KR102587666B1 - Cosmetic composition comprising stabilized glutathione by lipid nanoparticles and physiologically active materials - Google Patents

Abstract

The present invention relates to a method for stabilizing glutathione and a cosmetic composition containing stabilized glutathione. Glutathione is stabilized by homogenizing it with other bioactive substances, eucalyptus and perilla lactic acid bacteria fermentation extract, using a microfluidic chip and a high-pressure homogenizer. It concerns technologies used as cosmetics for skin whitening, skin moisturizing, antioxidant, skin inflammation relief, skin barrier strengthening, and skin elasticity improvement.

Description

Cosmetic composition comprising stabilized glutathione by lipid nanoparticles and physiologically active materials}

The present invention relates to a method for stabilizing glutathione and a cosmetic composition containing stabilized glutathione. Specifically, glutathione is stabilized by manufacturing it into nanoparticles by applying microfluidic chip technology along with eucalyptus and perilla lactic acid bacteria fermentation extract, which It is about technology used as cosmetics.

Cosmetics have been used for the purpose of protecting the skin and giving it beauty. However, recently, with an emphasis on functionality, functional cosmetics that go beyond simple skin protection and moisturizing and have various functions such as improving skin wrinkles and elasticity, antioxidants, and whitening are in the spotlight. As a result, various active ingredients are contained, which can cause skin problems or inflammation.

In addition, in the case of cosmetics containing these various active ingredients, there is a problem in that it is difficult to maintain formulation stability and the skin absorption rate is low, making it difficult to expect sufficient skin improvement activity as desired when using cosmetics.

Among these active ingredients, glutathione is a tripeptide containing glutamate, cysteine, and glycine, and has powerful antioxidant activity, anti-inflammatory activity, toxic substance detoxification activity, and immunity strengthening activity. Eggplant is known as a biological nutrient. Accordingly, glutathione is used as a raw material for health functional foods and pharmaceuticals, and is also used as a raw material for cosmetics, which are external skin products.

However, glutathione has a problem in that its activity decreases over time in general formulations. There is a problem in that the thiol group of glutathione (GSH), which is also related to whitening activity, is easily oxidized to glutathione disulfide (GSSG), preventing its antioxidant activity from being maintained. Additionally, glutathione has a very low ability to penetrate cell membranes, making absorption difficult. Therefore, there has been a need to improve the stability and bioavailability of glutathione.

The present inventors studied the stabilization and improvement of skin absorption rate of glutathione in the formulation and converted glutathione, a skin-improving active ingredient, into nanoparticles by applying microfluidic chip technology along with other bioactive substances, eucalyptus and perilla lactic acid bacteria fermentation extract. The present invention was completed by confirming that when manufacturing, the stability of the active ingredient is improved, the skin absorption rate is improved, and the synergistic effect shows excellent skin improvement activity.

(0001) Republic of Korea Patent Publication No. 10-2016-0112681 (2016.09.28) (0002) Republic of Korea Patent Publication No. 10-2009-0109789 (2009.10.21)

The purpose of the present invention is to provide a method for stabilizing glutathione.

Another object of the present invention is to provide a cosmetic composition containing stabilized glutathione and exhibiting excellent skin whitening, skin moisturizing, antioxidant, inflammation relieving, skin barrier strengthening, and skin elasticity improving effects.

According to the present invention to achieve the above object,

(A) 2 to 8% by weight of heated Dipropylene Glycol and 65 to 75% by weight of purified water, 0.1 to 20% by weight of glutathione, and 0.5 to 1.5% by weight of fermented extracts of eucalyptus and perilla lactic acid bacteria, which are bioactive substances. Preparing an aqueous phase by mixing;

(B) Phosphatidyl choline, PCL (Poly-ε-caprolactone), PGA (Polyglycolic acid), PLLA (Poly-L-lactide) in 8 to 12% by weight of heated Dipropylene Glycol. , Sphingomyelin, Phosphatidyl serine, Phosphatidyl inositol, Phosphatidyl ethanolamine, Lecithin and Polylactic Acid. Preparing an oil phase by mixing and stirring 1 to 5% by weight of emulsified polymer;

(C) Homogenizing the oil phase and the aqueous phase at a ratio of 1:10 to 5:5, respectively, by passing them through a microfluidic chip, then mixing and stirring 1 to 5% by weight of polyglyceryl-10 laurate, a water-soluble solubilizer. ; and

(D) A method for stabilizing glutathione is provided including the step of forming nanoparticles with a size of 50 to 300 nm using a high pressure homogenizer (Microfluidizer) under conditions of 300 to 800 bar.

Preferably, in the step (A), the eucalyptus and perilla leaf lactic acid bacteria fermentation extract is eucalyptus and perilla leaf (Perilla Ocymoides) Lactobacillus Kepyranofaciens subsp. It is characterized by being extracted by fermentation with kefir granum (Lactobacillus kefiranofaciens subsp. kefirgranum).

More preferably, the eucalyptus and perilla leaf lactic acid bacteria fermentation extract is the lactic acid bacterium Lactobacillus Kefiranofaciens subsp. It is manufactured by inoculating kefir granum (Lactobacillus kefiranofaciens subsp. kefirgranum) into the culture medium, adding eucalyptus and Perilla Ocymoides, fermenting at 25-30°C for 20-30 hours, and centrifuging the fermented product. It is characterized by

Preferably, the homogenization in step (C) is characterized in that it is homogenized by repeatedly passing the chip 1 to 3 times under conditions of 1 to 10 bar and 15 to 40 ° C. using a microfluidic chip with a flow channel of 5 μm to 10 μm.

In order to achieve the above other object, according to the present invention, a cosmetic composition containing 0.0001 to 50.0% (w/w) of glutathione stabilized by the above method based on the total weight of the composition is provided.

The cosmetic composition is used for moisturizing skin, antioxidant, relieving skin inflammation, strengthening skin barrier, or improving skin elasticity.

The cosmetic composition includes skin lotion, skin toner, pack, nutritional cream, moisture cream, essence, body cream, body lotion, body oil, shampoo, rinse, hair conditioner, hair gel, cleansing foam, cleansing lotion, soap, patch, and foundation. It is characterized by having a formulation such as lipstick, makeup base, etc.

By homogenizing glutathione with other bioactive substances, such as eucalyptus and perilla leaf lactic acid bacteria fermentation extract, using a microfluidic chip and a high-pressure homogenizer, the antioxidant activity stability of glutathione is improved, and glutathione produced by the method for stabilizing glutathione of the present invention Nanoparticles containing the bioactive substances eucalyptus and perilla lactic acid bacteria fermentation extract have a high skin absorption rate and remain on the skin for a long time, stably absorbing the active substances, providing excellent moisturizing, antioxidant, skin inflammation relief, skin barrier strengthening, and elasticity improvement effects. Therefore, it can be usefully used as a skin improvement cosmetic.

Figure 1 is a graph showing the particle size analysis results of nanoparticles prepared in an example of the present invention.

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

The present invention stabilizes glutathione as an active ingredient with nanoliposomes and homogenizes it with other active ingredients, eucalyptus and perilla lactic acid bacteria fermentation extract, using a microfluidic chip and a high-pressure homogenizer to improve the stability and skin absorption rate of the active ingredient. It is a technical feature.

Nanoparticle capsules containing glutathione and other bioactive substances, such as eucalyptus and perilla lactic acid bacteria fermentation extract, are made up of a liposome double layer structure and have a high skin absorption rate. They stay on the skin for a long time and stably absorb the active substances into the skin efficiently. It has the advantage of being able to secure improvement effects.

Glutathione, an active ingredient contained in the nanoparticles, serves to reduce melanin pigment, that is, oxidized pigment in the skin, and excrete it out of the skin. By applying antioxidant technology to make glutathione active, it maintains the homeostasis and maintenance of glutathione. When the balance is right, it not only has a skin whitening effect, but also plays a role in helping cell regeneration along with bioactive substances. Glutathione is an antioxidant that maintains thiol status in the human body and plays an important role in detoxification. Reduced glutathione (GSH) is known to have a whitening effect.

Eucalyptus globulus is a myrtle plant native to subtropical highlands. Its most important ingredient is cineol, also called eucalyptol, which has antibacterial, antibacterial, antiseptic, and expectorant effects. great. In addition, alpha-pinene, a type of monoterpene contained in phytoncide that is effective in relieving fatigue, and limonene, which has the effect of increasing concentration and is contained in most herbal plants, relieves inflammation, has antiviral properties, and promotes nerve function. It contains citronella, which has a calming and insect-repelling effect.

Perilla Ocymoides is an annual herb from the Lamiaceae family and has been growing naturally in Korea for a long time. It is also called by names such as chazugi, perilla, and soma. 'Perilla leaves with the spicy taste of cinnamon' are also called cinnamon sesame seeds, sesame leaf leaves, and gye im (桂荏). In English, both lobule and perilla are called beefsteak plant or wild basil. It looks very similar to perilla seeds, but the difference between perilla seeds and perilla seeds is that the stems and leaves are purple. As you can see from the character meaning purple, both the front and back of the leaves are purple. Light purple flowers bloom in late summer. Perilla leaves provide abundant nutrients to the hair roots and are effective in keeping hair and scalp healthy. The various antioxidants contained in perilla leaves not only remove various harmful substances that cause cancer, but also inhibit the proliferation of cancer cells. It has anti-cancer action. In addition, perilla leaf contains excellent antioxidant components such as beta-carotene, anthocyanin, vitamin A, C, etc. It is effective in preventing aging by removing free radicals, which are major factors that accelerate aging, and has an antioxidant effect that prevents cell oxidation. It is also rich in vitamin K, which suppresses inflammation and helps blood coagulation. It is also rich in vitamins such as vitamins B1, B2, B6, and niacin, as well as calcium, potassium, magnesium, and zinc. It has effects such as strengthening immunity and improving atopic dermatitis and allergic constitution, and the perylaldehyde component contained in perilla leaves has an excellent antibacterial effect and is effective in removing harmful bacteria from the body. In addition, it is rich in linoleic acid, which has the effect of lowering cholesterol and body fat levels, and is also rich in EPA, an unsaturated fatty acid, which boosts immunity and helps prevent various adult diseases. In the production of perilla leaf exosomes of the present invention, leaves and stems are used, and preferably perilla leaf exosomes are used.

According to the present invention, (A) 2 to 8% by weight of heated Dipropylene Glycol and 65 to 75% by weight of purified water, 0.1 to 20% by weight of glutathione, and fermented extracts of eucalyptus and perilla leaf lactic acid bacteria, which are bioactive substances. Preparing an aqueous phase by mixing 0.5 to 1.5% by weight;

(B) Phosphatidyl choline, PCL (Poly-ε-caprolactone), PGA (Polyglycolic acid), PLLA (Poly-L-lactide) in 8 to 12% by weight of heated Dipropylene Glycol. , Sphingomyelin, Phosphatidyl serine, Phosphatidyl inositol, Phosphatidyl ethanolamine, Lecithin and Polylactic Acid. Preparing an oil phase by mixing and stirring 1 to 5% by weight of emulsified polymer;

(C) Homogenizing the oil phase and the aqueous phase at a ratio of 1:10 to 5:5, respectively, by passing them through a microfluidic chip, then mixing and stirring 1 to 5% by weight of polyglyceryl-10 laurate, a water-soluble solubilizer. ; and

(D) A method for stabilizing glutathione is provided including the step of forming nanoparticles with a size of 50 to 300 nm using a high pressure homogenizer (Microfluidizer) under conditions of 300 to 800 bar.

First, glutathione and the bioactive substances eucalyptus and perilla leaf lactic acid bacteria fermentation extract are added and mixed in a solvent of dipropylene glycol and purified water heated to 40-50°C to prepare the water phase.

At this time, based on the total weight of the composition including the water phase, oil phase, and water-soluble solubilizer, 2 to 8% by weight of warmed Dipropylene Glycol, 65 to 75% by weight of purified water, 0.1 to 20% by weight of glutathione, and physiological The water phase is prepared by mixing the active substances, eucalyptus and perilla leaf lactic acid bacteria fermentation extract, at a ratio of 0.5 to 1.5% by weight.

Preferably, the eucalyptus and perilla leaf lactic acid bacteria fermented extract is eucalyptus and perilla leaf Lactobacillus Kefiranopaciens subsp. It is characterized by being extracted by fermentation with kefir granum (Lactobacillus kefiranofaciens subsp. kefirgranum).

Lactobacillus Lactobacillus Kefiranofaciens subsp. Kefir granum (Lactobacillus kefiranofaciens subsp. kefirgranum) is isolated from kefir grain.

According to one embodiment of the present invention, the fermented extract of eucalyptus and perilla leaf lactic acid bacteria is Lactobacillus Kefiranopaciens subsp. It is manufactured by inoculating the culture medium with Lactobacillus kefiranofaciens subsp. kefirgranum, adding eucalyptus and perilla leaves, fermenting at 25-30°C for 20-30 hours, and centrifuging the fermented product. At this time, eucalyptus and perilla leaves are mixed and extracted by fermentation at a weight ratio of 1 to 5:1 to 5, respectively.

The stability of glutathione is further improved by adding the bioactive substances eucalyptus and perilla leaf lactic acid bacteria fermentation extract, and the eucalyptus and perilla leaf lactic acid bacteria fermentation extract has a high organic acid content, showing excellent skin improvement activity in itself. In addition, nanoliposomes produced by glutathione stabilization and containing glutathione and fermented eucalyptus and perilla lactic acid bacteria extracts exhibit excellent skin improvement activity due to their synergistic effect.

Next, an emulsified polymer is added to the heated dipropylene glycol and mixed to prepare an oil phase.

According to one embodiment of the present invention, phosphatidyl choline, PCL (Poly-ε-caprolactone), and PGA (Polyglycolic acid) are added to 8 to 12% by weight of Dipropylene Glycol heated to 40 to 50°C. ), PLLA (Poly-L-lactide), Sphingomyelin, Phosphatidyl serine, Phosphatidyl inositol, Phosphatidyl ethanolamine, Lecithin and Polylactic Acid. ) to prepare an oil phase by mixing and stirring 1 to 5% by weight of at least one emulsified polymer selected from the group consisting of.

Next, the prepared water phase and oil phase are mixed and emulsified to stabilize them. In the present invention, a microfluidic chip and a high-pressure homogenizer are used to manufacture an effective formulation.

A microfluidic chip is a chip that is based on microfluidics (microfluidics) technology and allows various experiments and diagnosis by flowing fluid through a micro channel. Microfluidic chip technology is also being applied to emulsion manufacturing. Republic of Korea Patent Publication No. 10-2015-0126561 discloses a liposome in which two types of solutions are injected after interposing a fine thin plate between the upper and lower plates to be stacked, and the uniformity and particle size are adjusted by adjusting the flow rate ratio of each solution. A microfluidic chip that enables the creation of liposomes and a liposome manufacturing method using the same are disclosed.

According to one embodiment of the present invention, the prepared oil phase and aqueous phase are homogenized by passing them through a microfluidic chip using a ratio of 1:10 to 5:5, respectively, and then mixed with polyglyceryl-10 laurate 1 to 1, which is a water-soluble solubilizer. Mix 5% by weight and stir to stabilize. The homogenization can be accomplished by repeatedly passing the chip 1 to 3 times under conditions of 1 to 10 bar and 15 to 40°C using a microfluidic chip with a flow channel of 5 μm to 10 μm.

Next, for secondary stabilization, a high-pressure homogenizer (Microfludizer) is used under conditions of 300 to 800 bar to produce nano liposomes with a size of 50 to 300 nm, thereby stabilizing the active ingredient glutathione.

In addition, solubilizers, stabilizers, fragrances, preservatives, pH adjusters, antioxidants, etc. can be additionally used within the range that do not impair the purpose of the present invention.

By this stabilization method, the stability of the antioxidant activity of the active ingredient glutathione was greatly improved (Test Example 7). In addition, nanoparticles manufactured by the stabilization method of the present invention, including glutathione and the bioactive substances eucalyptus and perilla leaf lactic acid bacteria fermentation extract, have excellent whitening effect (Test Examples 3 and 4) and skin moisturizing effect (Test Example 5, 10), antioxidant (Test Example 6), inflammation relief (Test Example 8), skin barrier strengthening (Test Example 9), and skin elasticity effect (Test Example 11), so it can be usefully used as a cosmetic for skin improvement.

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

The cosmetic composition is used for skin whitening, moisturizing, relieving skin inflammation, strengthening the skin barrier, or strengthening skin elasticity.

The cosmetic composition includes skin lotion, skin toner, pack, nutritional cream, moisture cream, essence, body cream, body lotion, body oil, shampoo, rinse, hair conditioner, hair gel, cleansing foam, cleansing lotion, soap, patch, and foundation. , lipstick, makeup base, etc.

[Example]

Hereinafter, the present invention will be described in more detail based on the following examples and test examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited by the following examples, and may be substituted or changed to other equivalent examples without departing from the technical spirit of the present invention. will be clear to those skilled in the art to which the present invention pertains.

Example 1: Preparation of nanocapsules containing glutathione and bioactive substances

Eucalyptus and perilla leaf fermented extract

10 g/L Sucrose, 20 g/L Glycerin, 5 g/L Monosodium Glutamate, 5 g/L Sodium Citrate, 10 g/L Yeast Extract, 0.5 g/L Potassium Phosphate, 1 g/L Magnesium Chloride, 0.5 g/L Lactobacillus kefiranofaciens subsp. was grown in a culture medium containing L manganese chloride. Kefirgranum was inoculated, eucalyptus leaves and perilla leaves were added, and fermented with stirring at 28°C for 24 hours.

After fermentation was completed, the fermented product was centrifuged at 3,000

The culture solution separated in the above step was centrifuged at 10,000 The recovered supernatant was frozen at -80°C for 20 hours and dried in a freeze dryer under vacuum for 100 hours to prepare eucalyptus and perilla leaf lactic acid bacteria fermented extracts. At this time, the vacuum state refers to the pressure state of the freeze dryer, and the freezing and drying time may vary depending on the volume of the solution.

Production of stabilized glutathione and bioactive substances

10 g of glutathione was mixed with 5 g of dipropylene glycol and 68.5 g of purified water heated to 40-50°C and stirred. An aqueous phase was prepared by adding 1 g of the bioactive substances, eucalyptus and perilla leaf lactic acid bacteria fermentation extract.

An oil phase was prepared by mixing 3 g of phosphatidyl choline with 10 g of dipropylene glycol heated to 40-50°C and stirring.

Nanoparticles were prepared by passing the prepared water phase and oil phase through a microfluidic chip.

A microfluidic chip with a 5 um flow channel was fabricated and used. 84.5% by weight of the water phase and 13% by weight of the oil phase were homogenized by passing through a microfluidic chip with a 5 um flow channel at 3 bar at room temperature. The process was repeated a total of three times to increase the degree of homogenization through self-assembly.

2.5 g of Polyglyceryl-10 Laurate, a water-soluble solubilizer, was added thereto and mixed and stirred. Here, nanoparticle capsules were manufactured under conditions of 800 bar using a high-pressure homogenizer (Microfludizer).

Comparative Example 1: Preparation of glutathione-containing nanocapsules

An aqueous phase was prepared by mixing 10 g of glutathione with 5 g of warmed Dipropylene Glycol and 69.5 g of purified water. The subsequent method was the same as Example 1 to obtain nanocapsules containing glutathione.

Comparative Example 2: Preparation of a mixture of glutathione and bioactive substances

A mixture was prepared by mixing 10 g of glutathione and 1 g of the bioactive substances, eucalyptus and perilla leaf lactic acid bacteria fermentation extract, with 10 g of warmed Dipropylene Glycol and 79 g of purified water.

Test Example 1: Particle size analysis of nanoparticles

To confirm the particle size of the nanoparticles prepared in Example 1, they were analyzed using a particle size analyzer.

Figure 1 is a graph showing the analysis results of particles according to Example 1. As a result of the analysis, the average size of the particles was confirmed to be 89.9 nm.

Test Example 2: Cytotoxicity evaluation

MTT assay evaluation was performed to confirm the cytotoxicity of nanoparticle capsules containing glutathione and bioactive substances. Human keratinocytes (HaCaT) were inoculated into a 96-well plate at a concentration of 1Х10 ⁵ cells/mL and cultured at 37°C for 18 hours under 5% CO ₂ . After incubation, the medium was removed and washed with PBS buffer, and then new medium and samples corresponding to Example 1 and Comparative Examples 1 and 2 were administered at different concentrations and cultured for another 24 hours. To measure cell viability, formazan formed over 4 hours after adding MTT solution (5 mg/mL) was dissolved with dimethyl sulfoxide (DMSO) and absorbance was measured at 570 nm using an ELISA reader.

The results are shown in Table 1 below.

Cell viability
(% of control) Example 1 Comparative Example 1 Comparative Example 2 Control 100 100 100 0.1% 115.40 125.50 115.50 One% 110.4 117.70 105.62 5% 106.10 110.52 100.15 10% 102.35 105.45 90.40

As confirmed in Table 1, cytotoxicity was not observed in all of the samples of Example 1 and Comparative Examples 1 and 2.

Test Example 3: Evaluation of whitening efficacy (inhibition of tyrosinase activity)

Pigmentation occurs in the skin when tyrosine is oxidized to dopaquinone by tyrosinase. To confirm the whitening efficacy of glutathione and bioactive substances, a test was conducted to inhibit the activity of tyrosinase enzyme, which darkens the skin, compared to the extract. Mushroom-derived tyrosinase and tyrosine were purchased and used from Sigma Chemical, and tyrosinase activity was measured in 150 μl of 0.1M phosphate buffer (pH 6.5) and 8 μl of mushroom tyrosinase (2100 units/ml, 0.05M phosphate buffer, pH 6.5), 1.5 μl. Samples corresponding to Example 1 and Comparative Examples 1 and 2 were treated with 36 μl of L-tyrosine at a mM concentration according to concentration. Tyrosinase inhibitory activity was confirmed by reacting the sample at 37°C for 15 minutes and measuring the absorbance at 490 nm. At this time, PBS was treated as a negative control, and Niacinamide 0.01% was treated as a positive control. The results are shown in Table 2 below.

Tyrosinase inhibition rate (% of control) Example 1 Comparative Example 1 Comparative Example 2 Positive Control 39.8% 0.1% 28.37 15.30 12.00 One% 83.62 63.62 30.50 5% 85.15 64.15 52.67 10% 89.80 64.40 53.85

As a result of the test, when the sample of Example 1 was treated, the tyrosinase inhibition rate increased in a concentration-dependent manner, and it was confirmed that the inhibition rate increased significantly in the treatment concentration range of Example 1 compared to the samples corresponding to Comparative Examples 1 and 2. At this time, when treated with Niacinamide 0.01%, the inhibition rate was 39.8%.

Test Example 4: Evaluation of whitening efficacy (inhibition of melanin production)

To confirm the whitening efficacy of glutathione and bioactive substances, a melanin production inhibition test was conducted compared to the extract. After inoculation of the mouse-derived B16F10 cell line (melanin-secreting cells), the cells were cultured in Dulbecco Modified Eagle Medium (DMEM) culture medium supplemented with 10% FBS at 37°C for 24 hours under 5% CO ₂ . After culturing, the medium was discarded and replaced with new medium, and Example 1 and Comparative Examples 1 and 2 were treated at different concentrations and cultured for 72 hours. After culturing, the cells were treated with Trypsin-EDTA and recovered by centrifugation. The recovered cells were washed, treated with 500 μL of 1N NaOH, and reacted at 100°C for 10 minutes to dissolve melanin. The amount of melanin was confirmed by measuring absorbance at 405 nm. At this time, PBS was treated as a negative control, and arbutin, a synthetic substance known as a whitening agent, was used as a standard sample and the results were converted to %.

Melanin production inhibition rate
(% of control) Example 1 Comparative Example 1 Comparative Example 2 Positive Control 20.6% 0.1% 36.70 20.87 15.80 One% 40.62 22.44 20.92 5% 58.60 30.72 25.22 10% 65.20 35.17 25.30

As a result of the test, it was confirmed that when the sample of Example 1 was treated, the inhibition rate of melanin production increased in a concentration-dependent manner, and the inhibition rate of melanin production increased more than the samples of Comparative Examples 1 and 2.

Test Example 5: Moisturizing efficacy evaluation (AQP3 expression)

To confirm the moisturizing effect of nanoparticle capsules containing glutathione and bioactive substances, the effect on AQP3 expression was evaluated. After inoculating human dermal fibroblast (HDFa), culture medium was cultured in Fibroblast Basal Medium (Medium 106) supplemented with 100 IU/mL penicillin and 100 μg/mL streptomycin at 37°C under 5% CO ₂ for 24 hours. Cultured. The medium from each well was removed and replaced with new serum-free medium. Samples corresponding to Example 1 and Comparative Examples 1 and 2 were administered to each well at different concentrations, and pretreatment was performed for 4 hours. Each well plate was irradiated with 15mJ/cm ² UVB using a UVB irradiation device (Vilber Loumet, France). The samples were treated with diluted serum-free medium and further cultured for 24 hours. At this time, PBS was treated as a negative control, and Hyaluronic acid 0.005% was treated as a positive control. Cells after cell culture were lysed using Ribo Ex TM Total RNA Isolation Solution (GeneAll Biotechnology, Korea) and a scraper, then 0.2 mL chloroform (Sigma-Aldrich, USA) was added and centrifuged (12,000 rpm, 4°C, 30 minutes). ) was done. The supernatant containing RNA was separated, inverted with the same amount of isopropanol (Merck-Millipore, Germany) as the supernatant, and then centrifuged (12,000 rpm, 4°C, 30 minutes). After precipitating RNA and discarding the supernatant except the precipitate, 70% ethanol (Merck-Millipore, Germany) was added to the remaining precipitate and washed by centrifugation (12,000 rpm, 4°C, 10 minutes). Ethanol was removed, dried at room temperature, dissolved in Nuclease-Free Water (Affymetrix, USA), and total RNA was extracted. After measuring the purity and concentration of RNA at the A260/A280 wavelength using a MaestroNano ^® Micro-volume Spectrophotometer (MN-913, Maestrogen, USA), it was confirmed that the ratio between 260nm and 280nm was in the range of 2.0-2.2. cDNA was prepared in a PCR tube with 1 μg RNA, Oligo dT (Bionics, Korea) dNTP (Takara, Korea), and nuclease free water in a total of 13 μL, reacted at 65°C for 5 minutes, and then incubated with M-MLV Reverse Transcriptase (Invitrogen, Thermo). It was synthesized by reacting at 37°C for 50 minutes using Fisher Scientific, Waltham, Massachusetts, USA. qRT-PCR was performed to quantitatively analyze the gene expression pattern occurring within each cell by sample. For qRT-PCR, prepare a reaction solution by mixing primer, cDNA, 2X SYBR green PCR Master Mix (Applied Biosystems, USA), and HPLC (J. T baker, USA) in a PCR tube with a total of 20 μL and use the StepOnePlus Real-Time PCR System ( PCR was performed using Applied Biosystems, USA).

The PCR primer sequence of the AQP3 gene is shown in Table 4 below, and the results of evaluating the moisturizing efficacy of the nanoparticle capsule containing glutathione and water-soluble bioactive substances prepared according to Example 1 in terms of AQP3 mRNA expression rate are shown in Table 5 below. .

gene Forward primer (5'→3') Reverse primer (5'→3') AQP3 5'-CCT TTG GCT TTG CTG CAC TC-3' 5'-ACG GGG TTG TTG TAA GGG TCA-3' GAPDH 5'-GTC TCC TCT GAC TTC AAC AGC G-3' 5'-ACC ACC CTG TTG CTG TAG CCA A-3'

Relative AQP3 expression
(% of control) Hyaluronic acid
(0.005%) Example 1 Comparative Example 1 Comparative Example 2 Control 130.25% 135.46 0.1% 103.56 100.25 103.53 One% 120.61 103.24 108.54 5% 138.52 117.53 110.60 10% 148.07 120.57 115.42

As a result of the test, when the sample of Example 1 was treated, AQP3 mRNA, a moisturizing factor involved in cell moisture movement and a protein that plays an important role in skin moisturization, increased in a concentration-dependent manner, compared to the samples corresponding to Comparative Examples 1 and 2. It was confirmed that the expression rate was significantly increased in the treatment concentration range of Example 1. At this time, when treated with 0.005% Hyaluronic acid, a positive control, an expression rate of 130.25% was observed.

Test Example 6: Antioxidant efficacy evaluation

A DPPH radical scavenging activity test was conducted to confirm the antioxidant efficacy of nanoparticle capsules containing glutathione and bioactive substances. DPPH radical scavenging activity was measured by the reducing power of the sample for DPPH radicals. DPPH (1,1-Diphenyl-2-picrylhydrazyl) reagent was dissolved in ethanol to prepare a 0.4mM solution, and samples corresponding to Example 1, Comparative Examples 1 and 2 or BHT (Butylated), a positive control, were prepared by concentration in a 96 well plate. 100 ㎕ of a solution of hydroxyl toluene (or 2,6-Di-tert-butyl-p-cresol) dissolved in ethanol and 100 ㎕ of a 0.4 mM DPPH solution were added and mixed. At this time, ethanol was used as a negative control, and after mixing, it was reacted at 37°C for 30 minutes. Absorbance was measured at 517 nm using an ELISA reader, and the DPPH radical scavenging ability (%) of the sample or positive control BHT was calculated using the formula below.

DPPH radical scavenging ability (%) = {1-(absorbance of positive control or sample/absorbance of control)} Х 100

Table 6 is a table showing the results of evaluating the antioxidant effect of each sample based on DPPH radical scavenging ability.

Concentration (%) DPPH radical scavenging activity (%) Example 1 Comparative Example 1 Comparative Example 2 0.1 32.98 19.72 18.55 1.0 38.59 25.40 25.04 5.0 49.72 33.08 30.00 10. 55.79 40.87 34.15 BHT (0.01%) 57.60

As a result of the test, it was confirmed that when the samples corresponding to Example 1 and Comparative Examples 1 and 2 were treated, the DPPH radical scavenging activity increased in a concentration-dependent manner, and that the highest activity was observed when the nanoparticles of Example 1 were treated.

Test Example 7: Antioxidant activity stability test

The antioxidant activity retention rate of the samples prepared in Example 1 and Comparative Examples was evaluated. The antioxidant activity test was performed in the same manner as in Test Example 6, and the antioxidant retention rates of Examples and Comparative Examples 1 and 2 were compared based on a standard concentration of 0.1%. Antioxidant activity was evaluated for each week at high temperature (45°C), based on 100% antioxidant results measured at week 0.

The results are shown in Table 7 below.

parking discrimination Example 1 Comparative Example 1 Comparative Example 2 0 parking 100 100 100 Week 1 91 85 87 Week 2 90 69 50 Week 4 82 65 45 Week 8 80 61 43

As shown in Table 7, when stabilizing glutathione with nano-liposomes, the antioxidant activity retention ability of the sample of Example 1 prepared using the bioactive substances eucalyptus and perilla lactic acid bacteria fermentation extract was higher than that of the samples of the comparative example. You can see that it is far superior.

Test Example 8: Anti-inflammatory efficacy evaluation

To confirm the anti-inflammatory effect, the NO (Nitric oxide) production inhibition rate was evaluated. Raw264.7 macrophages were distributed at 8Х10 ⁴ cells/well in a 96 well plate and cultured for 24 hours, and then the samples of Example 1 and Comparative Examples 1 and 2 were treated for 1 hour. After treatment, LPS was treated at a concentration of 100ng/ml and cultured for 24 hours to induce inflammation. Afterwards, 100 μL of cell culture supernatant and equal amounts of Griess reagent were mixed and reacted at room temperature in the dark for 10 minutes, and the absorbance was measured at 540 nm using an ELISA reader. Table 8 is a table showing the results of evaluating anti-inflammatory efficacy in terms of NO (Nitric oxide) production inhibition rate. As a result of the test, the amount of NO production decreased in a concentration-dependent manner in all samples, and it was confirmed that the sample of Example 1 had the best NO production inhibition activity.

Concentration (%) NO Production (% of control) Example 1 Comparative Example 1 Comparative Example 2 Control 100 100 100 0.1 89.60 92.27 89.01 1.0 72.26 89.63 85.50 5.0 55.11 85.80 84.62 10.0 49.70 70.70 79.75

Test Example 9: Evaluation of skin barrier strengthening efficacy (filaggrin expression)

Skin barrier protein is an important element of the skin structure and plays an important role in blocking external antigens from entering the body. Types of skin barrier proteins include filaggrin, involucrin, and loricrin. Among them, filaggrin plays an important role in the skin barrier by aggregating keratin filaments within keratinocytes. Therefore, to confirm the skin barrier strengthening effect of the nanoparticle capsule of Example 1 containing glutathione and bioactive substances, a filaggrin gene expression test was conducted compared with Comparative Examples 1 and 2.

After inoculation of human epidermal keratinocyte (HEKa), the cells were cultured in Dulbecco Modified Eagle Medium (DMEM) culture medium supplemented with 100 IU/mL penicillin and 100 μg/mL streptomycin at 37°C for 24 hours under 5% CO ₂ . . After culturing, the medium was discarded and the samples of Example 1 and Comparative Examples 1 and 2 were treated and cultured for 48 hours. Afterwards, RNA was extracted from the cultured cells using TransZol reagent and RT-PCR (real-time genetic polymerase chain reaction) was performed. The product generated by PCR was electrophoresed on a 1% agarose gel and confirmed using the Gel Documentation system. . At this time, PBS was treated as a negative control, and 20mM of CaCl ₂ was treated as a positive control.

Concentration (%) Relative FLG expression (% of control) Example 1 Comparative Example 1 Comparative Example 2 Control 100 100 100 0.1 105.73 102.18 100.18 1.0 119.40 103.60 102.61 5.0 125.06 107.67 104.76 10.0 129.30 113.90 110.69

As confirmed in Table 9, filaggrin expression increased in a concentration-dependent manner when the nanoparticle capsule of Example 1 was treated, and the increase rate was much higher than when the samples of Comparative Example were treated.

Example 2: Preparation of lotion

A lotion containing the nanoparticle capsule of Example 1 was prepared by a conventional method with the composition shown in Table 10 below.

Ingredients (% by weight) Content (% by weight) Nanoparticles containing glutathione and bioactive substances (Example 1) 1.00 glycerin 10.00 Butylene glycol 5.00 Glyceryl oleate 1.80 Cetearyl olivate 0.50 Sorbitan Olivate 0.50 Caprylic/Capric Triglyceride 5.00 Cetyl Ethyl Hexanoate 1.00 beeswax 0.50 squalane 0.20 1,2-hexanediol 2.00 Cholesteryl/Behenyl/Octyldodecyllauroylglutamate 1.00 Dimethicone 0.50 Cyclopentasilonsac/cyclohexasiloxane 2.00 Cetiaryl alcohol 1.00 mineral oil 2.50 Disodium EDTA 0.02 BHT 0.05 Tocopheryl Acetate 0.30 panthenol 0.20 Ethylhexylmethoxycinnamate 0.20 incense 0.01 Purified water remaining amount

Comparative Examples 3-4: Preparation of lotion

In Example 2, lotion was prepared in the same manner by adding Comparative Example 1 instead of the nanoparticles of Example 1 (Comparative Example 3). In addition, a lotion containing 3.00% hyaluronic acid, which is known to have a moisturizing effect, was prepared and used as Comparative Example 4.

Test Example 10: Effect of improving skin moisturizing ability

To determine the effect of improving skin moisturization, the following experiment was conducted. 60 people in their 20s to 40s without skin disease were divided into 3 groups of 20 people per group, and each group was treated with Example 2 and Comparative Example 3 above and a lotion containing hyaluronic acid, a substance known to have a moisturizing effect (comparison) Example 4) was applied to the face and forearm twice daily for 1 month.

Before starting application, skin conductivity was measured using a corneometer (CM820 courage Khazaka electronic GmbH) under constant temperature and humidity conditions (24°C, 40% humidity) and set as the default value, and skin conductivity after 1, 2, and 4 weeks. was measured to evaluate the rate of increase. The results are shown in Table 11 below.

Sample name 1 week later 2 weeks later 4 weeks later Example 2 30 42 47 Comparative Example 3 15 18 20 Comparative Example 4 44 40 42

As shown in the results in Table 11, the lotion of the formulation of Example 2 containing nanoparticle capsules containing glutathione and bioactive substances of the present invention had a very excellent skin conductivity increase rate compared to Comparative Example 3. In addition, compared to Comparative Example 4, which is a cosmetic containing hyaluronic acid, which is known to have a moisturizing effect, it showed a similar increase in skin conductivity.

In general, skin conductivity is proportional to the amount of skin moisture, so from the above results, it can be confirmed that the cosmetic contained in the present invention maintains a higher skin moisture content compared to the cosmetic containing purified water.

Test Example 11: Skin elasticity improvement effect

The skin elasticity improvement effect of Example 2 and Comparative Examples 3 and 4 was measured on 30 healthy women (average age 33.7 years) in a constant temperature and humidity room at 22°C and 45% relative humidity.

The above formulation was applied to the eye area once every night for 4 weeks, and the skin elasticity of each group was measured using a skin elasticity meter (Cutometer SEM 575, C+KElectronic Co., Germany). The change values are shown in Table 12 below.

division Example 2 Comparative Example 3 Comparative Example 4 Skin elasticity level 32.5 20.6 30.2

Through the results in Table 12, it was confirmed that when the lotion of Example 2 was used, the degree of skin elasticity improvement was higher than that of Comparative Examples 3 and 4, indicating that the skin elasticity improvement effect was excellent.

Claims (10)

(A) 2 to 8% by weight of heated Dipropylene Glycol and 65 to 75% by weight of purified water, 0.1 to 20% by weight of glutathione, and 0.5 to 1.5% by weight of fermented extracts of eucalyptus and perilla lactic acid bacteria, which are bioactive substances. Preparing an aqueous phase by mixing;
(B) Phosphatidyl choline, PCL (Poly-ε-caprolactone), PGA (Polyglycolic acid), PLLA (Poly-L-lactide) in 8 to 12% by weight of heated Dipropylene Glycol. , Sphingomyelin, Phosphatidyl serine, Phosphatidyl inositol, Phosphatidyl ethanolamine, Lecithin and Polylactic Acid. Preparing an oil phase by mixing 1 to 5% by weight of an emulsified polymer and emulsifying it;
(C) Homogenizing the oil phase and the aqueous phase at a ratio of 1:10 to 5:5, respectively, by passing them through a microfluidic chip, then mixing and stirring 1 to 5% by weight of polyglyceryl-10 laurate, a water-soluble solubilizer. ; and
(D) A method for stabilizing glutathione including the step of forming nanoparticles with a size of 50 to 300 nm using a high pressure homogenizer (Microfluidizer) under conditions of 300 to 800 bar. The method of claim 1, wherein in step (A), the eucalyptus and perilla leaf lactic acid bacteria fermented extract is eucalyptus and perilla leaf Lactobacillus kepyranofaciens subsp. A method for stabilizing glutathione, characterized in that it is extracted by fermentation with kefir granum (Lactobacillus kefiranofaciens subsp. kefirgranum). The method of claim 1, wherein the homogenization in step (C) is performed using a microfluidic chip with a flow channel of 5 μm to 10 μm and passing the chip repeatedly 1 to 3 times under conditions of 1 to 10 bar and 15 to 40°C. Method for stabilizing glutathione. A cosmetic composition containing 0.0001 to 50.0% (w/w) of glutathione stabilized by the method of any one of claims 1 to 3, based on the total weight of the composition. The cosmetic composition according to claim 4, wherein the cosmetic composition is for skin whitening. The cosmetic composition according to claim 4, wherein the cosmetic composition is for antioxidant purposes. The cosmetic composition according to claim 4, wherein the cosmetic composition is for moisturizing skin. The cosmetic composition according to claim 4, wherein the cosmetic composition is used to relieve skin inflammation. The cosmetic composition according to claim 4, wherein the cosmetic composition is used to strengthen the skin barrier. The cosmetic composition according to claim 4, wherein the cosmetic composition is used to improve skin elasticity.