Compositions for skin lightening comprising green onion-derived exosome-like nanoparticles as active ingredients

Abstract

The cosmetic composition for skin whitening containing exosome-like nanoparticles derived from green onions separated by the polyethylene glycol precipitation method of the present invention as an active ingredient has the effect of significantly inhibiting tyrosinase expression and reducing melanin production, and exhibits an enhanced whitening effect even at low concentrations compared to hot water extracts, so it can be widely utilized in the field of developing safe and effective natural whitening cosmetics.

Description

Technology Field

[0001] The present invention relates to a skin whitening composition comprising exosome-like nanoparticles derived from green onions as an active ingredient. Background Technology

[0002] Skin pigmentation is a skin condition caused by an excessive increase in melanin synthesis, primarily triggered by factors such as UV exposure, inflammation, and hormonal changes. Melanin is produced in melanosomes within skin cells and plays a crucial role in determining skin color and protecting against external stimuli. Tyrosinase, a key enzyme in the melanin synthesis process, oxidizes tyrosine and accelerates the initial stages of melanin production. Excessive activity of this enzyme can lead to pigmentation-related conditions such as melasma and freckles.

[0003] Representative ingredients in whitening cosmetics designed to inhibit tyrosinase activity or reduce melanin production include arbutin, vitamin C, and kojic acid; these ingredients work by reducing melanin production through the inhibition of tyrosinase. However, these cosmetic ingredients may cause skin irritation or side effects in sensitive skin. In particular, they have the disadvantage of potentially weakening the skin barrier with long-term use, necessitating the need for safer and more effective alternatives.

[0004] Exosomes are small extracellular vesicles, ranging in size from 30 to 200 nanometers, secreted by cells. They are surrounded by a lipid bilayer and contain various biomolecules, including proteins, nucleic acids, and lipids. Exosomes play a crucial role in intercellular communication and are involved in physiological and pathological processes. Recent studies have revealed that exosomes can be potentially utilized in the diagnosis and treatment of various diseases, such as cancer, neurodegenerative diseases, and cardiovascular diseases.

[0005] Methods for isolating and purifying exosomes include ultracentrifugation, density gradient centrifugation, chromatography, and immunomagnetic separation. Ultracentrifugation is a traditional method that precipitates exosomes using high rotational force; while it can yield relatively high-purity exosomes, it has the disadvantages of requiring a long processing time and expensive equipment. Density gradient centrifugation separates exosomes using density differences; although it offers high purity, the process is complex and time-consuming. Chromatography separates exosomes using a column, allowing for the rapid acquisition of relatively high-purity exosomes, but it requires specialized equipment and technology. Immunomagnetic separation is a method that selectively separates exosomes using antibodies against specific antigens on the exosome surface; while it offers high specificity, it is expensive and has limitations in mass production.

[0006] Even within the same plant species, the composition and ratio of components in plant extracts can vary depending on the extraction site, harvest time, cultivation environment, extraction solvent, and method. According to Non-Patent Literature 1, a comparison of biochemical components and phytochemical profiles extracted from the leaves of various aquatic plant species revealed that the metabolites of each species differ. Furthermore, according to Non-Patent Literature 2, it can be seen that even among plants belonging to the same Asteraceae family, the total phenolic substances and flavonoid content vary significantly depending on the species.

[0007] Against this background, the inventors confirmed that exosome-like nanoparticles (Green-Onion derived Exosome, GE) isolated from crushed green onions using a polyethylene glycol precipitation method have a whitening effect by inhibiting melanin and tyrosinase at low concentrations compared to hot water extracts, thereby completing the present invention. Prior art literature

[0008] Korean Patent Publication No. 10-2024-0120701 (2024.08.07) Korean Patent Publication No. 10-2024-0037208 (2024.03.21)

[0009] Nazima Rashid et al, “Comparative biochemical analysis and GC-MS phytochemical profiling in some aquatic plants”, Chemical Papers 78 (3) 1931-1946 (2024)JunHyeok Kim et al, “Antioxidant Activity of Asteraceae Plant Seed Extracts”, Journal of Life Science 2021 Vol. 31.No. 6. 543~549 The problem to be solved

[0010] The objective of the present invention is to provide a cosmetic composition for skin whitening comprising exosome-like nanoparticles derived from green onions as an active ingredient.

[0011] Another objective of the present invention is (a) a step of preparing a ground green onion by washing and then grinding the green onion;

[0012] (b) A step of filtering the above-mentioned crushed green onion;

[0013] (c) a step of adding polyethylene glycol (PEG) to the filtered pulverized material and then precipitating it;

[0014] (d) a step of dissolving the above precipitate in phosphate-buffered physiological saline (PBS) after centrifugation; and

[0015] (e) A step of separating exosome-like nanoparticles by filtering the dissolved solution;

[0016] The present invention provides a method for preparing a cosmetic composition for skin whitening comprising exosome-like nanoparticles. means of solving the problem

[0017] To achieve the above objective, the present invention provides a cosmetic composition for skin whitening comprising exosome-like nanoparticles derived from green onions as an active ingredient.

[0018] The present invention also includes the step of (a) washing and then grinding green onions to produce ground green onion material;

[0019] (b) A step of filtering the above-mentioned crushed green onion;

[0020] (c) a step of adding polyethylene glycol (PEG) to the filtered pulverized material and then precipitating it;

[0021] (d) a step of dissolving the above precipitate in phosphate-buffered physiological saline (PBS) after centrifugation; and

[0022] (e) A step of separating exosome-like nanoparticles by filtering the dissolved solution;

[0023] A method for preparing a cosmetic composition for skin whitening comprising exosome-like nanoparticles is provided. Effects of the invention

[0024] The cosmetic composition for skin whitening containing exosome-like nanoparticles derived from green onions separated by the polyethylene glycol precipitation method of the present invention as an active ingredient helps improve skin tone by effectively inhibiting the production of melanin and tyrosinase, and as a naturally derived ingredient, it causes less skin irritation and has superior safety compared to synthetic whitening ingredients. In addition, due to the nanometer-sized exosome structure, the ingredient can be delivered deep into the skin, so it can be widely utilized in the development of cosmetics for safe and effective skin whitening and improvement of pigmentation while reducing the side effects of synthetic ingredients. Brief explanation of the drawing

[0025] Figure 1 shows a schematic diagram of the separation of green-onion-derived exosome-like nanoparticles (Green-Onion derived Exosome, GE) using the PEG precipitation method. Figure 2 shows the results of the separation and analysis of the characteristics of GE, Figure 2A shows the results of GE particle size analysis using the dynamic light scattering method, Figure 2B shows an electron microscope image, and Figure 2C shows the GE zeta potential using the electrophoretic light scattering method. Figure 3 shows the results of the cytotoxicity evaluation of GE on B16F10 cells. Figure 4 shows the results of analyzing the inhibitory effect of GE on melanin production in B16F10 cells, and the results of measuring the intracellular melanin content after treatment with 200 nM α-MSH and 10 μg / mL GE for 48 hours. Figure 5 shows the results of analyzing the inhibitory effect of GE on tyrosinase activity in B16F10 cells, and the results of measuring the tyrosinase activity of the cells after treatment with 200 nM α-MSH and 10 μg / mL GE for 48 hours. Figure 6 shows the results of analyzing the concentration-dependent inhibitory effect of GE on tyrosinase activity in B16F10 cells. Figure 7 shows the results of measuring the cytotoxicity of green onion hot water extract (Green-Onion derived Water Extract, GWE) on B16F10 cells. Figure 8 shows the results of a comparative evaluation of tyrosinase activity of GE and GWE on B16F10 cells. Specific details for implementing the invention

[0026] The present invention will be described in detail below.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0028] In the present invention, the term "cosmetic composition" means an article used on the human body to cleanse and beautify the human body, to add attractiveness and brighten the appearance, or to maintain or promote the health of the skin or hair.

[0029] In the present invention, the term "skin whitening" refers to a process of improving skin tone by inhibiting the activity of the tyrosinase enzyme or promoting the reduction of already produced melanin pigment. Tyrosine, produced in melanosomes—melanocytes present in the epidermis—is a precursor of melanin and is synthesized into melanin via DOPA-quinone. Tyrosinase is the enzyme involved in the oxidation of tyrosine into DOPA and dopachrome during this process. Inhibiting the activity of tyrosinase reduces melanin production, thereby bringing about the effect of brightening the skin tone.

[0031] The present invention provides a cosmetic composition for skin whitening comprising exosome-like nanoparticles derived from green onions as an active ingredient.

[0032] The above-mentioned green onion (Allium fistulosum L.) refers to an annual root plant belonging to the genus Allium of the Liliaceae family. As a representative spice vegetable, it is cultivated worldwide and used in almost all dishes. In addition to its value as a food seasoning, green onions possess various pharmacological effects. Green onions are known to have antibacterial, anticancer, blood pressure-lowering, antioxidant, anti-aging, and antiviral properties. However, there is no known efficacy regarding the inhibition of melanin and / or tyrosinase production.

[0033] The exosome-like nanoparticles mentioned above may be isolated from one or more parts selected from the group consisting of the root part, the blanched part, and the green part of the green onion, preferably isolated from the blanched part, but are not limited thereto.

[0034] The above "root portion" refers to the root part of the green onion, "white stem portion" refers to the white stem portion, and "green leaf portion" refers to the green leaf portion.

[0035] The above composition may contain the exosome-like nanoparticles at a concentration of 0.1 to 20 μg / mL, preferably at a concentration of 1 to 15 μg / mL, but is not limited thereto.

[0036] The exosome-like nanoparticles described above are characterized by being separated from crushed green onion using a polyethylene glycol precipitation method.

[0037] The polyethylene glycol (PEG) precipitation method is a simple yet efficient method for effectively separating nanoparticles such as exosomes. In the present invention, a solution containing exosomes derived from green onions was treated with PEG 8000 and then precipitated at a low temperature for a certain period of time. Subsequently, the precipitated nanoparticles were recovered through centrifugation to separate the exosome-shaped nanoparticles. The above method is a combination of physical and chemical treatments, which has the advantage of obtaining exosomes of relatively high purity while preserving heat-sensitive components.

[0038] Hot water extracts are produced by heating plants at high temperatures to extract active ingredients. While green onion hot water extract contains various bioactive components with antioxidant and anti-inflammatory effects, there is a possibility that some of these components may be destroyed during the high-temperature processing.

[0039] In the present invention, it was confirmed that exosome-like nanoparticles extracted by the polyethylene glycol precipitation method showed an enhanced whitening effect at low concentrations compared to hot water extracts.

[0040] The molecular weight of the polyethylene glycol may be 6,000 to 10,000, preferably 7,000 to 9,000, but is not limited thereto.

[0041] The molecular weight of polyethylene glycol (PEG) plays a significant role in precipitation methods. Generally, as the molecular weight of PEG increases, it affects the viscosity and osmotic pressure of the solution, which can lead to changes in precipitation efficiency and the size of the separated particles. For example, PEG with a low molecular weight exists in a liquid state, while PEG with a high molecular weight exists in a solid state. These differences in physical properties influence solubility and interaction modes during the precipitation process. Therefore, it is important to select PEG with an appropriate molecular weight depending on the specific separation purpose or the characteristics of the target substance.

[0042] The size of the exosome-like nanoparticles may be 50 to 200 nm, but is not limited thereto.

[0043] The above composition is characterized by inhibiting the activity of melanin or tyrosinase.

[0044] The above composition may be one or more formulations selected from the group consisting of serum, lotion, essence, paste, mask pack, patch, gel, cream, lotion, nourishing lotion, nourishing cream, moisturizing cream, massage cream, powder, soap, cleanser, oil, foundation, makeup base, wax, and spray, but is not limited thereto.

[0045] The above cosmetic composition may be prepared in any formulation conventionally manufactured in the technical field to which the present invention belongs. For example, the composition may be formulated into a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, massage cream, essence, ampoule, skin adhesive type, spray, etc. Specifically, it can be manufactured in formulations such as lotions like softening lotions or nourishing lotions, emulsions like facial lotions and body lotions, creams like nourishing creams, moisturizing creams, and eye creams, essences, cosmetic ointments, sprays, gels, packs, sunscreens, makeup bases, foundations in liquid, solid, or spray types, powders, makeup removers such as cleansing creams, cleansing lotions, and cleansing oils, cleansers such as cleansing foams, soaps, and body washes, liquid or gel formulations of mesotherapy that can be injected into the skin layer, and formulations mixed with hyaluronic acid gel.

[0046] The above cosmetic composition may further comprise any conventional cosmetic ingredient selected from additional ingredients commonly used in cosmetics, such as excipients, thickeners, dispersants, fragrances, fillers, preservatives, antiseptics, neutralizing agents, sweeteners, vitamins, free radical scavengers, metal ion chelating agents, functional ingredients, and mixtures thereof. A person skilled in the art may select any additional ingredient and / or the amount thereof so that the advantageous properties of the composition according to one aspect are not adversely affected or are substantially affected by the expected addition.

[0047] For example, the above excipients may include surfactants, emulsifiers, saponic acid, solvents, coloring agents, preservatives, antioxidants, antifoaming agents, antibacterial agents, anti-redeposition agents, enzymes, plant or mineral oils, fats, fluorescent substances, fungicides, hydrotropy-inducing substances, humectants, fragrances, preservatives, proteins, silicones, solubilizers, sugar derivatives, sunblocks, vitamins, plant extracts, waxes, fatty acids, higher alcohols, hydrocarbon oils, ester oils, triglycerides, vegetable oils, etc.

[0048] The above cosmetic composition may additionally include ingredients that are typically added to cosmetic compositions, such as purified water, surfactants, moisturizers, lower alcohols, chelating agents, disinfectants, preservatives, antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0049] In addition, the above cosmetic composition may be appropriately formulated with ingredients commonly used in external skin preparations such as cosmetics or pharmaceuticals, for example, aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof as needed. The above cosmetic composition may also appropriately incorporate metal chelating agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, bellapamil, licorice extract, glablidin, hot water extract of the fruit of Carin, various herbal medicines, pharmaceuticals such as tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts, vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, trehalose, etc.

[0050] In the case where the above cosmetic composition is a surfactant-containing cleansing formulation, it may further include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic acid derivative, or ethoxylated glycerol fatty acid ester, etc., as a carrier component.

[0051] In the case where the cosmetic composition according to one aspect is in the form of a cream or gel, it may further include animal oil, vegetable oil, wax, paraffin, starch, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide as a carrier component.

[0052] In the case where the above cosmetic composition is in the form of a solution or emulsion, it may further include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, propylene glycol, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan as a solvent, solvating agent, or emulsifying agent.

[0053] In the case where the above cosmetic composition is in the form of a suspension, it may further include, as a carrier component, a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanth.

[0054] In the case where the above cosmetic composition is in the form of a powder or spray, it may further include lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder as a carrier component, and in particular, in the case of a spray formulation, it may further include a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether.

[0055] In addition, in one aspect, the cosmetic composition may further include an organic sunscreen and / or an inorganic sunscreen.

[0056] The above organic sunscreen may be ethylhexyl methoxycinnamate, bis-ethylhexyloxyphenol methoxyphenyl triazine, ethylhexyl salicylate, butyl methoxydibenzoyl methane, octocrylene, homosalate, isoamyl p-methoxycinnamate, diethylaminohydroxybenzoylhexyl benzoate, phenylbenzimidazole sulfonic acid, etc., and the above inorganic sunscreen may be titanium dioxide, zinc oxide, zinc oxide, etc.

[0057] The above cosmetic composition may be used by applying it alone or in combination, or by applying it in combination with other cosmetic compositions other than the cosmetic composition according to one aspect. Furthermore, all ingredients included in the above cosmetic composition do not exceed the regulations set by each country. Additionally, the cosmetic composition according to one aspect may be used according to a standard method of use, and the frequency of use may be varied depending on the user's skin condition or preference.

[0059] The present invention also includes the step of (a) washing and then grinding green onions to produce ground green onion material;

[0060] (b) A step of filtering the above-mentioned crushed green onion;

[0061] (c) a step of adding polyethylene glycol (PEG) to the filtered pulverized material and then precipitating it;

[0062] (d) a step of dissolving the above precipitate in phosphate-buffered physiological saline (PBS) after centrifugation; and

[0063] (e) A step of separating exosome-like nanoparticles by filtering the dissolved solution;

[0064] A method for preparing a cosmetic composition for skin whitening comprising exosome-like nanoparticles is provided.

[0065] The molecular weight of the polyethylene glycol may be 6,000 to 10,000, preferably 7,000 to 9,000, but is not limited thereto.

[0066] The precipitation in step (c) above may be carried out at 1 to 10°C for 1 to 30 hours, preferably at 2 to 6°C for 5 to 20 hours, but is not limited thereto.

[0068] Since the description of the above-mentioned manufacturing method, its effects, and all related descriptions are identical to those previously stated, such description is omitted to avoid excessive complexity in this specification due to redundancy.

[0070] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0072] Example 1. Experimental Materials and Methods

[0074] 1-1. Experimental Materials

[0076] Polyethylene glycol 8000, trypan blue solution (T8154), 3,4-dihydroxy-L-phenylalanine (L-Dopa) (D9628), and α-melanocyte-stimulating hormone (α-MSH) (M4135) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0078] 1-2. Cell Culture

[0080] B16F10 mouse melanoma cells were cultured in DMEM (Dulbecco′Modified Eagle′Medium) supplemented with 10% heat-treated fetal bovine serum and antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin) at 37°C and 5% CO2.

[0082] 1-3. Isolation of Green Onion-Derived Exosomes (GE)

[0084] Leek-derived exosomes (GE) were isolated using a modified polyethylene glycol (PEG) precipitation method based on previous studies. Leeks were purchased from a local market, and 500 g of leek stems were thoroughly washed with phosphate-buffered saline (PBS) and ground in an electric blender for a total of 3 minutes at intervals of 1 minute of operation followed by 30 seconds of rest. The ground leek was filtered through a cloth filter to remove large debris, and the filtrate was centrifuged sequentially at 3,000 g for 10 minutes, 7,000 g for 20 minutes, and 12,000 g for 30 minutes to remove impurities and small debris. The supernatant was filtered through a 0.45-μm syringe filter (Corning, Glendale, AZ, USA). PEG 8000 (Sigma-Aldrich) was added to a final concentration of 5%, and the mixture was precipitated at 4°C for 16 hours. Subsequently, the sample was centrifuged at 7,000 g for 30 minutes, the pellet was dissolved in PBS, and filtered through a 0.20-μm syringe filter (Corning). The separated GE was dispensed in 1 mL aliquots and stored at -80°C.

[0086] 1-4. Isolation of green onion hot water extract

[0088] Green-onion derived water extract (GWE) for comparison with green-onion-derived exosome-like nanoparticles (GE) was extracted through the following process. 500 g of green onion was thoroughly washed with phosphate-buffered saline (PBS), 1 L of distilled water (DW) was added, and the mixture was blended using an electric blender for a total of 3 minutes at intervals of 1 minute of operation followed by a 30-second rest. The blended green onion was heated at 95°C for 2 hours, and then centrifuged at 1000 g for 10 minutes to remove impurities, after which the supernatant was obtained. The supernatant was filtered using 3-M filter paper, and the filtrate was freeze-dried to obtain the green-onion hot water extract.

[0090] 1-5. Analysis of GE's Characteristics

[0092] The particle size and zeta potential of GE were analyzed using Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS) methods, respectively, with the Litesizer DLS (Litesizer DLS 100, Anton Paar, AUT) and ELSZneo (ELSZneo, Otsuka Electronics, JP) instruments. To measure particle size, 1 mL of GE solution was placed in a disposable cuvette, measured three times, and the average value was calculated. To measure zeta potential, 1 mL of GE solution was placed in a Zeta Cell (Otsuka Electronics), measured three times, and the average value was calculated. The morphology of GDENs was confirmed using a transmission electron microscope (TEM, JEM 1010, JEOL Ltd., Japan). Briefly, 5 μL of GE solution was placed on a Formba carbon-coated grid and left for 5 minutes. Subsequently, 5 μL of tungstic phosphate was added to the grid and reacted for 5 minutes for negative staining. After removing the remaining tungstic phosphate, the grid was dried overnight.

[0094] 1-6. Cytotoxicity Analysis

[0096] The cytotoxicity of GE was investigated using the trypan blue exclusion method. 1.14 × 10⁻⁶ 5 Cells were seeded into 35-mm culture dishes and cultured for 24 hours, after which they were treated with various concentrations of GE. After 48 hours, the cells were isolated by treatment with Trypsin-EDTA (TE) for 3 minutes, and an equal amount of medium was mixed with Trypan Blue and stained. 10 μL of the mixture was placed in a hemocytometer to count viable cells. The cytotoxicity of green onion hot water extract (GWE) was investigated using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. 1.14 × 10⁶ 5Canine cells were seeded into 35-mm culture dishes and cultured for 24 hours, after which various concentrations of GWE were added. After 48 hours, the medium was replaced with a medium containing 10% MTT and incubated at 37°C for 2 hours. After removing the medium, the resulting formazan was dissolved in isopropanol containing 4 mM HCl, and the absorbance was measured at 570 nm (Thermo Scientific, Waltham, USA).

[0098] 1-7. Measurement of Melanin Content

[0100] 7.5 × 10 5 Cells were seeded into 100-mm culture dishes and cultured for 24 hours. Subsequently, the cells were cultured for 48 hours under conditions of 200 nM α-MSH and GE. The cells were washed twice with cold PBS to remove the medium, and then collected using a cell scraper. The collected cells were lysed by sonication (Q55, Qsonica, Newtown, CT, USA) in a 1 M NaOH solution containing 10% DMSO. After heating the cell lysate at 85°C for 2 hours, 100 μL was dispensed into 96-well plates, and the absorbance was measured at 405 nm.

[0102] 1-8. Tyrosinase Activity Analysis

[0104] 2 × 10 5Cells were seeded into 60-mm culture dishes and cultured for 24 hours. Subsequently, the cells were treated with 200 nM α-MSH, GE, and Green-Onion Derived Water Extract (GWE) for 48 hours according to the conditions. After treatment, the cells were collected after treatment with TE. After washing with cold PBS, the cells were lysed by sonication (Qsonica) in PBS containing 1% Triton X-100 (0694-1L, Amresco, Solon, OH, USA). The lysate was centrifuged at 13,000 g for 20 minutes, and the supernatant was used for further analysis. After measuring the protein concentration, the supernatant corresponding to a protein amount of 30 μg was mixed with 0.1 M sodium phosphate buffer solution (pH 6.8) containing 2 mg / mL L-DOPA. After incubating the mixture at 37°C for 2 hours, the absorbance was measured at 405 nm (Thermo Scientific).

[0106] 1-9. Statistical Analysis

[0108] Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, Version 25; IBM Corp., New York, USA). Data were expressed as mean ± standard error, and statistical significance was determined using Student's t-test or Tukey's post-hoc test following one-way analysis of variance (ANOVA). Non-parametric statistics were used when the data did not follow normality.

[0110] Example 2. Characterization of exosome-like nanoparticles derived from green onions

[0112] The particle size of green onion-derived exosomes (GE) was analyzed using Dynamic Light Scattering (DLS) (Fig. 2A). As a result, the average hydrodynamic diameter of the particles was measured to be approximately 136.63 nm, and major peaks within the particle distribution appeared at 16.47 nm and 190.00 nm. This indicates that green onion-derived exosomes have a uniform nanometer-sized particle distribution.

[0113] The morphology of exosomes derived from green onions was confirmed using transmission electron microscopy (TEM) (Fig. 2B). Observation revealed that the exosomes derived from green onions exhibited a spherical nanoparticle structure, with a particle size of approximately 200 nm or less. This result is consistent with the particle size confirmed by DLS analysis and indicates that the exosomes maintain a constant size and shape.

[0114] The zeta potential of exosomes derived from green onions was analyzed using electrophoretic light scattering (ELS) (Fig. 2C). The average zeta potentials of samples GE-1, GE-2, and GE-3 were measured to be -12.64 mV, -12.89 mV, and -14.54 mV, respectively, with an overall average of -13.35 mV. This indicates that the exosomes derived from green onions are nanoparticles with appropriate stability and suggest high uniformity by exhibiting low potential dispersion.

[0116] Example 3. Cytotoxicity Evaluation

[0118] As a result of evaluating the cytotoxicity of leek-derived exosomes (GE) on B16F10 melanoma cells, no significant difference was observed compared to the control group (0 μg / mL) at concentrations of 1, 2.5, and 5 μg / mL, and cell viability was maintained at over 95%. At a concentration of 10 μg / mL, cell viability remained above 90%, and no toxicity was observed. However, at concentrations of 25 μg / mL or higher, cell viability decreased sharply, showing approximately 50% and 20% at concentrations of 50 μg / mL and 100 μg / mL, respectively. In particular, at concentrations of 25 μg / mL or higher, a significant decrease was observed compared to the control group ( ** p<0.01), it was confirmed that toxicity increased in a concentration-dependent manner. Through the above results, it can be confirmed that leek-derived exosomes do not exhibit cytotoxicity at low concentrations (10 μg / mL or less), but toxicity increases at high concentrations.

[0120] Example 4. Inhibitory effect of leek-derived exosomes on melanin production

[0122] The effect of leek-derived exosomes (GE) on melanin production was evaluated using B16F10 melanoma cells. In the experiment, melanin production was induced with α-MSH (200 nM), and then the melanin content was compared by treating with GE (10 μg / mL) or arbutin (200 μg), which was used as a positive control.

[0123] The α-MSH alone-treated group showed an increase in melanin production of approximately 20% or more compared to the control group ( **It was confirmed that α-MSH induces melanin synthesis (p<0.01). Arbutin (200 μg) inhibited melanin production induced by α-MSH (200 nM), but leek-derived exosomes (GE, 10 μg / mL) showed a stronger melanin inhibitory effect than arbutin. In the group treated with GE, the melanin content decreased more significantly compared to the group treated with α-MSH. Based on these results, it can be seen that leek-derived exosomes can effectively inhibit melanin production induced by α-MSH, and in particular, leek-derived exosomes exhibit excellent melanin inhibitory effects even at lower concentrations compared to arbutin.

[0125] Example 5. Inhibitory effect of leek-derived exosomes on tyrosinase production

[0127] The effect of leek-derived exosomes (GE) on tyrosinase production was evaluated using B16F10 melanoma cells. In the experiment, tyrosinase production was induced with α-MSH (200 nM), followed by treatment with GE (10 μg / mL) to compare melanin content. Analysis results showed that tyrosinase activity significantly increased in the group treated with α-MSH (200 nM), rising approximately 2.5 times compared to the control group. This suggests that α-MSH contributes to the promotion of melanin synthesis. Conversely, in the groups treated with GE (1, 5, and 10 μg / mL) along with α-MSH, tyrosinase activity showed a tendency to gradually decrease with increasing GE concentration. In particular, tyrosinase activity was significantly inhibited at concentrations of 5 μg / mL and 10 μg / mL, which showed a statistically significant difference compared to the α-MSH alone treatment group. The above results suggest that GE may inhibit melanin synthesis by inhibiting tyrosinase activity.

[0129] Example 6. Evaluation of cytotoxicity of green onion hot water extract

[0131] When melanoma cells were treated with green-onion derived water extract (GWE) at concentrations of 0, 10, 25, 50, 100, 250, 500, and 1000 μg / mL and cell viability was measured, cell viability remained above 100% at all concentrations, and no significant differences were observed. This suggests that GWE does not induce cytotoxicity within the experimental concentration range.

[0133] Example 7. Comparison of Tyrosinase Production Inhibitory Effects of Exosomes According to Extraction Method

[0135] In the group treated with α-MSH (200 nM), tyrosinase activity significantly increased by more than three times compared to the control group. In the group treated with α-MSH and 10 μg / mL of green-onion derived exosome-like nanoparticles (Green-Onion derived Exosome, GE), tyrosinase activity significantly decreased compared to the group treated with α-MSH alone ( ## , p < 0.01). It was confirmed that GE effectively inhibits tyrosinase activity induced by α-MSH.

[0136] In addition, tyrosinase activity was inhibited in the group treated with 500 μg / mL of green-onion derived water extract (GWE) together with α-MSH, but the inhibitory effect was relatively lower compared to the group treated with GE. This suggests that under the same experimental conditions, GE exhibits more effective tyrosinase inhibitory activity than GWE. Based on these results, it can be seen that GE has a superior effect in inhibiting melanin production.

Claims

Claim 1 A cosmetic composition for skin whitening containing exosome-like nanoparticles derived from green onions as an active ingredient. Claim 2 A cosmetic composition for skin whitening according to claim 1, characterized in that the exosome-like nanoparticles are isolated from one or more selected from the group consisting of the root portion, the blanched portion, and the green portion of a leek. Claim 3 A cosmetic composition for skin whitening according to claim 1, characterized in that the composition comprises the exosome-like nanoparticles at a concentration of 0.1 to 20 μg / mL. Claim 4 A cosmetic composition for skin whitening according to claim 1, characterized in that the exosome-like nanoparticles are separated from ground green onion using a polyethylene glycol precipitation method. Claim 5 A cosmetic composition for skin whitening according to claim 1, characterized in that the size of the exosome-like nanoparticles is 50 to 200 nm. Claim 6 A cosmetic composition for skin whitening according to claim 1, characterized in that the composition inhibits the activity of melanin or tyrosinase. Claim 7 A cosmetic composition for skin whitening according to claim 1, characterized in that the composition is one or more formulations selected from the group consisting of serum, lotion, essence, paste, mask pack, patch, gel, cream, lotion, nourishing lotion, nourishing cream, moisturizing cream, massage cream, powder, soap, cleanser, oil, foundation, makeup base, wax, and spray. Claim 8 (a) a step of preparing a chopped green onion by washing and grinding the green onion; (b) a step of filtering the chopped green onion; (c) a step of adding polyethylene glycol (PEG) to the filtered chopped green onion and allowing it to precipitate; (d) a step of centrifuging the precipitate and dissolving it in phosphate-buffered physiological saline (PBS); and (e) a step of separating exosome-like nanoparticles by filtering the dissolved solution; comprising a method for preparing a cosmetic composition for skin whitening containing exosome-like nanoparticles. Claim 9 A method for preparing a cosmetic composition for skin whitening, characterized in that, in claim 8, the molecular weight of the polyethylene glycol is 6,000 to 10,000. Claim 10 A method for preparing a cosmetic composition for skin whitening according to claim 8, characterized in that the precipitation in step (c) is carried out at 1 to 10 ℃ for 1 to 30 hours.

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