KR102622315B1 - Method for manufacturing nanovesicles stably capturing high content of high-molecular Polysaccharide - Google Patents

Abstract

The present invention relates to a method for producing nanovesicles that stably capture high molecular polysaccharides in a high content, and to a cosmetic composition containing the produced nanovesicles with skin moisturizing, elasticity, and soothing effects.
Conventional high-molecular polysaccharides have useful effects on the skin, but because they contain a lot of -OH groups, the intermolecular attraction is strong, so formulation stability is low when formulations containing high content are manufactured, and since they are water-soluble substances, penetration into lipid-based skin is limited. However, according to the present invention, by manufacturing a nanovesicle that stably captures a high content of polymer polysaccharide, not only does it solve the problems of the conventional polymer polysaccharide, but it also improves skin moisturization, elasticity, and elasticity compared to polymer polysaccharide not captured in nanovesicles. The sedative effect can be significantly increased.

Description

Method for manufacturing nanovesicles stably capturing high content of high-molecular polysaccharide}

The present invention relates to a method for producing nanovesicles that stably capture high molecular polysaccharides in a high content, and to a cosmetic composition containing the produced nanovesicles with skin moisturizing, elasticity, and soothing effects.

High molecular weight polysaccharides have a structure in which monosaccharide units are linked by glycosidic bonds, and polysaccharides generally contain 10 or more monosaccharides. In addition, it has a lot of -OH groups, forming hydrogen bonds with water molecules in aqueous solutions to form a huge matrix, and has a high water holding power, so it acts as a moisturizer. It has a unique stickiness and a moist and soft feeling as it dries after application to the skin. Therefore, it is used as a feeling improver.

High molecular polysaccharides are specifically one of those obtained from mycelium such as hyaluronic acid, xanthan gum, dextran, gellan gum, pullulan, beta-glucan, structural polysaccharides such as cellulose and chitin, and storage polysaccharides such as starch and glycogen. .

High-molecular polysaccharides have useful effects on the skin, but because they contain -OH groups in the monomer, the intermolecular attraction is strong, so when a formulation containing a high content is manufactured, formulation stability is reduced. Additionally, since it is a water-soluble substance, it has limitations in penetrating into skin made of lipids.

There are various types of carriers that can carry active ingredients, such as emulsions, Pickering emulsions, liposomes, and capsules, but they usually require a unique structure to carry oil-soluble ingredients and water-soluble active ingredients. Liposomes are composed of a spherical, closed bilayer composed of phospholipids as the main component, and have a structure with a spherical space inside that can contain water-soluble substances. What makes this possible is that phospholipids are amphipathic, and specifically, they consist of a polar (hydrophilic) head and a nonpolar (lipophilic) tail, which is an alkyl chain. In addition, liposomes have excellent affinity as phospholipids have a similar component structure to the cell membrane of biological cells, so they not only increase the fluidity of biological membranes, but also effectively and safely deliver oil-soluble and water-soluble active substances into cells at the same time, giving the effect to the desired area. There is.

Korean Patent No. 10-0530880 does not use chemically synthesized emulsifiers that tend to cause irritation to sensitive skin, but uses natural phospholipids extracted from soybeans as an emulsifier, so it is non-irritating to the skin and contains high-pressure emulsifiers for ingredients that were difficult to formulate. A cosmetic product made of nano-sized phospholipid liposomes, which is encapsulated in nano-sized phospholipid liposomes and configured to be stably delivered deep into the skin, and a method for producing the same are disclosed.

Korean Patent Publication No. 10-2015-0074390 involves preparing a phospholipid solution in which phospholipids are dissolved in a glycol solvent and a solution in which a cholesterol compound and an active material are dissolved in oil, and mixing the phospholipid solution with the solution containing the active material. Disclosed is a method for producing liposome nanoparticles encapsulated with an active material, including the step of preparing a solution, and a cosmetic composition for improving wrinkles.

Conventional high molecular polysaccharides are water-soluble substances and have high intermolecular attraction, which limits their absorption into lipid-based skin. Accordingly, the present inventors stabilized high-molecular polysaccharide into liposomes in a high content to facilitate transdermal absorption, confirmed that the cosmetic composition containing this improved skin moisturizing, elasticity, and soothing effects, and completed the present invention. It has been done.

KR 10-0530880 B KR 10-2015-0074390 A

Therefore, the main purpose of the present invention is to provide a method for producing nanovesicles that stably capture high molecular weight polysaccharides.

Another object of the present invention is to provide a cosmetic composition having skin moisturizing, elasticity, or soothing effects containing nanovesicles containing high molecular polysaccharide prepared by the above production method.

As used herein, the term “nanovesicle” refers to a nanoliposome made of a lipid bilayer as a vesicle structure with a nanoscale size. The size of the nanovesicles of the present invention has an average particle diameter of 100-500 nm , preferably 150-250 nm. am. Additionally, the term “entrapment” means that the nanovesicle contains an active ingredient such as a high molecular weight polysaccharide.

According to one aspect of the present invention, the present invention provides a method for producing nanovesicles containing polymeric polysaccharides comprising the following steps:

1) Preparing an active ingredient base by dispersing high molecular polysaccharide in polyol;

2) mixing the active ingredient base with a primary oil base containing an oil-based gelling agent and a non-ionic surfactant to perform primary emulsification; and

3) Mixing the primary emulsification result and purified water with a secondary oil base containing polyol, fatty acid, lecithin, and cholesterol, followed by secondary emulsification using a high-pressure emulsifier.

In the present invention, the polymer polysaccharide has an -OH group and thus has hydrophilic properties. Any polymer polysaccharide polymerized with a long chain of monosaccharide units can be used, but preferably the polymer polysaccharide is hyaluronic acid, xanthan gum, dextran, or gellan. a mycelium-derived polysaccharide selected from gum, pulludan, or beta-glucan; Structural polysaccharides selected from cellulose or chitin; and at least one selected from the group consisting of storage polysaccharides selected from starch or glycogen. In the present invention, the expression "polymer" is used in the sense that the polymer polysaccharide is polymerized into a long chain, so its molecular weight may vary depending on the monomer and degree of polymerization, but is preferably characterized as having a molecular weight of 3,000 to 5,000,000 daltons.

In an example of the present invention, hyaluronic acid or beta-glucan, as an example of the polymer polysaccharide, was captured in nanovesicles, and particle size, uniformity of size distribution, and long-term stability were confirmed. In the method for producing nanovesicles of the present invention, the polymer polysaccharide may contain 0.01% by weight to 10% by weight relative to the total weight of the nanovesicle composition, and preferably contains 0.1% by weight to 7% by weight. As shown in the examples of the present invention, a stable formulation can be maintained for a long period of time even if it contains a high content of 1% by weight or more of a high molecular weight polysaccharide with strong intermolecular attraction.

In the present invention, the polyol can be any polyol that has two or more -OH groups in the molecule and is compatible with hydrophilic components. Preferably, the polyol is propylene glycol, butylene glycol, or pentylene glycol. It provides a method for producing nanovesicles, characterized in that one or more polyols selected from the group consisting of hexylene glycol, glycerin, methyl propanediol, and dipropylene glycol. In the examples of the present invention, butylene glycol, glycerin, and methylpropanediol were used as examples of polyols in step 1), and dipropylene glycol was used as examples of polyols in step 3). In the method for producing nanovesicles of the present invention, in step 1), the polyol may contain 35% to 60% by weight relative to the total weight of the nanovesicle composition, and preferably contains 40% to 55% by weight. do. Additionally, in step 3), the polyol may contain 5% to 15% by weight, preferably 8% to 13% by weight, based on the total weight of the nanovesicle composition.

In the present invention, the oil-based gelling agent can be any oil-based gelling agent that has thickening properties and is compatible with lipophilic ingredients, but preferably, the oil-based gelling agent is processed from vegetable ingredients that do not contain petroleum-derived ingredients. It is preferable that it is a glyceryl ester system of vegetable ingredients produced through a process, and more preferably, the glyceryl ester system includes glyceryl behenate, glyceryl stearate, glyceryl stearate SE, glyceryl cocoate, glyceryl oleate, and glyceryl ester. A method for producing nanovesicles is provided, wherein the nanovesicles are at least one selected from the group consisting of lyl olivate, glyceryl citrate, glyceryl lactate, glyceryl linoleate, and trihydroxystearin. In the examples of the present invention, glyceryl stearate was used as an example of an oil-based gelling agent. In the method for producing nanovesicles of the present invention, the oil-based gelling agent may contain 0.5% by weight to 5% by weight, and preferably contains 1.5% by weight to 3% by weight, based on the total weight of the nanovesicle composition.

In the present invention, the nonionic surfactant has the characteristic of not generating ions in an aqueous solution and can be any nonionic surfactant that can be used as a wetting agent or emulsifier, but preferably the nonionic surfactant is polyethylene glycol. It is preferable that it is a polyglycerol fatty acid ester that does not contain polyglycerol fatty acid esters, and more preferably, the polyglyceryl fatty acid esters include polyglyceryl-2 caprate, polyglyceryl-3 caprate, polyglyceryl-4 caprate, and polyglyceryl-2 caprate. Oleate, polyglyceryl-4 oleate, polyglyceryl-5 oleate, polyglyceryl-6 oleate, polyglyceryl-2 sesquioleate, polyglyceryl-2 laurate, polyglyceryl-10 Laurate, polyglyceryl-10 myristate, polyglyceryl-10 oleate, polyglyceryl-10 palmitate, polyglyceryl-10 stearate, polyglyceryl-10 isostearate, polyglyceryl-10 diiso. Provided is a method for producing nanovesicles, characterized in that they contain at least one selected from the group consisting of stearate and polyglyceryl-10 distearate. In the examples of the present invention, polyglyceryl-10 myristrate was used as an example of the nonionic surfactant. In the method for producing nanovesicles of the present invention, the nonionic surfactant may contain 1% to 7% by weight relative to the total weight of the nanovesicle composition, and preferably contains 2% to 5% by weight. .

In the present invention, the fatty acid is a carboxylic acid with a long aliphatic chain, and can be any fatty acid that maintains a liquid state at room temperature. Preferably, the fatty acid is an unsaturated higher fatty acid such as palmitoleic acid, oleic acid, or linoleic acid. It provides a method for producing nanovesicles, characterized in that they contain at least one selected from the group consisting of linolenic acid, linolenic acid, and arachidonic acid. In the examples of the present invention, oleic acid was used as an example of the fatty acid. In the method for producing nanovesicles of the present invention, the fatty acid may contain 0.5% to 2% by weight, preferably 0.7% to 1.5% by weight, based on the total weight of the nanovesicle composition.

In the present invention, the lecithin added to the oil base in step 3) is an amphipathic polar lipid that not only has an emulsifying effect to mix oil and water, but also forms a nanovesicle composed of a bilayer together with cholesterol. It plays a stabilizing role. In the method for producing nanovesicles of the present invention, the lecithin may contain 3% to 10% by weight, preferably 4% to 7% by weight, based on the total weight of the nanovesicle composition. In addition, the cholesterol may contain 0.5% to 2% by weight based on the total weight of the nanovesicle composition, and preferably contains 0.7% to 1.5% by weight.

In the present invention, the dispersion of the polymer polysaccharide in step 1) is accomplished by stirring for 20-40 minutes using a general emulsification mixer (eg, Aji mixer, homo mixer) at 4-50°C; In step 2), the first emulsification is performed by stirring at 6-70°C for 5-20 minutes using a general emulsification mixer (eg, Aji mixer, homo mixer, etc.); In step 3), the secondary emulsification is first stirred at 6-70°C for 1-3 minutes using a general emulsification mixer (e.g., Aji mixer, homo mixer, etc.), and then stirred at 30-50°C using a high pressure emulsifier (e.g., high pressure). It is performed by high-pressure treatment for 2 to 4 cycles at a pressure of 500 to 1,500 bar using a mixer, high-pressure homogenizer, etc.

According to another aspect of the present invention, the present invention provides a cosmetic composition having skin moisturizing, elasticity, or soothing effects containing as an active ingredient a nanovesicle containing a high molecular polysaccharide prepared by the production method according to the present invention.

In the present invention, preferably, the nanovesicle contains 0.1 to 7% by weight of polymer polysaccharide, 47 to 68% by weight of polyol, 1.5 to 3% by weight of oil-based gelling agent, 2 to 5% by weight of nonionic surfactant, and 0.7 to 5% by weight of fatty acid. It provides a cosmetic composition comprising 1.5% by weight, 4 to 7% by weight of lecithin, 0.7 to 1.5% by weight of cholesterol, and 20 to 44% by weight of purified water.

In the present invention, a cosmetic composition is provided, wherein the polymer polysaccharide-containing nanovesicle is contained in an amount of 1 to 50 parts by weight based on 100 parts by weight of the cosmetic composition.

In the present invention, a cosmetic composition is preferably provided, wherein the composition has any one formulation selected from the group consisting of lotion, lotion, cream, essence, gel, pack, or ointment.

Conventional high-molecular polysaccharides have useful effects on the skin, but because they contain a lot of -OH groups, the intermolecular attraction is strong, so formulation stability is low when formulations containing high content are manufactured, and since they are water-soluble substances, penetration into lipid-based skin is limited. There was this. However, in the present invention, this problem was solved by stably capturing high molecular weight polysaccharides in nanovesicles. Specifically, the polymer polysaccharide was first emulsified by dispersing it in polyol and surfactant, and then secondarily emulsified by high-pressure emulsification. Furthermore, through in vitro experiments, it was confirmed that polymer polysaccharides captured in nanovesicles according to the present invention can significantly increase skin moisturizing, elasticity, and soothing effects compared to polymer polysaccharides not captured in nanovesicles.

1A to 1D are graphs showing the cytotoxicity evaluation of nanovesicles containing beta-glucan or hyaluronic acid according to the present invention against HaCaT.
Figures 2a to 2d are graphs showing the cytotoxicity evaluation of nanovesicles containing beta-glucan or hyaluronic acid according to the present invention on Fibroblast.
Figure 3a is a graph examining the expression of a skin moisturizing gene (Filaggrin) of beta-glucan-containing nanovesicles according to the present invention.
Figure 3b is a graph examining the expression of a skin moisturizing gene (Filaggrin) of hyaluronic acid-containing nanovesicles according to the present invention.
Figure 4 is a graph examining the expression of a gene (Caspase-14) related to skin moisturization of hyaluronic acid-containing nanovesicles according to the present invention.
Figure 5a is a graph examining the expression of skin elasticity-related genes (Collagen type I) in nanovesicles containing hyaluronic acid according to the present invention.
Figure 5b is a graph examining the expression of skin elasticity-related genes (Collagen type III) of beta-glucan-containing nanovesicles according to the present invention.
Figure 6 is a graph examining the expression of skin soothing-related gene (IL-6 mRNA) of beta-glucan-containing nanovesicles according to the present invention.
Figure 7a is a Western blot photograph examining the expression of a skin soothing-related gene (IL-6 protein) of beta-glucan-containing nanovesicles according to the present invention.
Figure 7b is a graph examining the expression of a skin soothing-related gene (IL-6 protein) of beta-glucan-containing nanovesicles according to the present invention.
Figure 7c is a Western blot photograph examining the expression of a skin soothing-related gene (IL-6 protein) of the hyaluronic acid-containing nanovesicle according to the present invention.
Figure 7d is a graph examining the expression of a skin soothing-related gene (IL-6 protein) of the hyaluronic acid-containing nanovesicle according to the present invention.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are merely for illustrating the present invention, the scope of the present invention is not to be construed as limited by these examples.

Examples and Comparative Examples: Preparation of nanovesicles

Vesicles containing the high molecular weight polysaccharide according to the present invention were prepared at the content according to Table 1. The method of producing a vesicle containing high molecular weight polysaccharide includes the following three steps.

1) Uniformly dispersing polymer polysaccharide in polyol (active ingredient base preparation step);

2) Mixing the active ingredient base with the first oil base to perform primary emulsification (Pre-mix);

3) mixing the primary emulsion, purified water, and secondary oil base and then performing secondary emulsification through high-pressure emulsification;

Ingredients Example 1 Example 2 Comparative Example 1 Comparative example 2 Active ingredient base Butylene glycol 5 5 5 5 glycerin - 40 40 40 Methylpropanediol 40 - - - high molecular weight polysaccharide 5 5 5 5 Total 50 50 50 50 1st oil painting Primary
paid glyceryl stearate 2 2 2 - Polyglyceryl-10 Myristrate 3 3 3 - Awards Active ingredient base 50 50 50 - Total 55 55 55 - 2nd oil painting Secondary
paid Dipropylene glycol 10 10 10 10 lecithin 5 5 5 5 Oleic Acid One One One One cholesterol One One One One glyceryl stearate - - - 2 Polyglyceryl-10 Myristrate - - - 3 Awards primary emulsion 55 55 55 Active ingredient base - - - 50 Purified water To 100 To 100 To 100 To 100 Manufacturing method high pressure oil painting high pressure oil painting simple mixing high pressure oil painting

As representative examples of high molecular weight polysaccharides, vesicles were manufactured using hyaluronic acid and beta-glucan. Examples 1 and 2 are intended to confirm differences depending on the type of polyol in step 1). Comparative Example 1 is to confirm the difference according to step 3) high pressure/simple emulsification, and Comparative Example 2 is to confirm the difference according to the presence or absence of step 2) (i.e., with and without primary emulsification).

The specific manufacturing methods of Examples 1 and 2 are as follows.

1) After mixing the components corresponding to the active ingredient base, slowly add the polymer polysaccharide and disperse it, then allow it to swell sufficiently (4-50℃, Azimixer, 30 minutes).

2) The ingredients corresponding to the first oil base are sequentially added and dissolved, then added to the active ingredient base and emulsified (6-70℃, homomixer, 10 minutes, first emulsification).

3) The components corresponding to the secondary oil base were sequentially added and dissolved, emulsified with the primary emulsion and purified water mixture (6-70℃, homomixer, 1 minute), and then mixed for the second time with a high-pressure mixer at around 40℃. Stable vesicles are prepared by emulsification. (High pressure homogenizer, 1000bar, 3cycle)

In Comparative Example 1, steps 1) and 2) are the same, and in step 3), the primary emulsion and purified water mixture are emulsified (6-70°C, homomixer, 10 minutes) and then cooled (40°C).

In Comparative Example 2, step 1) is followed by step 3). Each step is the same as Examples 1 and 2, except that step 2) is omitted, so the first oil base component of step 2) is included in the second oil base of step 3).

Experimental Example 1: Measurement of polymer polysaccharide vesicle particle size and PDI

The stability of the polymer polysaccharide vesicle was confirmed through particle size analysis. Particle size was analyzed using Zetasize advance (Malvern), and was measured after dispersing 1% of liposomes in water. Additionally, the particle size (unit: nm) was determined as the average value of five measurements.

In DLS analysis, PDI is an indicator of how uniform the sizes of colloidal particles dispersed in a colloidal solution are. The closer it is to 0, the more monodisperse (uniformity) the closer it is to 1, the more polydispersity (non-uniformity) it is. In general, if the value is less than 0.3, you can see that the size distribution is uniform, but if it is more than 0.7, it has a very wide distribution and is not suitable for DLS.

Particle size change 1 week later 2 weeks later 3 weeks later 4 weeks later Example 1 Particle size (nm) 231.2 354.1 377.2 392.8 425.1 PDI 0.2981 0.3275 0.3457 0.3624 0.3949 Example 2 Particle size (nm) 226.1 229.2 231.4 229.7 230.6 PDI 0.2898 0.2758 0.2985 0.2867 0.2967 Comparative Example 1 Particle size (nm) 798.4 854.3 945.4 1034.8 1185.3 PDI 0.8709 0.8624 0.8982 0.8525 0.8765 Comparative example 2 Particle size (nm) 245.1 374.9 424.6 493.7 573.9 PDI 0.2615 0.3445 0.3957 0.4274 0.4789

As shown in Examples 1 and 2, there is no significant difference in the size of the initial vesicle depending on the type of polyol that disperses the polymer polysaccharide, but it can be seen that as time passes, the PDI increases and the degree of dispersion decreases. It can be inferred that the dispersion force of the polymer polysaccharide is different from that of the polyol particles, and as a result, as time passes, the polymer polysaccharide aqueous solution in the vesicle pops out, causing collisions and coalescence between particles. However, it can be seen that in Examples 1 and 2, compared to Comparative Examples 1 and 2, not only did the particle size of the vesicle remain low over time, but the distribution was much more uniform, resulting in excellent formulation stability.

As shown in Comparative Example 1, it was confirmed that there was a difference in the stability of the vesicle depending on the emulsion strength. The stability of the vesicle in Example 2, which had a large emulsification strength, was significantly improved compared to Comparative Example 1, which had a low emulsification strength. This means that the greater the energy, the greater the force applied to the vesicle interface film, resulting in small, uniform, and stable vesicle particles. When the particle size is around 200 nm and the PDI value is around 0.3, the particles become thermodynamically stable and coalescence between particles decreases.

As shown in Comparative Example 2, it was confirmed that there was a difference in vesicle stability depending on the first emulsification (pre-mix). The stability of the vesicle in Example 2, which underwent primary emulsification, was significantly improved compared to Comparative Example 2, which omitted primary emulsification. This means that Example 2 creates self-assembled particles through primary emulsification and secondary high-pressure emulsification, which reduces the possibility of polymer polysaccharides sticking out of the vesicle and appears to be able to generate the final vesicle with less energy.

Experimental Example 2: Confirmation of long-term stability of polymer polysaccharide vesicles by temperature

It was confirmed that each composition continued to maintain formulation stability despite long-term temperature changes. The compositions of Examples 1 and 2 and Comparative Examples 1 and 2 were selected as test samples, and these compositions were manufactured under conditions of 4°C, room temperature, 45°C, and CYCLE (changed every 6 h from -20°C to 40°C). The formulation stability was evaluated every week for one month.

Example 1 Example 2 Comparative Example 1 Comparative example 2 1 week later 25℃ X X X X 45℃ X X O X CYCLE X X O X 4℃ X X X X 2 weeks later 25℃ X X X X 45℃ X X O X CYCLE X X O O 4℃ X X X X 3 weeks later 25℃ X X X X 45℃ O X O O CYCLE X X O O 4℃ X X X X 4 weeks later 25℃ X X X X 45℃ O X O O CYCLE O X O O 4℃ X X X X Phase separation occurs: O Phase separation does not occur:

As shown in the table above, compared to Comparative Examples 1 and 2, phase separation did not occur in the compositions of Examples 1 and 2 even with long-term temperature changes, showing that each composition continuously maintained formulation stability. This yielded similar results to DLS analysis.

Next, the in-vitro skin moisturizing, elasticity, and soothing effects of a cosmetic composition containing a polymer polysaccharide vesicle (Example 2) and a polymer polysaccharide not captured in the vesicle were compared. High molecular polysaccharides were tested separately into hyaluronic acid and beta-glucan.

Experimental Example 3: Cytotoxicity evaluation: MTT assay

Inoculate and culture human fibroblast or keratinocyte. Replace with serum-free medium, process the sample, and culture it. Replace the medium containing the prepared MTT solution, react for 4 hours, add DMSO, and measure the absorbance (570 nm) with an ELISA reader.

1A to 1D are graphs showing the cytotoxicity evaluation of nanovesicles containing beta-glucan or hyaluronic acid according to the present invention against HaCaT.

Both nanovesicles containing beta-glucan (LPS-Glucan) and beta-glucan aqueous solution (Glucan) showed no cytotoxicity up to 0.1-3%.

Nanovesicles containing hyaluronic acid (LPS-HA) showed no cytotoxicity up to 0.1-2%, and hyaluronic acid aqueous solution (HA) did not show cytotoxicity up to 0.1-3%.

Figures 2a to 2d are graphs showing the cytotoxicity evaluation of nanovesicles containing beta-glucan or hyaluronic acid according to the present invention on Fibroblast.

Both nanovesicles containing beta-glucan (LPS-Glucan) and beta-glucan aqueous solution (Glucan) showed no cytotoxicity up to 0.1-3%.

Nanovesicles containing hyaluronic acid (LPS-HA) showed no cytotoxicity up to 0.1-0.5%, and hyaluronic acid aqueous solution (HA) did not show cytotoxicity up to 0.1-3%.

Experimental Example 4: Investigation of gene expression related to skin moisturization: Real time PCR

Inoculate and culture human normal keratinocytes. Replace with serum-free medium, process the sample, and culture it. Collect RNA with QIAzol™ Lysis Reagent and extract RNA according to the manufacturer's method. After quantifying the extracted RNA, cDNA is synthesized, real-time PCR is performed, and gene expression (Filaggrin) is confirmed.

Figure 3a is a graph examining the expression of a skin moisturizing gene (Filaggrin) of beta-glucan-containing nanovesicles according to the present invention.

Both nanovesicles containing beta-glucan (LPS-Glucan) and beta-glucan aqueous solution (Glucan) showed similar efficacy in moisturizing the skin.

Figure 3b is a graph examining the expression of a skin moisturizing gene (Filaggrin) of hyaluronic acid-containing nanovesicles according to the present invention.

Nanovesicles containing hyaluronic acid (LPS-HA) showed more efficacy in moisturizing the skin than aqueous hyaluronic acid solution (HA).

Figure 4 is a graph examining the expression of a gene (Caspase-14) related to skin moisturization of hyaluronic acid-containing nanovesicles according to the present invention.

Nanovesicles containing hyaluronic acid (LPS-HA) showed more efficacy in moisturizing the skin than aqueous hyaluronic acid solution (HA).

Experimental Example 5: Investigation of skin elasticity-related gene expression: Real time PCR

After inoculating and culturing human fibroblasts, the medium is replaced with serum-free medium, and the samples are processed and cultured. Collect RNA with QIAzol™ Lysis Reagent and extract RNA according to the manufacturer's method. After quantifying the extracted RNA, cDNA is synthesized, real-time PCR is performed, and gene expression (Col-III) is confirmed.

Figure 5a is a graph examining the expression of skin elasticity-related genes (Collagen type I) in nanovesicles containing hyaluronic acid according to the present invention.

Nanovesicles containing hyaluronic acid (LPS-HA) showed greater efficacy in skin elasticity than aqueous hyaluronic acid solution (HA).

Figure 5b is a graph examining the expression of skin elasticity-related genes (Collagen type III) of beta-glucan-containing nanovesicles according to the present invention.

Nanovesicles containing beta-glucan (LPS-Glucan) showed better efficacy in terms of elasticity than beta-glucan aqueous solution (Glucan).

Experimental Example 6: Investigation of sedation-related gene expression: Real time PCR

Inoculate and culture human keratinocytes (HaCaT). After irradiating with UVB and replacing with serum-free medium, the sample is processed and cultured. Collect RNA with QIAzol™ Lysis Reagent and extract RNA according to the manufacturer's method. After quantifying the extracted RNA, cDNA is synthesized, real-time PCR is performed, and gene expression (IL-6) is confirmed.

Figure 6 is a graph examining the expression of skin soothing-related gene (IL-6 mRNA) of beta-glucan-containing nanovesicles according to the present invention.

Nanovesicles containing beta-glucan (LPS-Glucan) showed better efficacy in sedation than beta-glucan aqueous solution (Glucan).

Experimental Example 7: Investigation of sedation-related gene expression: Western blot

Inoculate and culture human keratinocytes (HaCaT). After irradiating with UVB and replacing with serum-free medium, the sample is processed and cultured. Cells are scraped with a cell scraper, centrifuged, and the supernatant is discarded. Then, protein extraction solution (iNtRON) is added, centrifuged, and the supernatant is collected. The extracted intracellular proteins were quantified and transferred to a membrane after SDS-PAGE. Then, it was blocked with 5% skim milk and treated with primary antibody [IL-6]. Washed with 1X TBST, treated with secondary antibody, and finally washed with 1X TBST, treated with ECL solution, and then confirmed the band with Chemi doc.

Figure 7a is a Western blot photograph examining the expression of a skin soothing-related gene (IL-6 protein) of the beta-glucan-containing nanovesicle according to the present invention.

Figure 7b is a graph examining the expression of a skin soothing-related gene (IL-6 protein) of beta-glucan-containing nanovesicles according to the present invention.

Vesicles containing beta-glucan (LPS-Glucan) showed better efficacy in sedation than beta-glucan aqueous solution (Glucan).

Figure 7c is a Western blot photograph examining the expression of a skin soothing-related gene (IL-6 protein) of the hyaluronic acid-containing nanovesicle according to the present invention.

Figure 7d is a graph examining the expression of a skin soothing-related gene (IL-6 protein) of the hyaluronic acid-containing nanovesicle according to the present invention.

Vesicles containing hyaluronic acid (LPS-HA) showed better efficacy in soothing than hyaluronic acid aqueous solution (HA).

Vesicles containing high molecular weight polysaccharides prepared by the method of the present invention can be formulated into various types of cosmetics, specifically lotions, cream packs, etc., and are not limited to those described below.

Formulation Example 1: Toner

ingredient content(%) Vesicles containing high molecular weight polysaccharides 10.0 Butylene glycol 6.0 glycerin 4.0 PEG 60 Hydrogenated Castor Oil 0.5 ethanol 3.0 EDTA-2Na 0.02 Fragrance, preservative q.s Purified water TO 100

Formulation Example 2: Lotion

ingredient content(%) Vesicles containing high molecular weight polysaccharides 20.0 Butylene glycol 6.0 glycerin 4.0 Polysorbate 60 2.0 Sorbitan sesquioleate 1.5 squalane 3.0 argan oil 2.0 carbomer 0.14 Triethanolamine 0.10 EDTA-2Na 0.02 Fragrance, preservative q.s Purified water TO 100

Formulation Example 3: Cream

ingredient content(%) Vesicles containing high molecular weight polysaccharides 30.0 Butylene glycol 8.0 glycerin 5.0 Glyceryl Stearate 1.0 Polyglyceryl-3 methylglucose distearate 3.0 Cetostearyl alcohol 1.5 Cetyl ethylhexanoin 3.0 squalane 5.0 xanthan gum 0.2 carbomer 0.25 Triethanolamine 0.20 EDTA-2Na 0.02 Fragrance, preservative q.s Purified water TO 100

Formulation Example 4: Pack

ingredient content(%) Vesicles containing high molecular weight polysaccharides 40.0 Butylene glycol 4.0 glycerin 3.0 PEG-40 Hydrogenated Castor Oil 0.5 Hydrogenated polyisobutene 3.0 xanthan gum 0.1 Carboxy vinyl polymer 0.3 EDTA-2Na 0.02 Fragrance, preservative q.s Purified water TO 100

Although the present invention has been described with reference to the above embodiments, this is merely illustrative, and those skilled in the art will understand that various modifications and other equivalent embodiments are possible therefrom. . Therefore, the true technical protection scope of the present invention should be determined by the technical details of the attached patent claims.

Claims (9)

A method for producing a nanovesicle containing a polymer polysaccharide comprising the following steps, wherein the nanovesicle is a nanoliposome made of a lipid bilayer and the average particle diameter of the nanovesicle is 100-500 nm:
1) Preparing an active ingredient base by dispersing high molecular polysaccharide in polyol;
2) mixing the active ingredient base with a primary oil base containing an oil-based gelling agent and a non-ionic surfactant to perform primary emulsification; and
3) Mixing the primary emulsification result and purified water with a secondary oil base containing polyol, fatty acid, lecithin, and cholesterol, followed by secondary emulsification using a high-pressure emulsifier. The method of claim 1, wherein the high molecular polysaccharide is a mycelium-derived polysaccharide selected from hyaluronic acid, xanthan gum, dextran, gellan gum, pulludan, or beta-glucan; Structural polysaccharides selected from cellulose or chitin; and a storage polysaccharide selected from starch or glycogen. The method of claim 1, wherein the polyol is at least one selected from the group consisting of propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, glycerin, methylpropanediol, and dipropylene glycol. Manufacturing method of nanovesicles characterized. The method of claim 1, wherein the oil-based gelling agent is a glyceryl ester type and includes glyceryl behenate, glyceryl stearate, glyceryl stearate SE, glyceryl cocoate, glyceryl oleate, glyceryl olivate, and glyceryl sheet. A method for producing nanovesicles, characterized in that at least one selected from the group consisting of glyceryl lactate, glyceryl linoleate, and trihydroxystearin. The method of claim 1, wherein the nonionic surfactant is polyglycerol fatty acid ester, and includes polyglyceryl-2 caprate, polyglyceryl-3 caprate, polyglyceryl-4 caprate, polyglyceryl-2 oleate, and polyglyceryl-2 caprate. Glyceryl-4 oleate, polyglyceryl-5 oleate, polyglyceryl-6 oleate, polyglyceryl-2 sesquioleate, polyglyceryl-2 laurate, polyglyceryl-10 laurate, poly Glyceryl-10 myristate, polyglyceryl-10 oleate, polyglyceryl-10 palmitate, polyglyceryl-10 stearate, polyglyceryl-10 isostearate, polyglyceryl-10 diisostearate and poly A method for producing nanovesicles, characterized in that at least one selected from the group consisting of glyceryl-10 distearate. The nanovesicle according to claim 1, wherein the fatty acid is an unsaturated higher fatty acid and is at least one selected from the group consisting of palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid. Manufacturing method. A cosmetic composition having skin moisturizing, elasticity, or soothing effects containing nanovesicles containing high molecular weight polysaccharides prepared by the production method according to any one of claims 1 to 6 as an active ingredient. The method of claim 7, wherein the nanovesicle contains 0.1 to 7% by weight of polymer polysaccharide, 47 to 68% by weight of polyol, 1.5 to 3% by weight of oil-based gelling agent, 2 to 5% by weight of nonionic surfactant, and 0.7 to 1.5% by weight of fatty acid. %, a cosmetic composition comprising 4 to 7% by weight of lecithin, 0.7 to 1.5% by weight of cholesterol, and 20 to 44% by weight of purified water. The cosmetic composition according to claim 7, wherein the polymer polysaccharide-containing vesicle is contained in an amount of 1 to 50 parts by weight based on 100 parts by weight of the cosmetic composition.