KR102015857B1 - Cosmetic composition for comprising crosslinked hyaluronic acid and prebiotics - Google Patents

Abstract

The present invention relates to a novel cosmetic composition that significantly increases the effect of conventional crosslinked hyaluronic acid-containing cosmetic composition using the synergistic effect of prebiotics on crosslinked hyaluronic acid. The cosmetic composition according to the present invention is remarkably superior in skin moisturizing, skin whitening, maintaining and improving skin elasticity, preventing skin wrinkles, and ultimately skin anti-aging effect, compared to when crosslinked hyaluronic acid is used alone.

Description

Cosmetic composition comprising cross-linked hyaluronic acid and prebiotics as active ingredient {Cosmetic composition for comprising crosslinked hyaluronic acid and prebiotics}

The present invention relates to a cosmetic composition comprising a crosslinked hyaluronic acid and a prebiotic as an active ingredient, and more particularly, to the effect of a conventional crosslinked hyaluronic acid-containing cosmetic composition using a synergistic effect of the prebiotics to the crosslinked hyaluronic acid. It relates to a new cosmetic composition with a significant increase.

Hyaluronic acid is a linear polymer polysaccharide in which β-D-N-acetylglucosamine and β-D-glucuronic acid are alternately bonded. Hyaluronic acid is distributed in connective tissues such as subcutaneous tissues and cartilage tissues of mammals, and is also present in the vitreous bodies and umbilical cords of eyes, and is also known to exist in streptococci and capillaries of bacillus. As a general method for obtaining hyaluronic acid, there is a method of extracting from chicken crust, umbilical cord, and the like, a method of culturing streptococci and bacilli, and then extracting and purifying them from them.

Natural hyaluronic acid, which has excellent biocompatibility, does not have species-specificity, does not have tissue or organ specificity, enhances skin's moisturizing ability, maintains skin elasticity, reduces damage to the lower part of the skin during skin damage, and collagen, a major component of skin cells, It also acts as a lubricating oil to facilitate the movement between. In addition, hyaluronic acid is known to induce the activation of skin cells and to help cell regeneration.

Hyaluronic acid has a weight average molecular weight different depending on the length of the oligosaccharide, and there is a difference in the skin area that acts according to the weight average molecular weight. In other words, the smaller the weight average molecular weight, the more easily penetrates the skin, hyaluronic acid having a small weight average molecular weight acts on the dermal layer, hyaluronic acid having a large weight average molecular weight acts in the stratum corneum, giving the skin moisturizing and regeneration.

On the other hand, if the natural hyaluronic acid is used as it is, the mechanical properties are not good as well, since it is easily decomposed and removed by an enzyme called hyaluronidase present in the body, there are limitations in various applications.

In order to make up for the shortcomings of such natural hyaluronic acid, efforts have been widely made to develop structurally stable hyaluronic acid derivatives (Laurent, TC "The chemistry, biology and medical applications of hyaluronan and its derivatives" Portland Press). Ltd., London, 1998).

Such hyaluronic acid derivatives are being developed for various purposes such as anti-adhesion agents after surgery, wrinkle improvers, molding aids, joint function improvers, drug carriers, and cell culture scaffolds. Especially, wrinkle improvers and molding aids are active for commercial purposes. Research is being done (F. Manna, M. Dentini, P. Desider, O. De Pita, E. Mortilla, B. Maras, Journal of European Academy of Dermatology and Venereology, 13 (1999) 183-192).

As one of the hyaluronic acid derivatives, hyaluronic acid crosslinked products which covalently bond hyaluronic acids with a crosslinking agent have excellent biocompatibility, physical stability and biodegradation, and are described in US Pat. Nos. 4,716,224, 4,963,666, 5,017,229, 5,356, 883 and the like disclose methods for their preparation.

Complementing the disadvantages of hyaluronic acid with the development of such cross-linked hyaluronic acid, as a cosmetic or transdermal application formulation, Korean Patent Publication No. 10-2018-0003443 "Cosmetic composition containing hyaluronic acids having different weight average molecular weights" and Korea Korean Patent Publication No. 10-2017-0076223 discloses a "hydrogel capsule, a preparation method thereof and a cosmetic composition comprising the same".

However, they have no limitation on the way to enhance the effect of cross-linked hyaluronic acid.

1. Republic of Korea Patent Publication No. 10-2018-0003443 "Cosmetic composition containing hyaluronic acids with different weight average molecular weight" 2. Korean Unexamined Patent Publication No. 10-2017-0076223 "Hydrogel capsules, a preparation method thereof and a cosmetic composition comprising the same"

Therefore, the technical problem to be achieved by the present invention is to provide a new cosmetic composition that significantly increases the effect of the conventional cross-linked hyaluronic acid-containing cosmetic composition using the synergistic effect of prebiotics to cross-linked hyaluronic acid.

In order to achieve the above technical problem, the present invention contains a cross-linked hyaluronic acid and prebiotics as an active ingredient, to provide a skin moisturizing, skin whitening, skin elasticity maintenance and improvement, skin anti-wrinkle cosmetic composition.

In the cosmetic composition according to the present invention as described above, the crosslinking agent of the crosslinked hyaluronic acid is divinyl sulfone, 1,3-butadiene diepoxide, 1,2,7,8-diepoxyoctane, 1,5-hexa Dienedioxide, ethylene glycol diglycidyl ether, bisphenol A diglycidyl ether, divinyl sulfone, epichlorohydrin, 1,4-butanediol diglycidyl ether (BDDE), 1,4-bis (2,3-epoxypropoxy) butane, 1,4-bisglycidyloxybutane, 1,2-bis (2,3-epoxypropoxy) ethylene and 1- (2,3-epoxypropyl) -2 It may be any one or more selected from the group consisting of, 3-epoxycyclohexane.

In the cosmetic composition according to the present invention as described above, the prebiotics are inulin, fructo- oligosaccharides, gluco-oligosaccharides, galacto- oligosaccharides, isomalto- oligosaccharides, xylo- oligosaccharides, mannan- oligosaccharides, betaglucan And lactosurose, lactulose, inulin and combinations thereof.

Cosmetic composition according to the present invention as described above comprises the cross-linked hyaluronic acid and prebiotics 0.001 to 20% by weight based on the total weight of the composition, respectively.

The cosmetic composition according to the present invention as described above further comprises 0.001 to 20% by weight of pure leaf hydrothermal extract based on the total weight of the composition.

The cosmetic composition according to the present invention possesses an advantage that the skin moisturizing, skin whitening, skin elasticity maintenance and improvement, skin wrinkle prevention effect, and ultimately skin anti-aging effect are significantly higher than when crosslinked hyaluronic acid is used alone.

Hereinafter will be described the specific content for practicing the present invention.

In order to achieve the above object, the present invention contains a cross-linked hyaluronic acid and prebiotics as an active ingredient, to provide a skin moisturizing, skin whitening, skin elasticity maintenance and improvement, skin anti-wrinkle effect cosmetic composition.

As the cross-linked hyaluronic acid is well known in the art to which the present invention belongs, the cross-linking reaction of the cross-linking agent and hyaluronic acid, and the step of cutting the cross-linked hyaluronic acid obtained, etc. can be used without particular limitation, Divinylsulfone, 1,3-butadiene diepoxide, 1,2,7,8-diepoxyoctane, 1,5-hexadiene diepoxide, ethylene glycol diglycidyl ether, bisphenol A diglycidyl ether , Divinylsulfone, epichlorhydrin, 1,4-butanediol diglycidyl ether (BDDE), 1,4-bis (2,3-epoxypropoxy) butane, 1,4-bisglycidyl Oxybutane, 1,2-bis (2,3-epoxypropoxy) ethylene and 1- (2,3-epoxypropyl) -2,3-epoxycyclohexane, the weight average of hyaluronic acid The molecular weight, degree of crosslinking, etc. can be determined appropriately according to the properties of the desired product. It is preferable to use divinyl sulfone as a crosslinking agent.

In addition, in the present invention, "crosslinked hyaluronic acid nanoparticles" may be used. For example, P-crosslinked hyaluronic acid nanoparticles may specifically include i) an oil phase in which a surfactant is dissolved and ii) a hyaluronic acid in a basic aqueous solution. And hyaluronic acid hydrogel nanoparticles characterized in that the water-soluble crosslinking agent is a hyaluronic acid hydrogel nanoparticle (PEGDG) crosslinked in a mixed w / o emulsion.

If the cross-linked hyaluronic acid used in the present invention is known to be used for skin moisturizing, skin regeneration, skin whitening, skin elasticity improvement, skin aging inhibition, the weight average molecular weight is not particularly limited, for example, 3,000 ~ One having a wide range of weight average molecular weights of up to 10,000,000 Da can be used.

Prebiotics, which are included as active ingredients in the cosmetic composition according to the present invention, for providing appropriate selective nutrients to such bacterial groups in order to increase the intestinal resistance, colonization ability and reproduction ability of certain bacterial groups called probiotics. As the material used, in chemical terms, the prebiotics material corresponds to carbohydrates and dietary fibers that are both digestible and non-digestible. After ingestion, these substances pass through almost all of the upper gastrointestinal tract intact without any digestive process and, when they reach the colon, provide the major nutritional substrate of healthy / symbiotic bacteria that are supported by them, As these substances are used and digested, they act as nutrient substrates.

Such prebiotics can be used without particular limitation as long as they are well known in the art, for example, glucose, galactose, xylose, maltose, sucrose, lactose, starch, xylan, guar Oligosaccharides produced from gums, gum arabic, dextran, hemicellulose, inulin, or mixtures thereof, more preferably inulin, fructo-oligosaccharides, gluco-oligosaccharides, galacto-oligosaccharides, isomalto-oligosaccharides, At least one selected from xylo-oligosaccharides, mannan-oligosaccharides, betaglucans, lactosurose, lactulose, inulin and combinations thereof.

The most widely available one is inulin, which was first described in the early nineteenth century and is found in many plants, but inulin extracted from chicory is preferred. Adding inulin to the product (which makes it "symbiotic") ensures the presence of nutrient substrates that are essential for the physiological balance of the entire microbial population. When inulin, a non-hydrolysable polysaccharide, is broken down (which can only be caused by bacterial action), it reduces intestinal pH and thus maintains an environment of the colon that is unsuitable for pathogen growth.

The cross-linked hyaluronic acid and prebiotics as described above are preferably included in an amount of 0.001 to 20% by weight based on the total weight of the composition, respectively, if less than the above, the effect can be exerted. It is also undesirable because it may affect the stability of the formulation.

The composition according to the present invention may further include a hydrothermal extract of chaekchae leaves to further increase the effect of cross-linked hyaluronic acid and prebiotics, preferably the composition according to the present invention is pure chaek leaf hydrothermal extract with respect to the total weight of the composition It contains 0.001 to 20% by weight.

Perennial pure vegetable is a perennial plant of the dicotyledon plant of the genus Amanaceae , its scientific name is Brasenia. As a schreberi , it grows in ponds, but in olden times it was cultivated in paddy fields to eat leaves and shoots. The stems grow sideways and grow to 50-100cm long and the leaves float on the surface. Leaves are alternate, oval, purple on the back, petioles in the center. When the leaves grow, they are surrounded by umbilical slime with young stems. Flowers bloom in May-August, grow in black reddish purple, and grow one by one at the end of long peduncle, which is about 2cm in diameter. Calyxes and three petals. Pistils are 6-18, with nipple-shaped projections, and many surgeries. Surgery encloses pistils and is longer than pistils and the central pistil is bent outward. After surgery, the pistil grows, causing sticky mucus on the head.

Cosmetic compositions in the present invention as described above may include components commonly used in cosmetic compositions, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers. Include.

Such cosmetic compositions may be prepared in any formulations commonly prepared in the art. In one embodiment, the formulation may be any one selected from the group consisting of a high viscosity emulsifier type, a low viscosity emulsifier type, and a solubilized formulation, but is not limited thereto. Depending on the formulation to be prepared may comprise a cosmetic composition blending components commonly used in the art. Examples include solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, packs, massage creams and sprays, etc. It may be, but is not limited thereto. More specifically, flexible lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, sun lotion, sun cream, makeup base, BB cream, cleansing cream, cleansing foam, cleansing water, pack, stick shape product, balm ( It can be prepared in the form of Balm) type products, sprays or powders.

Depending on the formulation to be prepared, water phase components, oil phase components, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, preservatives, fragrances and the like may be selected and added.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples. It is obvious that the present invention is not limited by the description of the above-described embodiments.

< Example  1: Preparation of the composition according to the present invention>

A composition according to the present invention was prepared by mixing a crosslinked hyaluronic acid having a weight average molecular weight of 10,000 to 30,000 Da with an inulin prepared by extraction from chicory with prebiotics in a 1: 1 weight ratio, using a vinyl sulfone as a crosslinking agent. It was.

< Example  2: preparation of a composition according to the invention>

A composition according to the present invention was prepared in the same manner as in Example 1 except that fructo-oligosaccharide was used as a prebiotic.

< Example  3: preparation of a composition according to the invention>

1,2,7,8-diepoxyoctane as a crosslinking agent, mixed with a crosslinked hyaluronic acid having a weight average molecular weight of 10,000 ~ 30,000 Da and inulin prepared by extraction from chicory with prebiotics in a 1: 1 weight ratio To prepare a composition according to the invention.

< Example  4: preparation of a composition according to the invention>

A composition according to the present invention was prepared in the same manner as in Example 3 except that fructo-oligosaccharide was used as a prebiotic.

< Example  5: Preparation of the composition according to the present invention>

After grinding the dried hot chaebol leaves with moisture content less than 5% to 20mesh size, 1000 parts by weight of purified water was added to 100 parts by weight of copper sprouts, heated to 95 ° C for 3 hours to extract hot water, and filtered to produce pure water leaf extract. The composition of Example 1 was further prepared by including the same weight as inulin in the composition of Example 1.

< Comparative example  Composition>

As a comparative example, the following materials were used.

Comparative Example 1: Crosslinked hyaluronic acid prepared by using divinyl sulfone as a crosslinking agent and having a weight average molecular weight of 10,000 to 30,000 Da

Comparative Example 2: Crosslinked hyaluronic acid prepared using 1,2,7,8-diepoxyoctane as a crosslinking agent and having a weight average molecular weight of 10,000 to 30,000 Da

Comparative Example 3; Inulin

Comparative Example 4: Fructo-Oligosaccharide

< Test Example  1: moisturize the skin>

1. Cytotoxicity Experiment

Cytotoxicity testing was performed using MTT (3-4) using Moseman's method (Moseman TJ, Immuno Methods, 65, 55 (1983)) and Skaper et al. (Skaper SD, et al, Cell Culture, 2, 17 (1990)). , 5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide) was used.

Human keratinocyte HaCaT was used by the Korea Cell Line Bank (Seoul, Korea). HaCaT was incubated in an animal cell incubator with 37 ° C., 5% CO 2 feed in the laboratory using DMEM medium containing 10% (V / V) FBS, 100 U / mL of penicillin and 100 g / mL of streptomycin.

Example and Comparative Example Compositions were dissolved in DMSO and added to the culture at 100 g / mL, and cells were cultured for 24 hours in the culture. After 4 hours of incubation with MTT solution (5 mg / mL), the amount of blue crystals, formazan, produced was measured at 595 nm using an ELISA reader. Toxicity to cells is expressed as a percentage of the mean absorbance value of each control.

When treated at 100 g / mL concentration to investigate cytotoxicity in HaCaT keratinocytes, cytotoxicity was investigated by calculating the degree of change in cell proliferation compared to the control group treated with DMSO only.

In all results, the cell viability was 95-98%, indicating that both the Example and Comparative Example compositions had no cytotoxicity.

2. Moisture when applied to human body Capacity  evaluation

In order to evaluate the skin moisturizing effect on the compositions of Examples and Comparative Examples, the moisture retention capacity was measured as follows on the skin to which each test substance of Examples and Comparative Examples were applied. Test substance was dissolved in 15% by weight in purified water.

Moisture retention capacity was measured by applying a certain amount (0.1 g / 4 cm 2) to the inside of 20 test subjects in a constant temperature and humidity room with a room temperature of 22 ° C. and a relative humidity of 45%, and before application, after 1 hour and after 2 hours of application. The moisture content of was measured. The measuring device is a moisture content measuring device (corneometer CM820, Courage + Khazaka, Cologne, Germany) to measure the moisturizing power by measuring the electrical capacity of the skin according to the moisture content of the skin is shown in Table 1 below.

Electrical conductivity Before application 1 hour after application 2 hours after application Example 1 50 83 77 Example 2 50 82 78 Example 3 50 84 78 Example 4 50 83 77 Example 5 50 88 83 Comparative Example 1 50 64 57 Comparative Example 2 50 63 57 Comparative Example 3 50 56 51 Comparative Example 4 50 56 51

From the Table 1, the composition according to the present invention was able to confirm a remarkably excellent water retention capacity compared to the composition of the comparative example, which proves to be a material having a very excellent moisturizing effect. In particular, the superior effect compared to Comparative Examples 1 and 2 is due to the synergistic effect on the cross-linked hyaluronic acid by the prebiotics and pure leaf hot water extract.

3. Aquaporin -3 and Involklin  Synthesis promoting effect experiment

In order to confirm the effect of increasing the synthesis of involuclin, which plays an important role in skin cell barrier and moisturizing function, and aquaporin-3, which plays an important role in water migration, the cells of Examples and Comparative Examples were prepared as follows. After each treatment, it was confirmed by western blotting.

First, 24 hours after inoculation of human keratinocytes HaCaT, Example and Comparative Example compositions were added, and cultured in DMEM medium, 37 ° C, and 5% CO2. After incubation for 24 hours, the cells were recovered and crushed to extract only proteins and stored at -40 ° C. The extracted protein was quantified by BCA saasy. Each sample was diluted with 30 g of protein, electrophoresed in 12% SDS-gel, and then transferred to PVDF membrane. After reacting with the primary antibody (rabbit anti-aquaporins-3 antibody), the remaining antibody was washed and removed and reacted with the secondary antibody (rabbit HRP-coupled anti-IgG antibody).

After removing the remaining antibody was added a reagent (peroxidase substrate and chromogen solution) that reacts with the antibody to generate a color reaction. The developed bands were measured and quantified through image analysis (BioRad, Universal Hood, USA) and the results are shown in Tables 2 and 3 below.

division Expression level of aquaporin-3 (%) Control 100 Example 1 148 Example 2 149 Example 3 148 Example 4 149 Example 5 157 Comparative Example 1 123 Comparative Example 2 122 Comparative Example 3 105 Comparative Example 4 103

As shown in Table 2, Comparative Examples 3 and 4 using only prebiotics showed little effect compared to the control group, and Comparative Examples 1 and 2 using only crosslinked hyaluronic acid showed a 20% increase effect compared to the control group. In Examples 1 to 5 according to the present invention showed a synergistic effect of about 50% showed a more significant effect than Comparative Examples 1 and 2.

division Expression level of involuclin (%) Control 100 Example 1 149 Example 2 149 Example 3 148 Example 4 149 Example 5 155 Comparative Example 1 121 Comparative Example 2 120 Comparative Example 3 102 Comparative Example 4 101

 When treated with HaCaT keratinocytes, the expression of involuclin, which plays an important role in skin barrier function and moisturization, showed little effect compared to the control examples 3 and 4 using only prebiotics as shown in Table 3 above. , Comparative Examples 1 and 2 using only cross-linked hyaluro acid showed an effect of about 20% increase compared to the control group, Examples 1 to 5 according to the present invention showed a synergistic effect of about 50% than Comparative Examples 1 and 2 The effect was remarkable.

Through the above experimental results, the composition of the present invention could confirm the excellent effect on the skin barrier function and moisturizing.

< Test Example  2: skin Mimac >

1. Confirmation of melanin production inhibitory effect

The compositions of Examples and Comparative Examples were added to the culture solution of B-16 mouse melanoma cells to test the whitening effect at the cellular level. (Lotan R, Lotan D Cancer Res 40: 3345-3350, 1980)

Before the experiment, rat melanoma cells were assessed for toxicity, and non-toxic concentrations were selected for whitening evaluation.

The final concentration was tested to 100 ㎍ / ml, and the control arbutin was added to the medium to 100 ㎍ / ml and treated with B-16 melanoma cells, respectively, and incubated for 3 days.

Then, cells were trypsin-treated (trypsin) was removed from the culture vessel and centrifuged to extract melanin. The detached cells were added with 1 ml of sodium hydroxide solution (1 N concentration), boiled for 10 minutes to dissolve the melanin, and the absorbance was measured at 400 nanometers (nm) using a spectrophotometer to measure the amount of melanin produced.

The melanin amount was measured by the absorbance of the unit cell count (10 ⁶ cells), the relative melanin production relative to the control group was calculated as the inhibition rate (%) and the results are summarized in Table 4. In addition, the experiment was repeated three times.

sample Melanin production % Inhibition Negative control 0.065 ± 0.002 - Positive control group 0.033 ± 0.001 49% Example 1 0.015 ± 0.003 77% Example 2 0.015 ± 0.001 77% Example 3 0.015 ± 0.002 77% Example 4 0.015 ± 0.003 77% Example 5 0.010 ± 0.001 85% Comparative Example 1 0.036 ± 0.003 44% Comparative Example 2 0.036 ± 0.003 44% Comparative Example 3 0.059 ± 0.001 10% Comparative Example 4 0.059 ± 0.001 10%

As can be seen from the results of Table 4, the composition of the present invention was found to have a superior melanin production inhibitory ability against the melanoma cells of the cultured mice compared with the known whitening material arbutin, only crosslinked haaluronic acid In Comparative Examples 1 and 2 used, the effect of less than arbutin was shown, while Comparative Examples 3 and 4 using only prebiotics showed insignificant effects of about 10% compared to the negative control, and the superiority of the composition according to the present invention was remarkably revealed. The synergistic effect of the prebiotics and the synergistic effect of the pure water hydrothermal extract on the crosslinked hyaluronic acid were also confirmed.

< Test Example  3: prevent skin wrinkle and maintain elasticity>

One. Elastase  Inhibition rate measurement

The inhibitory effect of Elastase, an enzyme that degrades elastin, was identified as follows.

Elastase was used as elastay derived from human leukocyte cells, and MeOSuc-Ala-Ala-Pro-Val-pNA, which is a synthetic substrate, was used as a substrate of elastays. 100 mM Tris (pH 7.5) solution was used as the buffer. Elastase used 0.2mU finally using a buffer solution. In addition, the synthetic substrate of elastays was diluted with a buffer solution to make a 100mM solution using DMSO to a final concentration of 05mM.

At this time, the positive control was set to put the quercetin (Quercetin) known as an elastase inhibitor at a concentration of 100ppm. Samples of Examples and Comparative Examples were also added to 100 ppm. The reaction was carried out in a 96-well plate, and reacted at room temperature for 20 minutes. Absorption was measured at 405 nm at 1 minute intervals using a spectrophotometer to determine the slope of absorbance versus time to determine the activity of the enzyme. Elastase inhibition rate was calculated as follows and the results are shown in Table 5.

sample Elastase Inhibition Rate (%) Positive control group 43.12% Example 1 77.29% Example 2 77.34% Example 3 77.18% Example 4 77.95% Example 5 85.21% Comparative Example 1 42.18% Comparative Example 2 42.36% Comparative Example 3 9.35% Comparative Example 4 9.38%

As can be seen from the results of Table 5, the composition of the present invention was found to have a very good elastase inhibitory activity, Comparative Examples 1 and 2 using only cross-linked haaluronic acid and Comparative Examples 3 and 4 using only prebiotics The composition of the present invention showed a marked difference, and the superiority of the composition according to the present invention was remarkably revealed. The synergistic effect of the prebiotics on the cross-linked hyaluronic acid and the synergistic effect of the pure hot water extract were also demonstrated.

2. Collagenase  Inhibition rate measurement

The inhibitory effect of collagenase, an enzyme that degrades collagen was confirmed as follows.

Collagenase was used as collagenase derived from mouse interest, and synthetic substrate N- (3- [2-Furyl] acryloyl) -Leu-Gly-Pro-Ala was used as a substrate of collagenase. As a buffer solution, a solution containing 50 mM Tricine, 10 mM CaCl ₂ · H ₂ O, and 400 mM NaCl (pH 7.5) was used. The synthetic substrate was dissolved in a concentration of 10 mM using a buffer solution and finally diluted to 75 μM. Collagenase was dissolved in 1 unit / ml concentration using cold tertiary distilled water at 4 degrees and finally diluted with a buffer solution to 0.05 unit / ml.

Samples in Examples and Comparative were tested at a final concentration of 100 ppm. At this time, the positive control set epigallocatechin gallate (EGCG) known collagenase inhibitory effect to a final concentration of 100ppm. The reaction was carried out in a 96-well plate, and reacted at room temperature for 10 minutes. Absorbance was measured at 345 nm at 1 minute intervals using a spectrophotometer to determine the maximum slope of absorbance over time to determine the activity of the enzyme. Collagenase inhibition rate was calculated as follows and the results are shown in Table 6.

sample Collagenase inhibition rate (%) Positive control group 42.12% Example 1 68.18% Example 2 67.34% Example 3 69.29% Example 4 69.35% Example 5 75.28% Comparative Example 1 39.15% Comparative Example 2 39.18% Comparative Example 3 7.52% Comparative Example 4 8.01%

As can be seen from the results of Table 6, the composition of the present invention was found to have a very good collagenase inhibitory activity, Comparative Examples 1, 2 using only cross-linked haaluronic acid and Comparative Examples 3, 4 using only prebiotics only The composition of the present invention showed a remarkable difference, and the superiority of the composition according to the present invention was remarkably revealed, and the synergistic effect of the prebiotics on the cross-linked hyaluronic acid and the synergistic effect of the pure hot water extract were demonstrated.

3. Measurement of Cell Proliferation Effect

To confirm skin cell proliferation activation, human fibroblasts were used to measure cell proliferation activity.

10 μg / ml of the compositions of Examples and Comparative Examples were added to the culture medium, followed by culturing human fibroblasts to perform the MTT reduction method, extracting the formazan product with isopropyl alcohol, and measuring the absorbance at 570 nm. By comparison, cell proliferation activity was compared. At this time, a culture medium containing no sample was used as the negative control group. The measured results were calculated numerically using the following formula and the results are shown in Table 7 below.

% Cell proliferation = absorbance in each sample group / absorbance in control group

sample Cell proliferation activity (%) Example 1 137.28% Example 2 137.10% Example 3 138.10% Example 4 136.41% Example 5 142.28% Comparative Example 1 112.15% Comparative Example 2 115.18% Comparative Example 3 100.52% Comparative Example 4 100.01%

As can be seen from the results of Table 7, the composition of the present invention showed a very good cell proliferation activity compared to the cell proliferation activity (100%) of the untreated control, Comparative Examples 1, 2 and only using crosslinked haaluronic acid In Comparative Examples 3 and 4 using only prebiotics, the composition of the present invention showed a remarkable difference, and the superiority of the composition according to the present invention was remarkably revealed. Synergy has been demonstrated.

As a result, the cosmetic composition according to the present invention was found to be the best cosmetic composition that can ultimately prevent skin aging due to excellent skin elasticity maintaining and improving effects and skin wrinkle prevention effect.

Taken together, the composition according to the present invention is significantly more effective in moisturizing skin, whitening skin, maintaining and improving skin elasticity, preventing skin wrinkles, and ultimately anti-aging effect than when crosslinked hyaluronic acid is used alone. It has excellent advantages.

Claims (5)

Crosslinked hyaluronic acid,
Inulin and as a prebiotics
Cosmetic composition for activating the barrier and moisturizing function of skin cells containing pure water extract of hot water as an active ingredient.
The crosslinking agent of the crosslinked hyaluronic acid according to claim 1, wherein the crosslinking agent of the crosslinked hyaluronic acid is divinyl sulfone, 1,3-butadiene diepoxide, 1,2,7,8-diepoxyoctane, 1,5-hexadiene diepoxide, ethylene glycol die Glycidyl ether, bisphenol A diglycidyl ether, divinyl sulfone, epichlorohydrin, 1,4-butanediol diglycidyl ether (BDDE), 1,4-bis (2,3-epoxypro Foxy) butane, 1,4-bisglycidyloxybutane, 1,2-bis (2,3-epoxypropoxy) ethylene and 1- (2,3-epoxypropyl) -2,3-epoxycyclohexane Cosmetic composition, characterized in that any one or more selected from the group consisting of.
The cosmetic composition according to claim 1 or 2, wherein the cross-linked hyaluronic acid, prebiotics and pure leaf hydrothermal extract each comprise 0.001 to 20% by weight based on the total weight of the composition.



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