Method for stabilizing ceramide and cosmetic composition containing stabilized ceramide

Abstract

The invention relates to a method for stabilizing ceramide and a cosmetic composition containing the stabilized ceramide, in particular to a method for stabilizing ceramide. The present invention relates to a method for homogenizing and stabilizing ceramide together with other physiologically active substances, namely beta-sitosterol, resveratrol, and allantoin, by self-assembly using a microfluidic chip and a high-pressure homogenizer, and a method for homogenizing and stabilizing a composition containing ceramide and beta-sitosterol prepared by the stabilization method, techniques for using nanoparticles of resveratrol and allantoin as cosmetics for moisturizing skin, resisting oxidation, relieving skin inflammation, enhancing skin barrier, and relieving skin itch.

Description

Technical Field

[0001] The present invention relates to a method for stabilizing ceramide and a cosmetic composition containing the stabilized ceramide, and more particularly to a technology for preparing ceramide together with β-sitosterol, resveratrol and allantoin as physiologically active substances into nanoparticles by applying a low-temperature microfluidic chip technology, stabilizing the ceramide and using the nanoparticles as a cosmetic. Background Art

[0002] Cosmetics have been used to protect the skin and give beauty. However, recently, functional cosmetics that emphasize functionality, such as wrinkle and elasticity improvement, anti-oxidation, whitening, etc., which go beyond simple skin protection and moisturizing, have attracted attention. Therefore, they contain ingredients with various activities, which induce skin problems or inflammation.

[0003] Furthermore, cosmetics containing multiple active ingredients are difficult to maintain stability and have low absorption rates, so that it is difficult to expect sufficient skin-improving activity when using the cosmetics.

[0004] Among these active ingredients, ceramide is one of the sphingolipids having a structure in which sphingosine or phytosphingosine is connected to fatty acids. Ceramide accounts for more than 40% of the intercellular lipids constituting the stratum corneum of the skin, and is a constituent of the double-layer cell membrane and the skin lipid membrane. In addition, ceramide is a component required for the structural formation or functional manifestation of the stratum corneum, and the ceramide present in the human body is divided into multiple types according to the degree of polarity. As an important element of the skin barrier, ceramide has the function of a lipid barrier that inhibits water evaporation and the function of maintaining the neat structure of the stratum corneum, and therefore, it has attracted much attention as a raw material for moisturizing cosmetics. It is known that the amount of the above-mentioned ceramide decreases as the skin ages, thereby reducing the function of the skin barrier and causing a decrease in the amount of water in the skin, an increase in the amount of water loss, and the shedding of the stratum corneum, thereby forming a damaged or unhealthy skin barrier. Such symptoms can be improved by supplying ceramide to the skin, but ceramide is limited in large amounts in cosmetics due to its structural specificity as a poorly soluble substance, and it is not easy to dissolve, and it is difficult to ensure the usability, transparency, and dosage form stability of cosmetics, and is limited in use.

[0005] The present inventors have studied the stability of ceramide in a dosage form and the improvement of skin absorption, and have confirmed that when ceramide, which is an active ingredient for improving the skin barrier and pruritus, is prepared as nanoparticles together with other physiologically active ingredients, such as β-sitosterol, resveratrol and allantoin, using a microfluidic chip, not only the stability of the active ingredient is improved and the skin absorption rate is increased, but also excellent skin improvement activity is exhibited by these enhancement effects, thereby completing the present invention.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent document 0001 (001): Korean Patent Publication No. 10-2016-0117075 (October 10, 2016)

[0009] Patent document 0002 (002): Korean Patent Publication No. 10-2018-0131876 (December 11, 2018) Summary of the invention

[0010] An object of the present invention is to provide a method for stabilizing ceramide.

[0011] Furthermore, another object of the present invention is to provide a cosmetic composition comprising stabilized ceramide, which exhibits excellent effects such as skin moisturizing, anti-oxidation, inflammation relief, skin calming, skin barrier enhancement, and skin itching relief.

[0012] In order to achieve the above object, the present invention provides a method for stabilizing ceramide, comprising: step (A), preparing a freeze-dried powder containing ceramide, resveratrol and β-sitosterol; step (B), comprising: step b1), dissolving the freeze-dried powder in dipropylene glycol (Dipropylene Glycol), and mixing an emulsified polymer to prepare an oil phase; and step b2), mixing purified water, dipropylene glycol, a water-soluble solubilizer and allantoin to prepare an aqueous phase; step (C), allowing the oil phase and the aqueous phase to self-assemble through a microfluidic chip; and step (D), using a high-pressure homogenizer (Microfluidizer) to form nanoparticles.

[0013] Preferably, the above step (A) comprises: step a1), adding ceramide to heated dipropylene glycol and ethanol to mix and dissolve resveratrol and β-sitosterol; step a2), removing precipitates by filtering at room temperature; and step a3), concentrating at a temperature of 40 to 50° C. and freeze-drying the concentrate to prepare a freeze-dried powder containing ceramide, resveratrol and β-sitosterol.

[0014] The emulsifying polymer is characterized in that it is at least one selected from the group consisting of phosphatidyl choline, poly-ε-caprolactone (PCL), polyglycolic acid (PGA), poly-L-lactide (PLLA), sphingomyelin, phosphatidylserine, phosphatidyl inositol, phosphatidylethanolamine and lecithin.

[0015] The ceramide is at least one selected from the group consisting of ceramide NP, ceramide EOP, ceramide NS, ceramide AS, ceramide EOS, and ceramide NDS.

[0016] Preferably, in the step a1), the ceramide, resveratrol and β-sitosterol are added and mixed at a weight ratio of 10:0.5-1.5:0.005-0.02, respectively.

[0017] Preferably, in the step b1), the oil phase is formed by mixing 0.5 to 1 weight percent of freeze-dried powder, 80 to 85 weight percent of dipropylene glycol and 14 to 18 weight percent of an emulsified polymer.

[0018] Preferably, in the above step b2), the aqueous phase is formed by mixing 88-90 weight percent of purified water, 8-10 weight percent of dipropylene glycol, 1-2 weight percent of a water-soluble solubilizer and 0.0005-0.002 weight percent of allantoin.

[0019] Preferably, in the above step (C), at 10-15° C., the oil phase and the water phase are passed through a microfluidic chip having a flow channel of 1 μm to 3 μm at a weight ratio of 1:7 to 12 for 1 to 5 times to prepare self-assembled liposomes.

[0020] The present invention is characterized in that, in the above step (D), the size of the above nanoparticles is 50 to 300 nm.

[0021] In order to achieve the above another object, the present invention provides a ceramide stabilized by the above method, comprising 0.0001 to 50.0 weight percent of the ceramide stabilized by the above method relative to the total weight of the composition.

[0022] The above cosmetic composition is characterized in that it is used for skin moisturizing, anti-oxidation, relieving skin inflammation, strengthening skin barrier or relieving skin itching.

[0023] The above-mentioned cosmetic composition is characterized in that it has dosage forms such as skin care lotion, toner, facial mask, nourishing cream, moisturizing cream, essence, body cream, body lotion, body oil, shampoo, conditioner, hair essence, hair gel, facial cleanser, cleansing lotion, soap, patch, foundation cream, lipstick, and isolation cream.

[0024] Effects of the Invention

[0025] The active stability of the ceramide dosage form is improved by homogenizing ceramide with other physiologically active ingredients such as β-sitosterol, resveratrol and allantoin using a microfluidic chip and a high-pressure homogenizer. The nanoparticles containing ceramide and β-sitosterol, resveratrol and allantoin prepared by the stabilization method of the present invention have a high skin absorption rate, stay in the skin for a long time and stably absorb the active substances, and show excellent moisturizing, anti-oxidation, skin inflammation relief, skin barrier enhancement and skin itching relief effects, so that they can be effectively used as skin improvement cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 4 is a graph showing the results of force analysis of nanoparticles prepared in Examples of the present invention. DETAILED DESCRIPTION

[0027] Hereinafter, the present invention will be described in more detail.

[0028] The technical feature of the present invention is that ceramide as an active ingredient is stabilized into liposomes, and is homogenized together with other active ingredients such as resveratrol, β-sitosterol and allantoin by freeze drying, microfluidic chips and high-pressure homogenizers to improve the stability and skin absorption rate of the active ingredients.

[0029] The advantage of the nanoparticle capsules prepared in this way, which contain ceramide and other physiologically active ingredients such as resveratrol, β-sitosterol and allantoin, is that they have a high skin absorption rate by forming a liposome bilayer structure, and the skin can stably absorb the active substances while staying in the skin for a long time, thereby effectively ensuring the skin improvement effect.

[0030] Resveratrol shows a variety of physiological and pharmacological effects such as strong antibacterial, antiviral, antioxidant, antithrombotic and anti-inflammatory effects. It is thus confirmed that it has cardiovascular protection and contributes to cancer prevention and anticancer activity, proving that it has the possibility of being a cancer preventive agent, and it has also attracted much attention as a component of food or cosmetics. However, resveratrol is greatly restricted in its use in water-soluble processing such as beverages because it is a poorly soluble component, causing structural instability. When exposed to light, heat, oxygen, etc., it changes from a cis-type to a trans-type with low biological activity, and is quickly metabolized and has a short half-life. Therefore, the biological utilization efficiency is very low when ingested. Therefore, in order to expand and develop the resveratrol industry, it is urgently needed to develop a technology that can increase the biological utilization rate of resveratrol and improve stability.

[0031] β-sitosterol is a fat-soluble substance in the human body with a chemical structure similar to cholesterol. It has anti-inflammatory, antibacterial and antioxidant effects and is used as a treatment for gum disease. TM However, β-sitosterol, as a fat-soluble substance, is not mixed in a conventional aqueous pharmaceutical composition and is separated to form agglomerated masses. When such a composition is applied to the skin, it does not spread throughout the skin but aggregates into masses, making it difficult to apply evenly to the skin and unstable, thus posing a problem in the development of dosage forms.

[0032] Allantoin removes dead skin cells by increasing the water content in the intercellular matrix, softens the skin by injecting moisture into the skin, and helps heal skin wounds by promoting cell proliferation. In addition, allantoin helps drugs penetrate better by dissolving dead skin cells, and helps heal wound tissues by supplying moisture, so it is also used as a drug or cosmetic ingredient that promotes metabolism.

[0033] The present invention provides a ceramide stabilization method, comprising: step (A), preparing a freeze-dried powder containing ceramide, resveratrol and β-sitosterol; step (B), comprising: step b1), dissolving the freeze-dried powder in dipropylene glycol and mixing an emulsified polymer to prepare an oil phase; and step b2), mixing purified water, dipropylene glycol, a water-soluble solubilizer and allantoin to prepare an aqueous phase; step (C), allowing the oil phase and the aqueous phase to self-assemble through a microfluidic chip; and step (D), using a high-pressure homogenizer to form nanoparticles.

[0034] Preferably, the above step (A) comprises: step a1), adding ceramide, resveratrol and β-sitosterol to heated dipropylene glycol and ethanol to mix and dissolve; step a2), removing precipitates by filtering at room temperature; and step a3), concentrating at a temperature of 40 to 50° C. and freeze-drying the concentrate to prepare a freeze-dried powder containing ceramide, resveratrol and β-sitosterol.

[0035] According to a specific example of the present invention, ceramide, resveratrol and β-sitosterol are first added and mixed into a mixed solvent of dipropylene glycol and ethanol heated to above 45° C., preferably, heated to 45-60° C., to dissolve.

[0036] The ceramide is one or more selected from the group consisting of ceramide NP, ceramide EOP, ceramide NS, ceramide AS, ceramide EOS and ceramide NDS. In this case, the ceramide, resveratrol and β-sitosterol are added and mixed at a weight ratio of 10:0.5-1.5:0.005-0.02, respectively.

[0037] After the solution was allowed to stand at room temperature for 20 to 30 hours at 15 to 25°C, the supernatant was recovered by centrifugation after filtration to remove the precipitate. After the supernatant was concentrated at 40 to 50°C, the concentrate was rapidly frozen to below -60°C for freeze-drying to obtain a high-purity powder, thereby preparing a freeze-dried powder containing ceramide, resveratrol and β-sitosterol.

[0038] Next, the freeze-dried powder is dissolved in dipropylene glycol and then mixed with an emulsified polymer to prepare an oil phase.

[0039] The emulsifying polymer may be at least one selected from the group consisting of phosphatidylcholine, poly-ε-caprolactone, polyglycolic acid, poly-L-lactide, sphingomyelin, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine.

[0040] According to a specific example of the present invention, the oil phase is formed by mixing 0.5 to 1 weight percent of freeze-dried powder, 80 to 85 weight percent of dipropylene glycol, and 14 to 18 weight percent of an emulsified polymer.

[0041] Next, purified water, dipropylene glycol, a water-soluble solubilizing agent, and allantoin are mixed to prepare an aqueous phase. According to a specific example of the present invention, the aqueous phase is formed by mixing 88 to 90 weight percent of purified water, 8 to 10 weight percent of dipropylene glycol, 1 to 2 weight percent of a water-soluble solubilizing agent, and 0.0005 to 0.002 weight percent of allantoin. Polyglyceryl-10 Laurate can be used as the water-soluble solubilizing agent.

[0042] Next, the aqueous phase and the oil phase prepared above are mixed and emulsified for stabilization. In the present invention, a microfluidic chip and a high-pressure homogenizer are used in the preparation of an effective stabilized dosage form.

[0043] Microfluidic chips are chips based on microfluidics technology that can flow fluids through microchannels to perform various experiments and diagnoses. Microfluidic chip technology is also used in emulsion preparation. Korean Publication Patent No. 10-2015-0126561 discloses the following microfluidic chip and a method for preparing liposomes using the same: a thin plate is inserted between the stacked upper and lower plates, and two solutions are injected. Liposomes with regulated uniformity and particle size can be generated by adjusting the flow rate ratio of each solution.

[0044] According to a specific example of the present invention, the oil phase and the aqueous phase are used in a ratio of 1:7 to 1:12 at a low temperature below 15°C, preferably at a temperature of 10 to 15°C, through a microfluidic chip having a microinjection outlet of 1 to 3 μm more than once, preferably, 1 to 5 times to prepare self-assembled liposomes.

[0045] Next, for secondary stabilization, the liposomes were formed into nanoparticles using a high-pressure homogenizer to stabilize ceramide.

[0046] According to one embodiment of the present invention, nanoparticles with a size of 50 to 300 nm are formed under the condition of 300 to 500 bar to stabilize ceramide.

[0047] When preparing nanoliposome particles, the stability of ceramide is further improved by adding resveratrol, β-sitosterol and allantoin as physiologically active substances, thereby improving the stability and skin permeability of resveratrol, β-sitosterol and allantoin. In addition, the ceramide, β-sitosterol, resveratrol and allantoin contained in the nanoliposome particles generated by stabilization of ceramide show more excellent skin improvement activity due to their ascending effect.

[0048] The nanoparticles containing ceramide and β-sitosterol, resveratrol and allantoin as physiologically active substances prepared by the above-mentioned stabilization method show excellent skin moisturizing effect (Test Example 3, Test Example 8), anti-oxidation (Test Example 4, Test Example 5), inflammation relief (Test Example 6), skin barrier enhancement (Test Example 7) and skin itching relief effect (Test Example 9, Test Example 10), and can be effectively used as cosmetics for skin improvement.

[0049] The stabilized ceramide is contained as an active ingredient in an amount of 0.0001 to 50.0% (w / w) based on the total weight of the cosmetic composition.

[0050] The above cosmetic composition is characterized in that it is used for skin whitening, for moisturizing, for relieving skin inflammation, for strengthening the skin barrier or for relieving skin itching.

[0051] Furthermore, solubilizers, stabilizers, flavoring agents, preservatives, pH adjusters, antioxidants, and the like may be used within the range not hindering the purpose of the present invention.

[0052] The above-mentioned cosmetic composition can be prepared into dosage forms such as skin care lotion, toner, facial mask, nourishing cream, moisturizing cream, essence, body cream, body lotion, body oil, shampoo, conditioner, hair essence, hair gel, facial cleanser, cleanser, soap, patch, foundation cream, lipstick, isolation cream, etc.

[0053] Example

[0054] The present invention is described in more detail below based on the following embodiments and experimental examples. However, the following embodiments are only used to illustrate the present invention, and the present invention is not limited to the following embodiments. It should be understood by those skilled in the art that the present invention can be changed to other replacement and equivalent embodiments without departing from the technical concept of the present invention.

[0055] Example 1: Preparation of ceramide and nanoparticles containing physiologically active substances

[0056] Preparation of high purity freeze-dried powder

[0057] In order to remove impurities, add 10 g of ceramide NP, 1 g of resveratrol, and 0.01 g of β-sitosterol to 100 g of dipropylene glycol and 900 g of ethanol heated to above 45°C, and stir. After the above solution is allowed to stand at room temperature for 24 hours, it is filtered to remove impurities and the supernatant is recovered by centrifugation. The recovered supernatant is concentrated at a temperature of 45°C to remove ethanol. Use a deep freezer to rapidly freeze for 3 hours to below -60°C. Powder the uniformly frozen powder by drying it in a freeze dryer at a temperature below 20°C in a vacuum state for 4 days (96 hours). In this case, the vacuum state refers to the pressure state of the freeze dryer, and the freezer drying time may vary depending on the volume of the solution.

[0058] Preparation of stabilized ceramide and physiologically active substances

[0059] After dissolving 0.1 g of the above freeze-dried powder containing ceramide, resveratrol and β-sitosterol in 10 g of dipropylene glycol, 2.0 g of phosphatidylcholine as a plant phospholipid was mixed and stirred to prepare an oil phase.

[0060] An aqueous phase was prepared by mixing 8.4 g of dipropylene glycol, 1.5 g of polyglyceryl-10 laurate as a solubilizing agent, and 0.001 g of allantoin in 80 g of purified water and stirring.

[0061] The aqueous phase and the oil phase prepared above were used in a weight ratio of 1:9 and passed through a 2 μm flow channel of an incubated microfluidic chip three times at a temperature below 15° C. to prepare self-assembled liposomes.

[0062] Nanoparticle capsules with uniform size were prepared by homogenization using a high-pressure homogenizer at 300 bar.

[0063] Comparative Example 1: Preparation of Nanocapsules Containing Ceramide

[0064] An oil phase was prepared by mixing 0.1 g of ceramide with 10 g of heated dipropylene glycol. Thereafter, ceramide-containing nanocapsules were obtained in the same manner as in Example 1.

[0065] Comparative Example 2: Preparation of a mixture of ceramide and physiologically active substances

[0066] 0.1 g of ceramide and 0.01 g each of β-sitosterol, resveratrol, and allantoin as physiologically active substances were mixed with 20 g of warmed dipropylene glycol and 80 g of purified water to prepare a mixture.

[0067] Test Example 1: Analysis of the particle size of nanoparticles

[0068] In order to confirm the particle size of the nanoparticles prepared in Example 1, analysis was performed using a particle size analyzer.

[0069] Figure 1 This is a graph showing the analysis results of the particles of Example 1. As a result of the analysis, it was confirmed that the average size of the particles was 134.2 nm.

[0070] Experimental Example 2: Evaluation of Cytotoxicity

[0071] In order to confirm the cytotoxicity of nanoparticle capsules containing ceramide and physiologically active substances, MTT assay was performed. 5 After inoculating human keratinocytes (HaCaT) at a concentration of cells / mL, the cells were cultured at 37°C and 5% CO2 for 18 hours. After culture, the culture medium was removed and washed with phosphate buffer solution (PBS buffer), and then new culture medium and samples corresponding to Example 1, Comparative Example 1 and Comparative Example 2 at different concentrations were applied and cultured for another 24 hours. In order to determine the cell viability, after adding MTT solution (5 mg / mL), formazan formed by dissolving it for 4 hours with dimethyl sulfoxide (DMSO) was used and the absorbance was measured at a wavelength of 570 nm using an ELISA reader.

[0072] The results are shown in Table 1 below.

[0073] Table 1

[0074]

[0075] As confirmed in Table 1, none of the samples of Example 1, Comparative Example 1, and Comparative Example 2 showed cytotoxicity.

[0076] Test Example 3: Evaluation of Moisturizing Efficacy (AQP3 Expression)

[0077] In order to confirm the moisturizing effect of nanoparticle capsules containing ceramide and physiologically active substances, the effect on AQP3 expression was evaluated. After inoculating human dermal fibroblasts (HDFa), they were cultured in Fibroblast Basal Medium (Medium 106) supplemented with 100 IU / mL penicillin and 100 μg / mL streptomycin at 37°C and 5% CO2 for 24 hours. The culture medium in each well was removed and replaced with a new serum-free culture medium. Samples corresponding to Example 1, Comparative Example 1 and Comparative Example 2 were applied to each well at different concentrations for pretreatment for 4 hours. Each well was irradiated with 15 mJ / cm by an outdoor ultraviolet (UVB) irradiation device (vilberloumet, France). 2 of outdoor ultraviolet rays. After treating the sample with diluted serum-free medium, additional culture was performed for 24 hours. In this case, phosphate buffered saline (PBS) was treated as a negative control group, and 0.01% hyaluronic acid was treated as a positive control group. After lysing the cells at the end of the culture using Ribo Ex TM Total RNA Isolation Solution (GeneAll Biotechnology, Korea) and a scraper, 0.2 mL of chloroform (Sigma-Aldrich, USA) was added and centrifuged (12000 rpm, 4°C, 30 minutes). The supernatant containing ribonucleic acid (RNA) was separated and placed in an equal amount of isopropanol (Merck-Millipore, Germany) to invert and centrifuge (12000 rpm, 4°C, 30 minutes). After the RNA was precipitated and the supernatant of the precipitate was removed, 70% ethanol (Merck-Millipore, Germany) was added to the remaining precipitate and centrifuged (12000rpm, 4°C, 10 minutes) to wash. After removing the ethanol and drying at room temperature, the total RNA was extracted by dissolving it in Nuclease-Free Water (Affymetrix, USA). The purity and concentration of RNA were measured by a micro-volume spectrophotometer (MN-913, Maestrogen, USA) at a wavelength of A260 / A280, and the ratio of 260 nm to 280 nm was confirmed to be in the range of 2.0-2.2. 1 μg of RNA was prepared in a polymerase chain reaction tube (PCR tube) with OligodT (Bionics, Korea), dNTP (Takara, Korea), and nuclease-free water to a total of 13 μL and reacted at 65°C for 5 minutes, and then M-MLV reverse transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to react at 37°C for 50 minutes to synthesize complementary deoxyribonucleic acid (cDNA). In order to quantitatively analyze the gene expression pattern caused by the sample in each cell, real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was performed. Real-time fluorescence quantitative polymerase chain reaction is to mix primers, complementary deoxyribonucleic acid, 2X SYBR green polymerase chain reaction premix (PCR Master Mix) (Applied Biosystems, USA), HPLC (JT baker, USA) in a polymerase chain reaction tube to prepare a total of 20 μL reaction solution, and then use StepOnePlus real-time polymerase chain reaction system (Real-Time PCR System) (Applied Biosystems, USA) to perform polymerase chain reaction.

[0078] The polymerase chain reaction primer (PCR primer) sequence of the AQP3 gene is shown in Table 2 below. The results of evaluating the moisturizing effect of the nanoparticle capsule containing ceramide and water-soluble physiologically active substances prepared according to Example 1 by the expression rate of AQP3 messenger ribonucleic acid (mRNA) are shown in Table 3 below.

[0079] Table 2

[0080]

[0081] Table 3

[0082]

[0083] The experimental results confirmed that when the sample of Example 1 was treated, the messenger RNA of AQP3, a protein that plays an important role in skin moisturizing and is a moisturizing factor related to cellular water movement, increased in a concentration-dependent manner, and the expression was significantly increased within the treatment concentration range of Example 1 compared to the samples of Comparative Examples 1 and 2. In this case, when 0.01% hyaluronic acid was treated as the positive control group, the expression rate was 149.52%.

[0084] Test Example 4: Evaluation of Antioxidant Efficacy

[0085] In order to confirm the antioxidant effect of the nanoparticle capsule containing ceramide and physiologically active substances, a DPPH radical scavenging activity test was performed. The DPPH radical scavenging activity was determined by the reducing power of the sample on the DPPH radical. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) reagent was dissolved in ethanol to prepare a 0.4 mM solution, and 100 μl of samples of Example 1, Comparative Example 1, and Comparative Example 2 of different concentrations or a solution of BHT (Butylated hydroxyl toluene) or 2,6-di-tert-butyl-p-cresol) dissolved in ethanol as a positive control group was placed in a 96-well plate and mixed with 100 μl of a 0.4 mM DPPH solution. In this case, ethanol was treated as a negative control group, and the mixture was reacted at 37°C for 30 minutes. The absorbance was measured at a wavelength of 517 nm using an ELISA plate reader, and the DPPH free radical scavenging ability (%) of the sample or BHT as a positive control group was calculated using a calculation formula.

[0086] DPPH free radical scavenging ability (%) = [1-(absorbance of positive control group or sample / absorbance of control group)] × 100

[0087] Table 4 is a table showing the results of evaluating the antioxidant effect of each sample based on the DPPH radical scavenging ability.

[0088] Table 4

[0089]

[0090] The test results showed that when the samples corresponding to Example 1, Comparative Example 1, and Comparative Example 2 were treated, the DPPH radical scavenging activity increased in a concentration-dependent manner, and the highest activity was shown when the nanoparticles of Example 1 were treated.

[0091] Test Example 5: Antioxidant Activity Stability Test

[0092] The antioxidant activity retention of the samples prepared in the above Example 1 and Comparative Example was evaluated. The antioxidant activity test was conducted in the same manner as in the above Test Example 4, and the antioxidant retention of the Example was compared with that of Comparative Example 1 and Comparative Example 2, based on a standard concentration of 5.0%. The antioxidant results measured at week 0 were taken as 100% and the antioxidant activity was evaluated at high temperature (45°C) by week.

[0093] The results are shown in Table 5 below.

[0094] Table 5

[0095] Week Example 1 Comparative Example 1 Comparative Example 2 Week 0 100 100 100 Week 1 91 30 82 Week 2 82 25 62 Week 4 80 13 35

[0096] As shown in Table 5, it was confirmed that when ceramide was stabilized by nanoliposomes, the sample of Example 1 prepared by using β-sitosterol, resveratrol and allantoin as physiologically active substances had significantly superior antioxidant activity compared to the sample of Comparative Example.

[0097] Test Example 6: Evaluation of anti-inflammatory efficacy

[0098] In order to confirm the anti-inflammatory effect, the inhibition rate of nitric oxide (NO) production was evaluated. 4 After inoculating Raw264.7 macrophages in a 96-well culture plate at a concentration of cells / well and culturing for 24 hours, the samples of Example 1, Comparative Example 1, and Comparative Example 2 were treated for 1 hour. After treatment, lipopolysaccharide (LPS) was treated at a concentration of 100ng / ml and cultured for 24 hours to induce inflammation. Then, 100μL of the cell culture supernatant was mixed with an equal amount of Griess reagent and reacted in a dark room at room temperature for 10 minutes, and the absorbance was measured at a wavelength of 540nm using an enzyme-linked immunosorbent assay (ELISA) plate reader. Table 6 shows the results of evaluating the anti-inflammatory efficacy by the inhibition rate of nitric oxide production. The test results confirmed that the amount of nitric oxide production was reduced in a concentration-dependent manner in all samples, and the nitric oxide inhibition activity was the best in the sample of Example 1.

[0099] Table 6

[0100]

[0101] Test Example 7: Evaluation of skin barrier enhancement efficacy (filaggrin expression)

[0102] Skin barrier proteins, as important elements in forming skin structure, play an important role in preventing external antigens from entering the body. The types of skin barrier proteins include filaggrin, involucrin, and loricrin, among which filaggrin plays an important role in the skin barrier by aggregating keratin filaments in keratinocytes. Therefore, in order to confirm the skin barrier enhancing effect of the nanoparticle capsules of Example 1 containing ceramide and physiologically active substances, a filaggrin gene expression test was performed by comparing with Comparative Examples 1 and 2.

[0103] After inoculating human epidermal keratinocyte cell line (hμman keratinocyte, HEKa), culture in Dulbecco Modified Eagle Medium (Dulbecco Modified EagleMediμm, DMEM) supplemented with 100IU / mL penicillin and 100μg / mL streptomycin at 37°C and 5% CO2 for 24 hours. After culture, remove the culture medium and treat the samples of Example 1, Comparative Example 1, and Comparative Example 2 and culture for 48 hours. Then, use TransZol reagent to extract RNA from the cultured cells and perform real-time gene polymerase chain reaction (RT-PCR). The products generated by the polymerase chain reaction are electrophoresed in 1% agarose gel and confirmed by a gel imaging acquisition and analysis system (GelDocumentation system). In this case, phosphate buffered saline (PBS) is treated as a negative control group, and 20mM calcium chloride (CaCl2) is treated as a positive control group.

[0104] Table 7

[0105]

[0106]

[0107] As confirmed in the above Table 7, when the nanoparticle capsules of Example 1 were treated, it was confirmed that the expression of filaggrin was increased in a concentration-dependent manner, and the increase rate was significantly higher than that when the samples of Comparative Examples were treated.

[0108] Example 2: Preparation of skin care lotion

[0109] A skin care lotion containing the nanoparticle capsules of Example 1 was prepared with the composition shown in Table 8 below by a conventional method.

[0110] Table 8

[0111]

[0112]

[0113] Comparative Examples 3 and 4: Preparation of Skin Care Lotion

[0114] A skin care lotion (Comparative Example 3) was prepared in the same manner as in Example 2 except that the nanoparticles of Comparative Example 1 were added instead of the nanoparticles of Example 1. In addition, a skin care lotion containing 3.00% of hyaluronic acid, which is known to have a moisturizing effect, was prepared as Comparative Example 4.

[0115] Test Example 8: Skin Moisturizing Improvement Effect

[0116] In order to understand the effect of improving the skin's moisturizing ability, the experiment was conducted as follows. 60 people aged 20 to 40 without skin diseases were divided into three groups of 20, and the skin care lotion of Example 2, Comparative Example 3 and hyaluronic acid (Comparative Example 4), a substance known to have a moisturizing effect, was applied to the face and forearm twice a day for one month.

[0117] Before the application, the skin electrical activity was measured using a corneometer (CM820, Courage Khazaka Electronic GmbH) under constant temperature and humidity conditions (24°C, humidity 40%) to serve as a basic value, and the skin electrical reflex was measured after 1 week, 2 weeks, and 4 weeks to evaluate the increase rate. The results are shown in Table 9 below.

[0118] Table 9

[0119] Sample name 1 week later 2 weeks later 4 weeks later Example 2 45 48 49 Comparative Example 3 22 27 35 Comparative Example 4 42 45 46

[0120] As shown in the results of Table 9, the skin care lotion of Example 2 containing the nanoparticle capsules containing ceramide and a physiologically active substance of the present invention showed a very excellent increase in galvanic skin reflex compared to Comparative Example 3. In addition, compared to the comparative example containing hyaluronic acid known to have a moisturizing effect, a similar increase in galvanic skin reflex was shown.

[0121] Generally, galvanic skin reflex is proportional to the moisture content of the skin. From the above results, it can be confirmed that the cosmetics contained in the present invention maintain a higher moisture content of the skin than cosmetics containing purified water.

[0122] Test Example 9: Skin itching relief and improvement effect

[0123] The skin pruritus alleviating effects of Example 2, Comparative Example 3 and Comparative Example 4 were compared and measured on 30 adults (average age 42.0 years) complaining of skin pruritus in a constant temperature and humidity chamber at 22°C and relative humidity of 45%.

[0124] Before using each dosage form in each group, the degree of skin itching was evaluated on a 10-point scale (1-3 points: unconscious itching (no interference with life and sleep), 4-6 points: itching to the extent that it interferes with life and sleep (not all day long), 7-9 points: itching to the extent that it interferes with life and sleep most of the time, 10 points: severe life and sleep disturbances caused by itching).

[0125] The samples were distributed to Example 2, Comparative Example 3, and Comparative Example 4 and used twice a day, morning and evening, for a total of 4 weeks. After 2 and 4 weeks, the subjective evaluation of skin pruritus was re-implemented to evaluate the improvement of pruritus. The changes compared with the initial values ​​are shown in Table 10.

[0126] Table 10

[0127] Sample name Before use 2 weeks later 4 weeks later Example 2 4.5 2.5 2.0 Comparative Example 3 5.0 4.5 4.5 Comparative Example 4 4.0 3.5 2.5

[0128] As shown in the results of Table 10, it was confirmed that the skin care lotion of Example 2 containing the ceramide of the present invention and a physiologically active substance has a superior skin itching alleviating effect compared to Comparative Example 3.

[0129] Test Example 10: Evaluation of skin irritation

[0130] The following experiment was conducted to evaluate the degree of skin irritation to confirm whether the formulation was safe and did not cause skin irritation or itching.

[0131] The test was conducted on 15 adults aged 20-40 years without skin diseases. After dropping 20ul into the patch chamber, the volatile substances were allowed to evaporate for 1 hour, and then the patch was attached to the back of the subject. After 24 hours, the patch was removed and the reading was performed after 30 minutes. The results are shown in Table 11.

[0132] Table 11

[0133]

[0134]

[0135] As shown in the results of Table 11, it was confirmed that, except for Comparative Example 2, the stimulation of Example 1, Example 2, Comparative Example 1, Comparative Example 3, and Comparative Example 4 was not high.

Claims

1. A method for stabilizing ceramide, characterized in that: include: Step (A), preparing a freeze-dried powder containing ceramide, resveratrol and β-sitosterol; Step (B) comprises: step b1), dissolving the freeze-dried powder in dipropylene glycol and mixing with an emulsified polymer to prepare an oil phase; and step b2), mixing purified water, dipropylene glycol, a water-soluble solubilizer and allantoin to prepare an aqueous phase; Step (C), allowing the oil phase and the water phase to self-assemble through a microfluidic chip; and In step (D), nanoparticles are formed using a high pressure homogenizer.

2. The method for stabilizing ceramide according to claim 1, characterized in that: The above step (A) comprises: Step a1), adding ceramide, resveratrol and β-sitosterol to heated dipropylene glycol and ethanol to mix and dissolve; Step a2), removing the precipitate by standing and filtering at room temperature; and Step a3), concentrating at a temperature of 40-50° C. and freeze-drying the concentrate to prepare a freeze-dried powder containing ceramide, resveratrol and β-sitosterol.

3. The method for stabilizing ceramide according to claim 1, characterized in that: The emulsifying polymer is at least one selected from the group consisting of phosphatidylcholine, poly-ε-caprolactone, polyglycolic acid, poly-L-lactide, sphingomyelin, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine.

4. The method for stabilizing ceramide according to claim 1, characterized in that: The ceramide is at least one selected from the group consisting of ceramide NP, ceramide EOP, ceramide NS, ceramide AS, ceramide EOS, and ceramide NDS.

5. The method for stabilizing ceramide according to claim 2, characterized in that: In the step a1), the ceramide, resveratrol and β-sitosterol are added and mixed at a weight ratio of 10:0.5-1.5:0.005-0.02, respectively.

6. The method for stabilizing ceramide according to claim 1, characterized in that: In the step b1), the oil phase is formed by mixing 0.5 to 1 weight percent of freeze-dried powder, 80 to 85 weight percent of dipropylene glycol and 14 to 18 weight percent of an emulsified polymer.

7. The method for stabilizing ceramide according to claim 1, characterized in that: In the step b2), the aqueous phase is formed by mixing 88-90 weight percent of purified water, 8-10 weight percent of dipropylene glycol, 1-2 weight percent of a water-soluble solubilizer and 0.0005-0.002 weight percent of allantoin.

8. The method for stabilizing ceramide according to claim 1, characterized in that: In the above step (C), at 10-15° C., the oil phase and the water phase are passed through a microfluidic chip having a flow channel of 1 μm to 3 μm at a weight ratio of 1:7 to 12 for 1 to 5 times to prepare self-assembled liposomes.

9. The method for stabilizing ceramide according to claim 1, characterized in that: In the above step (D), the size of the above nanoparticles is 50 to 300 nm.

10. A cosmetic composition, characterized in that The composition comprises 0.0001 to 50.0 weight percent of the ceramide stabilized by the method according to any one of claims 1 to 9, relative to the total weight of the composition.

11. The cosmetic composition according to claim 10, characterized in that The above cosmetic composition is used for moisturizing the skin.

12. The cosmetic composition according to claim 10, characterized in that The above cosmetic composition is used for anti-oxidation.

13. The cosmetic composition according to claim 10, characterized in that The above cosmetic composition is used for relieving skin inflammation.

14. The cosmetic composition according to claim 10, characterized in that The above-mentioned cosmetic composition is used to strengthen the skin barrier.

15. The cosmetic composition according to claim 10, characterized in that The above cosmetic composition is used to relieve skin itching.

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