Cosmetic composition containing snail mucus exosomes for anti-inflammation and skin regeneration

Abstract

The present invention relates to a cosmetic composition for anti-inflammatory and skin regeneration containing snail mucus exosomes. The exosomes inhibit the production of nitric oxide (NO), exhibit excellent skin cell regeneration efficacy, have moisturizing efficacy that increases the gene expression of Aquaporin-3, and have excellent skin barrier improvement efficacy that increases the gene expression of Filaggrin, Claudin-1, or Cadherin-1 (E-Cadherin, CDH1), making them easy to use as a multifunctional cosmetic composition.

Description

Technology Field

[0001] The present invention relates to a cosmetic composition for anti-inflammatory and skin regeneration containing snail mucus exosomes. Background Technology

[0002] All animal and plant cells secrete extracellular vesicles. Vesicles are a general term for membrane-bound vesicles secreted by cells for intercellular information exchange, and include exosomes, ectosomes, microvesicles, microparticles, and apoptotic bodies. Exosomes are vesicles tens to hundreds of nanometers in size composed of a phospholipid bilayer identical to the structure of a cell membrane. They contain cytokines, growth factors, miRNAs, DNA, proteins, etc., and because they reflect the state of the originating cell (donor cell), they contain specific genetic material and bioactive factors depending on the properties and state of the cell from which they originated.

[0003] Although research using these exosomes has been conducted in the pharmaceutical and cosmetics industries, various studies are being more actively carried out to extract exosomes with improved quality, stability, safety, high-efficiency functionality, and delivery capabilities from the same materials.

[0004] Snails belong to the class Gastropoda, and snail mucus contains allantoin, glycolic acid, collagen, elastin, mucin, etc., and has already been used as a functional cosmetic ingredient.

[0005] Accordingly, the inventors confirmed that exosomes extracted from snail mucus have superior skin-improving efficacy compared to existing snail mucus extracts or snail mucus exosomes extracted by other methods, thereby completing the present invention. Prior art literature

[0006] (Patent Document 0001) KR 2701915 B1(Patent Document 0002) KR 2058294 B1 The problem to be solved

[0007] The objective of the present invention is to provide a cosmetic composition for anti-inflammatory and skin regeneration containing snail mucus exosomes. means of solving the problem

[0008] The present invention relates to an anti-inflammatory cosmetic composition containing snail mucus exosomes.

[0009] The snail mucus exosome of the present invention is characterized by having an average particle size of 100 to 120 nm and a particle size distribution of 90% or more (D90) of 150 nm or less.

[0010] The above exosomes inhibit the production of NO (Nitric oxide).

[0011] The above exosomes may have the efficacy to regenerate skin cells.

[0012] The above exosomes have moisturizing effects and, preferably, increase the gene expression of Aquaporin-3 (AQP-3) in skin cells.

[0013] The above exosomes have skin barrier strengthening efficacy and, preferably, enhance the gene expression of filaggrin (FLG), Claudin-1 (CLDN1), or cadherin-1 (Cadherin 1 / E-Cadherin, CDH1).

[0014] The present invention will be described in detail below.

[0015] To recover exosomes according to the present invention, after obtaining snail mucus, centrifuge at 8,000 to 15,000 rpm for 5 to 30 minutes, and recover only the supernatant. The recovered supernatant is then ultrafiltration to remove the culture medium components, and macroparticles are removed through membrane filtration of 0.1 to 10 μm, for example, membrane filtration with pore sizes of 10, 5, 2, 1, 0.6, 0.45, and 0.1 μm, and exosomes according to the present invention can be obtained.

[0016] The snail mucus exosomes of the present invention can increase AQP-3 gene expression by 1.3 to 1.5 times at 0.75 to 3.00 ppm. In addition, the exosomes increase CDH1, CLDN1, and FLG mRNA expression by 1.5 to 1.7 times, 1.5 to 1.8 times, and 1.7 to 2.0 times, respectively, at 3.0 ppm. The NO inhibition rate is 90 to 95% at 3.0 ppm, and cell regeneration is shown to increase by approximately 1.2 to 1.3 times at 0.75 to 3.0 ppm.

[0017] Snail mucus can be obtained by adding 1 to 5 times the weight of purified water to raw snails and extracting at 25 to 35°C for 3 to 10 hours.

[0018] The formulation of the cosmetic composition of the present invention may be any formulation conventionally manufactured in the art, and may be an essence, lotion, cream, emulsion, pack, hand cream, foot cream, body lotion, lip balm, lipstick, eyeshadow, eyeliner, eyebrow pencil, blusher, highlighter, general cosmetic water, skin toner, cream, serum, beauty soap, softening cosmetic water, medicinal cosmetic water, body cleanser, cleansing foam, cleansing lotion, gel, cleansing oil, cleansing cream, cleansing tissue, cleansing water, mask pack, and other hair products. In particular, when it is a various cosmetic product for hair growth, improvement of hair loss, improvement of scalp, etc., the formulation is preferably not particularly limited, and more specifically, hair soap, hair shampoo, hair rinse, hair serum, hair treatment, hair essence, hair water, hair tonic, hair lotion, hair emulsion, It can be selected from any formulation such as hair cream, hair massage cream, hair wax, hair pack, hair oil, hair dryer, hair preservative, hair dye, hair bleach, hair gel, hair glaze, hair mousse, hair spray, hair ampoule, etc.

[0019] More specifically, when the formulation of the cosmetic composition of the present invention is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane-butane, or dimethyl ether may be additionally included. In the case where the formulation of the cosmetic composition of the present invention is a solution or an emulsion, a solvent, a solvating agent, or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan. In the case where the formulation of the cosmetic composition of the present invention is a suspension, a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol or polyoxyethylene sorbitol ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanthera may be used as a carrier component. In the case where the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolin derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.The cosmetic composition of the present invention may additionally contain excipients including fluorescent substances, fungicides, hydrotropism-inducing substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunblocks, vitamins, plant extracts, etc. Depending on the formulation or purpose of use, the amount of the above ingredients added may be selected within a range that does not impair the inherent effects of the cosmetic composition. The amount of the above ingredients added may be, for example, 0.1 to 10 weight%, preferably 0.1 to 6 weight%, based on the total weight of the composition, but is not limited thereto. Effects of the invention

[0020] The present invention relates to a cosmetic composition for anti-inflammatory and skin regeneration containing snail mucus exosomes. The exosomes inhibit the production of nitric oxide (NO), exhibit excellent skin cell regeneration efficacy, have moisturizing efficacy that increases the gene expression of Aquaporin-3, and have excellent skin barrier improvement efficacy that increases the gene expression of Filaggrin, Claudin-1, or Cadherin-1 (E-Cadherin, CDH1), making them easy to use as a multifunctional cosmetic composition. Brief explanation of the drawing

[0021] FIG. 1 is a flowchart illustrating an example of a process for purifying snail mucus exosomes and snail mucus in the present invention. Figure 2 shows the NTA analysis results and Cryo-TEM image analysis results showing the structure of the snail mucus exosome of the present invention. Figure 3 shows the results of the MTT assay for snail mucus exosomes and snail mucus samples in HaCaT cells. Figure 4 shows the results confirming the efficacy of snail mucus exosomes and snail mucus samples in increasing AQP-3 gene expression. Figure 5 shows the results confirming the efficacy of snail mucus exosomes and snail mucus samples in increasing CDH1 gene expression. Figure 6 shows the results confirming the efficacy of snail mucus exosomes and snail mucus samples in increasing CLDN1 gene expression. Figure 7 shows the results confirming the efficacy of snail mucus exosomes and snail mucus samples in increasing FLG gene expression. Figure 8 shows the results of the MTT assay for snail mucus exosomes and snail mucus samples in RAW 264.7 cells. Figure 9 shows the results confirming the efficacy of snail mucus exosomes and snail mucus samples in inhibiting NO (Nitric oxide) production. Figure 10 shows the results of the MTT assay for snail mucus exosomes and snail mucus samples in HDF cells. Figure 11 shows the results of confirming cell proliferation for snail mucus exosomes and snail mucus samples in HDF cells. Specific details for implementing the invention

[0022] Preferred embodiments of the present invention will be described in detail below. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the content introduced herein is provided to fully convey the concept of the present invention to those skilled in the art, so that it may be thorough and complete.

[0023] <Example 1. Preparation of Snail Mucus Exosomes>

[0024] A snail in a living state ( Achatina fulica , Acatina pulica was washed with running purified water to remove impurities and prepared as a raw material.

[0025] 800g of snails were added to 1.6kg of purified water and extracted at 30℃ for 5 hours, and the liquid phase was collected. The collected liquid phase was centrifuged at 10,000 rpm for 10 min, ultrafiltered to remove low molecular weight substances of 150,000 Da or less, and then filtered sequentially through 0.22㎛ and 0.11㎛ membrane filters to obtain snail mucus exosomes, which were then freeze-dried to form a powder.

[0026] At this time, attempts were made to manufacture exosomes without performing ultrafiltration in the above process; however, since the freeze-dried powder product aggregated or did not dissolve well when rehydrated in various solvents for use as a cosmetic ingredient, ultrafiltration was subsequently made mandatory. Furthermore, because the ease of storage for mass production of exosomes was poor when used without powdering, NTA analysis of exosome nanoparticles after long-term storage of 1 to 6 months showed very large deviations in dispersion and other parameters compared to immediately after manufacturing.

[0027] In other words, it was confirmed that this leads to a decrease in the production efficiency of snail mucus exosomes.

[0028] <Comparative Example 1. Preparation of Snail Mucus (Extract)>

[0029] 800g of snails were added to 1.6kg of purified water and extracted at 30℃ for 5 hours, and the liquid was collected. The collected liquid was sequentially filtered through membrane filters of 5㎛, 1㎛, 0.45㎛, and 0.22㎛ to obtain snail mucus (extract), which was then freeze-dried to form a powder.

[0030] <Experimental Example 1. Analysis of Snail Mucus Exosomes - NTA, Cryo-TEM>

[0031] The snail mucus exosomes of Example 1 were analyzed using nanoparticle tracking analysis (NTA). Particle size distribution and concentration were measured using a NanoSight NS300 (Malvern Panalytical, UK). Additionally, the lipid bilayer structure of the exosomes was confirmed using cryo-transmission electron microscopy (Cryo-TEM) to distinguish similarly sized particles separated from the exosomes. After preparing frozen specimens using liquid nitrogen, they were observed at 150,000x magnification using a Tecnai G2 Spirit TWIN (FEI, USA).

[0032] As a result, as shown in Table 1 and Figure 2, when the exosome particles of Example 1 were measured by Nano particle Tracking Analysis, the particle size was approximately 107 nm, with more than 90% of the particle size distribution (D90) being 150 nm or less, and the average particle concentration was 5.28 x 10⁻⁶ 9 It is confirmed to be particle / mL, and the presence of vesicles with a lipid bilayer structure can be confirmed by cryo-TEM image analysis.

[0033] Analysis sample name Average value (nm) Mode (nm) Standard deviation (nm) D90(nm) Particle count concentration (particles / mL) Snail mucus exosomes 107 84.5 40 150 5.28 x 10 9

[0034] <Experimental Example 2. Confirmation of Cytotoxicity against HaCaT>

[0035] Cell viability against HaCaT was measured using the MTT assay method. Cells were seeded into a 96-well plate and cultured for 24 hours in a humid incubator at 37°C supplied with 5% CO2. After treating with series dilutions of the exosomes from Example 1 at different concentrations, the cells were cultured for 24 hours in a humid incubator at 37°C supplied with 5% CO2.

[0036] After treating with an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution and reacting at 37 °C for 2 h, the supernatant was removed and the generated formazan was completely dissolved in 100 µl of DMSO. The absorbance was measured at 590 nm using a microplate reader.

[0037] MTT assay (HaCaT) Cell viability (%) SD Sample concentration (ppm) Untreated 100.00 2.40 Snail mucus exosomes 0.75 99.30 1.44 1.50 98.21 0.81 3.00 97.16 0.60 Snail mucus 0.75 99.14 3.75 1.50 100.28 0.79 3.00 101.14 2.60

[0038] The results for this are shown in Table 2 and Figure 3, where both the exosomes and mucus of Example 1 and Comparative Example 1 did not show cytotoxicity up to a concentration of 3 ppm.

[0039] <Experimental Example 3. Confirmation of skin moisturizing and skin barrier improvement efficacy - in vitro >

[0040] Experimental Example 3-1. AQP-3 verification

[0041] HaCaT cells 1.5 × 10 5 The cells were seeded into 60 mm culture dishes and cultured for 18 hours. The cultured HaCaT cells were replaced with serum-free DMEM medium, and each sample was treated with serial dilutions to non-cytotoxic concentrations. Harvesting was performed after 24 hours. The harvested cells were treated with NucleoZOL lysis buffer (MN, 740404), and RNA isolation was performed using the protocol provided by MN. The isolated RNA was quantified using a microplate reader, and cDNA was synthesized using the HiSenScript™ RH(-) RT PreMix Kit (intron, 25087). The gene was amplified using the 2X Real-Time PCR Master mix (Biofact, DQ385-40h), and the amplification product was quantified.

[0042] The conditions for each primer are as follows (Table 3).

[0043] Gene name Primer sequence AQP-3 Forward 5'-CCGTGACCTTTGCCATGTGCTT-3' Reverse 5'-TTGTCGGCGAAGTGCCAGATTG-3' GAPDH Forward 5'-GTCTCCTCTGACTTCAACAGCG-3' Reverse 5'-ACCACCCTGTTGCTGTAGCCAA-3'

[0044] AQP-3 Relative mRNA expression (%) SD Sample concentration (ppm) Untreated 100.00 1.19 Snail mucus exosomes 0.75 131.11 0.37 1.50 135.37 4.97 3.00 140.85 1.76 Snail mucus 0.75 105.21 5.86 1.50 108.09 1.41 3.00 110.51 2.64

[0045] As a result, as shown in Table 4 and Figure 4, snail mucus exosomes increased AQP-3 gene expression in a concentration-dependent manner, increasing it by approximately 1.41-fold at 3 ppm. Snail mucus increased AQP-3 gene expression by up to 1.11-fold at 3 ppm.

[0046] Experimental Example 3-2. Confirmation of CDH1, CLDN1, and FLG

[0047] Next, skin barrier strengthening factors CDH1, CLDN1, and FLG were identified using the same method as in Experimental Example 3-1.

[0048] Gene name Primer sequence CDH1 Forward 5'-TGAAGGTGACAGAGCCTCTGGAT-3' Reverse 5'-TGGGTGAATTCGGGCTTGTT-3' CLDN1 Forward 5'-CCGGCGACAACATCGTGAC-3' Reverse 5'-CGGGTTGCTTGCAATGTGC-3' FLG(Filaggrin) Forward 5'-GCTGAAGGAACTTCTGGAAAAGG-3' Reverse 5'-GTTGTGGTCTATATCCAAGTGATC-3' GAPDH Forward 5'-GTCTCCTCTGACTTCAACAGCG-3' Reverse 5'-ACCACCCTGTTGCTGTAGCCAA-3'

[0049] Sample CDH1 CLDN1 FLG Relative mRNA expression (%) SD Relative mRNA expression (%) SD Relative mRNA expression (%) SD Sample concentration (ppm) Untreated 100.00 3.00 100.00 0.88 100.00 0.10 Snail mucus exosomes 0.75 116.76 0.31 137.96 0.76 101.5 3.34 1.50 144.64 0.13 146.62 0.10 116.76 0.67 3.00 162.64 6.02 153.81 1.10 183.17 3.33 Snail mucus 0.75 100.17 5.56 99.04 2.56 100.14 2.96 1.50 107.00 2.98 101.02 2.47 102.78 4.64 3.00 115.62 2.27 100.97 2.32 108 1.46

[0050] The results are shown in Table 6 and Figures 5 to 7. At a concentration of 3 ppm, snail mucus exosomes increased CDH1, CLDN1, and FLG mRNA expression by 1.63, 1.54, and 1.83 times, respectively. Snail mucus increased the gene expression of CDH1 by 1.16 times at a concentration of 3 ppm. On the other hand, it had a negligible effect on claudin-1 and filaggrin.

[0051] <Experimental Example 4. Confirmation of Cytotoxicity against RAW 264.7>

[0052] Cytotoxicity was confirmed by performing an MTT assay on RAW 264.7 using the method of Experimental Example 2, and the results are shown in Table 7 and Figure 8 below.

[0053] MTT (RAW 264.7) Cell viability (%) SD Sample concentration (ppm) Untreated 100.00 4.20 Snail mucus exosomes 0.75 103.60 0.65 1.50 104.22 0.72 3.00 95.07 5.37 Snail mucus 0.75 99.75 2.61 1.50 99.24 1.64 3.00 104.82 2.13

[0054] Table 7 and Figure 8 show that each sample of the present invention has no cytotoxicity at RAW 264.7 up to 15 ppm.

[0055] <Experimental Example 5. Confirmation of Anti-inflammatory Efficacy - NO (Nitric Oxide) Assay>

[0056] The amount of NO (Nitric oxide) produced from RAW 264.7 cells was determined using Griess reagent for the NO present in the cell culture medium ₂- 1×10⁶ RAW 264.7 cells were measured using DMEM (10% FBS) medium. ⁴ After adjusting the cells / well ratio, the cells were seeded into 96-well plates and cultured in a humid incubator at 37°C with 5% CO₂. Cells were treated with the sample at the following concentrations, and after 2 hours, 0.5 μg / mL of LPS was added, followed by 24 hours of incubation. The cell culture supernatant was mixed with Griess reagent in a 1:1 ratio and reacted in 96-well plates for 10 minutes, after which the absorbance was measured at 540 nm. The NO inhibition rate was calculated using the following formula.

[0057] Infection rate = [(NO % of LPS-treated group - NO %) of sample / (NO % of LPS-treated group - NO % of untreated group)] x 100

[0058] NO assay NO contents (%) SD Inhibition rate (%) Sample concentration (ppm) Untreated 0.87 0.77 LPS 100.00 3.10 LPS + Spiny mucus exosomes 0.75 81.66 0.91 18.50 1.50 42.90 0.62 57.60 3.00 8.28 1.32 92.53 LPS + snail mucus 0.75 100.31 2.84 - 1.50 99.96 2.48 0.04 3.00 94.29 1.20 5.85

[0059] Through the results of Table 8 and Figure 9, it can be seen that snail mucus exosomes have excellent anti-inflammatory efficacy, with an inhibition rate of approximately 92.53% of NO production at a concentration of 15 ppm, whereas snail mucus has a very negligible NO inhibition rate of 5.85% at a concentration of 15 ppm, which is practically the same as having no anti-inflammatory efficacy.

[0061] <Experimental Example 6. Confirmation of Cytotoxicity to HDF>

[0062] Cytotoxicity was confirmed by performing an MTT assay on HDF using the method of Experimental Example 2, and the results are shown in Table 9 and Figure 10 below.

[0063] MTT (HDF) Cell viability (%) SD Sample concentration (ppm) Untreated 100.00 2.68 Snail mucus exosomes 0.75 99.05 2.56 1.50 100.20 1.19 3.00 97.23 1.98 Snail mucus 0.75 99.60 2.75 1.50 100.77 1.25 3.00 100.14 1.26

[0064] Table 9 and Figure 10 show that each sample of the present invention has no cytotoxicity in HDF up to 3 ppm.

[0065] <Experimental Example 7. Confirmation of Cell Regeneration Efficacy>

[0066] Human dermal fibroblast (HDF) cells were seeded into a 96-well plate at a density of 6.0 x 10³ cells / well and cultured for 18 hours. The medium was replaced with DMEM containing 2% FBS, and the sample was serially diluted to non-cytotoxic concentrations and reacted in an incubator at 37°C with 5% CO2. After 24 hours, when formazan was produced by adding 5 mg / ml of MTT sample, the supernatant was removed, and the formazan was dissolved in DMSO and reacted under light protection for 1 hour, after which the absorbance was measured at 590 nm.

[0067] Cell regeneration Cell Proliferation (%) SD Sample concentration (ppm) Untreated 100.00 1.96 Snail mucus exosomes 0.75 120.45 5.35 1.50 121.19 0.90 3.00 123.87 2.34 Snail mucus 0.75 100.60 0.83 1.50 98.68 1.71 3.00 103.65 0.82

[0068] As shown in the results of Table 10 and Figure 11, snail mucus exosomes exhibited excellent cell proliferation efficacy of 1.20 times at 0.75 ppm, 1.21 times at 1.5 ppm, and 1.24 times at 3 ppm, but snail mucus had no effect on the cell condition even at a concentration of 3 ppm.

Claims

Claim 1 Anti-inflammatory cosmetic composition characterized by containing snail mucus exosomes. Claim 2 An anti-inflammatory cosmetic composition according to claim 1, characterized in that the exosomes inhibit the production of NO (Nitric oxide). Claim 3 An anti-inflammatory cosmetic composition according to claim 1, characterized in that the exosomes have skin cell regenerative efficacy. Claim 4 An anti-inflammatory cosmetic composition according to claim 1, characterized in that the exosomes have moisturizing efficacy. Claim 5 An anti-inflammatory cosmetic composition according to claim 1, characterized in that the exosomes increase the protein expression of Aquaporin-3 in skin cells. Claim 6 An anti-inflammatory cosmetic composition according to claim 1, characterized in that the exosomes have a skin barrier strengthening effect. Claim 7 An anti-inflammatory cosmetic composition according to claim 1, characterized in that the exosomes enhance the gene expression of filaggrin (FLG), Claudin-1 (CLDN1), or Cadherin-1 (Cadherin 1 / E-Cadherin, CDH1).

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