KR20170030834A - External composition for antiaging comprising chelidonic acid - Google Patents

Abstract

The present invention relates to a composition for external application for skin comprising kelidonic acid as an active ingredient, and more specifically to a composition for preventing skin aging, antioxidation or anti-inflammation, which comprises kelidon acid as an active ingredient. The composition for external application for skin, which comprises the active ingredient of Kelvidonic acid according to the present invention, promotes hyaluronic acid production and expression of synthetic enzymes, thereby promoting skin elasticity and skin moisturization, promoting collagen synthesis in skin fibroblasts, Inhibits the activity of the skin, suppresses the wrinkles of the skin, promotes regeneration of the skin, abolishes the free radicals to exhibit antioxidative effect, inhibits NO production involved in the inflammatory reaction and is excellent in anti-inflammatory effect, ≪ / RTI >

Description

[0001] The present invention relates to an external composition for preventing skin aging, which comprises kelidonic acid,

The present invention relates to a composition for external application for skin comprising kelidonic acid as an active ingredient, and more specifically to a composition for preventing skin aging, antioxidation or anti-inflammation, which comprises kelidon acid as an active ingredient.

Hyaluronic acid is a kind of glycosaminoglycans, and is a chain-like polymer polysaccharide substance in which glucuronic acid and N-acetylglucosamine residues are repeatedly connected. It has high viscosity and elasticity because it has a property of making gel by combining with a large amount of water. Hyaluronic acid is a major component of the extracellular matrix and has been reported to be involved not only in the retention of water content, intercellular spacing, storage and spread of cell growth factors and nutrients, but also in cell division, differentiation and migration have.

In skin, more than 50% of the hyaluronic acid present in the mammalian body has been reported to be distributed in the skin, especially intercellular spaces of the epidermis and the connective tissue of the dermis. Such hyaluronic acid is mainly synthesized by keratinocytes and fibroblasts . The amount of hyaluronic acid in human skin has been reported to decrease with aging, and the reduction of hyaluronic acid in the skin is considered to be one of the direct causes of decreased skin elasticity or moisture content due to aging (Biochem Biophys Acta 279, 265-275 ; Carbohydr Res 159, 127-136; Int J Dermatol 33, 119-122). It is also known that hyaluronic acid is involved in maintaining the structure of the stratum corneum and maintaining skin barrier function (J Cosmet Dermatol. 2007 Jun, 6 (2), 75-82).

However, the hyaluronic acid having the above effect has a large molecular weight and is not well absorbed into the skin. In addition, although a method of injecting hyaluronic acid into the skin is currently performed, A method of increasing hyaluronic acid synthesis in skin cells is more effective. Therefore, studies on a method for increasing the production of hyaluronic acid in the human body are actively conducted, but the remarkable results of the study are not yet known.

On the other hand, collagen, which is a major component of the extracellular matrix, is a major substrate protein produced in the fibroblasts of the skin and exists in extracellular epilepsy. It is also an important protein that accounts for about 30% of the total weight of bioproteins, and has a solid triple helix structure. Collagen forms most of the organic matter in the skin, tendons, bones and teeth, especially in the bones and skin (dermis). In most other structures, it exists as a fibrous inclusion.

The functions of collagen present in the skin are known as mechanical rigidity of the skin, resistance of connective tissues and binding force of tissues, support of cell adhesion, induction of cell division and differentiation (when organism grows or healing) (van der Rest et al., 1990). Such collagen is related to regeneration of skin, elasticity of skin, prevention of aging of skin, wound healing, moisturization, and particularly, it is reduced by aging and photoaging by ultraviolet irradiation, and it is known to be closely related to wrinkling of skin (Arthur K "Aging and the Skin", 1989), and recent advances in extensive research on skin aging have revealed important functions of collagen in the skin.

In the past, products that combine collagen in cosmetics have been released from the improvement of skin wrinkles and moisturizing effects of collagen. However, these products apply collagen to the surface of skin, and collagen, which is a polymer, is hardly absorbed percutaneously, There was no.

There are active ingredients known to promote the synthesis of collagen and thus to improve wrinkles. In the past, retinoic acid, proteins derived from animal placenta (Japanese Patent Publication No. 8-231370), betulinic acid (Japanese Patent Publication No. 8-208424), chlorella extract Japanese Patent Publication No. 9-40523, Japanese Patent Application Laid-Open No. 10-36283, fibroblast proliferation promoting action) and the like have been known. However, since the problem of safety such as irritation and redness is applied to the skin, It is impossible to expect an effect of improving skin function by promoting collagen synthesis of the skin. Therefore, it is urgently required to develop a new collagen expression promoter which is safe for living body and is more effective than the existing collagen expression promoting substance.

On the other hand, active oxygen introduced from the outside of the living body or generated in the living body causes many problems such as promoting aging of the living body, cancer, and the like. Therefore, the development and research of antioxidants that inhibit oxidation by active oxygen have been performed. Antioxidants are widely distributed in copper and plants, and many phenolic compounds, flavonoids, tocopherols, vitamin C, and selenium are known in fruits and vegetables. However, the antioxidant substances present in nature can not be expected to have practically sufficient effects in skin application. Therefore, although synthetic antioxidants having excellent antioxidant ability and low cost are widely used, their use is restricted due to safety concerns such as human side effects.

In addition, inflammation is an immune response of a human body in response to a wound or disease, and oxidative stress such as ultraviolet rays, active oxygen, free radicals, etc. activate inflammatory factors and cause aging of various diseases and skin. The vasoactive polypeptide, kinin, plasmin and complement, have vasodilatory and contraction and chemotaxis effects, as well as lymphokines such as interleukin-6 (IL-6) Phosphorus and arachidonic acid are responsible for inflammation. Arachidonic acid is metabolized through inflammatory mediators such as prostaglandin and lukotrienes via two pathways: cyclooxygenase or lipooxygenase, mediating a variety of inflammatory responses.

On the other hand, in order to eliminate inflammation, elimination of inflammation source, reduction of vital reaction and symptoms is called anti-inflammatory. To date, substances used for antiinflammatory purposes have been classified as nonsteroidal agents such as flufenamic acid, ibuprofen, benzydamine, or indomethacin, and steroids such as prednisolone ) Or dexamethasone. In addition, it is known that allantoin, azene, or hydrocortisone is effective for anti-inflammation. However, these substances are limited in the usage amount due to the safety of skin, or the effect is insignificant, There is a problem.

Therefore, development of a functional ingredient which is safe in the living body, stable in the active ingredient, and most effective in the skin elasticity, skin moisturizing, skin aging prevention, skin regeneration, skin wrinkle improvement, antioxidant, or anti- This is desperately desired.

A problem to be solved by the present invention is to provide a composition for external application for skin for preventing skin aging comprising kelidon acid as an active ingredient.

Another object of the present invention is to provide an antioxidant or antiinflammatory composition for external application for skin comprising Kelvidonic acid as an active ingredient.

In order to achieve the above object, the present invention provides a skin external composition for preventing skin aging comprising chelidonic acid represented by the following formula (1) as an active ingredient. The term "skin aging prevention" of the present invention means improvement of skin elasticity, skin moisturization, promotion of skin regeneration, or improvement of skin wrinkles. The present invention also provides an antioxidant or antiinflammatory composition for external application for skin comprising, as an active ingredient, a chelidonate represented by the following formula (1).

[Chemical Formula 1]

Hereinafter, the configuration of the present invention will be described in detail.

In order to exert an excellent effect when the skin condition improving ingredient is applied to the actual skin, it exhibits a highly active skin condition improving activity at a low concentration and should be excellent in the ability to permeate and absorb the skin. In addition, it is preferable that it is low in volatility so as to be able to stay for a sufficient time to exhibit a skin condition improving effect, the active ingredient remains stable on the composition or skin, is easy to be formulated into cosmetics, and is safe for skin.

However, among the known components, components that satisfy all of the above characteristics are not common. For example, some skin condition-improving ingredients have excellent skin-improving activity even at low concentrations in in vitro experiments, but they are difficult to apply to real skin because of their ability to permeate through the skin. Other functional ingredients are poor in hydrophilicity and are difficult to formulate into cosmetics, or when they are exposed to heat, light, or oxygen, the active ingredient may be degraded or transformed into other compounds and the effect may already be lost before application to the skin.

As a result of intensive researches to overcome the problems of the prior art, the present inventors have found that a composition for external application for skin comprising chelidonic acid promotes the production of hyaluronic acid in keratinocyte, and hyaluronic acid synthase , HAS) and increase the synthesis of hyaluronic acid in the body. It also promotes collagen synthesis by inhibiting the collagenase activity of fibroblasts of the skin, promotes skin regeneration, improves wrinkles, inhibits free radicals and inhibits the production of antioxidative and NO (nitrogen monoxide) The present invention has been accomplished on the basis of these findings.

The chelidonic acid used in the present invention is Chelidonium There is majus . asiaticum ) extract.

As can be seen in the following examples, the chelidonate shows remarkably excellent hyaluronic acid production promoting effect, collagen production promoting effect, antioxidative effect, or anti-inflammatory effect at a low concentration. Therefore, the kelidon acid can be used as an active ingredient of a skin external composition for skin anti-aging effect and antioxidant or anti-inflammatory effect such as skin elasticity improvement, skin moisturizing, skin regeneration and skin wrinkle improvement effect.

The present invention provides a skin external composition for preventing skin aging comprising kelidon acid as an active ingredient. The skin external composition for preventing skin aging may be used for improving skin elasticity, for moisturizing skin, for promoting skin regeneration, for improving skin wrinkles, but is not limited thereto. In addition, the composition for external application for skin has excellent skin regeneration ability and can be used for wound healing of skin surface.

The term "skin aging" of the present invention refers to the appearance of symptoms such as reduction of elasticity, reduction of gloss, wrinkle formation, weakening of regeneration ability or severe drying of skin, and may be caused by time or external environment, and hyaluronic acid or collagen Decrease may cause skin elasticity to decrease or wrinkles of skin to be produced, which can be caused by reduction of hyaluronic acid or collagen.

The term "skin aging prevention" of the present invention means prevention or improvement of skin aging. The term "prophylactic " means any action that inhibits or delays skin aging or skin wrinkles upon administration of the compositions of the present invention. Further, the term " improvement "means any action in which skin elasticity or skin wrinkle is improved or changed by administration of the composition of the present invention, thereby inhibiting skin aging.

The term "skin elasticity" of the present invention is represented by elastic fibers composed of elastin existing in the dermal layer. Such elastic fibers have a very low elastic modulus such as rubber and are easily deformed by a small force, When the force is removed, it easily returns to the original shape. When the amount of hyaluronic acid between the elastin and the collagen is decreased, the skin elasticity is lowered.

The term "skin moisturizing" of the present invention refers to maintaining the homeostasis of a living body by appropriately adjusting water loss (moisture evaporation) and the like. When hyaluronic acid or collagen in the skin decreases, moisture is lost, hyaluronic acid or collagen If it is increased, moisture may increase.

The term "skin regeneration effect" of the present invention refers to the recovery of skin tissue against damage caused by external or internal causes of the skin. Damage due to external causes may include ultraviolet rays, external contaminants, wound, trauma, etc. The damage caused by the internal causes may be stress.

The term "skin wrinkles" of the present invention refers to a scarring caused by skin degeneration, which may be caused by a cause of a gene, reduction of collagen present in the skin dermis, external environment, etc., and hyaluronic acid between elastin and collagen is reduced Skin wrinkles are created.

The skin external composition for skin anti-aging composition comprising the present invention as an active ingredient is characterized in promoting the production of hyaluronic acid. In addition, the composition is characterized in that the expression of hyaluronic acid synthase (HAS) is increased.

The present invention is based on the finding that It is based on the fact that expression of hyaluronic acid protein can be increased in human keratinocytes. In the present invention, experiments were conducted to investigate the effect of promoting the production of hyaluronic acid in kelidonate, and as shown in Examples 2 to 4 of the present invention, the concentration of hyaluronic acid was increased when treating with kelidonate, The expression of hyaluronic acid protein was increased by gene expression of the enzyme.

The term "hyaluronic acid" according to the present invention is a kind of glycosaminoglycans, and refers to a chain-like polymer polysaccharide substance in which glucuronic acid and N-acetylglucosamine residues are repeatedly connected.

In addition, the composition for external application for skin for preventing skin aging comprising kelidonic acid of the present invention as an active ingredient is characterized by promoting the production of collagen. In addition, the composition is characterized by promoting collagen production of dermal fibroblasts and inhibiting collagenase activity, thereby enhancing the amount of collagen synthesis in the body.

In the present invention, an experiment was conducted to examine the effect of promoting collagen production of kelidon acid. As shown in Examples 5 and 6 of the present invention, when kelidic acid was treated, the amount of collagen expression increased and the production of collagenase was inhibited Respectively.

The present invention also provides an antioxidant composition for external application for skin comprising kelidon acid as an active ingredient. The composition for external application for skin for antioxidation is characterized by exhibiting an antioxidative effect by inhibiting radical formation. The composition for external application for skin for antioxidation can effectively remove active oxygen generated from outside the living body or generated in living body, and thus can be used for skin anti-aging or anticancer.

In the present invention, an experiment was conducted to investigate the effect of inhibiting the radical formation of kelidonate. As shown in Example 7 of the present invention, the kallidic acid treatment showed higher radical scavenging activity than ascorbic acid, Excellent.

In addition, the present invention provides a composition for external application for skin for anti-inflammation, which comprises kelidon acid as an active ingredient. The composition for external application for skin for anti-inflammation is characterized by exhibiting an anti-inflammatory effect by inhibiting the formation of NO (Nitrogen monoxide). The composition for external application for skin for antiinflammation may be used, for example, for skin troubles.

In the present invention, an experiment was conducted to investigate the inhibitory effect of kelidonate on NO production. As shown in Example 8 of the present invention, when kelidic acid was treated, the effect of suppressing NO production was excellent, It was confirmed that there was a remarkable effect.

The term "antioxidant" effect of the present invention inhibits the oxidation of cells with highly reactive free radicals or reactive oxygen species (ROS) according to the oxidative stress caused by intracellular metabolism or ultraviolet rays. And includes removal of free radicals or reactive oxygen species, thereby reducing damage to the cells.

The term "anti-inflammatory" effect of the present invention refers to inhibition of inflammation. The inflammation is one of biological tissue defense responses to a certain stimulus, and includes complex lesions involving tissue degeneration, circulatory disorders and exudates, and tissue proliferation. It says. More specifically, inflammation is part of congenital immunity and, like in other animals, human congenital immunity recognizes a pattern of cell surfaces that are specifically present in a pathogen. Phagocytes recognize cells with such surfaces as non-magnetic and attack pathogens. If pathogens break through the physical barriers of the body, an inflammatory reaction occurs. Inflammation is a nonspecific defense that creates hostile environments for microorganisms entering the wound. In the inflammatory response, white blood cells that are responsible for the early stage of immune response, when wounded or infected, enter the body to express cytokines. Therefore, the expression level of intracellular cytokine is an index of inflammatory response activation. Examples of skin diseases associated with inflammation include atopic dermatitis, psoriasis, radiation, chemical substances, inflammatory diseases caused by burns, acid burns, vesicular dermatosis, visceral type diseases, itching due to allergies, seborrheic eczema, rose acne, Inflammatory hair loss such as pemphigus vulgaris, polymorphous exudative erythema, erythema nodosum, erythematosus, pharyngitis, alopecia areata, skin T-cell lymphoma, and the like.

The composition for preventing skin aging, antioxidation or anti-inflammation according to the present invention can be used as a cosmetic composition for improving skin condition or as a pharmaceutical composition for preventing or treating skin diseases.

In the present invention, the composition for external application for skin comprises 0.001 to 10% by weight, preferably 0.0001 to 7% by weight, more preferably 0.001 to 5% by weight, and most preferably 0.01 to 3% by weight, %. When the concentration of the chelidonate is less than 0.00001% by weight based on the total weight of the composition for external application for skin, it is difficult to obtain the effect. When the concentration exceeds 10% by weight, there is a problem in formulation stability.

The chelidonate of the present invention increases protein expression of hyaluronic acid in the skin to promote the production, increases the production of collagen, has anti-inflammatory and antioxidative effects, and thus can be effectively used as an effective ingredient of various external preparations for skin. When the present invention is used as a composition for external application for skin, it can be used in any form that can be applied to a skin such as liquid, oil, cream, ointment, stick, pack, pasta or powder.

More specifically, the formulation of the composition for external application for skin is a solution, an external ointment, a cream, a foam, a nutritional lotion, a soft lotion, a perfume, a pack, a soft water, a latex, a makeup base, an essence, Or a powder selected from the group consisting of sunflow oil, suspension, emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing oil, powder foundation, emulsion foundation, wax foundation, patch, Can be used.

The composition for external application for skin containing chelidonate according to the present invention may contain cosmetic or pharmacologically acceptable carriers, diluents, adjuvants, coloring agents, stabilizers, stabilizers and the like to facilitate the use and handling, , A flavoring agent, a surfactant, an oil, a humectant, an alcohol, a thickening agent, an antioxidant, a pH adjusting agent, or an ultraviolet screening agent.

The composition for external application for skin, which comprises the active ingredient of Kelvidonic acid according to the present invention, promotes the production of hyaluronic acid and the expression of synthetic enzymes, thereby promoting skin elasticity and skin moisturization, promoting collagen synthesis in skin fibroblasts, Inhibits the activity of the skin, suppresses the wrinkles of the skin, promotes regeneration of the skin, abolishes the free radicals to exhibit antioxidative effect, inhibits NO production involved in the inflammatory reaction and is excellent in anti-inflammatory effect, ≪ / RTI >

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Preparation of materials

The chelidonosaccharide used in the following examples was purchased from Shaanxi Shipyard Biological Co., Ltd. in China.

Example  One: Kelly's  Cytotoxicity Assessment

Cell viability of human keratinocytes (HaCaT) was measured by Cell Counting Kit-8 (Dojindo Lab, USA) for the evaluation of cytotoxicity of Keliddon.

First, a uniform number of cells were plated on a 96-well plate, stabilized, treated with 0.5 or 1 ppm of Kelidonic acid, and further incubated for 24 hours. After that, CCK-8 was treated and the absorbance was measured at 450 nm for 2 hours. The relative cell viability of cells treated with chelidonate versus no treatment (control) was analyzed as follows, and the results are shown in Table 1 below.

sample Untreated group Glucosamine hydrochloride
1000 ppm Kelly Donsan
0.5 ppm Kelly Donsan
1 ppm Survival rate (%) 100 96 99 95

* Number of iterations n = 3

* Relative cell survival rate = (extract treated group) / (untreated group) X 100

As shown in Table 1, it was found that the number of cells was not significantly changed by treatment with the kelidonate. This means that the chelidonate does not cause cytotoxicity.

Example  2: Enzyme-linked immunosorbent assay (ELISA) was used in human keratinocytes To Kelly Donsan  Increased hyaluronic acid concentration by

Human keratinocytes were cultured in DMEM medium containing 10% fetal bovine serum at a concentration of 1 × 10 ⁵ cells / ml and 500 μl / well in a 24-well plate. After incubation for 18 hours, the serum was not contained Washed twice with non-DMEM medium, and added with Kelvidonic acid (0.5 and 1 ppm) by concentration. Negative controls were treated with the same amount of ethanol and treated with 100 ppm of glucosamine hydrochloride (Sigma-aldrich), a positive control known to promote hyaluronic acid production. After culturing for 24 hours, cell culture medium was recovered and the concentration of hyaluronic acid was measured using a Hyaluronan ELISA kit (R & D Systems).

sample No treatment
(Negative control) Glucosamine hydrochloride
100 ppm Kelly Donsan
0.5 ppm Kelly Donsan
1 ppm Hyaluronic acid
Relative concentration (%) 100 136 152 168

* Number of iterations n = 3

* The concentration of hyaluronic acid in the test group treated with the chelidonate was quantified based on the hyaluronic acid concentration of the negative control group (100%).

As a result, the concentration of kelidon acid increased hyaluronic acid production in keratinocytes derived from human body.

Example  3: From keratinocytes derived from human body To Kelly Donsan  Of hyaluronic acid synthase (HAS) gene

Human keratinocytes were cultured in DMEM medium containing 10% fetal bovine serum at a concentration of 1 × 10 ⁶ cells / ml, and 3000 μl of each was dispensed into a 6-well plate for 18 hours. Washed twice with DMEM medium, and treated with 0.5 ppm and 1 ppm of chelidonate for 24 hours. Cells were collected, and total RNA was extracted with RNeasy mini kit (Qiagen), and then 1 μg of RNA was quantified and reverse transcribed using GeneAmp ^® RNA PCR kit (Applied Biosystems). The reverse transcription reaction was performed using a Mycycler ^® PCR instrument (Biorad).

After using the reverse transcription reaction solution 2 ㎕ and HAS-2, HAS-3, Taqman ® probe (Invitrogen, USA, HS02511055_S1, HS03929097_g1) of GAPDH (Glyceraldehyde-3- phosphate dehydrogenase) was performed quantitative real time PCR. Real time PCR was performed using a CFX96 Touch ^TM Real-Time PCR Detection System instrument (Biorad). Experimental results obtained by real time PCR were calculated by the method described in Livak KJ et al. (Methods, 2001, 25, 402-408) based on the intracellular control gene GAPDH.

The mRNA expression level of the test group treated with the chelidonate was quantified by using the amount of the mRNA expression of the negative control as a reference (100%), and is shown in Table 3 below.

sample No treatment
(Negative control) Glucosamine hydrochloride
100 ppm Kelly Donsan
0.5 ppm Kelly Donsan
1 ppm HAS-2
Expression level (%) 100 117 109 125 HAS-3
Expression level (%) 100 123 99 113

* Number of iterations n = 3

As a result, expression of hyaluronic acid synthase, particularly HAS-2 and HAS-3, was increased in human keratinocytes by treatment with kelidonate.

Example  4: From human-derived fibroblasts To Kelly Donsan  Increased hyaluronic acid concentration by

Human Dermal Fibroblast was cultured in DMEM medium containing 10% fetal bovine serum at 1 × 10 ⁵ cells / ml and 500 μl / well in a 24-well plate. After 18 hours of culture, Lt; RTI ID = 0.0 > DMEM < / RTI > Kellydonate was added by concentration (0.5 and 1 ppm). Negative controls were treated with the same amount of ethanol and treated with 100 ppm of glucosamine hydrochloride (Sigma-aldrich), a positive control known to promote hyaluronic acid production. After culturing for 24 hours, the cell culture medium was recovered and the concentration of hyaluronic acid was measured using a Hyaluronan ELISA kit (R & D Systems).

sample No treatment
(Negative control) Glucosamine hydrochloride
100 ppm Kelly Donsan
0.5 ppm Kelly Donsan
1 ppm Hyaluronic acid
Relative concentration (%) 100 163 203 214

* Number of iterations n = 3

From the experimental results shown in Table 4, the kelidon acid increased hyaluronic acid production in human-derived fibroblasts.

Example  5: To Kelly Donsan  Collagen Expression level  increase

The above-mentioned kelidonate was added to a culture solution of human-derived fibroblasts to determine the amount of collagen expression at the cellular level. The expression of collagen was quantitated using a PICP EIA kit (Procollagen Type I C-Peptide Enzyme Immuno Assay, TAKARA Korea).

After culturing for 24 hours at a concentration of 1 ppm and 10 ppm, the culture broth was taken and the degree of collagen expression at each concentration was measured at 450 nm using a spectrophotometer at each concentration using the PICP EIA Kit. The negative control group was untreated and the positive control group was treated with 50 nM vitamin C. The amount of collagen expression was expressed relative to the negative control, and the increase rate was shown in Table 5.

Name of sample Negative control group Positive control group
Vitamin C 50nM Kelly Donsan
1 ppm Kelly Donsan
10 ppm Increase 100% 138% 115% 124%

* Number of iterations n = 3

Example  6: To Kelly Donsan  Collagenase MMP -1 inhibitory effect

In Table 6, the effect of inhibiting the production of collagenase (MMP-1) by the chelidonate was confirmed.

To evaluate the cytotoxicity of human fibroblasts before the experiment in the concentration range of 0.001 to 10 ppm of the test substance (MTT test by culturing fibroblasts) [Reference: Mossman T. (1983). Rapid Colorimetric Assay for Cellular Growth & Survival: application to proliferation & cytotoxicity assays. Journal of Immunological Methods 65, 55-63]. MMP-1 production inhibition assay was performed by selecting concentrations without cytotoxicity.

Human normal skin cells, fibroblasts, were inoculated into 24-well microplates at 2.5 × 10 ⁴ cells per well, cultured in 10% serum DMEM medium and 37 ° C. for 24 hours, and 10% serum DMEM medium was removed The cells were washed once with phosphate buffered saline and cultured for 30 min in serum-free DMEM supplemented with chelidonate and in serum-free DMEM medium containing no chelidonate.

After 30 minutes of sample treatment, the cells were cultured for 24 hours after stimulation with 50 ng / ml TNF-α (tumor necrosis factor-α), a substance known to produce MMP-1. TNF-α-treated group and TNF-α-treated group were not treated with TNF-α and TNF-α-treated group.

The supernatant of each well was collected and the amount (ng / ml) of the newly synthesized MMP-1 was measured using an MMP-1 assay kit (Amersham, USA), and the inhibition rate (%) of MMP-1 production was calculated.

MMP-1 inhibitory effect of kelidon acid (repeated number n = 3) sample The amount of MMP-1 (ng / ml) Inhibition rate (%) Negative control (TNF-alpha no treatment) 6.4 The positive control (TNF-a treatment) 20.5 Kellydonic acid 1 ppm 18.0 17.7% Kellydonic acid 10 ppm 15.6 34.8%

Example  7: To Kelly Donsan  Antioxidant Effect - Free Radical Scavenging Rate

Free radical scavenging activity was measured to confirm the antioxidative activity of kelidonic acid. The free radical scavenging activity was determined by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) method as follows (Blois, MS Nature 181, 1190, 1958). DPPH is a relatively stable free radical, exhibiting maximum absorption at 517 nm in the presence of radicals, and loses its absorbance when cleaved. DPPH was purchased from Sigma, and dissolved in methyl alcohol at a concentration of 0.15 mM. First, 100 쨉 l of ascorbic acid as a positive control and 96 well plate were added to each well. 100 μl of DPPH solution was added thereto, and the mixture was allowed to stand at room temperature for 30 minutes. Then, the absorbance at 517 nm was measured using a microplate reader (BioTek EL-340). At this time, methyl alcohol was used instead of kelidone as a control group. The effect of free radical scavenging activity was determined with the absorbance measured for the experimental group and the control group, and the results are shown in Table 7 below.

Free radical scavenging rate (%) sample Free radical scavenging rate (%) Kelly Donsan 54.2% Ascorbic acid 79.9% Methyl alcohol 6.7%

As can be seen from the results in Table 7, it can be seen that the chelidonate has high antioxidative effect because it shows high activity compared with ascorbic acid which is a known antioxidant.

Example  8: To Kelly Donsan  Anti-inflammatory effect - inhibitory effect on NO production

In order to confirm the anti-inflammatory effect and the skin trouble relieving effect of the kelidon acid, nitric oxide (NO) formation inhibition experiment was performed by the GRIESS method using RAW264.7 cell line (ATCC number: CRL-2278).

Specifically, RAW264.7 cells, macrophages of mice, were subcultured several times, placed in 24-well plates at 3 × 10 ⁵ per well, and cultured for 24 hours. Then, the cell culture medium containing the diluted Keliddon acid was substituted with the concentrations shown in Table 8 below. At this time, L-NMMA (L-NG-Monomethylarginine), which is an inhibitor of NO production, was treated together as a positive control, and cultured for 30 minutes. Lipopolysaccharide (LPS) . 100 μl of the supernatant was transferred to a 96-well plate, and 100 μl of GRIESS solution was added thereto at room temperature for 10 minutes. The absorbance at 540 nm was measured to determine the NO inhibitory effect. The results are shown in Table 8 Respectively.

NO generation inhibitory effect sample NO production inhibition rate (%) L-NMMA (positive control) 40.1 Keldonic acid 0.5 ppm 12.4 Kellydonic acid 1 ppm 22.5 Kellydonic acid 10 ppm 30.8

Example  9: To Kelly Donsan  Increased skin moisture by

In order to apply the skin moisture content increasing effect to the external skin preparation containing kelidic acid to human skin, it was prepared according to the composition of the nutritional cream of Preparation Example 1 below. In a constant temperature and humidity chamber at 22 캜 and 50% relative humidity, (2 mg / ㎠) was applied to the inside of the habock, and the moisture content of the skin was measured before application, one hour after application and two hours after application. In the control group, nutritional cream containing the same amount of ethanol instead of Kellydon was prepared by the same method, and normal skin without any treatment was used as a non - treated group. The water conductivity was measured by using a Corneometer (Courage + Khazaka, GmbH, Germany). The results are shown in Table 9 below.

Units (corneometer units) Production Example 1
Nourishing cream Control group
Nourishing cream Untreated group Before application 42 39 45 After 1 hour of application 77 67 48 After 2 hours of application 74 51 41

* n = 20

As a result, the nutritional cream containing kelidon acid increased the water content of human skin and showed excellent water holding ability even after 2 hours of application.

As a result, it has been confirmed that Kelidonic acid has an excellent effect of increasing skin moisture content and water retention ability, and thus can be useful for improving skin elasticity, preventing aging, preventing wrinkles or treating skin, and moisturizing skin.

Example  10: To Kelly Donsan  Promoting skin elasticity

In order to apply the skin elasticity enhancement effect to the external skin preparation containing kelidic acid to human skin, it was prepared according to the composition of the nutritional cream of Preparation Example 1 below. In a constant temperature and humidity chamber at 22 ° C and a relative humidity of 50% After applying for 4 weeks, the elasticity of the skin before and after application was measured and the skin elasticity improvement effect was compared and evaluated.

The nutritional cream containing Kellydon acid was used on the left side of the subject and the nutrient cream containing the same amount of ethanol instead of Kellydon was used on the right side for 4 weeks after cleansing twice a day in the morning and evening. After that, the degree of improvement in elasticity at each site was measured using a skin elasticity tester (Cutometer, Germany). Ur / Uf was used as a parameter and the results are shown in Table 10.

Production Example 1
Nourishing cream Control group
Nourishing cream Elasticity improvement rate (%) 21.8 5.3

* n = 20

As a result, nutritional creams containing kelidonic acid improved the elasticity of human skin and showed no side effects in the skin.

As a result, it has been confirmed that the effect of improving skin elasticity is excellent, and thus, it can be effectively used for improving skin elasticity, preventing aging, and preventing or treating skin wrinkles.

The production example of the composition according to the present invention will be described in detail based on the above-mentioned effect test results. However, it should be apparent to those skilled in the art that the following preparations are merely examples of the composition of the present invention, and that the composition of the present invention is not limited to the following formulations. Hereinafter, a prescription example of the external preparation for skin containing chelidonate is shown by the production example.

Manufacturing example  1: Manufacture of nutritional cream

Nutritional creams containing the chelidonate as an active ingredient in the composition shown in the following Table 11 were prepared by a conventional method used in the field of cosmetics.

Ingredients Content (% by weight) Kelly Donsan 1.0 Beta-1,3-glucan 5.0 Wax 10.0 Polysorbate 60 1.5 Piggy 60 hardened castor oil 2.0 Sorbitan sesquioleate 0.5 Liquid paraffin 10.0 Squalane 5.0 Caprylic / capric triglyceride 5.0 glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 antiseptic 0.05 Pigment 0.05 Spices 0.05 Purified water To 100

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (8)

A composition for external application for skin for preventing skin aging comprising chelidonic acid as an active ingredient. [2] The composition according to claim 1, wherein the composition is used for improving skin elasticity, moisturizing skin, promoting skin regeneration, or improving skin wrinkles. The composition for external application for skin for preventing skin aging according to claim 1, wherein the composition promotes hyaluronic acid production. 2. The composition for external application for skin against aging according to claim 1, wherein the composition promotes collagen production. A composition for external application for skin for antioxidation comprising chelidonic acid as an active ingredient. A composition for external application for skin for antiinflammation comprising chelidonic acid as an active ingredient. 7. The composition for external application for skin according to any one of claims 1 to 6, wherein the composition comprises 0.001 to 10% by weight of chelidonate based on the total weight of the composition. The composition according to any one of claims 1 to 6, wherein the formulation is a solution, an external ointment, a cream, a foam, a nutritional lotion, a flexible lotion, a perfume, a pack, a soft water, a latex, a makeup base, The compositions of the present invention may be formulated as tablets, pills, powders, creams, bath salts, sunscreen creams, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant- containing cleansers, oils, powder foundations, emulsion foundations, waxes, Wherein the composition is any one selected from the group consisting of: