KR101589002B1 - Cosmetic composition containing sargassum serratifolium extract for improving skin wrinkle - Google Patents

Abstract

The present invention relates to a cosmetic composition for reducing skin wrinkles and, more specifically, to a cosmetic composition effective in reducing skin wrinkles by containing a Sargassum serratifolium extract or a fraction thereof as an active ingredient. The Sargassum serratifolium extract or the fraction thereof in accordance with the present invention is far superior in an MMP-1 inhibitory activity by suppressing the phosphorylation of MAPKs (JNK, ERK, AKT) and the expression of activator protein-1 (AP-1) which are important for the expression of MMP-1, so the composition containing the same as an active ingredient can be effectively used as a cosmetic composition for preventing skin aging induced by UV light and reducing skin wrinkles. Particularly, Sargassum serratifolium is edible as a natural substance, so the cosmetic composition for reducing skin wrinkles, containing an extract derived from the Sargassum serratifolium or the fraction thereof, is safe for long-term use.

Description

TECHNICAL FIELD The present invention relates to a cosmetic composition containing sargassum serratifolium extract for improving skin wrinkle comprising, as an active ingredient,

The present invention relates to a cosmetic composition for improving wrinkles, and more particularly to a cosmetic composition having an effect of improving wrinkles, comprising an extract of Sawatil mushroom as an active ingredient or a fraction thereof.

Skin aging, one of the aging phenomena of human beings, is largely divided into internal and external factors. Among them, UV irradiation by sunlight is known to be the main cause of physiological and morphological changes of skin aging . Ultraviolet rays, which directly affect the skin, penetrate deeply into the epidermis and dermis of the skin and undergo various processes including several enzymatic reactions to generate oxygen free radicals in cells and tissues. Excessive active oxygen Mutation, inflammation, cell killing, carcinogenesis and skin aging. In the skin, the active oxygen attacks the cell membrane and oxidizes it, and the oxidized lipids damage the cell membrane and cause loss of normal skin cell function. In addition, the balance of antioxidant defense system of the skin is broken, and the skin is not recovered from the persistent oxidation state, and the skin becomes rough and unglamorous, and the repetition of this process causes wrinkle formation.

In addition, an increase in collagenase is one of the important causes of denaturation and destruction of collagen fibers. The active oxygen generated in vivo accelerates the activation of collagenase such as MMPs (matrix metalloproteinases), collagen Thereby suppressing the elasticity of the skin and promoting the generation of wrinkles.

MMPs (Matrix metalloproteinases) are a group of enzymes involved in extracellular matrix (ECM) and basement membrane degradation. They are classified into interstitial collagenase, stromelysin, gelatinase gelatinase, and membrane-type MMP (MT-MMP). MMPs are known to more than 20 species in mammals, birds, amphibians, plants and nematodes to date.

MMPs also have structural and functional similarities, although they differ in substrate specificity. Therefore, the names of all MMPs are generally numbered. The collagenases-1, 2 and 3 are MMP-1, 8 and 13, the gelatinoids-A and B are MMP-2 and 9, the stromelysin-1, 2 and 3 are MMP- 10, 11, metalloelastase, MMP-12, matrilysin, MMP-7, and membrane-type MMP (MT-MMPs) , 24, and enamelysin is called MMP-20, MMP-19, MMP-23, and the like.

These enzymes can degrade proteins such as collagen, fibronectin, laminin, proteoglycan, entatin and elastin, respectively. MMP is a metalloproteinase with zinc at the active center and is secreted in the form of an inactive precursor (zymogen) in vivo. In order to have enzymatic activity, structural modification must occur and amino terminal region should be cleaved. Activated MMP is regulated by inhibitors such as 2-macroglobulin or TIMPs (Tissue inhibitors of metalloproteinase). The vast majority of cells, including keratinocytes and fibroblasts, secrete MMPs. MMP activity is reported to be a major factor in photo-aging since the activity of MMP in the skin is increased and collagen can be remarkably reduced even at one time of UV irradiation.

To date, the most effective method for improving wrinkles is retinol and retinoic acid. Such retinol and retinoids, such as retinoic acid, have been well known for improving wrinkles or improving elasticity (Dermatology therapy, 1998, 16, 357-364). These retinoids, however, have a disadvantage in that they are stimulated even when only a small amount of skin is applied to the skin, and they are easily oxidized and deteriorated when exposed to air due to instability, . Therefore, studies for stabilizing retinoids have been continuing, but the problem of safety for the skin has not yet been solved.

What is proposed as a substitute for this is to inhibit the activity of enzymes that degrade collagen and elastin, and studies on inhibitors capable of inhibiting the activity of these enzymes have been actively conducted. Recently, a remarkable increase in the activity of collagenase, which is an enzyme capable of degrading collagen, and substrate metalloprotease has been found to be one of the causes of skin wrinkles.

In recent years, attention has been focused on functional cosmetics having functions of alleviating wrinkles and whitening obtained from natural extracts in order to reduce skin irritation caused by various chemical substances and the like. Natural products have a small adverse effect on the skin, and consumers are increasingly responding to skin external preparations using natural materials, so that development value as a raw material for anti-aging cosmetic compositions is further increased. For example, Japanese Patent Application Laid-Open No. 9-672662 describes algae extracts having hyaluronidase inhibitory activity. Japanese Patent Application Laid-Open No. 10-85905 discloses an effect of improving skin texture of fucoidan extracted from Wakame. Japanese Patent Laid-Open No. 2-245087 describes the antioxidative effect of Sargassum sp. Extract. Japanese Patent Laid-Open No. 3-294384 discloses an antioxidant composition extracted from a genus of Hizikia. Japanese Patent Application Laid-Open No. 7-10769 discloses Phaeophyceae extract having an astringent effect. U.S. Pat. No. 5,508,033 describes a cosmetic with anti-radical activity, including cap moiety. Korean Patent No. 810134 discloses a process for washing and demineralizing a moth or tortoise; Primary enzymatic degradation consisting of endo-β1,4-glucanase, alginic acid lyase and exocellulose; And a secondary enzyme consisting of arabinase, cellulase and? 1, 4-glucanase. Also disclosed is a method for producing the same, And it can be used as a cosmetic or the like.

The inventors of the present invention have studied the possibility of application to cosmetic compositions for improving wrinkles in various natural materials which have not been known so far. As a result, the present invention has been made to select moths belonging to the moths and to produce extracts. Various kinds of moths such as Sargassum serratifolium Of the present invention is remarkably excellent in wrinkle-reducing effect compared to other herbicides known in the art.

Korean Patent No. 10-0810134 Korean Patent Publication No. 10-2013-0023326

Accordingly, an object of the present invention is to provide a cosmetic composition which is stable to the human body and has excellent wrinkle-reducing activity even after prolonged use by using an extract of natural seaweed.

In order to achieve the above-mentioned object of the present invention,

The present invention provides a cosmetic composition for improving wrinkles comprising an extract of Tootsuna mash or a fraction thereof as an active ingredient.

In one embodiment of the present invention, the extract may be extracted with at least one solvent selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, water and hexane.

In one embodiment of the present invention, the lower alcohol having 1 to 4 carbon atoms may be ethanol.

In one embodiment of the present invention, the extract or the fraction thereof may have a wrinkle-reducing effect through the inhibition of MMP-1 expression.

In one embodiment of the present invention, the cosmetic composition may be at least one selected from the group consisting of a soft lotion, a gel, a water-soluble liquid, a milk lotion, a nutritional cream, a massage cream, an essence, an oil in water emulsion, , Oil dispersions in aqueous phase using microspheres, ionic lipid vesicles, nonionic lipid vesicles, ointments, cleansing foams, cleansing waters, packs, body oils, oil-in-oil makeup bases, watershed makeup bases, foundations, skins And may be one type of formulation selected from the group consisting of a cover, a lipstick, a lip gloss, a face powder, a two-way cake, an eye shadow, a mascara, a cheek color and an eyebrow pencil.

The Sargassum serratifolium extract or its fractions according to the present invention inhibit phosphorylation of MAPKs (JNK, ERK, AKT) and inhibit AP-1 (activator protein-1) expression, which play an important role in the expression of MMP-1 Since the MMP-1 inhibitory activity is remarkably excellent, a composition containing it as an active ingredient can be usefully used as a cosmetic composition for preventing aging and improving wrinkles by UV light. In particular, since the sawtooth moth is edible as a natural substance, the cosmetic composition for improving wrinkles of the present invention, including the extract derived therefrom and its fractions, has a safety advantage over long-term use.

FIG. 1 is a graph showing the cell viability of human epidermal keratinocytes of Haemophilus influenzae extract and hexane fraction of Haemophilus influenzae according to the present invention.
FIG. 2 shows the results obtained after treatment of HaCaT cells with UVB (35 mJ / cm 2) irradiation and the hexane fraction of the present invention with the concentrations of 1.0, 2.5 and 5.0 μg / ml for 3 days. The MMP- The expression level was measured by Western blotting.
FIG. 3 is a graph showing the degree of phosphorylation of MAPKs (JNK, ERK, AKT) by Western blotting with HaCaT cells treated with concentrations of 1.0, 2.5, and 5.0 μg / (3a: Teaspoon juice extract, 3b: Hexane fraction).
FIG. 4 shows the degree of phosphorylation of c-Jun, a component of AP-1, by treatment of Hacca T cells with the concentrations of 1.0, 2.5, and 5.0 μg / ml of the extracts of Tephalan myrtle extract and hexane fraction of the present invention after UVB irradiation, (3a: Teaspoon juice extract, 3b: Hexane fraction).

The present invention is characterized by providing a cosmetic composition for improving wrinkles comprising an extract of Tootsuna mash or a fraction thereof as an active ingredient.

The saw blade of the present invention ( Sargassum serratifolium ) is a marine algae with mosquito and mosquito . It is distributed in southern coast and Jeju region in Korea. Plants are 1 ~ 4m in diameter, 4 ~ 5cm in diameter, conical, rubbery and ringed.

There has been no report on researches such as anti-aging and wrinkle improvement until now.

The present inventors selected moths belonging to the cosmetics composition as a material of cosmetic composition and prepared extracts. Various kinds of moth moth moth moth moth moth moths suppress phosphorylation of MAPKs (JNK, ERK, AKT) The inhibition of AP-1 (activator protein-1) expression suppressed the MMP-1 inhibitory activity. Thus, it was confirmed that the extracts of lettuce or their fractions can effectively improve the skin wrinkles.

In detail, the inventors of the present invention evaluated the MMP-1 expression-inhibiting activity of various mothballs such as dumpling bean moth, horn bean moth, and staff moth, in addition to the sawtooth moth, (95% ethanol) showed a suppression rate of 42.3% of MMP-1 expression at a concentration of 10 ppm, whereas the inhibition rate of MMP-1 expression was about 80% at a concentration of 1 ppm, Water extracts showed inhibitory activity on MMP-1 expression at 50 ppm, and the activity of the extracts of hornblende moxibustion and moxibustion extracts was found to be low even at 100 ppm (see table below) ≪ / RTI > inhibition of MMP-1 protein expression).

For reference, the amount of MMP-1 protein produced was determined by Western blotting of the amount of MMP-1 protein expressed in the supernatant after 3 days of HaCaT cells were irradiated with UVB (35 mJ / cm 2)

From the above results, it has been objectively proved through experiments that the Saw Mab Bark Extract of the present invention and its fractions exhibit an activity of inhibiting MMP-1 expression by 5-fold to 100-fold over the other mulberry extracts extracted by the same method.

Therefore, the cosmetic composition of the present invention comprising the extract of Tootsum matsumana and the fraction thereof as an active ingredient can effectively improve skin wrinkles.

The extract of Tooth Motha according to the present invention can be obtained by extracting and isolating from nature using an extraction and separation method known in the art, and the extract defined in the present invention can be obtained by using a suitable solvent such as Sargassum serratifolium ), for example, crude extracts of sawtooth moth , polar solvent-soluble extracts or non-polar solvent-soluble extracts.

As an appropriate solvent for extracting the extract from the sawtooth mackerel, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used. Examples of the solvent include, but are not limited to, purified water, methanol alcohol having 1 to 4 carbon atoms, acetone, ether, benzene, chloroform (including methanol, ethanol, propanol, isopropanol, and butanol) various solvents such as chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. It is preferable to use ethanol (alcohol), more preferably 70% ethanol.

As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the production method of the sawtooth extract of the present invention, and any known method may be used.

For example, in the composition of the present invention, the first extract extracted with the hot water extraction method or the solvent extraction method can be prepared into a powder state by an additional process such as vacuum distillation, freeze drying or spray drying have. Further, the primary extract can be further purified by using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography and the like, You can get it.

Therefore, in the present invention, the teaspoon mallow extract is a concept including all the extract, fraction and purified product obtained in each step of extraction, fractionation or purification, and a diluted solution, a concentrate or a dried product thereof.

Hereinafter, a method of preparing the sawtooth extract according to an embodiment of the present invention will be described in more detail.

After the dried sawtooth mats are pulverized, the toothed mats are dried by reflux cooling using 70% ethanol as an extraction solvent, followed by filtration and concentration to extract.

These extracts of Tephritidae can be further fractionated using an n-hexane solvent. For example, it is possible to separate a water layer and a hexane layer by adding a mixed solvent of n-hexane, alcohol and water in a volume ratio of 10: 1: 9 to an extract of a teaspoon, The hexane fraction of the teaspoon mine extract can be prepared by repeating the process of adding n-hexane and separating out the n-hexane layer by allowing to stand and concentrating the obtained hexane fraction.

Accordingly, the composition of the present invention corresponds to a cosmetic composition for improving the wrinkles of the skin comprising an extract of Tootsum matsumana or a fraction thereof as an active ingredient.

In one embodiment of the present invention, the Sawmill Extract or the fraction thereof may be contained in the cosmetic composition at a concentration of 1 μg / ml to 1000 μg / ml (or 1 to 1000 ppm).

Examples of products to which the cosmetic composition of the present invention can be added include cosmetics such as astringent lotion, softening longevity lotion, nutrition lotion, various creams, essences, packs, foundation and the like, cleansing, cleanser, soap, .

Specific formulations of the cosmetic composition of the present invention may include, but are not limited to, softening lotion, gel, water-soluble liquid, milk lotion, nutritional cream, massage cream, essence, oil in water emulsion, water in oil type emulsion, facial anhydrous product, A lipid, a lipid, a lipid, a lipid, a lipid, a lipid, a lipid, a lipid, a lipid, a lipid, Lipstick, lip gloss, face powder, two-way cake, eye shadow, mascara, cheek color, and eyebrow pencil.

According to a preferred embodiment of the present invention, the content of the active ingredient (trehalose extract or fraction thereof) of the present invention is 0.00001-40 wt%, preferably 0.0005-40 wt%, more preferably 0.0005-40 wt% 0.0005-10% by weight. If the content of the active ingredient (saponin extract or fraction thereof) is less than 0.00001% by weight, the effect of improving skin wrinkles is greatly reduced. If it exceeds 40% by weight, skin irritation may be caused, have.

On the other hand, the cosmetic composition according to the present invention may be formulated by stabilizing the active ingredient (saponin extract or fraction thereof) contained in the nanoliposome. When the above-mentioned active ingredient (saponin extract or fraction thereof) is contained in the inside of the nanoliposome, the active ingredient is stabilized, thereby solving problems such as formation of precipitates, discoloration and deterioration during formulation and solubility of components and transdermal absorption It is possible to maximize the efficacy expected from the extract.

In the present invention, nanoliposome refers to a liposome having a typical liposome form and having an average particle diameter of 10 to 500 nm. According to a preferred embodiment of the present invention, the average particle diameter of the nanoliposome is 50 to 300 nm, more preferably 100 to 200 nm. When the average particle diameter of the nanoliposome exceeds 500 nm, improvement of skin penetration and improvement of formulation stability is very weak among technical effects to be achieved in the present invention.

The nanoliposomes used to stabilize the extract according to the present invention may be prepared by a mixture comprising a polyol, an oily component, a surfactant, a phospholipid, a fatty acid and water.

The polyol used in the nanoliposome of the present invention is not particularly limited and is preferably a polyol such as propylene glycol, dipropylene glycol, 1,3-butylene glycol, glycerin, methylpropanediol, isopropylene glycol, pentylene glycol, erythritol, , Sorbitol, and mixtures thereof. The amount thereof is 10 to 80% by weight, preferably 30 to 70% by weight, based on the total weight of the nanoliposome.

The oil component used in the preparation of the nanoliposome of the present invention may be selected from a variety of oils known in the art and is preferably a hydrocarbon oil such as hexadecane and paraffin oil, Vegetable oils such as silicon oil such as dimethicone and cyclomethicone, sunflower oil, corn oil, soybean oil, avocado oil, sesame oil and fish oil, ethoxylated alkyl ether oils, propoxylated alkyl ether oils ₄₀ is a fatty alcohol, and mixtures thereof -, phytosphingosine, sphingosine, and scan non-ping the same scan pinggo cannabinoid lipid, cholesterol side-by celebrity, sitosterol cholesteryl sulfate, cholesteryl sulfate, cytokines, C _10. The amount thereof may be 1.0 to 30.0% by weight, and preferably 3.0 to 20.0% by weight based on the total weight of the nanoliposome.

Any surfactant known in the art may be used as the surfactant used in the preparation of the nanoliposome of the present invention. For example, anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants can be used. Preferred are anionic surfactants and nonionic surfactants. Specific examples of anionic surfactants include alkyl acyl glutamates, alkyl phosphates, alkyl lactylates, dialkyl phosphates and trialkyl phosphates. Specific examples of nonionic surfactants include alkoxylated alkyl ethers, alkoxylated alkyl esters, alkyl polyglycosides, polyglyceryl esters and sugar esters. Particularly preferred surfactants are polysorbates belonging to nonionic surfactants. The amount thereof may be 0.1 to 10% by weight, preferably 0.5 to 5.0% by weight, based on the total weight of the nanoliposome.

The phospholipid, another component used in the preparation of the nanoliposomes of the present invention, is a lipophilic lipid that is used with both affinity lipids and is composed of natural phospholipids (e.g., egg yolk lecithin or soy lecithin, sphingomyelin) Dipalmitoyl phosphatidylcholine or hydrogenated lecithin), preferably lecithin. In particular, unsaturated lecithin or saturated lecithin derived from natural origin extracted from soybean or egg yolk is preferable. Normally, the amount of phosphatidylcholine is 23 to 95% and the amount of phosphatidylethanolamine is 20% or less in natural derived lecithin. In the production of the nanoliposome of the present invention, the amount of the phospholipid to be used is 0.5 to 20.0% by weight, preferably 2.0 to 8.0% by weight based on the total weight of the nanoliposome.

Fatty acid to be used in nano-liposome preparation of the present invention is a higher fatty acid, preferably a C ₁₂ - ₂₂ as a saturated or unsaturated fatty acid of the alkyl chain, e.g., lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid . The amount thereof may be 0.05 to 3.0% by weight, preferably 0.1 to 1.0% by weight, based on the total weight of the nanoliposome.

The water used in the preparation of the nanoliposome of the present invention is generally deionized distilled water, and the amount thereof may be 5.0-40 wt% based on the total weight of the nanoliposome.

The preparation of nanoliposomes can be accomplished through a variety of methods known in the art, but most preferably is made by applying a mixture comprising the ingredients to a high pressure homogenizer. The preparation of a nanoliposome by a high pressure homogenizer can be carried out under various conditions (for example, pressure, number of times, etc.) depending on a desired particle size, and preferably at a high pressure homogenizer So that the nanoliposome can be produced.

The cosmetic composition of the present invention may be used alone or in combination, or it may be used in combination with other cosmetic compositions other than the present invention. The cosmetic composition having an excellent wrinkle-reducing effect according to the present invention can be used according to a conventional method of use and can be used in a number of times depending on the skin condition or taste of the user.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example >

Cell culture

Ultraviolet rays of 20 ~ 90 mJ / cm ² as UVB were irradiated using CUBE 401 lamp (Sankyo ultraviolet). Human keratinocyte HaCat cells were cultured in 6 wells at a concentration of 4 × 10 ⁵ cells / ml and cultured until reaching 80% confluency. Before the UV irradiation, the original medium was removed and the serum was completely removed by washing with PBS. After irradiation with UV, the cells were treated with free serum medium.

< Example  1>

Manufacture of teaspoon mushroom extract

&Lt; 1-1 > Preparation of extracts of teaspoon mackerel

The sawtooth moths used in the present invention were obtained by scuba diving using natural scavengers obtained from Gijang-gun and Geonan Tongyeong City in Busan. Professor Chang-Keun Choi of Ecological Engineering Department, Pukyong National University, Professor Kim Nam-gil performed this. The toothed mushroom was naturally dried in sunshine, and 1.5 kg of the powder obtained by grinding was added to 6 L of the alcohol, and the mixture was refluxed and cooled twice at 70 캜 for 3 hours, and then the foreign substance was removed with an ultrafilter (MWCO 50 kDa). The filtrate was extracted with a vacuum rotary condenser at 40 ° C, and 160 g of a crude extract was obtained as an extracted residue (hereinafter, abbreviated as "ESS" in the present invention). The thus obtained Saw Moth extract was used as a sample in the following experiment while being stored at 4 ° C in a refrigerator.

<1-2> n- Hexane Fraction  Produce

4 L of a mixed solvent of n-hexane, alcohol, and water in a volume ratio of 10: 1: 9 was added to 100 g of the extract of the Tooth Moth extract prepared in Example <1-1> The n-hexane layer was allowed to separate. After 3-5 hours, the mixture was separated into a 2 L water layer and a 2 L hexane layer. 2 L of n-hexane was added again to the separated 2 L water layer, and the process of separating 2 L of n-hexane layer was repeated 3 times. As a result, a total of 8 L of n-hexane layer and 2 L of water layer were finally obtained. The 8 L n-hexane layer obtained as described above was concentrated under reduced pressure at 40 캜 by a vacuum rotary condenser to remove the solvent, and 79 g of the residue was obtained as a n-hexane fraction. The n-hexane fraction thus obtained was used as a sample in the following experiment while being stored at 4 ° C in a refrigerator.

< Experimental Example  1>

The toothed pan extract of the present invention and Hexane fraction  Cytotoxicity Assessment

In this experiment, in order to confirm cytotoxicity of the extracts of the teaspoon of the present invention and the hexane fractions prepared in Example 1, human skin keratinocytes were treated with the extracts or fractions of the present invention at different concentrations, The survival rate was analyzed by MTS assay. Human dermal keratinocytes used in the experiments were HaCaT.

In detail, HaCaT cells were cultured in 10% FBS-DMEM (Dulbecco's Modified Eagle's Medium, Gibco), and then 200ul each was added to a 96-well plate at a cell concentration of 5 x 10 ⁴ cells / ml. in% CO ₂ incubator and incubated for 24 hours. After 24 hours of incubation, the medium was removed and 100 ul of each of the sera extract and hexane fractions and 100 ul of FBS-free DMEM (0, 1.0, 2.5, 5.0 μg / ml) prepared by 10% FBS- Lt; / RTI &gt; for 24 hours. At this time, the experimental group using only FBS-free DMEM was used as a control of the sample. 3 hours before completion of the reaction, 5 mg / ml MTS solution (in 1 x PBS (-), (3- (4,5-dimethylthiazol- 2- yl) -5- (3- carboxymethoxyphenyl) -2- -2H tetrazolium) was added in an amount of 5 ul, followed by further reaction for 3 hours. After removing the MTS reaction solution, DMSO (Dimethyl sulfoxide) was added in an amount of 150. After stirring for 5 minutes (at room temperature), the absorbance was measured at 570 nm using a microplate reader.

As a result, as shown in Fig. 1, the extracts of teaspoon mine extract and hexane fraction showed no cytotoxicity up to a concentration of 5 μg / mL.

< Experimental Example  2>

The toothed pan extract of the present invention and Hexane fraction  Wrinkle improvement effect

In order to examine the wrinkle-reducing activity of the extracts of teaspoon mugwort and hexane according to the present invention prepared in Example 1, MMP-1 expression inhibitory effect, MAPKs inhibitory effect and AP-1 inhibitory effect were examined.

As a reference, MMP-1 is a fibroblast type collagenase which is an enzyme of epilepsy. Collagen type I, II, III, VII, VIII, X and gelatin are the substrates and originally 3/4 or 1/4 To facilitate the action of nonspecific enzyme (gelatinase). By inhibiting the activity and biosynthesis of MMP-1, which breaks down type collagen, which occupies the largest proportion of binding proteins, it can protect skin binding proteins and prevent wrinkle formation and elasticity degradation.

On the other hand, when the extracellular-regulated protein kinase (ERK), Jun-N-terminal kinase (JNK) and AKT belong to mitogen-activated protein kinases (MAPKs) The activity of the protein-1 is induced (Xu Y, Fisher GJ., J Dermatol Sci. Suppl. 2005; 1: S1 S8), resulting in MMPs (matrix metalloproteinase) secretion and collagen degradation. C-Jun and c-Fos are known to affect DNA binding with AP-1 (Waskiewicz AJ, Cooper JA, Curr Opin Cell Biol., 1995; 7: 798 805).

In order to examine the wrinkle-reducing activity of the extracts of extracts of hexane and hexane of the present invention produced through Example 1, the present inventors examined the effect of MMP-1 on the inhibition of type-collagen degradation, (JNK, ERK, AKT) and MMP-1 expression, thereby inhibiting AP-1 inhibiting activity of degrading collagen and promoting skin wrinkle formation, Respectively.

<2-1> Cell culture

Ultraviolet rays of 35 mJ / cm ² were irradiated with UVB using CUBE 401 lamp. HaCaT cells were cultured at 4 × 10 ⁵ cells / ml in 6 wells and cultured until reaching 80% confluency.

<2-2> Western Blat

HaCaT cells were cultured for 24 hours in a concentration-dependent manner and then treated with UVB for 30 minutes, followed by treatment with RIPA (protease inhibitor) containing 1% protease inhibitor Protein was extracted from the buffer and centrifuged at 4 to 12,000 rpm for 25 minutes to obtain the supernatant. Protein content was quantified by using bovine serum albumin as a standard substance using a BCA protein assay kit (Pierce, Rockford, Ill., USA). 20-40 μg of the protein was put into a 5 × SDS sample buffer and denatured by heating at 100 ° C. for 10 minutes and then subjected to electrophoresis. After electrophoresis in a 10% SDS polyacrylamide gel at 120 V for 2 hours, the membrane was transferred to nitrocellulose membrane and blocked in a blocking solution containing 5% skim milk. Lt; / RTI &gt; The primary antibody was diluted 1: 500, reacted at room temperature for 1 hour, and washed three times for 10 minutes with Tris-Buffered Saline Tween-20 (TBS-T). Then, the reaction was performed with a secondary antibody diluted at a ratio of 1: 5000 at room temperature for 1 hour and then washed with TBS-T four times for 10 minutes each. The difference in protein expression was observed using a luminoimager (ChemiDoc XRS +, BIO-RAD) using an ECL western blotting substrate (Thermo Scientific).

<2-3> MMP -1 expression inhibitory effect

HaCaT cells were cultured in 6 wells at a concentration of 4 × 10 ⁵ cells / ml and cultured until an 80% confluency was reached.

HaCaT cells were treated for 24 hours at concentrations of 0, 1.0, 2.5 and 5.0 μg / ml, respectively, according to the present invention. After 24 hours, the medium containing the sample was removed and washed with PBS to completely remove the serum from the medium. The cells were cultured for 72 hours in UV-irradiated medium (35 mJ / cm ² ) and then cultured in free serum medium. Cell culture medium was collected and MMP-1 in the medium was analyzed by Western blotting.

As a result, as shown in the following Table 1 and FIG. 2, when the HaCaT cells were irradiated with UVB, the expression of MMP-1 was all increased, but the extracts of Tephalan myrtle extract and hexane of the present invention were 1.0, 2.5 and 5.0 μg / ml It was confirmed that the concentration of MMP-1 in the cell culture solution was remarkably reduced compared with the no-added group.

Table 1 below quantitatively quantifies the amount of MMP-1 protein expression analyzed by Western blotting.

Amount of MMP-1 protein expression by treatment concentration of ethanol extract and hexane fraction UVB (-) UVB (+) - - 1.0 μg / ml 2.5 μg / ml 5.0 μg / ml Alcohol extract 1.00 ± 0.05 52.0 ± 0.78 30.58 + - 0.39 17.22 + - 0.51 10.54 + - 0.48 Hexane fraction 1.00 ± 0.05 13.3 ± 1.51 5.81 ± 0.97 2.74 ± 0.29 1.52 + 0.17

<2-4> MAPKs  Inhibitory efficacy

In the experiment example <2-3>, MMP-1 inhibitory activity was examined. In order to confirm the signaling cascade that causes the inhibitory activity of MMP-1, MMP-1 expression (JNK, ERK, AKT), which plays an important role in the inhibition of phosphorylation.

HaCaT cells were treated with 24 hour dilutions of the extracts of Horseradish peroxidase and hexane fraction in serum-free media. After 24 hours, the medium containing the sample was removed and washed with PBS to completely remove the serum from the medium. After UV irradiation (35 mJ / cm ² ), the cells were cultured for 30 min in a free serum medium.

As a result, as shown in Tables 2, 3 and 3, phosphorylation of all MAPKs was markedly increased when UVB was applied to cells, whereas in the experiment group in which the extracts of Tephalan myrtle extract and hexane fraction of the present invention were added, The phosphorylation of AKT, ERK and JNK was markedly decreased. In particular, AKT and ERK were found to inhibit phosphorylation to a similar degree to that of UVB-exposed cells at all concentrations of the extracts of Schnabelmaschinensis and hexane fractions. On the other hand, it was confirmed that there was no change in the intracellular expression amounts of JNK, ERK and AKT due to UVB irradiation and addition of the extracts of the extracts of teaspoon of the present invention and the hexane fraction of the present invention.

As a result, it was confirmed that the tobacco extract and hexane fraction of the present invention inhibited the expression of MMP-1 by inhibiting UVB-induced phosphorylation of MAPKs that regulate the activation of AP-1.

Tables 2 and 3 below quantitatively quantify the amount of phosphorylated JNK, ERK, and AKT protein expressed by Western blot analysis.

Expression of phosphorylated JNK, ERK, AKT protein by treatment concentration UVB (-) UVB (+) - - 1.0 μg / ml 2.5 μg / ml 5.0 μg / ml pAKT 1.00 ± 0.05 11.19 ± 0.78 4.14 + 0.48 3.89 ± 0.51 3.69 ± 0.58 pERK 1.00 + - 0.01 3.01 ± 0.64 0.54 + - 0.37 0.72 + - 0.50 0.86 ± 0.31 pJNK 1.00 0.35 5.02 + 0.78 3.75 ± 0.52 1.92 + - 0.27 1.86 ± 0.27

Amount of phosphorylated JNK, ERK, AKT protein expressed by treatment concentration of hexane fraction UVB (-) UVB (+) - - 1.0 μg / ml 2.5 μg / ml 5.0 μg / ml pAKT 1.00 + 0.02 14.19 ± 0.78 3.79 ± 0.22 3.89 ± 0.48 3.79 ± 0.23 pERK 1.00 ± 0.05 4.19 ± 0.68 1.06 + - 0.33 1.09 + - 0.47 1.33 + - 0.33 pJNK 1.00 + 0.04 4.72 ± 0.75 6.03 ± 0.27 8.04 0.32 8.91 ± 0.39

<2-5> AP-1 inhibitory effect

AP-1 is a transcription factor that binds c-Jun and c-Fos proteins, mediates the expression of MMP-1 and ultimately degrades collagen to promote skin wrinkling. In this experiment, the effect of inhibiting phosphorylation of c-Jun, a component of AP-1, was examined in order to determine whether the extract of the present invention had an inhibitory effect on the activation of AP-1 by UVB.

HaCaT cells were treated with 24 hour dilutions of the extracts of Horseradish peroxidase and hexane fraction in serum-free media. After 24 hours, the medium containing the sample was removed and washed with PBS to completely remove the serum from the medium. After UV irradiation (35 mJ / cm ² ), the cells were cultured for 30 min in a free serum medium.

As a result, as shown in Table 4 and FIG. 4, the UVB irradiation of HaCaT significantly increased the phosphorylation of c-Jun compared to the control group. However, when the extract of the present invention was added to the extracts of hexane and hexane, it was confirmed that the phosphorylation of c-Jun was inhibited. Table 4 below quantitatively quantifies the amount of phosphorylated c-Jun protein expressed through Western blot analysis.

As a result, it was confirmed that the ethanol extract of hexane and the hexane fraction inhibited the expression of MMP-1 induced by UVB by inhibiting the activity of AP-1.

The amount of phosphorylated c-Jun protein expressed by the treatment concentration of the ethanol extract and hexane fraction UVB (-) UVB (+) - - 1.0 μg / ml 2.5 μg / ml 5.0 μg / ml Alcohol extract 1.00 ± 0.05 2.53 + - 0.24 0.87 ± 0.06 0.78 ± 0.05 0.67 ± 0.08 Hexane fraction 1.00 + - 0.01 4.71 ± 0.35 1.26 ± 0.15 1.10 ± 0.12 1.12 ± 0.11

< Manufacturing example  1>

The wrinkle improvement of the present invention Cosmetics  Composition manufacturing

<1-1> Softened skin (skin)

The softened skin containing the teaspoon juice extract or hexane fraction of the present invention was prepared in a conventional manner at the following proportions:

Examples of the softening longevity formulation of the present invention ingredient Content (% by weight) Sawtooth extract or hexane fraction 1-3 glycerin 5.0 1,3-butylene glycol 3.0 REG 1500 1.0 Allantoin 0.1 DL-Panthenol 0.3 EDTA-2Na 0.02 Benzophenone-9 0.04 Sodium hyaruronate 5.0 ethanol 10.0 Octyldodec-16 0.2 Polysorbate 20 0.2 Unicide - Yu 13 0.01 incense a very small amount Distilled water Balance Sum 100

<1-2> Nourishing lotion

Nutritional lotions containing the teaspoons alcohol extract or hexane fraction of the present invention were prepared in a conventional manner at the following proportions:

The nutritional lotion formulation of the present invention ingredient Content (% by weight) Sawtooth extract or hexane fraction 1-3 Glyceryl stearate SE 1.5 Cetearyl alcohol 1.5 lanolin 1.5 Polysorbate 60 1.3 Sorbitastarate 0.5 Hardened vegetable oil 4.0 Mineral oil 5.0 Trioctanoin 2.0 Dimethicone 0.8 Tocopheryl acetate 0.5 Carboxyvinyl polymer 0.12 glycerin 5.0 1,3-butylene glycol 3.0 Sodium hyaruronate 5.0 Triethanolamine 0.12 Unicide - Yu 13 0.02 incense a very small amount Distilled water Balance Sum 100

<1-3> Nourishing cream

Nutritional creams containing the teaspoon juice extract or hexane fraction of the present invention were prepared in a conventional manner at the following proportions.

The nutritional cream formulation of the present invention ingredient Content (% by weight) Sawtooth extract or hexane fraction 1 to 10 Pro-type glycerin monostearate 1.5 Cetearyl alcohol 1.5 Stearic acid 1.0 Polysorbate 60 1.5 Sorbitastarate 0.6 Isostearyl isoserate 5.0 Squalane 5.0 Mineral oil 35.0 Dimethicone 0.5 Hydroxyethylcellulose 0.12 glycerin 6.0 Triethanolamine 0.7 Unicide - Yu 13 0.02 incense a very small amount Distilled water Balance Sum 100

<1-4> Massage Cream

Massage creams containing the teaspoons alcohol extract or hexane fraction of the present invention were prepared in a conventional manner at the following proportions.

Massage Cream Formulation Example of the Present Invention ingredient Content (% by weight) Sawtooth extract or hexane fraction 1-3 Propylene glycol 6 glycerin 4.0 Triethanolamine 0.5 Wax 2.0 Tocopheryl acetate 0.1 Polysorbate 60 3.0 Sorbitan sesquioleate 2.5 Cetearyl alcohol 2.0 Liquid paraffin 30.0 Carboxyvinyl polymer 0.5 incense a very small amount Distilled water Balance Sum 100

<1-5> Pack

A pack containing the teaspoons alcohol extract or hexane fraction of the present invention was prepared in a conventional manner at the following proportions.

Pack Formulation Example of the Present Invention ingredient Content (% by weight) Sawtooth extract or hexane fraction 1-3 glycerin 10.0 Betaine 5.0 REG 1500 2.0 Allantoin 0.1 DL-Panthenol 0.3 EDTA-2Na 0.02 Benzophenone-9 0.04 Hydroxyethylcellulose 0.1 Sodium hyaruronate 80. Carboxyvinyl polymer 0.2 Triethanolamine 0.18 Octyldodecanol 0.3 Octyldodec-16 0.4 ethanol 6.0 Unicide - Yu 13 0.01 incense a very small amount Distilled water Balance Sum 100

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (5)

Which comprises as an active ingredient,
The toothed mugwort extract comprises: a) natural drying of the sawtooth moth in sunny place, followed by grinding and pulverizing; b) adding the alcohol to the powder, refluxing and cooling at 70 ° C, and removing foreign matter through an ultrafilter using a 50 kDa filtration membrane; And c) removing the alcohol from the filtrate extract obtained through step b). &Lt; Desc / Clms Page number 20 &gt; A cosmetic composition for improving wrinkles comprising hexane fraction of alcohol extract of teaspoon as an active ingredient. delete 3. The method according to claim 1 or 2,
The cosmetic composition for improving wrinkles is characterized in that the toothed mackerel extract or its hexane fraction has MMP-1 expression inhibiting activity. The method according to claim 1, 2, or 4,
The cosmetic composition may be an oil in an aqueous phase using a softening lotion, a gel, a water-soluble liquid, a milk lotion, a nutritional cream, a massage cream, an essence, an oil in water emulsion, a water in oil emulsion, a facer anhydrous product, Lipid, lip gloss, face powder, lipstick, lipstick, lipstick, lipstick, lipstick, lipstick, lipstick, lipstick, lipstick, lipstick, , Two-way cake, eye shadow, mascara, cheek color, eyebrow pencil, and the like.

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