Cosmetic composition containing a fermented yacon callus culture extract and a pomegranate-derived exosome mixture

Abstract

This invention relates to a composition comprising a mixture of fermented Edelweiss callus culture extract and pomegranate-derived exosomes. This invention provides a cosmetic composition that, compared to using fermented Edelweiss callus culture extract or pomegranate-derived exosomes alone, has superior skin moisturizing, anti-inflammatory, anti-aging, and wrinkle-improving effects.

Description

Technical Field

[0001] This invention relates to a cosmetic composition comprising a mixture of fermented Edelweiss callus culture extract and pomegranate-derived exosomes, which has skin moisturizing, anti-inflammatory, anti-aging or wrinkle-improving effects. Background Technology

[0002] Anti-inflammatory, skin-whitening, skin-moisturizing, and anti-wrinkle properties, as hallmarks of beauty and anti-aging, have always been a focus of attention. Driven by this focus, the cosmetics market is actively launching whitening, moisturizing, and anti-aging products. As consumer attention increases, demand continues to rise, and the related cosmetics industry is growing into a high-value-added industry.

[0003] Recently, according to many studies, materials are being developed that can not only maximize anti-inflammatory effects and promote skin hydration, but also maximize anti-wrinkle effects by maximizing changes in the activity of collagen synthesis-related elements that cause wrinkles. Research and consumer demand are also increasing.

[0004] The endoplasmic reticulum refers to membrane-bound microvesicles with diameters ranging from approximately 20 nm to 5 μm. Based on their size and composition, they are classified into exosomes, extranuclear granules, microvesicles, and microparticles. Exosomes, in particular, are substances secreted extracellularly in all living cells—animal cells, microorganisms, plants, and body fluids—serving as a means of intercellular communication; they are 50 nm to 200 nm in size. Exosomes consist of a phospholipid bilayer similar to the cell membrane. They efficiently and easily transmit signals within cells by delivering proteins to target cells. Furthermore, because they can simultaneously transport multiple secondary metabolites and genetically related substances, they can transmit complex signals. However, their disadvantages include high manufacturing costs and poor price competitiveness.

[0005] In addition, existing cosmetic material development utilizes plant cells cultured from Edelweiss, adventitious root cultures, or their extracts, as well as fermentation processes. However, unlike these existing studies, research on efficacy differences based on exosome mixing ratios is minimal.

[0006] Therefore, in this invention, it was confirmed that when a complex was prepared by co-fermenting Edelweiss callus culture extract and pomegranate-derived exosomes, it could promote and enhance wrinkle improvement, skin moisturizing, and anti-inflammatory effects compared to using either fermented Edelweiss callus culture extract or pomegranate-derived exosomes alone, thus completing this invention.

[0007] Existing technical documents

[0008] Patent documents

[0009] Korean Patent Publication No. 10-2016-0043551

[0010] Korean Patent Publication No. 10-1842700 Summary of the Invention

[0011] The problem that the invention aims to solve

[0012] The purpose of this invention is to provide a cosmetic composition comprising a mixture of fermented edelweiss callus culture extract and pomegranate-derived exosomes as active ingredients, and having the effects of improving skin hydration, anti-inflammation, anti-aging and wrinkle reduction.

[0013] Solution for solving the problem

[0014] In order to achieve the above-mentioned objectives, the inventors confirmed that when fermented Edelweiss callus culture extract and pomegranate-derived exosomes are used in a certain proportion, the results show that it can improve wrinkles, moisturize the skin and reduce inflammation. Moreover, compared with using Edelweiss callus culture extract or pomegranate-derived exosomes alone, the wrinkle improvement, skin moisturizing and anti-inflammatory effects are improved, thereby completing the present invention.

[0015] The present invention provides a cosmetic composition comprising a mixture of edelweiss callus culture extract and pomegranate pulp-derived exosomes as active ingredients.

[0016] The cosmetic composition of the present invention contains natural extracts as active ingredients, which not only have excellent biocompatibility but also minimize skin irritation. In particular, since the above-mentioned Edelweiss plant cell culture extract and pomegranate-derived exosomes act on the skin simultaneously, their synergistic effect can significantly enhance anti-inflammatory, anti-aging, wrinkle-improving, and skin-moisturizing functions.

[0017] The mixture of the above-mentioned fermented Edelweiss callus culture extract and pomegranate-derived exosomes can be mixed at a weight of 0.1% to 10% relative to the total weight of the cosmetic composition.

[0018] Furthermore, the above mixture can be prepared by mixing fermented Edelweiss callus culture extract and pomegranate-derived exosomes in a weight ratio of 1:9 to 9:1. In specific embodiments, the inventors confirmed in the results of testing the mixture of fermented Edelweiss callus culture extract and pomegranate-derived exosomes in weight ratios of 9:1, 7:3, 5:5, and 3:7 that when the mixture contains the same or more fermented Edelweiss callus culture extract and pomegranate pulp-derived exosomes, the effects of improving wrinkles, moisturizing the skin, and anti-inflammation are even better.

[0019] In the above-mentioned Edelweiss callus culture extract, the term "callus" refers to plant cells or adventitious root cultures, which can refer to plant cell clusters formed through the division of Edelweiss plant cells.

[0020] The above-mentioned Edelweiss callus culture extract can be a fermentation extract obtained by inoculating Lactobacillus sp. strains.

[0021] The aforementioned fermentation refers to the process of using enzymes possessed by microorganisms to decompose organic matter. The metabolic products of fermentation contain various amino acids, organic acids, and antioxidants beneficial to the skin, thereby promoting skin metabolism and making the skin more elastic and smooth. Furthermore, the fermentation process reduces particle size, not only improving absorption but also alleviating skin problems or allergic side effects.

[0022] The above-mentioned fermented extract can be prepared by the following method: the extract is dried naturally or by using a device such as a rotary vacuum concentrator or a freeze dryer, and then the culture or extract is diluted in a solvent at a certain concentration, preferably in pure water, and then inoculated with fermentation microorganisms, that is, inoculated with Lactobacillus sp. strains and fermented.

[0023] The aforementioned Lactobacillus strains may be Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei, or Lactobacillus acidophilus, but are not limited to these.

[0024] The cosmetic compositions of the present invention can be used for skin moisturizing, anti-inflammatory, anti-aging or improvement of skin wrinkles.

[0025] The aforementioned "skin moisturizing" refers to all behaviors that maintain skin tissue homeostasis by appropriately regulating the loss of skin moisture (water evaporation). The skin moisturizing effect may be accompanied by various additional skin-improving effects, such as improved keratinization and reduced skin irritation.

[0026] The aforementioned "anti-inflammatory" effect refers to the inhibition of inflammation. As is well known, the regulation of inflammatory responses is highly complex, which enhances the body's recovery system and reduces damage. However, if repeated tissue damage or regeneration leads to a persistent inflammatory response, excessive reactive oxygen species (ROS) are generated in inflammation-related cells, resulting in permanent gene mutations. In other words, ROS are closely related to the inflammatory response, which regulates various cellular functions in the body.

[0027] The above-mentioned "improvement of wrinkles" refers to the phenomenon of inhibiting or preventing the formation of wrinkles on the skin, or alleviating wrinkles that have already formed.

[0028] Furthermore, the present invention provides a method for preparing a mixture of fermented Edelweiss callus culture extract and pomegranate-derived exosomes, comprising the following steps: preparing Edelweiss callus culture extract; fermenting the Edelweiss callus culture extract; preparing exosomes using pomegranate pulp; and mixing the fermented Edelweiss callus culture extract and the exosomes isolated from pomegranate pulp in a certain proportion to prepare the mixture.

[0029] In the step of separating exosomes from pomegranate pulp, after crushing the pomegranate pulp, distilled water is used as a solvent, and the mixture is heat-treated at 60°C to 70°C for 2 to 3 hours for hot water extraction. The resulting product is then cooled and filtered to obtain the exosomes. Specifically, the pomegranate pulp-derived exosomes can be obtained through the following steps: Step (a), adding distilled water to the pomegranate pulp raw material and crushing it; Step (b), centrifuging the crushed material and filtering the supernatant to separate and remove dead cells and foreign matter; Step (c), ultracentrifuging the filtered supernatant to form a primary particle layer containing exosomes; Step (d), centrifuging the supernatant from step (c) again to form a secondary particle layer containing exosomes; and Step (e), suspending the primary and secondary particle layers again in purified water to separate the exosomes from the pomegranate pulp. More specifically, the pomegranate pulp exosomes obtained above can be obtained through the following steps: Step (a) adding 20 times the weight of distilled water to the pomegranate pulp raw material and pulverizing it, then stabilizing the pulverized material at 4°C for 24 hours; Step (b) centrifuging the pulverized material using a centrifuge, and only taking the supernatant of the pulverized liquid to separate and remove dead cells and foreign matter; Step (c) filtering the supernatant to remove residues; Step (d) centrifuging the supernatant using an ultra-high speed centrifuge to precipitate the exosomes to form a primary particle layer; Step (e) centrifuging the supernatant again using an ultra-high speed centrifuge to precipitate additional exosomes to form a secondary particle layer; Step (f) repeating steps (d) and (e) above 2 to 3 times to separate high-purity exosomes; and Step (g) suspending the particle layer formed in the above steps in pure water again to separate exosomes from the pomegranate pulp.

[0030] The mixture of Edelweiss callus culture extract and pomegranate-derived exosomes prepared by the above method can be formulated into various forms of cosmetic compositions using conventional methods.

[0031] The cosmetic compositions described above may have dosage forms such as moisturizing lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, eye cream, eye serum, serum, makeup remover cream, makeup remover milk, facial cleanser, makeup remover water, facial mask, loose powder, body lotion, body cream, body serum, shower gel, hair dye, shampoo, conditioner, hair styling agent, hair growth agent, ointment, gel, cream, patch, spray, powder, and skin adhesive type, but are not limited thereto.

[0032] In addition to the necessary ingredients mentioned above, for various dosage forms, ordinary technicians may appropriately select and combine other ingredients according to the type of other dosage form or the purpose of use.

[0033] The above-described cosmetic compositions can be provided in all dosage forms suitable for topical application. For example, they can be provided in the form of solutions, oil-in-water emulsions, water-in-oil emulsions, suspensions, solids, gels, powders, pastes, microneedles, foams, or aerosol compositions. Such dosage forms can be prepared according to conventional methods in the art.

[0034] The cosmetic compositions described herein may also contain functional additives other than the compounds described herein, as well as ingredients commonly found in conventional cosmetic compositions. These functional additives may include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular-weight peptides, high-molecular-weight polysaccharides, sphingolipids, and seaweed extracts. Preferably, the cosmetic compositions according to this specification may contain other ingredients that synergistically enhance the main effects, without impairing the primary efficacy. Furthermore, the cosmetic compositions according to this specification may also contain moisturizers, emollients, surfactants, UV absorbers, preservatives, sterilizing agents, antioxidants, pH adjusters, organic and inorganic pigments, fragrances, coolants, or antiperspirants. Those skilled in the art can readily select the amounts of the above ingredients without impairing the purpose and effects of this specification, and the amounts may range from 0.001% by weight to 10% by weight, specifically from 0.01% by weight to 3% by weight, based on the total weight of the composition.

[0035] Invention Effects

[0036] Embodiments of the present invention can provide a cosmetic composition with excellent skin moisturizing, anti-inflammatory, anti-aging and wrinkle-improving effects by preparing a mixture of fermented edelweiss callus culture extract and pomegranate-derived exosomes.

[0037] The mixture of fermented Edelweiss callus culture extract and pomegranate-derived exosomes provided by this invention, compared with the fermented Edelweiss callus culture extract or pomegranate-derived exosomes used alone, not only has excellent wrinkle-improving and anti-inflammatory effects, but also excellent skin moisturizing effects. Therefore, when used as an active ingredient in cosmetic substrates, it has excellent effects as a cosmetic composition. Attached Figure Description

[0038] Figure 1 Charts for evaluating the moisturizing and lasting moisturizing power of dosage form example 2 and comparative dosage form examples 1 to 3 according to the present invention.

[0039] Figure 2 Charts for evaluating the moisturizing and lasting moisturizing power of dosage forms 1 to 6 according to the present invention.

[0040] Figure 3 Charts were prepared to evaluate the moisturizing and sustained moisturizing effects of dosage form 8 and comparative dosage forms 4 to 6 according to the present invention.

[0041] Figure 4 Charts for evaluating the moisturizing and lasting moisturizing power of dosage forms 7 to 12 according to the present invention.

[0042] Figure 5 Charts were created to evaluate the improvement in transdermal water loss according to dosage form example 2 and comparative dosage form examples 1 to 3 based on the present invention.

[0043] Figure 6 Charts for evaluating the improvement in transdermal water loss according to dosage forms 1 to 6 of the present invention.

[0044] Figure 7 Charts were created to evaluate the improvement in transdermal water loss according to dosage form 8 and comparative dosage forms 4 to 6 based on the present invention.

[0045] Figure 8 Charts for evaluating the improvement in transdermal water loss according to dosage forms 7 to 12 of the present invention.

[0046] Figure 9 The graph shows the changes in the activity of nitric oxide (NO), an inflammation-related factor, in Examples 1 to 4 and Comparative Examples 1 and 2 of the present invention.

[0047] Figure 10 The graph shows the changes in the inhibitory activity of TNF-α production of inflammation-related factors, which are Examples 1 to 4 and Comparative Examples 1 and 2 of the present invention.

[0048] Figure 11 A graph to confirm the changes in activity of pro-collagen type 1, a skin elasticity-related protein, as described in Examples 1 to 4 and Comparative Examples 1 and 2 of the present invention.

[0049] Figure 12 The graph shows the changes in activity of MMP-1, a skin elasticity-related protein, which is used in Examples 1 to 4 and Comparative Examples 1 and 2 of the present invention. Detailed Implementation

[0050] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described herein, and can be embodied in other forms. The embodiments described herein are provided to make the disclosure more detailed and complete, and to fully convey the spirit of the invention to those skilled in the art. In the following description of the present invention, detailed descriptions of relevant well-known functions or components will be omitted where it is determined that such specific descriptions would unnecessarily obscure the essence of the invention.

[0051] <Comparative Example 1: Preparation of Fermented Edelweiss Callus Culture Extract>

[0052] Plant cells cultured from the stems, flowers, or leaves of Edelweiss, or from germinating seedlings of Edelweiss seeds, were washed, dehydrated, and dried to obtain 100 g of dried Edelweiss callus powder. This powder was added to 1 L of purified water, stirred at 50°C for 8 hours, and subjected to a primary extraction followed by hot water extraction at 98°C for 10 minutes. After filtering the extract, it was concentrated under reduced pressure at 30°C and freeze-dried to obtain a solid. The obtained Edelweiss callus culture extract was mixed with purified water and inoculated with *Lactobacillus plantarum* as a fermentation strain, and fermented at 15°C–25°C for 3 days. After fermentation, the extract was filtered, and 1,2-hexanediol was added to obtain the fermented extract. Table 1 below shows the components used to prepare the fermented Edelweiss callus culture extract according to the present invention.

[0053] Table 1

[0054] Element Comparative Example 1 (by weight percentage) Edelweiss callus culture extract 0.1 Lactobacillus ferment 0.1 1,2-Hexanediol 2 purified water margin total 100

[0055] <Comparative Example 2: Preparation of pomegranate-derived exosomes>

[0056] To isolate exosomes from pomegranate pulp, the pulp was soaked in 20 times its weight of purified water and then pulverized. To improve the purity of the exosomes, the pulverized pomegranate pulp was centrifuged at 3000 x g to 4000 x g for 10 minutes, and only the supernatant was collected, excluding the granular layer, to separate and remove residues. The supernatant obtained in this process was then centrifuged at 10000 x g to 15000 x g for 20 to 30 minutes using an ultracentrifuge. To further improve the purity of the supernatant, it was filtered, and residues were separated and removed. Exosomes present in the high-purity supernatant were then centrifuged at 100000 x g to 150000 x g for 2 to 4 hours using an ultracentrifuge, allowing the exosomes to precipitate in the granular layer. Exosomes were prepared by resuspending the exosomes, which served as the final particle layer, in sterile distilled water to disperse them.

[0057] <Examples 1-4: Preparation of a mixture of fermented Edelweiss callus culture extract and pomegranate exosomes>

[0058] The final mixture was prepared by mixing fermented Edelweiss callus culture extract and pomegranate-derived exosomes obtained in Comparative Examples 1 and 2 above. Figure 2 shows the components used to prepare the mixture of fermented Edelweiss callus culture extract and pomegranate-derived exosomes according to the present invention.

[0059] Table 2

[0060] Comparative Example 1 (by weight percentage) Comparative Example 2 (by weight percentage) Example 1 9 1 Example 2 7 3 Example 3 5 5 Example 4 3 7

[0061] <Dosage Form Examples 1-6 and Comparative Dosage Form Examples 1-3: Preparation of Serum>

[0062] Disodium ethylenediaminetetraacetate, glycerol, propylene glycol, and carbomer were dispersed in purified water, and an emulsion system dissolved in the aqueous phase and heated to 70°C to 75°C was added. The mixture was then stirred for 5 minutes using an AGI mixer. Tromethamine was added at 60°C to 65°C, and the mixture was stirred and neutralized for 3 minutes using an AGI mixer. 1,2-Hexanediol and ethylhexylglycerin were added at 45°C, and the mixture was stirred for 3 minutes before cooling to 30°C. Then, cosmetic materials comprising a mixture of fermented Edelweiss callus culture extract and pomegranate-derived exosomes from Examples 1 to 3 (dosage form examples 1 to 6), and cosmetic materials comprising fermented Edelweiss callus culture extract and pomegranate-derived exosomes from Comparative Examples 1 and 2 (comparative dosage forms 1 and 2), were added, and the mixture was stirred and defoamed for 3 minutes to prepare the serum dosage form. Table 3 below shows the ingredients used to prepare serums containing or entirely excluding those of Examples 1 to 3 and Comparative Examples 1 and 2 according to the present invention.

[0063] Table 3

[0064]

[0065] <Dosage Form Examples 7-12 and Comparative Dosage Form Examples 4-6: Preparation of Creams>

[0066] A transparent emulsion system was prepared by heating ethyl hexadecyl hexanoate, cetearyl alcohol, and glyceryl stearate to 75°C to 80°C and dissolving them. Disodium EDTA, glycerol, propylene glycol, cetearyl alcohol olive oil ester, sorbitan olive oil ester, and carbomer were dispersed in purified water. The emulsion system dissolved in the aqueous phase and heated to 70°C to 75°C was then added, and emulsification was carried out for 5 minutes using a high-speed mixer at 3500 rpm to 5000 rpm. Tromethamine was added at 60°C to 65°C, and the mixture was stirred and neutralized for 3 minutes using a high-speed mixer at 3000 rpm to 3500 rpm. 1,2-Hexanediol and ethylhexylglycerin were added at 45°C and stirred for 3 minutes, then cooled to 30°C. Subsequently, cosmetic materials comprising a mixture of fermented Edelweiss callus culture extract and pomegranate exosomes from Examples 1 to 3 (dosage form examples 7 to 12), and cosmetic materials comprising fermented Edelweiss callus culture extract and pomegranate-derived exosomes from Comparative Examples 1 and 2 (comparative dosage form examples 4 and 5), were added, and the mixture was stirred and defoamed for 3 minutes to prepare a cream. Table 4 below shows the ingredients used to prepare creams comprising or entirely excluding those of Examples 1 to 3 and Comparative Examples 1 and 2 according to the present invention.

[0067] Table 4

[0068]

[0069] <Experimental Example 1. Evaluation of Skin Patch Test>

[0070] After applying the products of dosage forms 1 to 12 and comparative dosage forms 1 to 6 according to the present invention to the skin, the initial irritation to human skin was evaluated by visual assessment, and skin patch tests were performed for objective verification. The experiment was conducted on 10 healthy adults. After applying 25 mg of the sample to the chamber of an IQ Ultra patch tester, it was sealed and applied to the test site—the inner lower arm—for 24 hours. Skin reactions were observed after 1 hour, 24 hours, and 48 hours after patch removal. The results of the human skin irritation test were evaluated according to the standards of the International Contact Dermatitis Research Group (ICDRG). As shown in Table 5 below, the evaluation confirmed that dosage forms 1 to 12 and comparative dosage forms 1 to 6 do not cause skin irritation and are safe for application to the skin.

[0071] Table 5

[0072]

[0073]

[0074] <Experimental Example 2. Evaluation of Moisturizing Power and Lasting Moisturizing Power>

[0075] During the preparation phase, to ensure identical testing conditions for all subjects, the test site was kept clean and dry, and the skin was stabilized for at least 30 minutes in a constant temperature and humidity environment (22±2℃, RH 40%–60%) before testing. During the testing phase, 2 mg / cm² of the solution was applied to a selected test site (5cm × 4cm) on the forearm using a micropipeter. 2 The test product was tested. Measurements were taken before product use, after use, and 3 hours later, for a total of three measurements. The average value was calculated from these three values. Skin moisture was measured using the Aphrodite MC-1000 moisture checker, and the results are shown in Table 6 below. Figures 1 to 4 The results are shown.

[0076] Table 6

[0077] sample Moisturizing effect immediately after application (%) Moisturizing effect (%) 3 hours after application Dosage Form Example 1 127.2 70.4 Dosage Form Example 2 139.0 80.3 Dosage Form Example 3 119.8 56.7 Dosage form example 4 116.7 52.8 Dosage form example 5 122.2 60.6 Dosage form example 6 112.0 50.1 Dosage Form Example 7 121.6 53.5 Dosage form example 8 138.1 65.0 Dosage form example 9 95.1 56.9 Dosage Form Example 10 104.1 50.5 Dosage Form Example 11 121.1 59.5 Dosage Form Example 12 80.9 23.5 Comparative Dosage Form Example 1 109.6 47.4 Comparative Dosage Form Example 2 90.5 34.0 Comparative dosage form example 3 61.9 7.2 Comparative dosage form example 4 84.4 38.6 Comparative dosage form example 5 53.7 15.5 Comparative dosage form example 6 30.6 4.0

[0078] Through the above Table 6 and Figures 1 to 4 It is evident that, compared to comparative formulations 1, 2, 4, and 5 which individually contain fermented Edelweiss callus culture extract and pomegranate-derived exosomes, the formulations 1 to 12 of the present invention, which contain a mixture of fermented Edelweiss callus culture extract and pomegranate-derived exosomes, exhibit increased and sustained moisturizing power immediately after application and after 3 hours of application. In particular, formulations 2 and 8, containing 10% of Example 2, demonstrate the most superior moisturizing power immediately after application and after 3 hours of application. Therefore, a mixture of fermented Edelweiss callus culture extract and pomegranate-derived exosomes is preferable to each containing only fermented Edelweiss callus culture extract and pomegranate-derived exosomes, with the optimal moisturizing effect confirmed when the ratio of fermented Edelweiss callus culture extract to pomegranate-derived exosomes is 7:3.

[0079] <Experimental Example 3: Assessment of Transdermal Moisture Loss>

[0080] During the preparation phase, to ensure identical testing conditions for all subjects, the test site was kept clean and dry, and the skin was stabilized for at least 30 minutes in a constant temperature and humidity environment (22±2℃, RH 40%–60%) before testing. During the testing phase, 2 mg / cm² of the solution was applied to a selected test site (5cm × 4cm) on the forearm using a micropipeter. 2The test product was tested. Measurements were taken three times: before application, immediately after application, and 3 hours after application. The average value was calculated from these three values. A transdermal water loss meter was used. The transepidermal water loss was determined using TM 300 and is shown in Table 7 below. Figures 5 to 8 The results are shown.

[0081] Table 7

[0082]

[0083] Through the above Table 7 and Figures 5 to 8 It is evident that, compared to comparative formulations 1, 2, 4, and 5 which individually contained fermented Edelweiss callus culture extract and pomegranate-derived exosomes, the transdermal water loss immediately after application and 3 hours after application, as shown in formulations 1 to 12 of the present invention containing a mixture of fermented Edelweiss callus culture extract and pomegranate-derived exosomes, was improved. In particular, formulations 2 and 8, containing 10% of Example 2, showed the most significant improvement in transdermal water loss immediately after application and 3 hours after application. Therefore, a mixture of fermented Edelweiss callus culture extract and pomegranate-derived exosomes is preferable to each containing only fermented Edelweiss callus culture extract and pomegranate-derived exosomes, and the improvement in transdermal water loss was most significant when the ratio of fermented Edelweiss callus culture extract to pomegranate-derived exosomes was 7:3.

[0084] <Experimental Example 4: Cytotoxicity Assessment>

[0085] In a 96-well plate, at 6×10 3 Normal human fibroblasts (NHF) were seeded at a rate of 1.5 × 10⁶ cells / well. 4 Human immortalized keratinocytes (HaCaT) were seeded at a rate of 1 × 10⁶ cells / well. 5 After inoculating mouse mononuclear macrophage leukemia cells (RAW 264.7) into wells, cells were cultured under cell culture conditions. After 24 hours, the culture medium was removed and the cells were washed with phosphate-buffered saline (PBS). For NHF cells, fibroblast basal medium (FBM) without supplement was used, while for HaCaT and RAW264.7 cells, Dalberg modified Eagle medium (DMEM) without fetal bovine serum (FBS) was used, and the cells were starved. The next day, a specific concentration of test material was added, and the cells were cultured for 24 hours. 100 μl of water-soluble tetrazolium salt reagent WST-1, diluted 10-fold in the culture medium, was added to each well, and after 2 hours of incubation, the absorbance was measured at 450 nm.

[0086] For cytotoxicity testing, tests were conducted at concentrations ranging from a minimum of 0.1% to a maximum of 10% of the test substance. The results showed that, at the highest concentration of 10%, the cell viability of Comparative Examples 1 and 2, and Examples 1 to 4 treated with NHF, HaCaT, and RAW264.7, respectively, was over 90%.

[0087] The concentrations selected for this efficacy test were non-cytotoxic to confirm the concentration-dependent nature of the efficacy, and are shown in Table 8.

[0088] Table 8

[0089]

[0090] <Experimental Example 5. Evaluation of NO generation inhibition ability>

[0091] In a 96-well plate, at 5×10 4 Cells were seeded at RAW 264.7 cells / well and cultured under cell culture conditions. After 24 hours, the culture medium was removed and the cells were washed with PBS, then starved in FBS-free DMEM medium. The next day, the cells were treated with a specific concentration of the test substance along with 5 μg / ml lipopolysaccharide (LPS) and cultured. After 24 hours, the same amount of cell culture medium and Griess reagent were added and mixed, and the mixture was reacted at room temperature for 15 minutes. The absorbance was measured at 560 nm, and the amount of nitrogen oxides (NO), an inflammatory mediator, was determined using a standard curve obtained from sodium nitrite. The final NO amount was converted to the amount of NO per specific protein and compared with the negative control group. Figure 9 As shown in the image.

[0092] The experimental results confirmed that the NO inhibition activity was excellent when the fermented Edelweiss callus culture extract and pomegranate-derived exosomes were mixed. In particular, the NO inhibition activity was most excellent when the mixing ratio of the fermented Edelweiss callus culture extract and pomegranate-derived exosomes was 7:3 (Example 2). Therefore, compared with using the fermented Edelweiss callus culture extract and pomegranate-derived exosomes alone, the NO inhibition effect of the mixed use of fermented Edelweiss callus culture extract and pomegranate-derived exosomes was better, and the efficacy of the optimal mixing ratio was confirmed, which is shown in Table 9.

[0093] Table 9

[0094]

[0095]

[0096] <Experimental Example 6. Evaluation of TNF-α Production Inhibition Activity>

[0097] RAW264.7 macrophages were cultured in Dulbecco's modified Eagle' medium (DMEM) supplemented with 10% (v / v) fetal bovine serum, streptomycin, and penicillin at approximately 37°C and 5% CO2, and then cultured at 1×10⁻⁶ cells / mL. 5 Cells were seeded per well in a 96-well plate.

[0098] Next, the compositions prepared according to Examples 1 to 4 and Comparative Examples 1 and 2 were treated according to their respective concentrations. After stimulation with LPS (5 μg / mL) and incubation for 24 hours, the cell culture medium was recovered, and the amount of TNF-α was determined using an enzyme-linked immunosorbent assay kit (ELISA kit) (R&D System Inc., Minneapolis, Minnesota, USA). The amount of TNF-α was quantified using the TNF-α standard curves for each concentration included in the kit.

[0099] In addition, indomethacin (20 μg / mL) was used as a positive control group instead of the sample and was used to compare the TNF-α inhibitory activity with that of the sample.

[0100] The experimental results confirmed that the LPS treatment group induced the production of 929.3 pg / mL of TNF-α, which was approximately 4.1-fold higher than the negative control group (226.3 pg / mL). Examples 2 to 4 showed TNF-α production of 579.0 pg / mL, 644.38 pg / mL, and 811.23 pg / mL, respectively. In particular, Example 2 demonstrated an inhibitory activity with approximately 37.7% statistical significance compared to the LPS control group. Furthermore, the mixed fermentation of Edelweiss callus culture extract and pomegranate-derived exosomes showed a concentration-dependent tendency to increase TNF-α inhibitory activity. Finally, it was confirmed that Example 2 exhibited the most superior TNF-α inhibitory activity among Examples 2 to 4.

[0101] Therefore, it can be confirmed that the TNF-α production inhibition effect is better when fermented Edelweiss callus culture extract and pomegranate-derived exosomes are used in combination compared with the use of fermented Edelweiss callus culture extract alone (Comparative Example 1) and pomegranate-derived exosomes alone (Comparative Example 2). The mixing ratio is very important and is shown in Table 10.

[0102] Table 10

[0103]

[0104] <Experiment 7. Evaluation of Procollagen Type 1 Production Capacity>

[0105] In a 96-well plate, at 6×10 3 Cells were seeded at NHF at a specified concentration and cultured under cell culture conditions. After 24 hours, the culture medium was removed and the cells were washed with PBS, then starved using FBM medium without supplementation. The next day, a specific concentration of the test substance was added and the cells were cultured for 24 hours. The absorbance was measured at 450 nm using a type 1 procollagen ELISA reagent. The final procollagen content was converted to the amount of procollagen per specific protein and compared with a negative control group. Figure 11 As shown in the image.

[0106] The experimental results confirmed that, under the same concentration and conditions, compared to using fermented Edelweiss callus culture extract and pomegranate-derived exosomes alone, the combined use of fermented Edelweiss callus culture extract and pomegranate-derived exosomes increased type 1 procollagen activity, resulting in superior collagen activity. In particular, the type 1 procollagen activity was significantly increased when the mixing ratio of fermented Edelweiss callus culture extract and pomegranate-derived exosomes was 7:3 (Example 2). Therefore, it can be confirmed that compared to using fermented Edelweiss callus culture extract and pomegranate-derived exosomes alone, the combined use of fermented Edelweiss callus culture extract and pomegranate-derived exosomes is more effective in improving wrinkles, and the mixing ratio is very important, as shown in Table 11.

[0107] Table 11

[0108]

[0109] <Experimental Example 8. Evaluation of Inhibition of Matrix Metalloproteinase 1 (MMP-1) Production>

[0110] In a 96-well plate, at 2.5 × 10 5 After seeding HaCaT cells at a cell / well ratio, they were cultured under cell culture conditions. After 24 hours, the culture medium was removed and the cells were washed with PBS, then starved in FBS-free DMEM medium (serum-free medium). The next day, the cells were cultured by irradiation with UVB.

[0111] In a 96-well plate at 6×10 3Cells were seeded at NHF levels and cultured under cell culture conditions. After 24 hours, the cells were starved using FBM medium without supplementation. The next day, human fibroblasts were treated with UVB-stimulated HaCaT culture medium along with the samples and cultured. After 24 hours of culture, the absorbance was measured at 450 nm using an MMP-1 ELISA reagent. The final MMP-1 level was converted to the MMP-1 level in each specific protein and compared with the negative control group. Figure 12 As shown.

[0112] The experimental results confirmed that, under the same concentration and conditions, the MMP-1 inhibitory activity was significantly better when the fermented Edelweiss callus culture extract and pomegranate-derived exosomes were used in combination, compared to using them alone. In particular, the increase in MMP-1 inhibitory activity was substantial when the ratio of fermented Edelweiss callus culture extract to pomegranate-derived exosomes was 7:3 (Example 2). Therefore, it can be confirmed that the wrinkle-improving effect is better when the fermented Edelweiss callus culture extract and pomegranate-derived exosomes are used in combination compared to using them alone; the mixing ratio is very important and is shown in Table 12.

[0113] Table 12

[0114]

[0115]

[0116] The above description refers to one embodiment of the present invention. However, those skilled in the art can make various modifications and alterations to the present invention during implementation without departing from the spirit and field of the invention as set forth in the claims. Therefore, when the modified embodiment substantially includes the technical features of the claims of the present invention, it should also be considered as being included within the technical scope of the present invention.

Claims

1. A cosmetic composition, characterized in that, The active ingredient is a mixture of Edelweiss callus culture extract and pomegranate-derived exosomes. The mixture contains the Edelweiss callus culture extract and the pomegranate-derived exosomes in a weight ratio of 7:

3.

2. The cosmetic composition according to claim 1, characterized in that, The mixture comprises 0.1 to 10.0% by weight relative to the total weight of the composition.

3. The cosmetic composition according to claim 1, characterized in that, The Edelweiss callus culture extract is a fermentation extract obtained by inoculating with Lactobacillus strains.

4. The cosmetic composition according to claim 1, characterized in that, The pomegranate-derived exosomes were prepared by the following method: Step (a): Add distilled water to the pomegranate pulp raw material and crush it to prepare a crushed pomegranate pulp raw material; Step (b): After centrifuging the pulverized material, filter the supernatant to separate and remove dead cells and foreign matter; Step (c) involves ultracentrifuging the filtered supernatant to form a primary particle layer containing exosomes. Step (d) involves centrifuging the supernatant from step (c) again to form a secondary particle layer containing exosomes. as well as Step (e) involves resuspending the primary and secondary particle layers in purified water to separate exosomes from the pomegranate pulp.

5. The cosmetic composition according to claim 4, characterized in that, Repeat steps (c) and (d) two to three times to separate high-purity exosomes.

6. The cosmetic composition according to any one of claims 1 to 5, characterized in that, The composition is formulated as one or more of the following: moisturizing lotion, nourishing lotion, hydrating cream, nourishing cream, massage cream, serum, ampoule, gel, eye cream, makeup remover, facial cleanser, makeup remover, mask, spray, and loose powder.

7. The cosmetic composition according to any one of claims 1 to 5, characterized in that, The composition is used for skin moisturizing, anti-inflammatory, anti-aging or improvement of skin wrinkles.

Images