerations when purchasing cosmetics, about 30% of women have a safety item indicating whether they have used safe ingredients or ingredients. They responded that they regard it as important (Sumi Lee, 2014). In addition, there are four certified substances for anti-wrinkle effect designated by the Ministry of Food and Drug Safety, including adenosine and retinol. Although these chemicals are frequently used in cosmetics worldwide, skin irritation has been reported (Elsner et al., 2005, Nohynek et al., 2010).

As consumers' awareness of the safety soybeans and ingredient labeling of cosmetics has increased, the present inventors have made diligent efforts to select natural substances for skin anti-aging and anti-wrinkle effects that are safe for the human body, have no side effects, and have excellent effects. 7,8-dihydroxyflavones promote collagen synthesis in human skin cells and inhibit the expression of collagenase, an enzyme that degrades collagen, and also reactive oxygen species (ROS) and related inflammation-related signals It has the effect of regulating (MAPK and Akt) and antioxidant enzymes, and thus it was confirmed that it can be used as a composition for preventing skin aging and improving skin wrinkles, and the present invention was completed.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

Republic of Korea Patent Publication No. 10-2014-0125946

An object of the present invention is to provide a cosmetic composition for preventing skin aging and improving skin wrinkles, comprising 7,8-dihydroxyflavone or a salt thereof as an active ingredient.

Another object of the present invention is to provide a food composition for preventing skin aging and improving skin wrinkles, comprising 7,8-dihydroxyflavone or a salt thereof as an active ingredient.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

In order to solve the above problems, the present invention provides a cosmetic composition for preventing skin aging and improving skin wrinkles comprising 7,8-dihydroxyflavone represented by the following Formula I or a salt thereof as an active ingredient.

[Formula I]

According to a preferred embodiment of the present invention, the cosmetic composition is (a) increased collagen synthesis; (b) inhibiting the expression of matrix metalloproteinse-1 (MMP-1); (c) inhibiting the accumulation of reactive oxygen species (ROS); (d) an increase in the expression level of antioxidant enzymes; And (e) reducing mitogen-activated protein kinase (MAPKs) and phosphorylation of Akt protein.

According to another preferred embodiment of the present invention, the cosmetic composition may contain 7,8-dihydroxyflavone or a salt thereof in a concentration of 0.1 μM to 10 μM.

According to another preferred embodiment of the present invention, the cosmetic composition may be any one selected from the group consisting of a flexible lotion, a nutritional lotion, a gel, a cream, an essence, an emulsion, a pack, a face wash, and a bath powder.

The present invention also provides a food composition for preventing skin aging and improving skin wrinkles comprising 7,8-dihydroxyflavone represented by the following formula (I) or a salt thereof as an active ingredient.

[Formula I]

According to a preferred embodiment of the present invention, the food composition comprises (a) increasing collagen synthesis; (b) inhibiting the expression of matrix metalloproteinse-1 (MMP-1); (c) inhibiting the accumulation of reactive oxygen species (ROS); (d) an increase in the expression level of antioxidant enzymes; And (e) reducing mitogen-activated protein kinase (MAPKs) and phosphorylation of Akt protein.

According to another preferred embodiment of the present invention, the food composition may contain 7,8-dihydroxyflavone or a salt thereof in a concentration of 0.1 μM to 10 μM.

The cosmetic composition and food composition of the present invention contains 7,8-dihydroxyflavone, a flavonoid-based compound as a natural substance, so that it is safe for the human body, has no side effects, and has excellent anti-aging and anti-wrinkle effects.

Specifically, the 7,8-dihydroxyflavone promotes collagen synthesis in human skin cells; Inhibits the expression of collagenase, an enzyme that degrades collagen; Inhibit the accumulation of reactive oxygen species in cells; Increase the expression level of antioxidant enzymes such as catalase, Mn-SOD and HO-1; It is useful to induce phosphorylation of MAPKs (mitogen-activated protein kinase) and Akt protein to decrease, thereby preventing skin aging and providing cosmetic and food compositions with excellent effects on skin wrinkles.

1 is a graph showing the results of measuring cytotoxicity using an MTT assay after treating and culturing 7,8-dihydroxyflavone on human skin cells (Hs68 cells).
2A shows 7,8-dihydroxyflavones were treated with human skin cells (Hs68 cells), and after 1 hour incubated with TNF-α (tumor necrosis factor-α) for 18 hours, the culture medium was collected and the amount of collagen secretion was obtained. It is a graph showing the change measured.
2B shows 7,8-dihydroxyflavones were treated with human skin cells (Hs68 cells), and 1 hour later incubated with TNF-α (tumor necrosis factor-α) for 18 hours, and then polymerase chain reaction (polymerase Chain reaction, PCR) was used to analyze the changes in the expression levels of collagen and MMP-1 (matrix metalloproteinse-1, collagenase) in skin cells.
FIG. 2C shows 7,8-dihydroxyflavones were treated with human skin cells (Hs68 cells), and after 1 hour, incubated with TNF-α (tumor necrosis factor-α) for 18 hours, Western blot This is the result of analyzing the change in the expression level of MMP-1 in skin cells using the method.
3A is a graph showing the DPPH radical scavenging ability of 7,8-dihydroxyflavone.
3B shows 7,8-dihydroxyflavones were treated with human skin cells (Hs68 cells), and after 1 hour incubated with TNF-α (tumor necrosis factor-α) for 18 hours, ROS in skin cells (Reactive Oxygen Species) level was analyzed with a fluorescence photometer.
3C shows 7,8-dihydroxyflavones were treated with human skin cells (Hs68 cells), and after 1 hour, after incubation with TNF-α (tumor necrosis factor-α) for 18 hours, ROS in skin cells (Reactive Oxygen Species) level observed with a fluorescence microscope.
4 shows 7,8-dihydroxyflavones were treated with human skin cells (Hs68 cells) and cultured with TNF-α (tumor necrosis factor-α) 1 hour later for 18 hours, and then Western blot This is the result of analyzing the change in the expression level of skin cell antioxidant enzymes (CAT, Mn-SOD and HO-1) using the method.
5 shows 7,8-Dihydroxyflavone was treated on human skin cells (Hs68 cells), and after 1 hour incubated with TNF-α (tumor necrosis factor-α) for 6 hours, using a western blot method This is the result of analyzing the changes in phosphorylation of p38, ERK, JNK and Akt proteins in skin cells.

As described above, there are four types of conventional anti-wrinkle-improving substances designated by the Ministry of Food and Drug Safety, such as adenosine and retinol, but these chemicals have been reported to cause skin irritation, and the safety of cosmetics. As consumers' awareness of soybeans and ingredient labeling increases, there is a demand for the development of a material that is safe for the human body, has no side effects, and has excellent skin anti-aging and anti-wrinkle effects.

Accordingly, the present invention sought a solution to the above-described problem by providing a cosmetic composition and a food composition for preventing skin aging and improving skin wrinkles, including the natural substance 7,8-dihydroxyflavone or a salt thereof as an active ingredient. The cosmetic composition and food composition for preventing skin aging and improving skin wrinkles comprising 7,8-dihydroxyflavone or a salt thereof of the present invention as an active ingredient, is a coke, an enzyme that promotes collagen synthesis and decomposes collagen in human skin cells. It inhibits the expression of genease, and also has the effect of regulating reactive oxygen species (ROS) and related inflammation-related signals (MAPK and Akt) and antioxidant enzymes (catalase, Mn-SOD and HO-1). It is excellent in preventing skin aging and improving skin wrinkles.

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

The present invention provides a cosmetic composition for preventing skin aging and improving skin wrinkles comprising 7,8-dihydroxyflavone represented by the following formula (I) or a salt thereof as an active ingredient.

[Formula I]

The 7,8-dihydroxyflavone (7,8-DHF; 7,8-Dihydroxyflavone) is a naturally-occuring flavone, but it may be prepared by a conventional organic synthesis method or commercially available.

The "salt" includes both silver acid and base addition salts. Exemplary salts are sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tart. Rate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, Glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts Including, but not limited to. Salts may include the inclusion of other molecules such as acetate ions, succinate ions or other counter ions. The counter ion can be any organic or inorganic moiety that stabilizes the charge in the parent compound.

As a result of performing the MTT assay to measure the cytotoxicity of the 7,8-dihydroxyflavone, as shown in FIG. 1, when the treatment was performed at a concentration of 10 μM or less, it did not show toxicity to human skin cells (Hs68 cells). , When treated with a concentration of 20 μM, cytotoxicity of 19% could be confirmed.

Human skin cells (Hs68 cells) were treated with 7,8-dihydroxyflavone by concentration (0.01, 0.1, 1, 2, 5, 10 and 20 μM), and cultured with TNF-α after 1 hour for 18 hours. Then, as a result of collecting the culture medium and measuring the amount of collagen secretion, as shown in FIG. 2A, the amount of collagen secretion increased as the concentration of 7,8-dihydroxyflavone increased from 0.1 μM to 10 μM.

In addition, after culturing human skin cells (Hs68 cells) in the same process as above, the expression level of collagen in the skin cells was measured using a polymerase chain reaction. As the concentration of oxyflavone increased from 0.1 μM to 10 μM, it was confirmed that the collagen mRNA level also increased.

Matrix metalloproteinase-1 (MMP-1), a protease that specifically acts on collagen and degrades collagen, has a concentration of 7,8-dihydroxyflavone of 0.1 as shown in FIGS. 2B and 2C. It was confirmed that the mRNA and protein expression levels decreased with increasing from μM to 10 μM.

Through this, the cosmetic composition for preventing skin aging and improving skin wrinkles comprising 7,8-dihydroxyflavone or a salt thereof of the present invention as an active ingredient, is a coke, an enzyme that promotes collagen synthesis and decomposes collagen in human skin cells. It was confirmed that there is an excellent effect on wrinkle improvement by suppressing the expression of genease.

In addition, 78-dihydroxyflavone solution prepared by concentration from 0 μM to 300 μM was mixed with DPPH (1,1-diphenyl-2-picrylhydrazyl) solution and reacted for 10 minutes while blocking light. As a result of measuring the absorbance at nm, it was confirmed that 300 μM of a 7,8-dihydroxyflavone solution scavenging 66.2% of DPPH radicals as shown in FIG. 3A.

The DPPH radical scavenging ability is expressed in% by the following calculation formula I by comparing the absorbance of the sample addition to the control.

[Calculation I]

Reactive oxygen species (ROS), which act as a cause of skin aging and wrinkles, induce inflammation by activating MAPKs (mitogen-activated protein kinases) and Akt signaling and destroy the extracellular matrix. , In order to confirm whether it is eliminated by 8-dihydroxyflavone, 7,8-dihydroxyflavone was treated on human skin cells as described in Examples 3-2 and 3-3, and after 1 hour, TNF-α and After incubation for 18 hours, the level of ROS (Reactive Oxygen Species) in skin cells was observed with a fluorescence photometer and a fluorescence microscope, respectively.

As a result, as shown in Fig. 3b, the group treated with 7,8-dihydroxyflavone with 5 μM and 10 μM, respectively, compared with the control group treated with only TNF-α (20 ng/ml), the amount of ROS accumulation in cells. It was confirmed that the ROS accumulation level of the untreated control group that was not treated with TNF-α decreased by 11% and 13%, respectively, and 7,8-dihydroxyflavones were respectively 5 μM, 10 μM and as shown in FIG. 3C. In the group treated with 20 μM, the amount of ROS accumulation in the cell matrix decreased in a concentration-dependent manner compared to the control group treated with only TNF-α (20 ng/ml), down to the level of ROS accumulation in the untreated control group not treated with TNF-α. It was confirmed to be.

Through this, the cosmetic composition comprising 7,8-dihydroxyflavone or a salt thereof of the present invention as an active ingredient prevents skin aging and wrinkles by inhibiting the accumulation of reactive oxygen species (ROS) in human skin cells. It was confirmed that it showed an excellent effect on improvement.

Furthermore, as described in Example 4, in order to analyze the expression level of the antioxidant enzyme according to the 7,8-dihydroxyflavone treatment, 0.1 μM and 1 of 7,8-dihydroxyflavone were added to human skin cells (Hs68 cells), respectively. After treatment with μM, 5 μM and 10 μM by concentration, and after 1 hour incubation with TNF-α for 18 hours, catalase (CAT), Mn-SOD and HO-1 and The expression level of the same antioxidant enzyme was measured.

As a result, as shown in FIG. 4, it was confirmed that the expression levels of catalase, Mn-SOD and HO-1 increased as the concentration of 7,8-dihydroxyflavone increased.

Through this, the cosmetic composition comprising 7,8-dihydroxyflavone or a salt thereof of the present invention as an active ingredient increases the expression of antioxidant enzymes such as catalase, Mn-SOD and HO-1, thereby reducing oxidative stress. It was confirmed that it can suppress the skin aging effect.

Finally, in order to confirm the anti-aging and anti-wrinkle efficacy according to the 7,8-dihydroxyflavone treatment, 0.1 7,8-dihydroxyflavone was added to human skin cells (Hs68 cells) as described in Example 5, respectively. After treatment at each concentration in μM, 1 μM, 5 μM and 10 μM, incubation with NF-α for 6 hours, phosphorylation levels of p38, ERK, JNK and Akt proteins in skin cells were measured using a Western blot method. .

As a result, as shown in FIG. 5, in the control group treated with only TNF-α (20 ng/ml), p38, ERK, JNK, and Phosphorylation of Akt protein was significantly increased.P38, ERK, JNK and Akt protein phosphorylation levels in the groups treated with 7,8-dihydroxyflavones 0.1 μM, 1 μM, 5 μM and 10 μM, respectively, compared to the control group. It was reduced in a concentration dependent manner.

As described above, the cosmetic composition comprising 7,8-dihydroxyflavone or a salt thereof of the present invention as an active ingredient can improve skin aging, skin inflammation, and wrinkles by controlling the phosphorylation of MAPKs and Akt proteins. It can be usefully used as an anti-aging, anti-wrinkle, and anti-inflammatory cosmetic composition.

Thus, the cosmetic composition of the present invention (a) increased collagen synthesis; (b) inhibiting the expression of matrix metalloproteinse-1 (MMP-1); (c) inhibiting the accumulation of reactive oxygen species (ROS); (d) an increase in the expression level of antioxidant enzymes; And (e) reducing mitogen-activated protein kinase (MAPKs) and phosphorylation of Akt protein.

In addition, the cosmetic composition of the present invention preferably contains 7,8-dihydroxyflavone or a salt thereof in a concentration of 0.1 μM to 10 μM. If the cosmetic composition contains 7,8-dihydroxyflavone or a salt thereof at a concentration of less than 0.1 μM, the concentration of the active ingredient is too low to exhibit the effects described in (a) to (e), If 7,8-dihydroxyflavone or a salt thereof is included in a concentration exceeding 10 μM, the increase in the expected effect according to the increase in the concentration of the active ingredient is insufficient.In particular, when the concentration is more than 20 μM, human skin cells Becomes toxic.

In addition to the active ingredients, the cosmetic composition of the present invention may further include conventional auxiliary agents and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, etc. that are commonly used in cosmetic compositions. For example, the cosmetic composition may further include auxiliary ingredients such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, and allantoin. .

The 7,8-dihydroxyflavone or salt thereof may be included in 0.0001 to 15% by weight, more preferably 0.0001 to 10% by weight, most preferably 0.0001 to 5% by weight, based on the total weight of the cosmetic composition. It is not limited and the content can be freely selected according to the dosage form, use and purpose.

In the cosmetic of the present invention, in addition to the essential ingredients, other ingredients that are usually blended in the cosmetic may be blended if necessary.

Other ingredients that can be added include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, Perfumes, blood circulation accelerators, cooling agents, restrictors, and purified water.

Examples of oil and fat components include ester oils, hydrocarbon oils, silicone oils, fluorine oils, animal oils, and vegetable oils.

Ester-based fats and oils include tri2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, stearic acid. Butyl acid, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl sebacate, adi Diisopropyl pinate, isoalkyl neopentanoate, tri(caprylic, capric acid) glyceryl, tri2-ethylhexanoate trimethylolpropane, triisostearate trimethylolpropane, tetra2-ethylhexanoate pentaellisli Toll, cetyl caprylate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, ricinooleate cetyl, isosyl laurate Thearyl, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, seto 2-ethylhexanoate Stearyl, 2-ethylhexanoate stearyl, isostearate hexyl, dioctanoate ethylene glycol, dioleate ethylene glycol, dicaprylic acid propylene glycol, di(caprylic, capric acid) propylene glycol, dicaprylic acid propylene glycol, Neopentyl glycol dicapric acid, neopentyl glycol dioctanoate, glyceryl tricaprylate, glyceryl tricaprylate, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostea octanoate Reel, isononanoate octyl, neodecanoate hexyldecyl, neodecanoate octyldodecyl, isostearate, isostearic acid isostearyl, isostearate octyldecyl, polyglycerololeic acid ester, polyglycerin isostearic acid ester , Triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, horse Diisostearyl acid, 2-ethylhexyl hydroxystearate, 2-ethylhexyl succinate, diisobutyl adipic acid, diisopropyl sebacate, dioctyl sebacate, stear Acid cholesteryl, isostearic acid cholesteryl, hydroxystearate cholesteryl, oleic acid cholesteryl, oleic acid dihydrocholesteryl, isostearic acid pitsteryl, pitsteryl oleate, 12-stealoylhydride And esters such as isocetyl oxystearate, stearyl 12-stealoylhydroxystearate, and isostearyl 12-stealoylhydroxystearate.

Hydrocarbon-based fats and oils include squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybuden, microcrystalline wax, and hydrocarbon-based fats such as petrolatum.

Silicone-based fats and oils include polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane/methylcetyloxysiloxane copolymer, dimethylsiloxane/methylsteaoxysiloxane copolymer, And alkyl-modified silicone oils and amino-modified silicone oils.

As fluorine-based fats and oils, perfluoropolyether, etc. are mentioned.

Animal or vegetable oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, new flower oil, soybean oil, corn oil, rapeseed oil, almond oil, palm kernel oil, palm oil, castor oil, sunflower oil, grapes. Seed oil, cottonseed oil, palm oil, cucuine nut oil, wheat germ oil, rice germ oil, shea butter, walnut colostrum oil, marker deimia nut oil, meadow home oil, egg yolk oil, beef tallow, horse oil, mink oil, orange rape oil, jojoba And animal or plant fats such as oil, canderry wax, carnauba wax, liquid lanolin, and hydrogenated castor oil.

Examples of the moisturizing agent include a water-soluble low-molecular moisturizer, a fat-soluble molecular moisturizer, a water-soluble polymer, and a fat-soluble polymer.

Water-soluble low molecular weight moisturizers include serine, glutamine, sorbitol, mannitol, pyrrolidone-sodium carboxylic acid, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (polymerization degree n = 2 or more), poly Propylene glycol (polymerization degree n=2 or more), polyglycerin B (polymerization degree n=2 or more), lactic acid, lactate, etc. are mentioned.

Examples of the fat-soluble low-molecular moisturizer include cholesterol and cholesterol esters.

Water-soluble polymers include carboxyvinyl polymer, polyaspartic acid salt, tragacanth, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble chitin, chitosan, dextrin, etc. Can be lifted.

Examples of the oil-soluble polymer include polyvinylpyrrolidone/eicosene copolymer, polyvinylpyrrolidone/hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, and polymer silicone.

Examples of the emollient agent include long-chain acyl glutamate cholesteryl ester, hydroxystearate cholesteryl, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesteryl ester, and the like.

Surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and the like.

Nonionic surfactants include self-emulsifying glycerin monostearate, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbit fatty acid ester, POE glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hydrogenated castor oil, POE castor oil, POE·POP (polyoxyethylene·polyoxypropylene) copolymer, POE·POP alkyl ether, polyether-modified silicone, laurine Acid alkanolamide, alkylamine oxide, hydrogenated soybean phospholipid, and the like.

Anionic surfactants include fatty acid soap, alpha-acyl sulfonate, alkyl sulfonate, alkyl allyl sulfonate, alkyl naphthalene sulfonate, alkyl sulfate, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE alkyl phosphate, alkylamide Phosphate, alkyloylalkyltaurine salt, N-acylamino acid salt, POE alkyl ether carboxylate, alkyl sulfosuccinate, sodium alkylsulfoacetate, acylated hydrolyzed collagen peptide salt, perfluoroalkyl phosphate, etc. have.

Cationic surfactants include alkyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium bromide, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethylbenzyl ammonium chloride, behenyl trimethyl ammonium bromide, Benzalkonium chloride, diethylaminoethyl amide stearate, dimethylaminopropyl amide stearate, lanolin derivative quaternary ammonium salt, and the like.

Amphoteric surfactants include carboxybetaine type, amidebetaine type, sulfobetaine type, hydroxysulfobetaine type, amide sulfobetaine type, phosphobetaine type, aminocarboxylate type, imidazoline derivative type, amideamine type, etc. And amphoteric surfactants.

Organic and inorganic pigments include silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium coated mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, oxide. Inorganic pigments such as aluminum, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine, chromium oxide, chromium hydroxide, calamine, and complexes thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene-styrene copolymer, Organic pigments, such as silk powder, cellulose, CI pigment yellow, and CI pigment orange, and complex pigments of these inorganic pigments and organic pigments, etc. are mentioned.

Examples of the organic powder include metal soaps such as calcium stearate; Alkyl phosphate metal salts, such as sodium zinc cetylate, zinc laurylate, and calcium laurylate; Acylamino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc, and N-lauroylglycine calcium; Amide sulfonic acid polyvalent metal salts such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; N, such as N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoyl lizine, N-alpha-paritoylolnitine, N-alpha-lauroylarginine, N-alpha-hardened tallow fatty acid acylarginine, etc. -Acyl basic amino acids; N-acyl polypeptides such as N-lauroylglycylglycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; Polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene/styrene copolymer, ethylene tetrafluoride, and the like.

UV absorbers include paraaminobenzoic acid, ethyl paraaminobenzoate, amyl paraaminobenzoate, octyl paraaminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicate, butyl phenyl salicylate, homomentyl salicylate, cinnamic acid Benzyl, paramethoxycinnamic acid-2-ethoxyethyl, paramethoxycinnamic acid octyl, diparamethoxycinnamic acid mono-2-ethylhexane glyceryl, paramethoxycinnamic acid isopropyl, diisopropyl/diisopropyl cinnamic acid ester mixture, Urocanic acid, ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and salts thereof, dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenone disulfonate, dihydroxybenzo Phenone, tetrahydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane,2,4,6-trianilino-p-(carbo-2'-ethylhexyl-1'-oxy)- 1,3,5-triazine, 2-(2-hydroxy-5-methylphenyl)benzotriazole, etc. are mentioned.

As a disinfectant, hinokitiol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zinphylithione, benzalkonium chloride, Photosensitizer No. 301, mononitrowire sodium, undecylenic acid, etc. are mentioned.

Examples of antioxidants include butylhydroxyanisole, propyl gallic acid, and lysorbic acid.

Examples of the pH adjuster include citric acid, sodium citrate, malic acid, sodium malate, pmalic acid, sodium pmaate, succinic acid, sodium succinate, sodium hydroxide, sodium monohydrogen phosphate, and the like.

Examples of alcohol include higher alcohols such as cetyl alcohol.

In addition, the blending components that can be added are not limited thereto, and any of the above components can be blended within a range that does not impair the object and effect of the present invention, but preferably 0.01-5% by weight based on the total weight, more It is preferably blended in an amount of 0.01-3% by weight.

The cosmetic composition of the present invention may take the shape of a solution, an emulsion, a viscous mixture, or the like.

The cosmetic composition of the present invention may be prepared in any formulation that is conventionally prepared with reference to known techniques in the art. For example, solution, suspension, emulsion, softening lotion, nutrient lotion, paste, gel, cream, lotion, essence, emulsion, pack, face wash, gel, powder, soap, surfactant-containing cleansing, oil, powder foundation, It may be formulated as an emulsion foundation, wax foundation, and spray, but is not limited thereto. In more detail, it may be prepared in the form of a flexible lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a mask pack, a spray or a powder.

In order to use the 7,8-dihydroxyflavone or salt thereof as a cosmetic composition, additional fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents ), fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipids It may contain adjuvants commonly used in the field of dermatology, such as vesicles or any other ingredients commonly used in cosmetic compositions. In addition, the ingredients may be introduced in an amount generally used in the field of dermatology.

If the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. May be included.

When the formulation of the cosmetic composition of the present invention is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be included as carrier components. It may contain propellants such as carbon, propane/butane, and dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent, an emulsifying agent, etc. may be included as a carrier component. Specifically, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene Glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, and the like may be included.

When the formulation of the cosmetic composition of the present invention is a suspension, liquid diluents such as water, ethanol, and propylene glycol as carrier components; Suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tracant, and the like may be included.

When the formulation of the cosmetic composition of the present invention is a surfactant containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, Fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, ethoxylated glycerol fatty acid ester, and the like may be included.

The present invention also provides a food composition for preventing skin aging and improving skin wrinkles comprising 7,8-dihydroxyflavone represented by the following formula (I) or a salt thereof as an active ingredient.

[Formula I]

The food composition, like the cosmetic composition of the present invention (a) increasing collagen synthesis; (b) inhibiting the expression of matrix metalloproteinse-1 (MMP-1); (c) inhibiting the accumulation of reactive oxygen species (ROS); (d) an increase in the expression level of antioxidant enzymes; And (e) reducing mitogen-activated protein kinase (MAPKs) and phosphorylation of Akt protein.

In addition, the food composition, like the cosmetic composition of the present invention, preferably contains 7,8-dihydroxyflavone or a salt thereof in a concentration of 0.1 μM to 10 μM. If the food composition contains 7,8-dihydroxyflavone or a salt thereof at a concentration of less than 0.1 μM, the concentration of the active ingredient is too low to exhibit the effects described in (a) to (e), If 7,8-dihydroxyflavone or a salt thereof is included in a concentration exceeding 10 μM, the increase in the expected effect according to the increase in the concentration of the active ingredient is insufficient.In particular, when the concentration is more than 20 μM, human skin cells Becomes toxic.

When the 7,8-dihydroxyflavone or salt thereof of the present invention is used as a food additive, the 7,8-dihydroxyflavone or salt thereof is added as it is, or used by mixing with other foods or food ingredients, etc. It can be appropriately used according to the phosphorus method.

In addition, the mixed amount of the 7,8-dihydroxyflavone or a salt thereof may be suitably changed according to the purpose of use (prevention, health, or therapeutic treatment), as well as the 7,8-dihydroxyflavone or its salt. Salt may be included in 0.0001 to 15% by weight, more preferably 0.0001 to 10% by weight, most preferably 0.0001 to 5% by weight based on the total weight of the food composition, but is not limited thereto, and Therefore, the content can be freely selected.

However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, it may be added in an amount less than the above range, and may be freely used within a concentration range that does not have a problem with safety.

There is no particular limitation on the type of the food. Examples of foods to which 7,8-dihydroxyflavones or salts thereof of the present invention can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, and ice cream. There are dairy products, various soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes, etc., and all health foods in the usual sense are included.

When the food composition of the present invention is prepared as a beverage, it may include additional ingredients such as various flavoring agents or natural carbohydrates, like a conventional beverage. The natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame may be used. The natural carbohydrate may be included in 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on the total weight of the food composition of the present invention.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols. , Carbonating agents used in carbonated beverages, and the like may be included. In addition, the composition of the present invention may include flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination.

The ratio of the additives is not largely limited, but is preferably included in the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Preparation example One.

7,8- Dihydroxyflavone And human Skin cell Ready

The 7,8-dihydroxyflavone used in the following examples of the present invention was obtained from Sigma-Aldrich (D5446, USA), and Hs68 cells, which are human skin cells, were obtained from ATCC (CRL-1635, USA).

7,8- Of dihydroxyflavones Cytotoxicity measurement

MTT assay (methylthiazolyldiphenyl tetrazolium bromide assay) was performed to measure the cytotoxicity of 7,8-dihydroxyflavone against human skin cells (Hs68 cells, human skin fibroblast).

Hs68 cells were dispensed into a 24-well plate at 1×10 ⁴ cells/ml and _{cultured in a 5% CO 2} , 37°C incubator for 24 hours. After cultivation, the culture medium was removed, and DMEM medium and 7,8-dihydroxyflavone were treated at different concentrations (0, 0.01, 0.1, 1, 2, 5, 10 and 20 μM) for 24 hours. Afterwards, a solution of MTT (3-[4,5-dimethyl-thiazol]-2,5-diphenyl-tetrazolium bromide) dissolved in 5 mg/ml in DPBS (Dulbecco's Phosphate-Buffered Saline, 1X) was added to each well. Each 120 µl was treated and incubated in an incubator at 37°C for 4 hours with 5% CO _2. After incubation, the culture medium was removed, and then 200 µl/well of dimethyl sulfoxide (DMSO) was added to each well and stirred well using a pipette. Then, the absorbance was measured at 570 nm with an ELISA reader. The measurement results are shown in FIG. 1.

As a result, as shown in FIG. 1, when 7,8-dihydroxyflavones were treated at concentrations of 0, 0.01, 0.1, 1, 2, 5, 10 and 20 μM, in skin cells treated with 10 μM It did not show toxicity, and the skin cells treated with 20 μM were able to confirm 19% cytotoxicity.

Of 7,8-dihydroxy skin cells according to the hydroxy flavone treatment collagen and collagenase of Expression level analysis

2-1. Measurement of changes in collagen biosynthesis

Collagen is a component of the connective tissue of the dermis and is a major protein that makes up the skin. Therefore, the increase in collagen biosynthesis in fibroblasts directly affects the elasticity and wrinkles of the connective tissue of skin cells. Collagen is synthesized in the form of a precursor called procollagen, and contains a propeptide sequence at the end. When the collagen polymerization reaction occurs, the propeptide at the end is cleaved and separated to aid in folding of collagen molecules to aid in collagen synthesis. Therefore, it is possible to measure the degree of collagen biosynthesis by measuring the amount of propeptide. In addition, TNF-α, which is expressed in skin cells when irradiated with ultraviolet rays, is an inflammatory cytokine and is known to cause skin aging through skin wrinkles and inflammatory reactions by decomposing collagen through activation of collagen-degrading enzymes (Borg et al., 2013).

Therefore, in order to investigate the effect of 7,8-dihydroxyflavone on collagen biosynthesis, Hs68 cells were aliquoted into a 6-well plate at 6.25 x 10 ⁴ cells/ml, cultured for 8 hours, and 78-dihydrogenated. After pretreatment with oxyflavone for 1 hour, it was incubated with 20 ng/ml of TNF-α for 18 hours. After culture, the medium was collected and the amount of collagen was measured with Propeptide Collagen Type 1 kit (Takara, Shiga, Japan).

As a result, as shown in Figure 2a, in the control group treated with only TNF-α (20 ng/ml), the amount of collagen secretion was 40 at 52 ng/ml compared to the untreated control group not treated with TNF-α. ng/ml, but in the group treated with 7,8-dihydroxyflavone 5 μM and 10 μM, respectively, the amount of collagen secretion increased to 45 ng/ml and 56 ng/ml, respectively, compared to the control group, and TNF-α was treated. It recovered to the level of collagen secretion of the untreated control group.

2-2. Investigation of effects on the expression levels of collagen and collagenase MMP -1 ( collagenase)

To investigate the effect of 7,8-dihydroxyflavone on collagen expression, Hs68 cells were aliquoted into a 6-well plate at 6.25 x 10 ⁴ cells/ml, incubated for 8 hours, and the sample solution was added for 1 hour. After pretreatment, TNF-α was incubated with 20 ng/ml for 18 hours. After removing the medium, cells were disrupted to quantify total RNA, and then cDNA was synthesized using a cDNA synthesis kit. Human type 1 procollagen primer (F-CTC GAG GTG GAC ACC ACC CT; R-CAG CTG GAT GGC CAC ATC GG, Cosmogenetech, Seoul, Korea) was performed with RT-PCR and then agarose gel electrophoresis was performed to measure the amount of mRNA expression.

As a result, as shown in Fig. 2b, in the control group treated with only TNF-α (20 ng/ml), the mRNA level of collagen decreased compared to the untreated control group (UC) that was not treated with TNF-α. In the group treated with 0.1, 1, 5 and 10 μM of 7,8-dihydroxyflavone, respectively, it was confirmed that the mRNA level of collagen was increased in a concentration-dependent manner compared to the control group.

MMP-1 is one of the MMPs that specifically act on collagen, and is a protease that breaks down collagen that makes up the connective tissue of skin cells. When collagen is decomposed by MMP-1, the skin loses elasticity and wrinkles are generated. The association between MMP-1 and wrinkles was confirmed by H. Nagase et al . (J. Biol . Chem., 274, 1999), and MMP-1. It has been reported that suppressing collagen degradation can reduce collagen degradation and improve wrinkle formation.

Therefore, in order to investigate whether 7,8-dihydroxyflavone inhibits the expression of MMP-1, which is a collagenase, it was analyzed using RT-PCR and western blot method.

Hs68 cells were aliquoted into a 6-well plate at 6.25 x 10 ⁴ cells/ml, incubated for 8 hours, and the sample solution was pretreated for 1 hour, followed by incubation with 20 ng/ml TNF-α for 18 hours. After removing the medium, cells were disrupted to quantify total RNA, and then cDNA was synthesized using a cDNA synthesis kit, and Human MMP-1 primer (F-ATT CTA CTG ATA TCG GGG CTT TGA; R-ATG TCC TTG) was added to the synthesized cDNA. GGG TAT CCG TGT AG, Cosmogenetech, Seoul, Korea) after performing RT-PCR and agarose gel electrophoresis to measure the mRNA expression level.

In addition, in order to analyze the expression level of MMP-1 using the Western blot method, cells were cultured in the same manner as described above, washed with phosphate buffered saline, and centrifuged at 1,400 rpm for 5 minutes to obtain cells. After adding 20 µl of lysis buffer to the precipitated cells, the reaction was performed on ice for 30 minutes, followed by centrifugation at 14,000 rpm for 30 minutes to obtain a protein. The protein was denatured at 95° C. for 5 minutes by adding quantitative loading dye using Bradford analysis, and then electrophoresis using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) gel. After electrophoresis, it was transferred to a nitrocellulose membrane, and then the primary anti-MMP-1 antibody (1:1000) and anti-beta actin antibody (1:1000) were diluted in 5% skim milk at each ratio. Then, it was put into a nitrocellulose membrane and reacted at 4°C for 24 hours. After washing three times every 10 minutes with TBST (TBS, 0.1% Tween 20), a HRP-conjugated (horeseradish peroxidase-conjugated) secondary antibody was diluted 1:2000 in 5% skim milk powder and reacted at room temperature for 2 hours. After washing three times using TBST in the same manner as described above, the ECL (Enchanced Chemiluminescence) substrate solution was reacted to the film for 1 minute, and then developed on the X-ray film.

As a result, as shown in Figs. 2b and 2c, in the control group treated with only TNF-α (20 ng/ml), the mRNA and the mRNA of MMP-1 compared to the untreated control group not treated with TNF-α. The protein expression level was significantly increased, but it was confirmed that the mRNA and protein expression levels of MMP-1 decreased in a concentration-dependent manner compared to the control group in the group treated with 7,8-dihydroxyflavones of 0.1, 1, 5 and 10 μM, respectively. I did.

Through this, the cosmetic composition for preventing skin aging and improving skin wrinkles comprising 7,8-dihydroxyflavone or a salt thereof of the present invention as an active ingredient, is a coke, an enzyme that promotes collagen synthesis and decomposes collagen in human skin cells. It was confirmed that there is an excellent effect on wrinkle improvement by suppressing the expression of genease.

7,8- For dihydroxyflavone treatment Follow Skin cells of mine ROS Stock analysis

During the process of skin wrinkle development, reactive oxygen species (ROS) accumulate in skin cells stimulated by various factors including UV rays and TNF-α, and the accumulated ROS is MAPKs (mitogen-activated protein kinases) and By activating Akt signaling, it causes inflammation and destroys the extracellular matrix, resulting in skin aging and wrinkles. Therefore, substances with antioxidant effect that scavenging reactive oxygen species in skin cells are candidates for skin anti-aging and anti-wrinkle.

3-1. DPPH Free radical Erasing Measure

In order to measure the antioxidant effect of 7,8-dihydroxyflavone, DPPH radical scavenging ability was measured. Add 0.2 ml of 7,8-dihydroxyflavone solution prepared at different concentrations (0 ~300 μM) and 1.8 ml of 0.1 mM DPPH (1,1-diphenyl-2-picrylhydrazyl) solution, mix well, and block the light. After reacting for 10 minutes in one state, absorbance was measured at 518 nm. The DPPH radical scavenging ability was expressed in% by the following calculation formula I by comparing the absorbance of the sample addition group to the control.

[Calculation I]

As a result, as shown in Fig. 3a, it was confirmed that 300 μM of a 7,8-dihydroxyflavone solution scavenging 66.2% of DPPH radicals.

3- 2. Cell of mine ROS (Reactive Oxygen species) level measurement

In TNF-α-treated human skin cells, DCF-DA (cell permeable acetylated forms of 2'7'-dichlorofluorescein) was used to confirm whether accumulated ROS was eliminated by 7,8-dihydroxyflavones. I did.

Hs68 cells were dispensed into a 96-well plate shielded from light at 6.25 x 10 ⁴ cells/ml, incubated for 8 hours, and the sample solution was pretreated for 1 hour, followed by incubation with 20 ng/ml TNF-α for 18 hours. . After removing the medium, 10 μM of DCF-DA was treated and incubated at 37° C. for 30 minutes, and fluorescence intensity was measured at 492 nm excitation and 530 nm emission using a fluorescence photometer.

As can be seen in Figure 3b, compared to the control group (Control) treated with only TNF-α (20 ng/ml), the amount of ROS accumulation increased by 15% compared to the untreated control group (Untreated control, UC) that was not treated with TNF-α. , In the group treated with 7,8-dihydroxyflavone 5 μM and 10 μM, respectively, the ROS accumulation amount decreased by 11% and 13%, respectively, compared to the control group, to the level of ROS accumulation amount of the untreated control group not treated with TNF-α. Became.

3-3. Within the cell matrix ROS (Reactive Oxygen species) stock change measurement

In TNF-α-treated human skin cells, DCF-DA (cell permeable acetylated forms of 2'7'-dichlorofluorescein) was used to confirm that ROS accumulated in the matrix was eliminated by 7,8-dihydroxyflavones. Then, it was stained and confirmed using a fluorescence microscope.

Hs68 cells were dispensed into an 8-well chamber at 6.25 x 10 ⁴ cells/ml, incubated for 8 hours, and the sample solution was pretreated for 1 hour, followed by incubation with TNF-α 20 ng/ml for 18 hours. After removing the medium, 10 μM of DCF-DA was treated and incubated at 37° C. for 30 minutes, and then ROS accumulated in the cell substrate was observed using a fluorescence microscope (Magnification, ×100).

As can be seen in Figure 3c, compared to the control group (Control) treated with only TNF-α (20 ng/ml), the amount of ROS accumulation significantly increased compared to the untreated control group (Untreated control, UC) not treated with TNF-α, In the group treated with 7,8-dihydroxyflavone 5, 10 and 20 μM, respectively, the amount of ROS accumulation decreased in a concentration-dependent manner compared to the control group, and thus decreased to the level of the ROS accumulation amount of the untreated control group not treated with TNF-α .

Through this, the cosmetic composition comprising 7,8-dihydroxyflavone or a salt thereof of the present invention as an active ingredient prevents skin aging and wrinkles by inhibiting the accumulation of reactive oxygen species (ROS) in human skin cells. It was confirmed that it showed an excellent effect on improvement.

7,8- For dihydroxyflavone treatment Follow Skin cell Antioxidant enzyme expression level analysis

UV and skin cells, the active is accumulated by the inflammatory cytokines, reactive oxygen species (Reactive Oxygen Species, ROS), the superoxide ion ^{_{(superoxide anion, O 2 -)}} , hydrogen peroxide (Hydrogen peroxide) and hydroxyl radicals (hydroxyl radical, OH) Etc. are included. Superoxide ions are converted to hydrogen peroxide by Mn-SOD (Manganese Superoxide Dismutase), which is an intracellular antioxidant enzyme, and the converted hydrogen peroxide is converted to water by catalase enzyme to remove intracellular ROS. Plays a role. In addition, when cells are subjected to oxidative stress, free heme is exposed from heme proteins such as hemoglobin, and the exposed free heme further generates hydroxy radicals, thereby intensifying oxidative stress. Therefore, by controlling the expression level of HO-1 (heme oxygenases), an enzyme that degrades free heme, an effect of reducing intracellular oxidative stress can be expected.

Western blot was performed to investigate whether the expression levels of antioxidant enzymes such as Mn-SOD, catalase, and HO-1 increased as the TNF-α-treated human skin cells were treated with 7,8-dihydroxyflavone. Hs68 cells were dispensed into a 6-well plate at 6.25 x 10 ⁴ cells/ml, incubated for 8 hours, and the sample solution was pretreated for 1 hour, followed by incubation with 20 ng/ml TNF-α for 18 hours. After removing the medium, the cells were washed with phosphate buffered saline and centrifuged at 1,400 rpm for 5 minutes to obtain cells. After adding 20 µl of lysis buffer to the precipitated cells, the reaction was performed on ice for 30 minutes, followed by centrifugation at 14,000 rpm for 30 minutes to obtain a protein. After quantifying the protein using the Bradford method, it was denatured at 95° C. for 5 minutes by adding a loading dye, followed by electrophoresis using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) gel. After electrophoresis, transfer to a nitrocellulose membrane, primary anti-catalase antibody (1:1000), anti-Mn-SOD antibody (1:1000), anti-HO-1 antibody (1:1000), anti-beta Actin antibody (1:1000) was diluted in 5% skim milk at each ratio, put in a nitrocellulose membrane, and reacted at 4°C for 24 hours. After washing three times every 10 minutes with TBST (TBS, 0.1% Tween 20), a HRP-conjugated (horeseradish peroxidase-conjugated) secondary antibody was diluted 1:2000 in 5% skim milk powder and reacted at room temperature for 2 hours. After washing three times using TBST in the same manner as described above, the ECL (Enchanced Chemiluminescence) substrate solution was reacted to the film for 1 minute, and then developed on the X-ray film.

As can be seen in Figure 4, 7,8-dihydroxyflavone concentration (0.1, 1, 5 and 10 μM) treated human skin cells compared to the control group not treated with 7,8-dihydroxyflavone When doing so, it was confirmed that the expression levels of antioxidant enzymes such as catalase, Mn-SOD and HO-1 increased as the concentration of the treated 7,8-dihydroxyflavone increased.

Through this, the cosmetic composition comprising 7,8-dihydroxyflavone or a salt thereof of the present invention as an active ingredient increases the expression of antioxidant enzymes such as catalase, Mn-SOD and HO-1, thereby reducing oxidative stress. It was confirmed that the skin aging effect can be suppressed.

Investigation of the mechanism of anti-aging and anti-wrinkle efficacy of skin cells according to 7,8- dihydroxyflavone treatment

Reactive oxygen species in skin cells accumulated by ultraviolet rays and inflammatory cytokines activate signaling through phosphorylation of MAPKs (mitogen-activated protein kinase) and Akt protein, stimulating AP1 transcription factor, resulting in protein degradation in cells. It promotes the secretion of enzymes and inflammatory cytokines (Kumar et al., 2015). Increased proteolytic enzymes and inflammatory cytokines destroy the extracellular matrix, ultimately leading to skin aging and wrinkles. MAPKs include extracellular signal-regulated kinase (ERK), p38 MAPK, and Jun N-terminal kinase (JNK).

Therefore, whether the anti-wrinkle and anti-aging effects of 7,8-dihydroxyflavones on skin cells depend on mitogen-activated protein kinase (MAPKs) and regulation of Akt signal transduction by Western blot method.

Hs68 cells were aliquoted into a 6-well plate at 6.25 x 10 ⁴ cells/ml, incubated for 8 hours, and the sample solution was pretreated for 1 hour, followed by incubation with 20 ng/ml TNF-α for 6 hours. The medium was removed, washed with phosphate buffered saline, and centrifuged at 1,400 rpm for 5 minutes to obtain cells. After adding 20 µl of lysis buffer to the precipitated cells, the reaction was performed on ice for 30 minutes, followed by centrifugation at 14,000 rpm for 30 minutes to obtain a protein. After quantifying the protein using the Bradford method, the protein was denatured at 95° C. for 5 minutes by adding a loading dye, followed by electrophoresis using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) gel. After electrophoresis and transfer to a nitrocellulose membrane, primary anti-phospho p38 antibody (1:1000), anti-phospho ERK antibody (1:1000), anti-phospho JNK antibody (1:500), anti -Phospho Akt antibody (1:500), anti-total p38 antibody (1:1000), anti-total ERK antibody (1:1000), anti-total JNK antibody (1: 1000), anti-total Akt antibody (1:1000), and anti-beta actin antibody (1:1000) were diluted in 5% skim milk at each ratio and added to a nitrocellulose membrane at 4℃. Reacted for 24 hours. After washing three times every 10 minutes with TBST (TBS, 0.1% Tween 20), a HRP-conjugated (horeseradish peroxidase-conjugated) secondary antibody was diluted 1:2000 in 5% skim milk powder and reacted at room temperature for 2 hours. After washing three times using TBST in the same manner as described above, the ECL (Enchanced Chemiluminescence) substrate solution was reacted to the film for 1 minute, and then developed on the X-ray film.

As can be seen in Figure 5, in the control (Control) treated with only TNF-α (20 ng/ml), p38, ERK, JNK, and Akt proteins compared to the untreated control (UC) that was not treated with TNF-α. Phosphorylation of p38, ERK, JNK, and Akt protein decreased in a concentration-dependent manner compared to the control group in the group treated with 0.1, 1, 5 and 10 μM of 7,8-dihydroxyflavone, respectively. Became.

As described above, the cosmetic composition comprising 7,8-dihydroxyflavone or a salt thereof of the present invention as an active ingredient can improve skin aging, skin inflammation, and wrinkles by controlling the phosphorylation of MAPKs and Akt proteins. It can be usefully used as an anti-aging, anti-wrinkle, and anti-inflammatory cosmetic composition.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Cosmetic composition comprising 7,8-dihydroxyflavone or salt thereof for prevention and treatment of skin aging and wrinkle <130> 1042675 <150> KR 10-2015-0181491 <151> 2015-12-18 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human type 1 procollagen forward primer <400> 1 ctcgaggtgg acaccaccct 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human type 1 procollagen reverse primer <400> 2 cagctggatg gccacatcgg 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human MMP-1 forward primer <400> 3 attctactga tatcggggct ttga 24 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Human MMP-1 reverse primer <400> 4 atgtccttgg ggtatccgtg tag 23

Claims (7)

A cosmetic composition for preventing skin aging and improving skin wrinkles comprising 7,8-dihydroxyflavone represented by the following formula (I) or a salt thereof as an active ingredient.
(I)

The cosmetic composition for preventing skin aging and improving skin wrinkles according to claim 1, wherein the cosmetic composition has at least one effect selected from the following (a) to (e):
(a) increased collagen synthesis;
(b) inhibiting MMP-1 (matrix metalloproteinase-1) expression;
(c) Reactive oxygen species (ROS) accumulation inhibition;
(d) increased antioxidant enzyme expression; And
(e) Reduction of phosphorylation of mitogen-activated protein kinase (MAPKs) and Akt proteins.
The cosmetic composition according to claim 1, wherein the cosmetic composition comprises 7,8-dihydroxyflavone or a salt thereof in a concentration of 0.1 μM to 10 μM.
The cosmetic composition according to claim 1, wherein the cosmetic composition is any one selected from the group consisting of a soft lotion, a nutritional lotion, a gel, a cream, an essence, an emulsion, a pack, a cleanser and a bath powder. Cosmetic composition.
A food composition for preventing skin aging and improving skin wrinkles comprising 7,8-dihydroxyflavone represented by the following formula (I) as an active ingredient.
(I)

The food composition for preventing skin aging and improving skin wrinkles according to claim 5, wherein the food composition has at least one effect selected from the following (a) to (e):
(a) increased collagen synthesis;
(b) inhibiting MMP-1 (matrix metalloproteinase-1) expression;
(c) Reactive oxygen species (ROS) accumulation inhibition;
(d) increased antioxidant enzyme expression; And
(e) Reduction of phosphorylation of mitogen-activated protein kinase (MAPKs) and Akt proteins.
6. The composition for preventing skin aging and improving skin wrinkles according to claim 5, wherein the food composition comprises 7,8-dihydroxyflavone or a salt thereof at a concentration of 0.1 μM to 10 μM.

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