Cosmetic Composition For Improving Skin Improvement Containing a Plant Complex Extract Derived From Jeju

Abstract

The present invention relates to a cosmetic composition for improving skin wrinkles and moisturizing, containing as an active ingredient an extract of a complex of mixed Artemisia annua and Tetragonia tetragonoides. The present invention provides an antioxidant effect through free radical scavenging activity, a wrinkle improvement effect through the promotion of collagen production, inhibition of MMP-1 expression, and increased laminin-5 expression, and a skin moisturizing effect through increased aquaporin expression.

Description

Technology Field

[0001] The present invention relates to a cosmetic composition for skin improvement containing a complex extract of plants from Jeju Island. More specifically, it relates to a cosmetic composition for improving skin wrinkles or moisturizing the skin containing, as an active ingredient, an extract of a complex mixture of Artemisia annua and Tetragonia tetragonoides. Background Technology

[0003] The skin is the outermost part of the body, composed of a three-layer structure of the epidermis, dermis, and subcutaneous tissue. It serves as a physical barrier protecting the body from the external environment and performs major biological functions such as absorbing substances, regulating body temperature, and maintaining homeostasis. However, as we age, skin cells become damaged by pollutants, ultraviolet rays, and stress, and impaired cell proliferation leads to wrinkles, loss of elasticity, and keratinization.

[0004] Meanwhile, skin aging can be classified into intrinsic and extrinsic aging depending on the influencing factors. Intrinsic aging is a natural aging phenomenon that occurs as the skin's physiological functions decline with age, whereas extrinsic aging refers to aging caused by external factors such as ultraviolet rays, reactive oxygen species (ROS), and stress.

[0005] The generation of reactive oxygen species caused by UV exposure induces intracellular oxidative stress, leading to physical changes in the structure of the skin layers and promoting melanin synthesis to cause pigment deposition. Furthermore, UV rays reduce the synthesis of extracellular matrix proteins, such as collagen and elastin, which are components of skin connective tissue, and alter their structures, thereby decreasing skin elasticity.

[0006] Collagen is synthesized by fibroblasts and is a major component of the extracellular matrix, which makes up most of the dermis; it is a major matrix protein that is degraded by matrix metalloproteinases (MMPs). MMPs are secreted by many cells in the skin, including keratinocytes and fibroblasts. Among them, MMP-1 plays a major role in skin aging by primarily degrading collagen and promoting the breakdown of fragmented collagen, thereby inducing wrinkle formation. According to various studies, increased activity of MMPs in the skin caused by ultraviolet radiation and reactive oxygen species leads to the loss of extracellular matrix components, such as collagen, within the dermis. This weakens elasticity, flexibility, and the ability to generate connective tissue, resulting in wrinkles. It is known that if this phenomenon persists, it leads to damage to the skin barrier and skin dehydration.

[0007] Furthermore, aquaporin (AQP), a factor related to skin hydration, is a type of membrane protein that transports water and various substances, playing a crucial role in regulating skin moisture both inside and outside cells. As we age, the expression of AQP-3 decreases, and this reduced expression is considered to be one of the causes of skin dryness.

[0008] Existing approaches to preventing and improving skin aging have primarily utilized sunscreens, retinoids, and antioxidants (such as vitamins C and E), but these ingredients have limitations such as irritation, safety concerns, and usage restrictions.

[0009] Accordingly, there is an increasing demand for new functional cosmetic ingredients for anti-wrinkle improvement based on naturally derived materials, and interest is growing in the high value-added processing of recyclable materials, such as food processing by-products. Prior art literature

[0011] Korean Patent Publication No. 1020210068197 (June 9, 2021) describes a fermented mugwort extract, a method for improving the content of active ingredients in the fermented mugwort extract prepared therefrom, a composition containing the fermented extract that is effective for improving skin wrinkles and anti-aging, and a cosmetic composition composed of the fermented extract. Korean Patent Publication No. 1020250066994 (May 14, 2025) describes a cosmetic composition for strengthening the skin barrier and anti-inflammatory effects containing a fermented extract of *Trifolium repens*. The problem to be solved

[0012] The present invention aims to develop a natural skin-improving cosmetic composition using Jeju native plants, Artemisia annua and Artemisia vulgaris, which effectively improves skin wrinkle formation induced by ultraviolet rays and exhibits moisturizing efficacy. means of solving the problem

[0014] The present invention provides a cosmetic composition for improving skin containing an extract of a complex of mixed Artemisia vulgaris and Tetragonia tetragonoides as an active ingredient. In this case, the Artemisia vulgaris and Tetragonia tetragonoides are preferably mixed in a weight ratio of 0.1 to 10 : 0.1 to 10.

[0015] In the cosmetic composition of the present invention, the cosmetic composition may preferably be for antioxidant use, for improving skin wrinkles, or for moisturizing the skin. Effects of the invention

[0017] The cosmetic composition of the present invention exhibits an antioxidant effect through free radical scavenging activity, a wrinkle improvement effect by promoting the production of collagen—a component of the skin—and inhibiting the expression of MMP-1, a collagen-degrading enzyme, while increasing the expression of laminin-5, and a skin moisturizing effect by increasing the expression of aquaporin, a membrane protein that transports moisture. Brief explanation of the drawing

[0019] Figure 1 shows the results of measuring the efficacy of the plant complex extract of the present invention in increasing Laminin-5 protein expression in a Reconstructed skin model. Figure 2 shows the results of confirming the effect of the plant complex extract of the present invention on promoting Type-1 procollagen synthesis in a Reconstructed skin model. Figure 3 shows the results of confirming the effect of the plant complex extract of the present invention on increasing Elastin expression in a Reconstructed skin model. Specific details for implementing the invention

[0020] The present invention aims to develop and provide a cosmetic composition containing, as an active ingredient, an extract of a complex of mixed Artemisia annua and Artemisia vulgaris native to Jeju Island, which exhibits antioxidant, wrinkle improvement, and moisturizing effects through free radical scavenging activity, promotion of collagen synthesis, inhibition of MMP-1 expression, increase in laminin-5 expression, and increase in aquaporin expression.

[0021] All technical terms used in this invention, unless otherwise defined, have the following definitions and correspond to the meaning generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention.

[0022] The term “about” means an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by about 30, 25, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0023] Throughout this specification, unless otherwise required by the context, the terms “comprising (or containing)” and “comprising (or containing)” should be understood to imply that the presented steps or components, or groups of steps or components, are included, but any other steps or components, or groups of steps or components are not excluded.

[0024] The present invention provides a cosmetic composition for skin improvement containing, as an active ingredient, an extract of a complex mixture of Artemisia vulgaris and Tetragonia tetragonoides. However, since the active ingredient in the case of a "complex extract mixture of Artemisia vulgaris extract and Tetragonia tetragonoides extract" is identical to that of the present invention's "extract mixture of Artemisia vulgaris extract and Tetragonia tetragonoides extract," it can be inferred that the same effect as the present invention will be produced; therefore, the "complex extract mixture of Artemisia vulgaris extract and Tetragonia tetragonoides extract" is also considered to fall within the technical scope of the present invention as a modified example of the "extract mixture of Artemisia vulgaris extract and Tetragonia tetragonoides extract" of the present invention.

[0025] The true mugwort used in the present invention, which is also commonly referred to as 'mugwort' ( Artemisia princeps Artemisia annua is a perennial plant belonging to the genus Artemisia of the Asteraceae family that grows naturally throughout Korea and on Jeju Island. Artemisia annua has silvery-gray leaves and a distinctive aroma, and its leaves have a deeply divided, pinnate structure. Young leaves are widely used as ingredients in dishes such as rice cakes, seasoned greens, and pancakes, while dried leaves are used for moxibustion or as a medicinal herb. In particular, Artemisia annua is known for its warm nature, which is traditionally believed to aid in promoting blood circulation and improving digestion. It is also reported to exhibit anti-inflammatory, antioxidant, and skin barrier-strengthening effects due to its rich content of flavonoids, phenolic compounds, and essential oils.

[0026] In addition, the *Trifolium repens* used in the present invention ( Tetragonia tetragonoidesTetragonia is a coastal herbaceous plant belonging to the genus Tetragonia of the Portulacaceae family, native to the southern coast of Korea and the coastal regions of Jeju Island. In Korea, it is known as 'Tetragonia' or 'New Zealand spinach.' It possesses strong vitality, thriving even in sandy coastal soil or saline areas. Its leaves are thick, succulent, and triangular or rhomboid in shape. The young leaves and stems of Tetragonia are blanched like spinach and used in various dishes such as salads, soups, and pancakes; it is highly nutritious due to its richness in minerals and vitamins. Traditionally known for aiding in physical recovery and fever reduction, it has recently garnered attention as a health food ingredient. Furthermore, its bioactive substances are known to be effective for skin protection functions, such as soothing, anti-inflammation, and moisture retention, making it suitable for use as a functional ingredient in cosmetic compositions.

[0027] In the cosmetic composition of the present invention, the Artemisia annua and Tetragonia tetragonoides are preferably mixed in a weight ratio of 0.1 to 10 : 0.1 to 10. However, they are not necessarily limited to the above range.

[0028] The extract in the ‘mixture of Artemisia annua and Tetragonia tetragonoides complex’ according to the present invention can be prepared using general extraction, separation, and purification methods known in the art.

[0029] In the present invention, the extract may be prepared by extracting with an extraction solvent or by adding a fractionation solvent to an extract prepared by extraction with an extraction solvent to fractionate it. At this time, the extraction solvent is not limited to any specific type, but preferably, water, an organic solvent, or a mixture of these solvents may be used. The organic solvent may be an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, a non-polar solvent such as hexane or dichloromethane, or a mixture of these solvents. As for the extraction method, methods such as heat extraction, cold maceration extraction, reflux cooling extraction, ultrasonic extraction, supercritical extraction, or subcritical extraction may be used, but are not necessarily limited thereto. Preferably, in the 'extract of a complex of mixed Artemisia vulgaris and Artemisia annua' according to the present invention, the extract is preferably extracted using a subcritical extraction apparatus, and it is preferable to use a temperature of 50 to 200°C and a pressure of 1 bar (14.5 psi) to 100 bar (1800 psi). More preferably, it is recommended to use 100~150℃ and 50~100 bar. At this time, it is recommended to extract for 10~30 minutes using subcritical water as a solvent.

[0030] Meanwhile, the cosmetic composition of the present invention may preferably contain 0.001 to 30% by weight of an extract of a complex of mixed Artemisia vulgaris and Artemisia annua relative to the total weight of the composition. If the content is less than 0.001% by weight, no effect may be observed, and if it exceeds 30% by weight, the degree of increase in effect with increasing content is negligible, there are issues with the safety and stability of the formulation, and it is not economical.

[0031] Meanwhile, the cosmetic composition of the present invention may preferably be for antioxidant use, for improving skin wrinkles, or for moisturizing the skin.

[0032] In addition, the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the extract, and may include, for example, conventional auxiliary agents and carriers such as solvents, antioxidants, stabilizers, solubilizing agents, preservatives, vitamins, pigments, and fragrances, but is not limited thereto.

[0033] In addition, the cosmetic composition of the present invention may be prepared in any formulation conventionally manufactured in the industry, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, pack, massage cream, and spray. However, it is not necessarily limited thereto. More specifically, it may be prepared in the form of a softening lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

[0034] In the case where the formulation of the cosmetic composition of the present invention is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

[0035] When the formulation of the cosmetic composition of the present invention is a solution or an emulsion, a solvent, a solubilizing 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.

[0036] When 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, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanthenic acid may be used as a carrier component.

[0037] When the formulation of the cosmetic composition of the present invention is a powder or a 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, it may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether.

[0038] In the case where the formulation of the cosmetic composition of the present invention is a cleansing product containing a surfactant, 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, lanolin derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.

[0039] When the cosmetic composition of the present invention is a soap, a cleansing formulation containing a surfactant, or a cleansing formulation not containing a surfactant, it may be wiped off, peeled off, or washed off with water after application to the skin. As specific examples, the soap is a liquid soap, powder soap, solid soap, and oil soap; the cleansing formulation containing a surfactant is a cleansing foam, cleansing water, cleansing towel, and cleansing pack; and the cleansing formulation not containing a surfactant is a cleansing cream, cleansing lotion, cleansing water, and cleansing gel, but is not limited thereto.

[0041] The composition of the present invention will be specifically explained below through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes variations of equivalent technical concepts.

[0043] [Example 1: Preparation of Artemisia annua ethanol extract]

[0044] Dried Artemisia annua was immersed in 10 times (by weight) of 70% (v / v) ethanol and reflux extracted at 80°C for 5 hours. The extract was filtered through filter paper (Whatman No. 2) and concentrated to prepare Example 1.

[0046] [Example 2: Preparation of Ethanol Extract of *Trifolium repens*]

[0047] Dried Tetragonia tetragonoides was immersed in 10 times (by weight) of 70% (v / v) ethanol and reflux extracted at 80°C for 5 hours. The extract was filtered through filter paper (Whatman No. 2) and concentrated to prepare Example 2.

[0049] [Example 3: Preparation of Plant Complex Ethanol Extract]

[0050] Dried Artemisia annua and Artemisia vulgaris were mixed in a weight ratio of 6:4, immersed in 10 times (weight ratio) of 70% (v / v) ethanol, and reflux extracted at 80°C for 5 hours. The extract was filtered through filter paper (Whatman No. 2) and concentrated to prepare Example 3.

[0052] [Example 4: Preparation of Hot Water Extract of Artemisia annua]

[0053] Dried Artemisia annua was immersed in 10 times the weight of water and reflux-extracted at 100°C for 5 hours. The extract was filtered through filter paper (Whatman No. 2) and concentrated to prepare Example 4.

[0055] [Example 5: Preparation of Hot Water Extract of *Trifolium repens*]

[0056] Dried Tetragonia tetragonoides was immersed in 10 times the weight of water and reflux-extracted at 100°C for 5 hours. The extract was filtered through filter paper (Whatman No. 2) and concentrated to prepare Example 5.

[0058] [Example 6: Preparation of Plant Complex Hot Water Extract]

[0059] Dried Artemisia annua and Artemisia vulgaris were mixed in a weight ratio of 6:4, immersed in 10 times the weight of water, and reflux extracted at 100°C for 5 hours. The extract was filtered and concentrated using filter paper (Whatman No. 2) to prepare Example 6.

[0061] [Example 7: Preparation of Artemisia subcritical water extract]

[0062] Dried Artemisia annua was placed in a subcritical extraction apparatus, and 10 times the amount of water (by weight) was added. The bath temperature was heated to 150°C, pressure was applied to reach 100 bar, and extraction was performed for 30 minutes. The extract was filtered through filter paper (Whatman No. 2) and concentrated to prepare Example 7.

[0064] [Example 8: Preparation of Subcritical Water Extract of Arugula]

[0065] Dried Tetragonia tetragonoides was placed in a subcritical extraction apparatus, and 10 times the amount of water (by weight) was added. The bath temperature was heated to 150°C, pressure was applied to reach 100 bar, and extraction was performed for 30 minutes. The extract was filtered through filter paper (Whatman No. 2) and concentrated to prepare Example 8.

[0067] [Example 9: Preparation of Plant Complex Subcritical Water Extract]

[0068] Dried Artemisia annua and Artemisia vulgaris were mixed in a ratio of 6:4 (by weight) and placed in a subcritical extraction apparatus, and 10 times the amount of water (by weight) was added. The bath temperature was heated to 150°C, pressure was applied to reach 100 bar, and extraction was performed for 30 minutes. The extract was filtered through filter paper (Whatman No. 2) and concentrated to prepare Example 9.

[0070] [Experimental Example 1: Free Radical Scavenging Activity]

[0071] In this experimental example, to confirm the antioxidant effects of Examples 1 to 9, the effect on DPPH radical scavenging ability was confirmed.

[0072] 20 µl of the extracts prepared in Examples 1–9 above and 180 µl of 1,1-diphenyl-2-picryhydrazyl (DPPH) solution were added and thoroughly mixed. After reacting at room temperature for 10 minutes, the absorbance was measured at 565 nm. L-ascorbic acid was treated at a concentration of 100 µM as a positive control. Radical scavenging activity was expressed as a percentage using Equation 1 below.

[0073] [Mathematical Formula 1]

[0074]

[0075] Sample name Concentration (µg / ml) DPPH radical scavenging activity (%) Example 1 50 72.26 Example 2 50 56.51 Example 3 50 74.42 Example 4 50 59.16 Example 5 50 48.23 Example 6 50 64.30 Example 7 50 73.72 Example 8 50 60.10 Example 9 50 80.12 L-ascorbic acid 20 89.56

[0077] As a result of confirming DPPH radical scavenging ability, Examples 1 to 9 exhibited DPPH radical scavenging activity as shown in Table 1. In particular, the plant complex extracts Examples 3, 6, and 9 showed superior DPPH radical scavenging activity compared to single extracts, and Example 9, a plant complex subcritical water extract, showed a DPPH radical scavenging activity of 80.12%, demonstrating a superior antioxidant effect compared to the ethanol extract and hot water extract.

[0079] [Experimental Example 2: Confirmation of Collagen Production Promotion Effect]

[0080] In this experimental example, in order to confirm the collagen production-promoting effect of Examples 1 to 9, the effect on the amount of collagen expression was examined.

[0081] 1 x 10 human skin fibroblasts (HDFn) 5Cells were seeded into 24-well plates at a concentration of cells / well and stabilized in a 37°C, 5% CO2 incubator. The medium was changed to FBS-free medium, and each sample was treated at various concentrations; TGF-β at a concentration of 10 ng / ml was used as a positive control. After 24 hours, the supernatant was collected, and the remaining cells were treated with MTT solution to check for cytotoxicity. A procollagen type I peptide EIA kit was used to determine collagen expression levels using the collected supernatant. After the collagen ELISA assay, the absorbance was measured at a wavelength of 450 nm to calculate the amount of collagen produced, which was then expressed as a percentage increase in collagen expression compared to the control group.

[0082] Sample name Concentration (µg / ml) Collagen expression increase rate (%) Example 1 100 33.47 Example 2 100 25.39 Example 3 100 35.22 Example 4 100 24.16 Example 5 100 18.14 Example 6 100 25.35 Example 7 100 37.53 Example 8 100 23.75 Example 9 100 39.13 TGF-β 10 ng / ml 31.48

[0084] As a result of the experiment, Examples 1 to 9 showed an effect of increasing collagen expression, as shown in Table 2. In particular, Examples 3, 6, and 9, which are plant complex extracts, showed superior efficacy in increasing collagen expression compared to single extracts, and Example 9, which is a plant complex subcritical water extract, showed a collagen expression increase rate of 39.13%, demonstrating a superior collagen production promoting effect compared to ethanol extracts and hot water extracts.

[0086] [Experimental Example 3: Measurement of MMP-1 Expression Inhibitory Effect]

[0087] In this experimental example, to confirm the wrinkle-improving effects of Examples 1 to 9, the effect on MMP-1 was confirmed using human skin fibroblasts.

[0088] 2.5 x 10 human skin fibroblasts (HDFn) in a 12-well plate 5Cells were seeded into wells and stabilized for one day in a 37°C, 5% CO2 incubator, followed by treatment with UVA at 2 J / cm². Subsequently, the extracts prepared in Examples 1 to 9 were each treated at a concentration of 100 μg / ml, and adenosine (a functional ingredient for wrinkle reduction) was treated at a concentration of 100 μg / ml as a positive control. After reacting for 6 hours, the supernatant was removed, the cells were washed once with 1 x PBS, and collected by scraping with a scraper. Then, proteins were isolated using cell lysis buffer and quantified by the BCA method, after which a certain amount of protein was subjected to electrophoresis on a 10% SDS-PAGE gel. The isolated proteins on the gel were transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour. Subsequently, the samples were washed three times with 1 x TBST solution and reacted with the primary antibody overnight at 4°C, followed by reacting with the HRP-conjugated secondary antibody at room temperature for 2 hours. Afterward, the samples were washed three times with 1 x TBST solution and reacted with ECL solution, and bands were detected using a Chemi-Doc instrument. The results were quantified using Image J 1.47 software, and the MMP-1 expression inhibition rate (%) was expressed as a percentage compared to the UV-treated group.

[0089] Sample name Concentration (µg / ml) MMP-1 expression inhibition rate (%) Example 1 100 57.26 Example 2 100 53.75 Example 3 100 61.43 Example 4 100 48.33 Example 5 100 36.28 Example 6 100 50.71 Example 7 100 63.62 Example 8 100 59.72 Example 9 100 68.25 Adenosine 100 70.34

[0091] As a result of the experiment, Examples 1 to 9 showed an MMP-1 expression inhibitory effect, as shown in Table 3. In particular, Examples 3, 6, and 9, which are plant complex extracts, showed superior MMP-1 expression inhibitory ability compared to single extracts, and Example 9, which is a plant complex subcritical water extract, showed an MMP-1 expression inhibition rate of 68.25%, demonstrating a superior wrinkle improvement effect compared to ethanol extracts and hot water extracts.

[0093] [Experimental Example 4: Measurement of the effect of increasing AQP-3 expression]

[0094] In this experimental example, to confirm the moisturizing effect of Examples 1 to 9, the effect on AQP-3 was confirmed using human keratinocytes (HaCaT).

[0095] 4 x 10 human keratinocytes in a 6-well plate 5 Cells were seeded into wells and stabilized for one day in a 37°C, 5% CO2 incubator. The extracts prepared in Examples 1–9 were each treated at a concentration of 100 μg / ml, and after incubation for 6 hours in a 37°C, 5% CO2 incubator, RNA was isolated using the Trizol method. RNA was quantified using a nanodrop, PCR was performed, and bands were detected by electrophoresis on a 2% agarose gel. The bands were quantified using Image J 1.47 software, and the increase rate (%) of AQP-3 expression was expressed as a percentage relative to the control.

[0096] Sample name Concentration (µg / ml) AQP-3 expression increase rate (%) Example 1 100 41.72 Example 2 100 42.02 Example 3 100 49.45 Example 4 100 47.21 Example 5 100 56.12 Example 6 100 56.45 Example 7 100 50.11 Example 8 100 58.42 Example 9 100 62.38

[0098] As a result of the experiment, Examples 1 to 9 showed an effect of increasing AQP-3 expression, as shown in Table 4. In particular, Examples 3, 6, and 9, which are plant complex extracts, showed superior efficacy in increasing AQP-3 expression compared to single extracts, and Example 9, which is a plant complex subcritical water extract, showed an AQP-3 expression increase rate of 62.38%, demonstrating a superior moisturizing effect compared to ethanol extracts and hot water extracts.

[0100] [Formulation Example 1: Preparation of a cosmetic composition containing a plant complex subcritical water extract]

[0101] In this formulation example, a cosmetic composition for improving the skin barrier containing 3.0% by weight of the plant complex subcritical water extract of Example 9 was prepared with the mixing ratio shown in Table 5 below. The composition is described with the composition in the table below, but it can be applied to various formulations, and this is not intended to limit the invention but merely to provide a specific description.

[0102] division ingredient Formulation Example 1 (weight%) Comparative Formulation Example 1 (weight%) A purified water 10.00 10.00 Carbomer 0.20 0.20 B Example 9 3.00 0.00 Disodium EDTA 0.02 0.02 1,2-hexanediol 2.00 2.00 glycerin 10.00 10.00 Butylene glycol 5.00 5.00 Betaine 1.00 1.00 Glycereth-26 2.00 2.00 purified water to.100 to.100 C Glyceryl Stearate SE 2.00 2.00 cetearyl alcohol 2.00 2.00 Glyceryl stearate 0.75 0.75 PEG-100 Stearate 0.75 0.75 Caprylic / Capric Triglyceride 3.00 3.00 Cetylethylhexanoate 2.00 2.00 Shea butter 1.00 1.00 Arachidyl alcohol 0.30 0.30 Behenyl alcohol 0.30 0.30 Arachidylglucoside 0.40 0.40 Cyclopentasiloxane 1.80 1.80 Cyclohexasiloxane 1.20 1.20 Dimethicon 0.20 0.20 D purified water 2.00 2.00 Tromethamine 0.18 0.18 E spices 0.16 0.16

[0104] [Experimental Example 5: Confirmation of skin wrinkle improvement and moisturizing effects using a reconstructed skin model]

[0105] In this experiment, to confirm the wrinkle improvement and moisturizing effects of the cosmetic composition formulation Example 1 containing Example 9 above, an experiment was conducted to confirm the expression levels of Laminin-5, Collagen Type 1, and Elastin using a Reconstructed skin model.

[0106] (1) Preparation of reconstructed skin tissue

[0107] Fibroblasts, type 1 collagen aqueous solution, 10× DMEM:F12 (3:1) Media, 10× buffer, and hyaluronic acid were thoroughly mixed and placed inside a culture insert of a 12-well plate. The mixture was then reacted in a 5% CO2, 37℃ cell incubator for 2 hours to solidify, after which fibroblast culture medium was added to the inside and outside, and the mixture was cultured overnight. Subsequently, the dermal layer containing fibroblasts was prepared by culturing for 5 days while exchanging the medium. Pre-cultured primary keratinocytes and melanocytes were seeded onto the top of the prepared dermal layer using Keratinocyte-SFM medium containing Bovine Pituitary Extract (BPE), insulin, hydrocortisone, Vitamin C, and epidermal growth factor (EGF), and cultured overnight. After culturing for 5 days to induce epidermal cell proliferation, 2 mM Ca 2+ Keratinocyte differentiation was induced by adding medium supplemented with [substances] and culturing. Subsequently, all the medium was removed, and EGF, 2 mM Ca [substances] were placed on the outside of the culture insert. 2+Reconstructed skin tissue was prepared by introducing a culture medium containing [the substance] and culturing primary keratinocyte cells in air for 12 days to induce the formation of an epidermal layer.

[0109] (2) Treatment and dyeing of samples

[0110] Prepared Reconstructed skin tissue (RST) was treated with 20 µl of Example 9 once every 2 days and cultured for 5 days. Afterward, the tissue was fixed in a 10% neutral formalin solution for 24 hours, embedded in OCT Compound, sliced ​​to a thickness of 12 µm, attached to a gelatin-coated slide, and the slide was dried and stored.

[0111] For morphological observation, staining was performed using Masson's trichrome kit (MST-100T, BIOGNOST). The tissue sections prepared on the slide were hydrated in distilled water, treated with Bouin's solution at 56°C for 1 hour, cooled at room temperature for 20 minutes, and then washed with distilled water. Nuclei were stained with Weigert's iron hematoxylin for 20 minutes and washed, followed by staining with Biebroch scarlet-acid fuchsin solution for 10 minutes and washing with distilled water. After mordanting and decolorizing with Phosphomolybdic acid-orange G solution for 5 minutes, collagen fibers were stained with Aniline Blue. Subsequently, unbound light blue was decolorized using 0.1% acetic acid, followed by dehydration and clearing. The samples were then mounted in Canada balsam and observed under a microscope.

[0112] Collagen Type 1, Laminin-5, and Elastin, proteins involved in improving elasticity and moisturizing effects, were analyzed using the immunofluorescence staining method described below. Tissue sections prepared on slides were hydrated in distilled water, then placed in a 0.01M citric acid solution (pH 6.0) and boiled using a microwave for 15 minutes, followed by incubation at room temperature for 20 minutes. The samples were blocked using anti-goat serum and reacted overnight at 4°C with primary antibodies Laminin-5 (1:500, abcam), Collagen Type I (1:500, invitrogen), and Elastin (1:100). Subsequently, the samples were washed with PBS solution, reacted with the secondary antibody Goat anti-mouse IgG Alexa Fluor 488 (1:500) (abcam) at room temperature for 1 hour, washed with PBS solution, and mounted in mounting medium with DAPI for observation under a fluorescence microscope. The positive staining area was quantified using the Image J program from images taken at 200x magnification under a fluorescence microscope, and the rate of change was calculated.

[0114] (3) Confirmation of the effect of increasing Laminin-5 protein content

[0115] Laminin-5 is a protein that constitutes the Dermal-Epidermal Junction (DEJ), which is responsible for the connection between the epidermis and the dermis. As aging progresses, its expression decreases, hindering skin damage and nutrient supply. Therefore, an increase in Laminin-5 expression implies that skin aging can be prevented. Figure 1 shows the results of measuring the effect of increased Laminin-5 protein expression; the blue light represents the staining of the cell nucleus, and Laminin-5 was stained in a linear shape with red fluorescence at the Dermal-Epidermal Junction.

[0116] As a result of the experiment, it was confirmed that Formulation Example 1 significantly increased the expression of Laminin-5 protein by 101.13% compared to Comparative Formulation Example 1, as shown in Fig. 1. This can be interpreted to mean that the plant complex subcritical water extract of Example 9 contained in Formulation Example 1 can help with elasticity and moisturizing efficacy in actual skin.

[0118] (4) Confirmation of the effect of increasing Collagen Type 1 protein content

[0119] Collagen Type 1 accounts for 70% of the dermis and is a protein that plays an important role in skin wrinkles and elasticity; therefore, an increase in the expression of Collagen Type 1 protein implies that it can help improve skin wrinkles. Figure 2 shows the results of measuring the effect of increasing Collagen Type 1 protein expression, where blue light stains the cell nuclei, and Collagen Type 1 was stained with green fluorescence in the dermal layer.

[0120] As a result of the experiment, it was confirmed that Formulation Example 1 significantly increased the expression of Collagen Type 1 protein by 61.37% compared to Comparative Formulation Example 1, as shown in Fig. 2. This can be interpreted to mean that the plant complex subcritical water extract of Example 9 contained in Formulation Example 1 can help with elasticity and moisturizing effects on actual skin.

[0122] (5) Confirmation of increased elastin protein content

[0123] Elastin is a highly elastic fibrous protein that makes up the dermis, and an increase in Elastin protein expression implies that it can help improve skin wrinkles. Figure 3 shows the results of measuring the effect of increased Elastin protein expression, where the area around the fibroblasts in the dermis was stained in a fibrous shape with red fluorescence.

[0124] As a result of the experiment, it was confirmed that Formulation Example 1 significantly increased the expression of elastin protein by 32.23% compared to Comparative Formulation Example 1, as shown in Fig. 3. This can be interpreted to mean that the plant complex subcritical water extract of Example 9 contained in Formulation Example 1 can help with elasticity and moisturizing effects on actual skin.

Claims

Claim 1 A cosmetic composition containing an extract of a complex of mixed Artemisia vulgaris and Artemisia annua as an active ingredient, wherein the extract is characterized by being extracted using water in a subcritical state as a solvent. Claim 2 A cosmetic composition according to claim 1, characterized in that the mugwort and Tetragonia tetragonoides are mixed in a weight ratio of 0.1 to 10 : 0.1 to 10. Claim 3 A cosmetic composition according to claim 1, characterized in that the cosmetic composition is for antioxidant use. Claim 4 A cosmetic composition according to claim 1, characterized in that the cosmetic composition is for improving skin wrinkles. Claim 5 A cosmetic composition according to claim 1, characterized in that the cosmetic composition is for moisturizing the skin.

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