Supercritical extract of stellaria alsine, antioxidant, skin condition-improving and / or Anti-inflammatory composition comprising same, and extraction method for preparing same

Abstract

The present invention relates to an antioxidant, skin condition-improving and / or anti-inflammatory composition comprising an ultrasonic extract and / or a supercritical extract of Stellaria alsine, a supercritical extract of Stellaria alsine, obtained by extracting Stellaria alsine through a supercritical extraction method, and an extraction method for preparing same. The ultrasonic extract and the supercritical extract of Stellaria alsine have excellent antioxidant, skin condition-improving and / or anti-inflammatory effects.

Description

Supercritical extract of Styrax japonica, an antioxidant, skin condition improvement, and / or anti-inflammatory composition containing the same, and an extraction method for preparing the same

[0001] The present specification relates to an antioxidant, skin condition improvement and / or anti-inflammatory composition comprising a flea plant extract and / or a supercritical extract of a flea plant and an extraction method for preparing the same.

[0002] Starwort is a biennial herbaceous plant belonging to the Caryophyllaceae family that is native to Korea. It is also known as Beolgeumjari, Gaemibanul, Deulbyeolkkot, Byeorukbyeolkkot, and Boribang, and grows throughout the country. When fully grown, it reaches a height of 15 to 25 cm and grows in vacant lots of rural farmhouses, rice paddies, field edges, and mountainous areas. Its young shoots are edible as a vegetable, and due to its antipyretic, detoxifying, and anti-inflammatory properties, it was used as a natural detoxifier in folk medicine until the Joseon Dynasty to treat colds, hepatitis, bruises, anal fistulas, boils, and snake or insect bites.

[0003] Currently, the plant is used in small quantities as a spring vegetable for salads or bibimbap, but it is mostly considered a dominant winter weed and is removed in the spring. The story behind the plant's decline is that until the Joseon Dynasty, it was used as an important medicinal herb to treat bruises in the Joseon army. However, during the Japanese colonial period, the Japanese military changed the plant's name from *Jakseolcho* to *Byeoruknamul* to prevent it from being used for treatment or food, making the mere mention of the name repulsive and thus unusable. Since then, the plant name *Byeoruknamul* has been passed down to this day.

[0004] Against this background, the inventors confirmed the whitening, anti-wrinkle, anti-inflammatory, antioxidant, and antibacterial effects of the fleaweed extract, and confirmed that roseoside can be extracted with a content six times higher than that of conventional ultrasonic extraction when the fleaweed is extracted under supercritical conditions, thereby completing the present invention.

[0005] The necessity of this invention stems from the need to utilize novel natural resources for the purpose of sustainable use and to discover alternative raw materials that respond to environmental changes, due to climate change which is having a severe impact on ecosystems worldwide. In particular, plants with strong resilience, such as weeds, possess the characteristic of being able to adapt and grow rapidly even in extreme environments. As they present new ecological challenges while attracting attention as natural resources with potential for utilization, they can overcome the limitations of existing technologies and maximize the potential for industrial application.

[0006]

[0007] One example of the present specification provides a composition for antioxidant, skin condition improvement and / or anti-inflammatory purposes, comprising a flea plant extract.

[0008] Another example provides a supercritical extract of *Euonymus japonicus* extracted by supercritical extraction.

[0009] Another example provides a method for preparing a supercritical extract of *Echinochloa crus-galli*, comprising the step of extracting *Echinochloa crus-galli* using a supercritical extraction method.

[0010] One example of the present specification provides an antioxidant composition comprising an ultrasonic extract and / or supercritical extract of *Echinochloa crus-galli*.

[0011] Another example provides a composition for improving skin condition comprising an ultrasonic extract and / or supercritical extract of *Echinochloa crus-galli*.

[0012] Another example provides an anti-inflammatory composition comprising an ultrasonic extract and / or supercritical extract of *Echinochloa crus-galli*.

[0013] Another example is fleaweed

[0014] (1) Temperature conditions of 30 to 70℃;

[0015] (2) pressure conditions of 300 to 500 Bar; and

[0016] (3) Provides a supercritical extract of Sturgeon extracted under a time condition of 90 to 150 minutes.

[0017] Another example provides a method for preparing a supercritical extract of *Euonymus japonicus*, comprising the step of supercritical extracting *Euonymus japonicus* under the conditions (1) to (3):

[0018] (1) Temperature conditions of 30 to 70℃;

[0019] (2) pressure conditions of 300 to 500 Bar; and

[0020] (3) Time conditions of 90 to 150 minutes.

[0021] Another example provides a method for preparing a supercritical extract of *Euonymus japonicus* with increased roseoside content, comprising the step of extracting *Euonymus japonicus* under the conditions (1) to (3):

[0022] (1) Temperature conditions of 30 to 70℃;

[0023] (2) pressure conditions of 300 to 500 Bar; and

[0024] (3) Time conditions of 90 to 150 minutes.

[0025] Another example provides a supercritical extraction method for producing a supercritical extract of *Euonymus japonicus* with increased roseoside content, comprising the step of extracting *Euonymus japonicus* under the conditions (1) to (3):

[0026] (1) Temperature conditions of 30 to 70℃;

[0027] (2) pressure conditions of 300 to 500 Bar; and

[0028] (3) Time conditions of 90 to 150 minutes.

[0029] The supercritical extract of *Euonymus japonicus* with increased roseoside content may have increased roseoside content compared to *Euonymus japonicus* extracts extracted by other methods (e.g., ultrasonic extraction, immersion extraction, stirring extraction, heating extraction, reflux extraction, hot water extraction, steam distillation extraction, pressurized hot water extraction, etc.). The *Euonymus japonicus* extract and / or the *Euonymus japonicus* extract prepared by the above manufacturing method and / or extraction method may have increased roseoside content compared to *Euonymus japonicus* extracts extracted by other methods (e.g., ultrasonic extraction, immersion extraction, stirring extraction, heating extraction, reflux extraction, hot water extraction, steam distillation extraction, pressurized hot water extraction, etc.).

[0030] Another example provides the antioxidant, skin condition improvement, and / or anti-inflammatory uses of fleaweed extract.

[0031] Another example provides the use of fleaweed extract for preparing compositions for antioxidant, skin condition improvement, and / or anti-inflammatory purposes.

[0032] A composition comprising the ultrasonic extract and / or supercritical extract of the above-mentioned fleaweed has excellent antioxidant, skin condition improvement, and / or anti-inflammatory effects, and in particular, the extract obtained by extracting fleaweed using the supercritical extraction method has a high roseoside content and is excellent in terms of antioxidant, skin condition improvement, and anti-inflammatory effects.

[0033]

[0034] The present invention will be described in more detail below.

[0035]

[0036] fleaweed extract

[0037] One example of the present specification provides a composition for antioxidant, skin condition improvement and / or anti-inflammatory purposes, comprising a flea plant extract.

[0038] Another example provides a supercritical extract of Styrax japonica extracted from Styrax japonica under specific ranges of temperature, pressure, and / or time conditions.

[0039] Another example provides a method for preparing a supercritical extract of Styrax japonica, comprising the step of extracting Styrax japonica under specific ranges of temperature, pressure, and / or time conditions.

[0040] The extraction method for preparing the above extract may use conventional extraction methods, for example, methods such as ultrasonic, supercritical, extraction with an extraction solvent, supercritical fluid extraction, ultrasonic extraction, and / or hot water extraction may be used.

[0041] In this specification, "fleaweed" may include, but is not limited to, plants of the genus Stellaria belonging to the family Caryophyllaceae that are commonly recognized as fleaweed. The fleaweed may be in one or more forms from the group consisting of the whole plant, leaves, stems, flowers, roots, and / or seeds of the plant, and may be in the form of fresh, dried, ground, freeze-dried, and / or raw material for extraction.

[0042] In this specification, "extract" may be ultrasonic extraction, supercritical extraction, hot water extract, solvent extract, distillation extract, pressing extract, and / or ultra-high pressure extract, and may be extracted by methods of hot water extraction, solvent extraction, distillation extraction, press extraction, and / or ultra-high pressure extraction, but is not limited thereto, and an extract obtained by a specific extraction method may be referred to as "(the method) extract," for example, an extract obtained by ultrasonic extraction may be referred to as ultrasonic extract, and an extract obtained by supercritical extraction may be referred to as supercritical extract.

[0043] The above fleaweed extract can be powdered by one or more methods selected from the group consisting of freeze-drying, hot-air drying, natural drying, and microwave drying, and can be used in powder form, and may additionally undergo washing and / or grinding steps prior to the extraction step if necessary.

[0044] The above fleaweed extract can be prepared using fleaweed, dried fleaweed, and / or freeze-dried fleaweed, and if necessary, fleaweed, dried fleaweed, and / or freeze-dried fleaweed can be powdered and used.

[0045] The above-mentioned fleaweed extract may be a fleaweed ultrasonic extract. In this specification, "ultrasonic-assisted extraction" refers to a technique for extracting specific components by using ultrasound to promote the interaction between a solvent and a target substance. Ultrasonic extraction is based on the acoustic cavitation phenomenon generated by ultrasound (e.g., a frequency of 20 to 40 kHz suitable for plant tissue decomposition and the extraction of high-molecular-weight components), and the repeated changes in high and low pressure occurring during this process destroy cell walls or promote the release of intracellular substances.

[0046] The above ultrasonic extract may be extracted by an ultrasonic extraction method under frequency conditions of 20 to 100 KHz, 20 to 80 KHz, 20 to 60 KHz, 20 to 40 KHz, 20 to 30 KHz, 20 to 25 KHz, or 20 to 22 KHz, for example, 20 KHz, but is not limited thereto.

[0047] The above ultrasonic extract is 100 to 1000W, 100 to 800W, 100 to 700W, 100 to 650W, 100 to 600W, 100 to 550W, 300 to 1000W, 300 to 800W, 300 to 700W, 300 to 650W, 300 to 600W, 300 to 550W, 400 to 1000W, 400 to 800W, 400 to 700W, 400 to 650W, 400 to 600W, 400 to 550W, 450 to 1000W, 450 to 800W, 450 to 700W, 450 to It may be extracted by ultrasonic extraction under output conditions of 650W, 450 to 600W, or 450 to 550W, for example, 500W, but is not limited thereto.

[0048] The above ultrasonic extract may be extracted by an ultrasonic extraction method for 30 to 120 minutes, 30 to 110 minutes, 30 to 100 minutes, 30 to 95 minutes, 50 to 120 minutes, 50 to 110 minutes, 50 to 100 minutes, 50 to 95 minutes, 70 to 120 minutes, 70 to 110 minutes, 70 to 100 minutes, 70 to 95 minutes, 80 to 120 minutes, 80 to 110 minutes, 80 to 100 minutes, 80 to 95 minutes, 85 to 120 minutes, 85 to 110 minutes, 85 to 100 minutes, or 85 to 95 minutes, for example, 90 minutes, but is not limited thereto.

[0049] The above ultrasonic extract may be extracted by an ultrasonic extraction method under temperature conditions of 20 to 50°C, 20 to 45°C, 20 to 40°C, 25 to 50°C, 25 to 45°C, 25 to 40°C, 30 to 50°C, 30 to 45°C, 30 to 40°C, 35 to 50°C, 35 to 45°C, or 35 to 40°C, for example, 37°C, but is not limited thereto.

[0050] The above ultrasonic extract may be extracted by repeating ultrasonic extraction 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, for example, 3 times, but is not limited thereto.

[0051] The above-mentioned *Euonymus japonicus* extract may be a *Euonymus japonicus* supercritical extract. In this specification, "supercritical extraction method (supercritical fluid extraction method)" refers to a technique for extracting specific components using a fluid that has reached a supercritical state as a solvent. Here, a supercritical state refers to a state in which a substance exceeds a critical temperature and critical pressure and simultaneously possesses the properties of both liquid and gas. Supercritical fluids possess high diffusivity, low viscosity, and high solubility, which can be utilized to maximize extraction efficiency. Furthermore, by setting a pressure and temperature above the phase boundary point between the gaseous and liquid states of the substance, it enables the substance to possess the characteristics of solubility in the liquid state and the diffusion coefficient and viscosity in the gaseous state, thereby enabling rapid and selective extraction.

[0052] The above supercritical extract of Styrax japonica is subjected to temperature conditions of 30 to 70°C, 30 to 65°C, 30 to 60°C, 30 to 55°C, 30 to 52°C, 35 to 70°C, 35 to 65°C, 35 to 60°C, 35 to 55°C, 35 to 52°C, 40 to 70°C, 40 to 65°C, 40 to 60°C, 40 to 55°C, 40 to 52°C, 45 to 70°C, 45 to 65°C, 45 to 60°C, 45 to 55°C, 45 to 52°C, 48 to 70°C, 48 to 65°C, 48 to 60°C, 48 to 55°C, or 48 to 52°C, for example, 50°C. It may be extracted by supercritical extraction, but is not limited thereto.

[0053] The above supercritical extract of Styrax japonica is 300 to 500 Bar, 300 to 470 Bar, 300 to 450 Bar, 300 to 430 Bar, 300 to 410 Bar, 330 to 500 Bar, 330 to 470 Bar, 330 to 450 Bar, 330 to 430 Bar, 330 to 410 Bar, 350 to 500 Bar, 350 to 470 Bar, 350 to 450 Bar, 350 to 430 Bar, 350 to 410 Bar, 370 to 500 Bar, 370 to 470 Bar, 370 to 450 Bar, 370 to 430 Bar, 370 to 410 Bar, 390 to 500 Bar, 390 It may be extracted by supercritical extraction under pressure conditions of up to 470 Bar, 390 to 450 Bar, 390 to 430 Bar, or 390 to 410 Bar, for example, 400 Bar, but is not limited thereto.

[0054] The above supercritical extract of the fleaweed may be extracted by a supercritical extraction method under time conditions of 90 to 150 minutes, 90 to 140 minutes, 90 to 130 minutes, 90 to 120 minutes, 100 to 150 minutes, 100 to 140 minutes, 100 to 130 minutes, 100 to 120 minutes, 110 to 150 minutes, 110 to 140 minutes, 110 to 130 minutes, 110 to 120 minutes, 120 to 150 minutes, 120 to 140 minutes, or 120 to 130 minutes, for example, 120 minutes, but is not limited thereto.

[0055] Another example is fleaweed

[0056] (1) Temperature conditions of 30 to 70℃;

[0057] (2) pressure conditions of 300 to 500 Bar; and

[0058] (3) Provides a supercritical extract of Sturgeon extracted under a time condition of 90 to 150 minutes.

[0059] Another example provides a method for preparing a supercritical extract of *Euonymus japonicus*, comprising the step of extracting *Euonymus japonicus* under the above supercritical extraction conditions (supercritical extraction), specifically comprising the step of extracting under the conditions of (1) to (3) below:

[0060] (1) Temperature conditions of 30 to 70℃;

[0061] (2) pressure conditions of 300 to 500 Bar; and

[0062] (3) Time conditions of 90 to 150 minutes.

[0063] The above supercritical extraction may involve extracting using CO2 as a supercritical fluid, but is not limited thereto. Supercritical carbon dioxide is applied in extraction processes because it is primarily used under conditions near room temperature and poses little risk of thermal denaturation; compared to other gases, it can produce a supercritical fluid and possesses unique characteristics such as being colorless, odorless, non-toxic, non-explosive, and non-reactive with solutes.

[0064] The above-mentioned Sturgeon extract comprises Sturgeon powder in an amount of 0.01 to 10 (w / v)%, 0.01 to 5 (w / v)%, 0.01 to 3 (w / v)%, 0.01 to 2.5 (w / v)%, 0.01 to 2 (w / v)%, 0.05 to 10 (w / v)%, 0.05 to 5 (w / v)%, 0.05 to 3 (w / v)%, 0.05 to 2.5 (w / v)%, 0.05 to 2 (w / v)%, 0.1 to 10 (w / v)%, 0.1 to 5 (w / v)%, 0.1 to 3 (w / v)%, 0.1 to 2.5 (w / v)%, and 0.1 to 2 based on the total volume of the extract. It may be included at concentrations of (w / v)%, 0.15 to 10 (w / v)%, 0.15 to 5 (w / v)%, 0.15 to 3 (w / v)%, 0.15 to 2.5 (w / v)%, 0.15 to 2 (w / v)%, 0.2 to 10 (w / v)%, 0.2 to 5 (w / v)%, 0.2 to 3 (w / v)%, 0.2 to 2.5 (w / v)%, or 0.2 to 2 (w / v)%, e.g., 0.2, 0.5, 0.8, 1.1, 1.25, 1.4, 1.7, or 2 (w / v)%, but is not limited thereto.

[0065] The above-mentioned fleaweed extract may contain fleaweed powder at concentrations of 0.5 to 40 μg / mL, 0.5 to 35 μg / mL, 0.5 to 32 μg / mL, 0.7 to 40 μg / mL, 0.7 to 35 μg / mL, 0.7 to 32 μg / mL, 0.9 to 40 μg / mL, 0.9 to 35 μg / mL, or 0.9 to 32 μg / mL, for example, 0.98, 1.95, 3.9, 7.8, 15.6, or 31.3 μg / mL, based on the total volume of the extract, but is not limited thereto.

[0066] The above extraction may be performed by selecting an appropriate extraction solvent as needed, and the extraction solvent may be one or more selected from the group consisting of alcohols having 1 (C1) to 4 (C4) carbon atoms and water. For example, it may be water, straight-chain or branched-chain alcohols having 1 to 4 carbon atoms (e.g., ethanol), and mixtures thereof.

[0067] The above extraction solvent may be ethanol, specifically 60 to 99%, 60 to 95%, 60 to 90%, 60 to 85%, 60 to 82%, 60 to 80%, 65 to 99%, 65 to 95%, 65 to 90%, 65 to 85%, 65 to 82%, 65 to 80%, 70 to 99%, 70 to 95%, 70 to 90%, 70 to 85%, 70 to 82%, 70 to 80%, 75 to 99%, 75 to 95%, 75 to 90%, 75 to 85%, 75 to 82%, 75 to 80%, 78 to 99%, 78 to 95%, It may be 78 to 90%, 78 to 85%, 78 to 82%, 78 to 80%, 80 to 99%, 80 to 95%, 80 to 90%, 80 to 85%, or 80 to 82%, for example, 80% ethanol, but is not limited thereto.

[0068] The above supercritical extraction may be performed by selecting an appropriate auxiliary solvent as needed, and the auxiliary solvent may be one or more selected from the group consisting of alcohols having 1 (C1) to 4 (C4) carbon atoms and water. For example, it may be water, straight-chain or branched-chain alcohols having 1 to 4 carbon atoms (e.g., methanol, ethanol, etc.), and mixtures thereof. The auxiliary solvent may be an alkane (e.g., methane, propane, etc.) or a ketone (e.g., acetone, etc.), but is not limited thereto.

[0069] The above auxiliary solvent may be ethanol, specifically 60 to 99%, 60 to 95%, 60 to 90%, 60 to 85%, 60 to 80%, 60 to 78%, 60 to 75%, 65 to 99%, 65 to 95%, 65 to 90%, 65 to 85%, 65 to 80%, 65 to 78%, 65 to 75%, 68 to 99%, 68 to 95%, 68 to 90%, 68 to 85%, 68 to 80%, 68 to 78%, 68 to 75%, 70 to 99%, 70 to 95%, 70 to 90%, 70 to 85%, 70 to 80%, It may be 70 to 78% or 70 to 75% ethanol, but is not limited thereto.

[0070] In the above extraction (e.g., supercritical extraction and / or ultrasonic extraction), the extraction solvent and / or auxiliary solvent may be administered and / or added before, after, and / or during the extraction process. Additionally, the extraction solvent and / or auxiliary solvent may be administered and / or added once, twice, three times, four times, five times, or more times, and may be administered and / or added in a constant amount per minute and / or hour (e.g., 1 to 10 ml, 1 to 8 ml, 1 to 6 ml, 3 to 10 ml, 3 to 8 ml, 3 to 6 ml, 4 to 10 ml, 4 to 8 ml, or 4 to 6 ml, e.g., 5 ml).

[0071] The above flea plant extract may contain roseoside.

[0072] In this specification, "roseoside" refers to a compound of the sesquiterpenoid series having the following chemical formula 1:

[0073] [Chemical Formula 1]

[0074]

[0075] In addition, it has strong antioxidant capacity and representative activity that inhibits lipid oxidation, and is also reported to exhibit various other activities such as insulin-stimulating activity, delay of carcinogenesis, and antihistamine activity, so the above-mentioned fleaweed extract may have the corresponding effects.

[0076] Specifically, reactive oxygen species (ROS; e.g., hydrogen peroxide (H2O2), superoxide (O2) - Oxidative stress can be generated in the skin by various external stimuli (e.g., ultraviolet rays, air pollutants, etc.), and such oxidative stress can be associated with a decline in skin function, abnormal collagen metabolism, and skin aging. Roseoside and / or the above-mentioned fleaweed extract containing it can reduce ROS production in cells with oxidative stress and increase the activity of antioxidant enzymes such as catalase and SOD (antioxidant effect).

[0077] An increase in inflammatory cytokines (e.g., TNF-α, IL-1α, IL-8, etc.) and chemokines / growth factors (e.g., MCP-1, TGF-β, etc.) along with oxidative stress can be linked to inflammatory responses and tissue dysfunction, and roseoside and / or the above-mentioned *Echinochloa crus-galli* extract containing it contains ROS (H2O2, O2 - It may exhibit properties that inhibit the production and expression / production of inflammatory factors (TNF-α, MCP-1, TGF-β) linked to the NADPH oxidase pathway. In addition, roseoside and / or the above-mentioned fleaweed extract containing it may have an anti-inflammatory effect by exhibiting activity that inhibits the release of leukotrienes that cause allergic and inflammatory reactions.

[0078] ROS and inflammatory cytokines induced by external environmental factors (e.g., atmospheric fine particles, ultraviolet rays, etc.) may promote an increase in MMPs and collagen degradation, or contribute to skin aging (e.g., wrinkles, loss of elasticity, etc.) and the exacerbation of inflammatory skin diseases. Roseoside and / or the above-mentioned fleaweed extract containing it exhibit antioxidant and / or anti-inflammatory effects, and through these actions, may contribute to the improvement of skin condition (e.g., anti-aging, improvement of skin elasticity, improvement of skin barrier, improvement of keratinization and / or skin moisturization). In addition, roseoside and / or the above-mentioned fleaweed extract containing it may exhibit a depigmentation effect (whitening effect) by inhibiting melanin synthesis in melanocytes.

[0079] The above-mentioned *Euphorbia pekinensis* extract has a high roseoside content and is associated with inhibiting oxidative stress (ROS), increasing antioxidant enzyme activity, and regulating inflammatory mediators, and may have antioxidant uses (effects), uses for improving skin condition (effects) (e.g., skin whitening, anti-aging, skin elasticity improvement, antioxidant, skin barrier improvement, exfoliation improvement and / or skin moisturization), and / or anti-inflammatory uses (effects). In addition, the extraction efficiency of an extract can vary depending on extraction conditions (e.g., type and composition of solvent, temperature, pressure, extraction time, whether stirring or ultrasonic treatment is performed, etc.), and accordingly, the content of specific indicator components (e.g., roseoside) may fluctuate significantly. The supercritical and / or ultrasonic extract of the above-mentioned fleaweed can secure a relatively high roseoside content when compared to other types of extracts (e.g., immersion extraction, stirring extraction, heating extraction, reflux extraction, hot water extraction, steam distillation extraction, pressurized hot water extraction, etc.), and accordingly, the applicability of the roseoside-based antioxidant composition, skin condition improvement composition (e.g., for skin whitening, anti-aging, skin elasticity improvement, antioxidant, skin barrier improvement, exfoliation improvement and / or skin moisturization), and / or anti-inflammatory composition can be enhanced.

[0080] The above-mentioned Sturgeon extract, the supercritical extract of Sturgeon with increased roseoside content in the method for preparing the above-mentioned supercritical extract of Sturgeon, and / or the supercritical extract of Sturgeon with increased roseoside content in the above-mentioned supercritical extraction method, wherein the roseoside is present in an amount of 0.01 to 0.7 mg / g, 0.01 to 0.65 mg / g, 0.01 to 0.6 mg / g, 0.01 to 0.55 mg / g, 0.01 to 0.5 mg / g, 0.01 to 0.45 mg / g, 0.01 to 0.4 mg / g, 0.01 to 0.35 mg / g, 0.01 to 0.3 mg / g, 0.01 to 0.25 mg / g, 0.01 to 0.2 mg / g, 0.01 to 0.15 mg / g, 0.01 to 0.1 mg / g, 0.04 to 0.7 mg / g, 0.04 to 0.65 mg / g, 0.04 to 0.6 mg / g, 0.04 to 0.55 mg / g, 0.04 to 0.5 mg / g, 0.04 to 0.45 mg / g, 0.01 to 0.4 mg / g, 0.04 to 0.35 mg / g, 0.04 to 0.3 mg / g, 0.04 to 0.25 mg / g, 0.04 to 0.2 mg / g, 0.04 to 0.15 mg / g, 0.04 to 0.1 mg / g, 0.06 to 0.7 mg / g, 0.06 to 0.65 mg / g, 0.06 to 0.6 mg / g, 0.06 to 0.55 mg / g, 0.06 to 0.5 mg / g, 0.06 to 0.45 mg / g, 0.06 to 0.4 mg / g, 0.06 to 0.35 mg / g, 0.06 to 0.3 mg / g, 0.06 to 0.25 mg / g, 0.06 to 0.2 mg / g, 0.06 to 0.15 mg / g, 0.06 to 0.1 mg / g, 0.1 to 0.7 mg / g, 0.1 to 0.65 mg / g, 0.1 to 0.6 mg / g, 0.1 to 0.55 mg / g, 0.1 to 0.5 mg / g, 0.1 to 0.45 mg / g, 0.2 to 0.7 mg / g, 0.2 to 0.65 mg / g, 0.2 to 0.6 mg / g, 0.2 to 0.55 mg / g, 0.2 to 0.5 mg / g, 0.2 to 0.45 mg / g, 0.3 to 0.7 mg / g, 0.3 to 0.65 mg / g, 0.3 to 0.6 mg / g, 0.3 to 0.55 mg / g, 0.3 to 0.5 mg / g, 0.3 to 0.45 mg / g, 0.35 to 0.7 mg / g, 0.35 to 0.65 mg / g, 0.35 to 0.6 mg / g, 0.35 to 0.55 mg / g, 0.35 to 0.5 mg / g, 0.35 to 0.45 mg / g, 0.4 to 0.7 mg / g, 0.4 It may be included at concentrations of up to 0.65 mg / g, 0.4 to 0.6 mg / g, 0.4 to 0.55 mg / g, 0.4 to 0.5 mg / g, or 0.4 to 0.45 mg / g, e.g., 0.07 or 0.41 mg / g, but is not limited thereto.

[0081]

[0082] Antioxidant uses

[0083] One example of the present specification provides an antioxidant composition comprising a flea plant extract.

[0084] In this specification, "antioxidant" refers to a concept that includes the action of inhibiting or preventing oxidative damage induced by reactive oxygen species (ROS), free radicals, or other oxidizing agents. Oxidative damage results in the alteration and destruction of major biomolecules, such as DNA, proteins, and lipids, within cells and tissues, and is known to be one of the major causes of aging, inflammatory diseases, cancer, cardiovascular diseases, and other chronic diseases.

[0085] The above composition may have one or more characteristics selected from the group consisting of (1) and (2) below, but is not limited thereto:

[0086] (1) DPPH radical scavenging activity; and

[0087] (2) Active inhibitory activity against the production of reactive oxygen species.

[0088] The composition containing the above-mentioned fleaweed extract can have strong antioxidant activity through a multi-antioxidant mechanism in which various types of antioxidant substances (e.g., flavonoids, polyphenols, amino acids, etc.) act complementarily, thereby preventing cell damage and alleviating oxidative stress, and can have a superior antioxidant effect compared to ascorbic acid, which performs antioxidant action through a single mechanism (e.g., electron transfer, etc.).

[0089] Another example provides a method for antioxidant and / or skin condition improvement, comprising the step of administering the above-mentioned antioxidant composition to a subject in need of antioxidant.

[0090] The above-mentioned target requiring antioxidants may refer to a living organism damaged or negatively affected by oxidative stress, etc., or a cell, tissue, or culture thereof isolated therefrom, and the above-mentioned target and / or organism may be animals including, but not limited to, birds (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finch, hawk, crow, ostrich, emu, and cassowary, etc.), cattle (e.g., cattle), pigs (e.g., pigs), canids (e.g., dogs, wolves, foxes, coyotes, jackals, etc.), felines (e.g., lions, tigers, domestic cats, wild cats, other big cats, cheetahs, lynxes, etc.), horses (e.g., horses, etc.), sheep (e.g., sheep, goats, lambs, bison, etc.), and primates (e.g., humans, prosimians, tarsiers, monkeys, gibbons, apes). The above "animal" may include, but is not limited to, individual animals at all developmental stages, such as embryonic and fetal stages. Additionally, humans may be excluded from the above animals.

[0091]

[0092] Use for improving skin condition

[0093] One example of the present specification provides a composition for improving skin condition comprising a flea plant extract.

[0094] In this specification, "improvement of skin condition" may be used to include all of the following: alleviation or improvement of symptoms such as wrinkles, melasma, acne, etc. of the skin; delay or alleviation of the progression of skin-related diseases (e.g., pigmentation and / or related diseases); improvement, alleviation, or stabilization of skin-related disease conditions or symptoms. Specifically, it may be one or more selected from the group consisting of skin whitening, anti-aging, improvement of skin elasticity (e.g., strengthening of skin elasticity), antioxidant, improvement of skin barrier (e.g., strengthening of skin barrier), improvement of exfoliation, and skin moisturization, but is not limited thereto. The skin-related diseases may be one or more selected from the group consisting of wrinkles, melasma, acne, freckles, age spots, pigmented scars, and dandruff, but are not limited thereto.

[0095] In this specification, the "skin whitening" effect means not only brightening the skin tone by inhibiting the synthesis of melanin pigment, but also improving skin hyperpigmentation such as melasma or freckles caused by ultraviolet rays, hormones, or heredity.

[0096] In this specification, "anti-aging" refers to a concept encompassing all actions that delay, prevent, alleviate, or improve structural and functional changes caused by the biological aging process. Here, the aging process refers to changes that occur over time at the skin, tissue, cellular, or molecular level, and may include wrinkles, loss of elasticity, pigmentation, cell damage, increased inflammatory response, accumulation of oxidative stress, etc.

[0097] In this specification, "melasma" refers to a condition in which brown spots of irregular shape and various sizes appear on exposed areas, particularly the face.

[0098] In this specification, "freckles" refers to small, yellowish-brown pigmented spots that appear mainly on the skin in areas exposed to sunlight, and occur mainly on the cheeks, upper arms, chest, and upper back.

[0099] In this specification, "elasticity" means a force that bounces like a spring or holds taut.

[0100] The above composition may have one or more characteristics selected from the group consisting of (1) and (2) below, but is not limited thereto:

[0101] (1) Tyrosinase inhibitory activity; and

[0102] (2) Elastase inhibitory activity.

[0103] The composition containing the above-mentioned *Euonymus japonicus* extract includes various types of antioxidants and enzyme inhibitors, and exerts enzyme inhibitory effects through multiple mechanisms, so that a single component can possess broad biological activity compared to ursolic acid. In this specification, "ursolic acid" is a naturally derived compound that possesses various biological activities related to skin health, is used as a representative comparative standard in elastase inhibition studies, and is a triterpene compound commonly found in plants that exhibits potent antioxidant and anti-inflammatory effects.

[0104] Another example provides a method for improving skin condition, comprising the step of administering the above-mentioned composition for improving skin condition to a subject who requires improvement of skin condition.

[0105] The subject requiring improvement of the skin condition may refer to a living organism or cells, tissues, or cultures isolated therefrom, and the subject and / or organism may be animals including, but not limited to, birds (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finch, hawk, crow, ostrich, emu, and cassowary, etc.), cattle (e.g., cattle), pigs (e.g., pigs), canids (e.g., dogs, wolves, foxes, coyotes, jackals, etc.), felines (e.g., lions, tigers, domestic cats, wild cats, other big cats, cheetahs, lynxes, etc.), horses (e.g., horses, etc.), sheep (e.g., sheep, goats, lambs, bison, etc.), and primates (e.g., humans, prosimians, tarsiers, monkeys, gibbons, apes). The above "animal" may include, but is not limited to, individual animals at all developmental stages, such as embryonic and fetal stages. Additionally, humans may be excluded from the above animals.

[0106]

[0107] Anti-inflammatory use

[0108] One example of the present specification provides an anti-inflammatory composition comprising a flea plant extract.

[0109] In this specification, "inflammation" refers to the reaction of cells or blood vessels that occurs when a living organism is damaged or stimulated.

[0110] In this specification, "anti-inflammatory" may mean suppressing or eliminating inflammation, and specifically, may mean preventing, improving, and / or treating inflammation. The anti-inflammatory composition may be used interchangeably with compositions for preventing, improving, and / or treating inflammation.

[0111] The above composition may have preventive, improving, and / or therapeutic effects for skin inflammation-related diseases. The skin inflammation refers to inflammation occurring on the skin and may include eczema, which appears moist among skin diseases. Skin inflammation can be divided into acute, subacute, and chronic types; acute and subacute types may progress through erythema (red spots) and / or papules (protrusions the size of millet or rice grains) to vesicles (small blisters), while chronic types may appear dry.

[0112] The above skin inflammation-related diseases may be one or more selected from the group consisting of acne, atopic dermatitis, seborrheic dermatitis, dry dermatitis, contact dermatitis, nummular dermatitis, and allergic dermatitis, but are not limited thereto.

[0113] The above composition may have NO production inhibitory activity, but is not limited thereto.

[0114] Another example provides a method for anti-inflammatory and / or skin condition improvement, comprising the step of administering the above anti-inflammatory composition to a subject in need of anti-inflammatory treatment.

[0115] The subject requiring anti-inflammatory treatment may refer to a living organism that may or has developed inflammation, or cells, tissues, or cultures isolated therefrom, and the subject and / or organism may be animals including, but not limited to, birds (e.g., chickens, ducks, geese, turkeys, quails, pheasants, parrots, finches, hawks, crows, ostriches, emus, and cassowaries), cattle (e.g., cattle), pigs (e.g., pigs), canids (e.g., dogs, wolves, foxes, coyotes, jackals, etc.), felines (e.g., lions, tigers, domestic cats, wild cats, other big cats, cheetahs, lynxes, etc.), horses (e.g., horses, etc.), sheep (e.g., sheep, goats, lambs, bison, etc.), and primates (e.g., humans, prosimians, tarsiers, monkeys, gibbons, apes). The above "animal" may include, but is not limited to, individual animals at all developmental stages, such as embryonic and fetal stages. Additionally, humans may be excluded from the above animals.

[0116]

[0117] Cosmetic composition containing fleaweed extract

[0118] One example of the present specification provides a cosmetic composition for antioxidant, skin condition improvement and / or anti-inflammatory purposes, comprising a flea plant extract.

[0119] The “cosmetic composition” and / or “cosmetic” provided in this specification may be any formulation form commonly manufactured in the art, and may be formulated, for example, as a capsule, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, pack, massage cream and spray, but is not limited thereto. Specifically, it may be one or more formulations selected from the group consisting of lotions (e.g., softening lotions, nourishing lotions, etc.), emulsions, lotions (e.g., body lotions, etc.), essences, creams (e.g., gel creams, massage creams, body creams, sunscreens, eye creams, cleansing creams, etc.), gels, packs, oils, makeup bases, foundations (e.g., powder foundations, emulsion foundations, wax foundations, etc.), lipsticks, patches, aerosols (sprays), cleansers, mouthwashes, skin conditioners, cleansing foams, cleansing waters, and / or powders, but is not limited thereto.

[0120] In one embodiment, the concentration of *Echinochloa crus-galli* extract included in the cosmetic composition is, based on the total volume of the composition, 0.1 to 10,000 µg / mL, 0.1 to 5,000 µg / mL, 0.1 to 3,000 µg / mL, 0.1 to 2,000 µg / mL, 0.1 to 1,000 µg / mL, 0.1 to 500 µg / mL, 0.1 to 300 µg / mL, 0.1 to 200 µg / mL, 0.1 to 100 µg / mL, 1 to 10,000 µg / mL, 1 to 5,000 µg / mL, 1 to 3,000 µg / mL, 1 to 2,000 µg / mL, 1 to 1,000 µg / mL, 1 to 500 µg / mL, 1 to 300 µg / mL, 1 to 200 µg / mL, It may be 1 to 100 ug / mL, 10 to 10000 ug / mL, 10 to 5000 ug / mL, 10 to 3000 ug / mL, 10 to 2000 ug / mL, 10 to 1000 ug / mL, 10 to 500 ug / mL, 10 to 300 ug / mL, 10 to 200 ug / mL, 10 to 100 ug / mL, 100 to 10000 ug / mL, 100 to 5000 ug / mL, 100 to 3000 ug / mL, 100 to 2000 ug / mL, 100 to 1000 ug / mL, 100 to 500 ug / mL, 100 to 300 ug / mL, or 100 to 200 ug / mL.

[0121] In one embodiment, a cosmetic product comprising the above cosmetic composition is provided.

[0122] The above cosmetic composition and / or cosmetic may additionally include a carrier, excipient, and / or additive acceptable to the cosmetic in addition to the fleaweed extract.

[0123] The above cosmetic or cosmetic composition may be used alone or in combination with other cosmetic compositions. Additionally, the cosmetic composition of the present invention may be used according to conventional methods of use, but the frequency of use may be varied depending on the user's skin condition or preference.

[0124]

[0125] Pharmaceutical composition containing fleaweed extract

[0126] One example of the present specification provides a pharmaceutical composition for antioxidant, skin condition improvement and / or anti-inflammatory purposes, comprising a flea plant extract.

[0127] Another example provides a pharmaceutical composition for the prevention, improvement, and / or treatment of skin-related diseases, comprising a flea plant extract.

[0128] The above skin-related diseases may be one or more selected from the group consisting of wrinkles, melasma, acne, freckles, age spots, pigmented scars, atopic dermatitis, seborrheic dermatitis, dry dermatitis, contact dermatitis, nummular dermatitis, and allergic dermatitis, but are not limited thereto.

[0129] In this specification, "treatment" may be used to include all of the following: alleviation or improvement of symptoms (e.g., skin-related diseases, etc.); delay or alleviation of the progression of a disease (e.g., skin-related diseases, etc.); improvement, alleviation, or stabilization of the disease state or symptoms; partial or complete recovery or elimination; and other beneficial treatment results. "Improvement" may be used to include all of the following: alleviation or improvement of (e.g., skin-related diseases, etc.); delay or alleviation of the progression of a disease (e.g., skin-related diseases, etc.); improvement, alleviation, or stabilization of the disease state or symptoms. "Prevention" is used to include all mechanisms and / or effects that act on a subject who does not have a specific disease to prevent the specific disease from developing or to delay the onset of the disease.

[0130] The mode of administration of the pharmaceutical composition provided in this specification may be any commonly used mode, and may be administered, for example, by oral administration, or by a non-parenteral route such as intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, local administration to a lesion site (e.g., a site requiring prevention, treatment, or improvement of inflammation, etc.), or intraperitoneal injection.

[0131] The above pharmaceutical composition may be administered in a pharmaceutically effective amount. The dosage of the above pharmaceutical composition may be prescribed differently depending on factors such as the formulation method, method of administration, patient's age, weight, sex, pathological condition, food, time of administration, interval of administration, route of administration, excretion rate, and response sensitivity. The dosage may vary depending on the patient's age, weight, sex, form of administration, health condition, and degree of disease, and may be divided into one to several doses per day at regular intervals at the discretion of a physician or pharmacist. For example, a single dose or daily dose of a pharmaceutical composition is 0.001 to 10,000 mg / kg based on the weight of the solid content of the active ingredient (Echinochloa crus-galli extract), specifically, 0.01 to 10,000 mg / kg, 0.01 to 5,000 mg / kg, 0.01 to 3,000 mg / kg, 0.01 to 2,500 mg / kg, 0.01 to 2,000 mg / kg, 0.01 to 1,800 mg / kg, 0.1 to 10,000 mg / kg, 0.1 to 5,000 mg / kg, 0.1 to 3,000 mg / kg, 0.1 to 2,500 mg / kg, 0.1 to 2,000 mg / kg, 0.1 to 1,800 mg / kg, 1 to 10,000 mg / kg, 1 to The range may be 5000 mg / kg, 1 to 3000 mg / kg, 1 to 2500 mg / kg, 1 to 2000 mg / kg, 1 to 1800 mg / kg, 10 to 10000 mg / kg, 10 to 5000 mg / kg, 10 to 3000 mg / kg, 10 to 2500 mg / kg, 10 to 2000 mg / kg, 10 to 1800 mg / kg, 100 to 10000 mg / kg, 100 to 5000 mg / kg, 100 to 3000 mg / kg, 100 to 2500 mg / kg, 100 to 2000 mg / kg, or 100 to 1800 mg / kg, but is not limited thereto. The above single dose or daily dose may be formulated as a single preparation in the form of a unit dose, formulated in appropriate quantities, or manufactured by being contained in a multi-dose container.The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences.

[0132] The above pharmaceutical compositions may each be formulated and used according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or parenteral formulations such as sterile injectable solutions.

[0133] In addition to the active ingredient described above, the pharmaceutical composition may be administered by mixing it with one or more adjuvants selected from the group consisting of pharmaceutically and / or physiologically acceptable carriers, excipients, and diluents, selected in consideration of the method of administration and standard pharmaceutical practice. For example, the pharmaceutically and / or physiologically acceptable carrier may include one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto, and may be any carrier commonly used in the pharmaceutical field.

[0134] The above pharmaceutically and / or physiologically acceptable diluents and / or excipients may be one or more selected from the group consisting of all fillers, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, etc., commonly used in the proper formulation of pharmaceutical compositions.

[0135] For example, in the case of solid formulations for oral administration such as tablets, pills, powders, granules, or capsules, the excipient may be one or more selected from the group consisting of at least one substance for the formulation of such solid formulations, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition, lubricants such as magnesium styrate talc may also be used. Furthermore, in the case of formulation of liquid formulations for oral administration such as suspensions, liquid formulations, emulsions, or syrups, one or more selected from the group consisting of commonly used simple diluents such as water and liquid paraffin may be used, and in addition, various commonly used excipients, such as humectants, sweeteners, flavorings, and / or preservatives, may be included, but are not limited thereto.

[0136] For example, the above pharmaceutical composition may be administered orally, intraorally, or sublingually in the form of tablets containing starch or lactose, in the form of capsules containing suitable excipients, or in the form of elixirs or suspensions containing chemicals for flavoring or coloring. These liquid formulations may be formulated with pharmaceutically acceptable additives such as suspensions (e.g., glyceride mixtures such as methylcellulose, semi-synthetic glycerides such as Witepsol, or mixtures of apricot kernel oil and PEG-6 esters, or mixtures of PEG-8 and caprylic / capric glycerides), but are not limited thereto.

[0137]

[0138] Food composition containing fleaweed extract

[0139] One example of the present specification provides a food composition for antioxidant, skin condition improvement and / or anti-inflammatory purposes, comprising a flea plant extract.

[0140] In the food compositions provided in this specification, the term "food" refers to an edible natural or processed product containing one or more nutrients, and in a conventional sense, may be used to refer to one or more selected from the group consisting of various general foods, health functional foods, beverages, food additives, and beverage additives. The term "food composition" may refer to a combination of materials for manufacturing said food.

[0141] The content of the herbal ingredient in the above food composition can be appropriately adjusted according to the form of food use, purpose, etc., and, for example, may be 0.00001 to 99.9 weight%, 0.0001 to 99.9 weight%, 0.001 to 99.9 weight%, or 0.001 to 50 weight% based on the weight of the solid content, but is not limited thereto.

[0142] The above-mentioned health functional food refers to any food manufactured using nutrients that are easily deficient in daily meals or raw materials that have functions useful to the human body (hereinafter referred to as "functional raw materials"), which helps maintain health or prevent and / or improve (alleviate) certain diseases or symptoms, and there are no special restrictions on the final product form. For example, the above-mentioned health functional food may be selected from a group consisting of various ordinary foods, food supplements, beverages, food additives, etc., but is not limited thereto. The above-mentioned health functional food may be in solid, semi-solid, or liquid form, and specifically may be formulated as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., but is not limited thereto.

[0143] The above-mentioned health functional food may additionally contain one or more selected from the group consisting of various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic or natural flavoring agents, coloring agents, thickening agents (cheese, chocolate, etc.), pectic acid or its salt, alginic acid or its salt, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. The proportion of such additives is generally selected in the range of 0.001 to about 20 parts by weight per 100 parts by weight of the total health functional food, but is not limited thereto.

[0144] The present invention relates to an antioxidant, skin condition improvement and / or anti-inflammatory composition comprising a flea plant extract and / or a supercritical extract of the flea plant and an extraction method for preparing the same, which has excellent antioxidant, skin condition improvement and / or anti-inflammatory effects.

[0145] Figure 1 is a photograph showing the shape of the fleaweed used in the preparation of fleaweed extract.

[0146] Figure 2 is a graph showing the results of measuring the DPPH radical scavenging activity of the ultrasonic extract of *Echinochloa crus-galli*.

[0147] Figure 3 is a graph showing the results of measuring the SOD-like activity of the ultrasonic extract of *Echinochloa crus-galli*.

[0148] Figure 4 is a graph showing the results of measuring the tyrosinase inhibitory activity of the ultrasonic extract of *Echinochloa crus-galli*.

[0149] Figure 5 is a graph showing the results of measuring the elastase inhibitory activity of the ultrasonic extract of *Echinochloa crus-galli*.

[0150] Figures 6 and 7, respectively, are graphs showing cell viability measured after administering an ultrasonic extract of *Echinochloa crus-galli* to RAW264.7 cells and culturing them for 24 or 48 hours.

[0151] Figure 8 is a graph showing the NO production amount measured after administering the ultrasonic extract of *Echinochloa crus-galli* to RAW264.7 cells and culturing them for 24 or 48 hours.

[0152] Figure 9 is a graph showing the results of measuring the roseoside content in ultrasonic and supercritical extracts of Styrax japonica.

[0153] Figure 10 shows the results of UHPLC-MS chromatogram analysis performed when measuring the roseoside content in the ultrasonic and supercritical extracts of *Echinochloa crus-galli*.

[0154] Figure 11 shows the results of UHPLC-UV (254 nm) chromatogram analysis performed when measuring the roseoside content in the ultrasonic and supercritical extracts of *Echinochloa crus-galli*.

[0155] The present invention will be explained in more detail below through examples. However, the following examples are merely for illustrating the content of the present invention, and the scope of the present invention is not limited by the following examples.

[0156]

[0157] Example 1. Preparation of Ultrasonic Extract of Stinkhorn

[0158] Whole plants of *Stellaria alsine var. undulata* from Jangseong, Jeonnam, harvested from open fields in March to May were obtained, washed with purified water, dried at room temperature, and pretreated by finely grinding them to a particle size of 0.2 to 0.5 mm using a grinder. The *Stellaria alsine var. undulata* used is shown in Figure 1, where a is the appearance of the whole plant before harvesting, b is the appearance before drying after washing, c is the appearance before grinding after drying, and d is the appearance of the dried *Stellaria alsine var. undulata* after grinding to 0.2 to 0.5 mm.

[0159] 200 g of the pretreated dried fleaweed powder from Example 1 was added to 80% ethanol at a concentration of 1.25 (w / v)%, and extracted three times for 90 minutes at a frequency of 20 KHz, 500W output, and 37℃ temperature using an ultrasonic extractor Labtech (NE-1000Z). Centrifugation was then performed at 4,000 rpm for 10 minutes to obtain the supernatant of the fleaweed extract.

[0160] The above supernatant was concentrated under reduced pressure using a rotary evaporator and then freeze-dried using a freeze dryer to prepare the final extract powder.

[0161]

[0162] Test Example 1. Measurement of the antioxidant activity of *Echinochloa crus-galli* extract

[0163] Test Example 1-1. Measurement of phenolic content in *Echinochloa crus-galli* extract

[0164] The phenol content in the fleaweed extract of Example 1 above was measured by the College of Pharmacy at Suncheon National University.

[0165] Specifically, 100 μl of the ultrasonic extract from Example 1 prior to freeze-drying, 100 μl of Folin & Ciocalteau's phenol reagent (Sigma), and 300 μl of 20% Na2CO3 were combined and reacted at room temperature for 15 minutes, then 1 ml of triple-distilled water was added and stirred at 10,000 rpm for 5 minutes. As a standard substance, tannic acid was prepared by dissolving it in distilled water at various concentrations.

[0166] The absorbance of the above sample was measured at 640 nm using a Spectrophotometer (Switzerland, Tecan, Sunrise instrument), a calibration curve (standard curve) was prepared from the above standard substance, and the phenol content was measured in units of g tannic acid equivalent sample dry weight (mgTAE / g), and the results are shown in Table 1 below.

[0167] Type of extract Total phenolic content (mg TAE / g extract) Example 117.49

[0168] As a result of measuring the phenol content, the phenol content of Example 1, which is an ultrasonic extract of Styrax japonica, was measured to be 17.49 mg TAE / g extract, indicating that it contains a high amount of phenol.

[0169]

[0170] Test Example 1-2. Measurement of DPPH radical scavenging activity of *Echinochloa crus-galli* extract

[0171] The DPPH radical scavenging activity of the fleaweed extract of Example 1 above was measured by the College of Pharmacy at Suncheon National University in accordance with the method of Ikeda et al.

[0172] Specifically, 200 μL of the extract of *Euphorbia pekinensis* was prepared by adding the extract powder of Example 1 to methanol at concentrations of 2, 5, 8, 11, 14, 17, or 20 mg / mL. 200 μL of 0.1 mM DPPH solution (Sigma Chemicals Co., USA) was added to the methanol containing the extract powder, stirred, left at room temperature for 30 minutes, and the absorbance (519 nm) was measured using a Spectrophotometer (Switzerland, Tecan, Sunrise instrument). The DPPH radical scavenging activity was calculated by the following Equation 1, and the results are shown in Figure 2.

[0173] As a positive control, ascorbic acid was added to methanol at concentrations of 0, 10, 20, 40, 60, or 80 μg / mL instead of the above extract powder, and the DPPH radical scavenging activity was calculated in the same way, and the results are shown in Table 2 below.

[0174] [Mathematical Formula 1]

[0175] DPPH radical scavenging activity (%) = {1-(A sample / A control )} X 100%

[0176] (In the above mathematical formula 1, A sample / is the absorbance when fleaweed extract or ascorbic acid is added, A control (This refers to the absorbance when neither of the two substances is added)

[0177] Ascorbic acid concentration (μg / ml) Free radical scavenging activity (%) 0 0 10 4.6 4 20 11.7 9 40 21.7 4 6 0 3 4.6 28 0 4 7.6 1

[0178] As a result of measuring DPPH radical scavenging activity, the scavenging activity of Example 1, which was an ultrasonic extract of Styrax japonica, was high, and in particular, it showed a scavenging activity of 75.12% at 20 mg / mL, having a high scavenging activity and excellent antioxidant effect.

[0179]

[0180] Test Example 1-3. Measurement of SOD-like activity of *Echinochloa crus-galli* extract

[0181] The SOD (Superoxide Dismutase)-like activity of the fleaweed extract of Example 1 above was commissioned to the College of Pharmacy at Suncheon National University to measure and calculate the degree of inhibition of the auto-oxidation of pyrogallol.

[0182] Specifically, 100 μl of ultrasonic extract of *Euphorbia pekinensis*, prepared by adding the *Euphorbia pekinensis* extract powder of Example 1 to 80% ethanol at concentrations of 2, 5, 8, 11, 14, 17, or 20 mg / mL, 500 μl of 50 mM Tris-HCl buffer (pH 8.5), 100 μl of 80% *Euphorbia pekinensis*, and 21 mM pyrogallol in 1 mM EDTA (DW) were reacted at 37°C for 10 minutes. Afterward, 10 μl of 1 M HCl was added to terminate the reaction, and the absorbance (440 nm) was measured using a Spectrophotometer (Switzerland, Tecan, Sunrise instrument). The SOD-like activity was calculated using the following Equation 2, and the results are shown in Figure 3.

[0183] As a positive control, ascorbic acid was added to 80% ethanol at concentrations of 0, 50, 100, 200, 400, 600, 800, or 1000 μg / mL instead of the above extract powder, and SOD-like activity was measured in the same way, and the results are shown in Table 3 below.

[0184] [Mathematical Formula 2]

[0185] SOD-like activity (%) = {1-(A sample / A control )} X 100%

[0186] (In the above mathematical formula 2, A sample ε is the absorbance when fleaweed extract or ascorbic acid is added, A control (This refers to the absorbance when neither of the two substances is added)

[0187] Ascorbic acid concentration (μg / ml) SOD-like activity (%) 0 0 5 0 7.1 1 10 0 11.4 9 20 0 19.8 6 40 0 ​​3 4.9 1 60 0 4 6.9 38 0 0 6 0.5 6 10 0 6 5.04

[0188] As a result of measuring SOD-like activity, the ultrasonic extract of *Euonymus japonicus* in Example 1 showed high SOD-like activity, and in particular, it had the highest SOD-like activity of 48.89% at 20 mg / mL, demonstrating excellent antioxidant effects.

[0189]

[0190] Test Example 2. Measurement of skin whitening and anti-aging effects of Sturgeon extract

[0191] Test Example 2-1. Measurement of tyrosinase inhibitory activity of *Echinochloa crus-galli* extract

[0192] The tyrosinase inhibitory activity of the fleaweed extract of Example 1 above was measured by the College of Pharmacy at Suncheon National University.

[0193] Specifically, the amount of DOPA chrome formed from DOPA by tyrosinase was measured by adding the fleaweed extract powder of Example 1 to 80% ethanol at 2, 5, 8, 11, 14, 17, or 20 mg / mL, reacting 60 μL of 300 units / mL of tyrosinase, and 120 μL of 100 mM Sodium phosphate buffer (pH 6.7) containing 2 mM L-DOPA dissolved in it at 37°C for 20 minutes, and measuring the absorbance (490 nm) using a Spectrophotometer (Switzerland, Tecan, Sunrise instrument).

[0194] Additionally, to correct the color effect of the extract itself, the above reaction was carried out at 37°C for 20 minutes without tyrosinase and the absorbance was measured. The corrected absorbance was then measured by subtracting the absorbance from the absorbance measured with tyrosinase included, and the tyrosinase inhibitory activity was calculated by the following mathematical formula 3, and the results are shown in Figure 4.

[0195] As a positive control, ascorbic acid was added to 80% ethanol at concentrations of 0, 50, 100, 200, 400, or 600 μg / ml instead of the above extract powder, and tyrosinase inhibitory activity was measured in the same way, and the results are shown in Table 4 below.

[0196] [Mathematical Formula 3]

[0197] Tyrosinase inhibitory activity (%) = {1-(A sample / A control )} X 100%

[0198] (In the above mathematical formula 3, A sample ε is the corrected absorbance when fleaweed extract or ascorbic acid is added, A control represents the corrected absorbance when the two substances are not added.

[0199] Ascorbic acid concentration (μg / ml)Tyrosinase inhibition activity (%)00503.2010010.2720023.6040055.8060091.45

[0200] As a result of measuring tyrosinase inhibitory activity, the ultrasonic extract of *Euonymus japonicus* in Example 1 showed high tyrosinase inhibitory activity, and in particular, it had the highest tyrosinase inhibitory activity of 17.16% at 17 mg / mL.

[0201]

[0202] Test Example 2-2. Measurement of Elastase Inhibitory Activity of Styrax Extract

[0203] The elastase inhibitory activity of the fleaweed extract of Example 1 above was measured by the College of Pharmacy at Sunchon National University.

[0204] Specifically, 0.5 mL of each prepared extract was placed in a test tube and the *Euphorbia pekinensis* extract powder of Example 1 was added to 80% ethanol at concentrations of 2, 5, 8, 11, 14, 17, or 20 mg / mL. Then, 0.5 mL of a solution containing porcine pancreas elastase dissolved in 50 mM tris-HCl buffer (pH 8.6) at a concentration of 2.5 U / mL was added, and 0.5 mL of a solution containing N-succinyl-(L-Ala)3-p-nitroanilide dissolved as a substrate in 50 mM tris-HCl buffer (pH 8.6) at a concentration of 0.5 mg / mL was added. The mixture was reacted for 20 minutes, the absorbance (at 410 nm) was measured using a Spectrophotometer (Switzerland, Tecan, Sunrise instrument), and the elastase inhibition was determined according to the following Equation 4. The activity was calculated, and the results are shown in Figure 5.

[0205] As a positive control, ursolic acid, which generally acts as an elastase inhibitor, was added to 80% ethanol at concentrations of 0, 25, 50, 100, 200, 400, or 800 μg / ml instead of the above extract powder, and the elastase inhibitory activity was measured in the same manner, and the results are shown in Table 5 below.

[0206] [Mathematical Formula 4]

[0207] Elastase inhibitory activity (%) = {1-(A sample / A control )} X 100%

[0208] (In the above mathematical formula 4, A sample ε is the absorbance when fleaweed extract or ascorbic acid is added, A control (This refers to the absorbance when neither of the two substances is added)

[0209] Ursolic acid concentration (μg / ml) Elastase inhibition activity (%) 0 0 25 6.5 5 5 0 10.4 9 10 0 13.6 5 20 0 24.7 2 40 0 ​​35.0 48 0 0 39.27

[0210] As a result of measuring elastase inhibitory activity, the ultrasonic extract of *Echinochloa crus-galli* in Example 1 showed high elastase inhibitory activity, and in particular, it had the highest tyrosinase inhibitory activity of 111.12% at 17 mg / mL.

[0211]

[0212] Test Example 3. Measurement of the anti-inflammatory effect of Styrax japonica extract

[0213] Test Example 3-1. Cell culture for measuring anti-inflammatory effect

[0214] RAW264.7 mouse-derived macrophages were obtained from the Korea Cell Line Bank and prepared. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GibcoBRL) supplemented with 10% fetal bovine serum (FBS, GibcoBRL, USA) and 1% penicillin / streptomycin (GibcoBRL) at a temperature of 37°C, a CO2 concentration of 5%, and a humidity of 95%, and then cultured in a Sanyo CO2 incubator (Japan, 5% CO2, 37°C) for 24 hours.

[0215]

[0216] Test Example 3-2. Measurement of cytotoxicity of Styrax japonica extract

[0217] The cytotoxicity of the fleaweed extract of Example 1 above was measured by the College of Pharmacy at Suncheon National University using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay.

[0218] Specifically, 1 x 10⁶ macrophages cultured in Test Example 3-1 were placed in a 96-well flat-bottomed culture plate (Nunc, Life Technologies, UK). 4 Cells were inoculated into each well, and 200 μL of ultrasonic extract prepared by adding the *Echinochloa crus-galli* extract powder of Example 1 to 80% ethanol at concentrations of 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 μg / mL was inoculated and cultured for 48 hours. After culture, 50 μL of MTT solution (3 mg / mL) dissolved in PBS was added, and the mixture was placed in a CO2 incubator and reacted for 4 hours.

[0219] After the reaction, the supernatant of the culture medium was removed, and 150 μL of dimethylsulfoxide (DMSO) was added. The absorbance was measured at 595 nm using a Spectrophotometer (Switzerland, Tecan, Sunrise instrument) at 24 or 48 hours after the administration of the succulent plant extract. Cell viability was measured according to the following Equation 5, and the results of cell viability after 24 or 48 hours are shown in Figures 6 and 7, respectively.

[0220] [Mathematical Formula 5]

[0221] Cell viability (%) = (A sample / A control ) X 100%

[0222] (In the above mathematical formula 5, A sample ε is the absorbance when fleaweed extract or ascorbic acid is added, A control (This refers to the absorbance when neither of the two substances is added)

[0223] As a result of measuring cell viability, a viability of over 70% was maintained at a concentration of 31.3 μg / mL or lower.

[0224]

[0225] Test Example 3-3. Measurement of the NO production inhibitory effect of Styrax japonica extract

[0226] The inhibitory effect of the fleaweed extract of Example 1 on Nitric Oxide (NO) production was confirmed by measuring the nitrite concentration in a culture medium using the Griess reagent system at the College of Pharmacy, Suncheon National University.

[0227] Specifically, 1 x 10⁶ macrophages cultured in Test Example 3-1 were placed in a 96-well flat-bottomed culture plate (Nunc, Life Technologies, UK). 4Cells were inoculated at a rate of cells / well, and 100 μl of ultrasonic extract of *Euonymus japonicus* was prepared by adding the *Euonymus japonicus* extract powder of Example 1 to 80% ethanol at concentrations of 0.98, 1.95, 3.9, 7.8, 15.6, or 31.3 μg / mL. After treatment with LPS (lipopolysaccharide, a substance that induces NO production) at a concentration of 1 μg / mL, the cells were cultured for 24 or 48 hours. As controls, a group treated with neither *Euonymus japonicus* extract nor LPS (LPS-) and a group treated only with LPS (LPS+) were prepared.

[0228] 100 μl of the supernatant of the above culture medium was reacted with 100 μl of Griess reagent (0.1% N-(1-naphtyl) ethylene-diamine, 1% sulfanilamide, 5% phosphoric acid), left at room temperature for 10 minutes, and the absorbance was measured at 570 nm using a microplate reader (Sunrise, Tecan) at 48 hours after administration of the fleaweed extract. The NO production was measured according to the following Equation 6, and the NO production results after 48 hours are shown in Figure 8.

[0229] As a result of measuring NO production, Example 1, which is an ultrasonic extract of Styrax japonica, showed a decrease and had an anti-inflammatory effect.

[0230]

[0231] Example 2. Preparation of Supercritical Extract of Sturgeon

[0232] 200g of the pretreated dried powder of *Euonymus japonicus* from Example 1 was injected into a supercritical extraction device (Ilshin Autoclave, Korea), and supercritical extraction was performed for 120 minutes under supercritical CO2 critical extraction conditions at a temperature of 50℃ and a pressure of 400 Bar to prepare a supercritical extract of *Euonymus japonicus*. During supercritical extraction, 70-75% ethanol (food additive grade (FA)) as an auxiliary solvent was injected at a rate of 5 ml per minute.

[0233] The above-mentioned extract was concentrated under reduced pressure using a rotary evaporator and then freeze-dried using a freeze-dryer to prepare the final extract powder.

[0234]

[0235] Test Example 4. Measurement of roseoside content in *Euonymus japonicus* according to extraction method

[0236] For each of the above Examples 1 (80% EtOH) and 2 (SFE EtOH), the extract powders of *Echinochloa crus-galli* were pretreated under the conditions of Table 6 below, and the content of roseoside, a standard substance, was analyzed using a high-speed liquid chromatography analyzer (UHPLC-DAD-ESI-Orbitrap-MS, Orbitrap Exploris 120 mass spectrometer, Thermo Scientific).

[0237] Specifically, for the LC analysis conditions, the system used was the Thermo scientific Vanquish UHPLC system, the column used was the Waters Acquity UPLC HSS T3 (Column Size: 4.6 mm x 100 mm, 1.8 μm), and the analysis was performed under the mobile phase conditions of Table 7 below with a maximum absorption wavelength of 254 nm. The roseoside content and concentration information of the two extracts measured through the analysis are shown in Figure 9 and Table 8 below, the UHPLC-MS chromatogram analysis results of the two extracts are shown in Figure 10, and the UHPLC-UV (254 nm) chromatogram analysis results are shown in Figure 11.

[0238] Sequence Sample Pretreatment Conditions 1. Add 20 mg of dried *Euonymus japonicus* sample to a 5 mL vial with 80% EtOH. 2. Vortex for 30 seconds, then perform sonication at room temperature for 90 minutes. 3. Centrifuge at 4,000 RPM for 10 minutes. 4. After collecting the supernatant, filter it through a 0.2 μm PTFE syringe filter (Thermo Scientific). 5. Prepare the analytical sample in an LC-MS vial.

[0239] Mobile phase conditions: (A) water / (B) ACN (each containing 0.1% formic acid) Injection volume: 4 μL Sample concentration: 60 mg / mL in 80% EtOH

[0240] Extract Type Roseoside Content (ppm) Roseoside Concentration (mg / g) Example 1 7.7 0.07 Example 2 4 3.6 10.41

[0241] As a result of measuring the content and concentration of roseoside, the roseoside concentration of Example 2 (SFE EtOH), which is a supercritical extract, was measured to be 0.41 mg / g (standard deviation: 0.02), and the concentration of Example 1, which is an ultrasonic extract, was measured to be 0.07 mg / g (standard deviation: 0.03). This indicates that the roseoside concentration in the supercritical extract of *Echinochloa crus-galli* is about 6 times higher, and thus possesses superior antioxidant and anti-inflammatory effects, as well as effects such as improvement of skin condition.

[0242] In addition, as a result of chromatography measurements, the roseoside peaks of the two extracts were detected at approximately 5.39 minutes in the UHPLC-MS chromatogram, and in UHPLC-UV (254 nm), the peak of Example 2, which is a supercritical extract, was detected at 5.37 minutes, and the peak of Example 1, which is an ultrasonic extract, was detected at 5.36 minutes, so the roseoside peaks were detected at the same time, proving that the analysis results of the roseoside content and concentration are appropriate.

Claims

1. Fleaweed (1) Temperature conditions of 30 to 70℃; (2) pressure conditions of 300 to 500 Bar; and (3) Supercritical extract of Sturgeon extracted under time conditions of 90 to 150 minutes.

2. The extract according to claim 1, wherein the extract is prepared by extracting using CO2 as a supercritical fluid.

3. The extract according to paragraph 2, wherein the extract is prepared by extraction using water, a straight-chain or branched-chain alcohol having 1 to 4 carbon atoms, or a mixture thereof as an auxiliary solvent.

4. An extract comprising roseoside in any one of paragraphs 1 to 3.

5. A method for preparing a supercritical extract of *Euonymus japonicus*, comprising the step of extracting *Euonymus japonicus* extract under the conditions of (1) to (3) below: (1) Temperature conditions of 30 to 70℃; (2) pressure conditions of 300 to 500 Bar; and (3) Time conditions of 90 to 150 minutes.

6. A manufacturing method according to claim 5, wherein the extraction is performed using CO2 as a supercritical fluid.

7. A method of preparation according to claim 5, wherein the extraction is performed using water, a straight-chain or branched-chain alcohol having 1 to 4 carbon atoms, or a mixture thereof as an auxiliary solvent.

8. A method of manufacturing in which the extract produced by the method of any one of paragraphs 5 to 7 comprises roseoside.

9. An antioxidant composition comprising an ultrasonic extract or supercritical extract of Styrax japonica.

10. A composition according to claim 9, wherein the ultrasonic extract is extracted under the conditions of (1) to (4) below: (1) Frequency conditions of 20 to 100 KHz; (2) Output conditions of 100 to 1000W; (3) Time conditions of 30 to 120 minutes; and (4) Temperature conditions of 20 to 50℃.

11. A composition according to claim 10, wherein the extract is prepared by extraction using water, a straight-chain or branched-chain alcohol having 1 to 4 carbon atoms, or a mixture thereof as an auxiliary solvent.

12. A composition according to claim 9, wherein the supercritical extract is extracted from fleaweed under the following (1) to (3) conditions: (1) Temperature conditions of 30 to 70℃; (2) pressure conditions of 300 to 500 Bar; and (3) Time conditions of 90 to 150 minutes.

13. A composition according to claim 12, wherein the extract is prepared by extracting using CO2 as a supercritical fluid.

14. A composition having one or more characteristics selected from the group consisting of (1) and (2) below, in any one of claims 9 to 13: (1) DPPH radical scavenging activity; and (2) Active inhibitory activity against the production of reactive oxygen species.

15. A composition for improving skin condition comprising an ultrasonic extract or supercritical extract of *Echinochloa crus-galli*.

16. A composition according to claim 15, wherein the ultrasonic extract is extracted under the conditions of (1) to (4) below: (1) Frequency conditions of 20 to 100 KHz; (2) Output conditions of 100 to 1000W; (3) Time conditions of 30 to 120 minutes; and (4) Temperature conditions of 20 to 50℃.

17. A composition according to claim 16, wherein the extract is prepared by extraction using water, a straight-chain or branched-chain alcohol having 1 to 4 carbon atoms, or a mixture thereof as an auxiliary solvent.

18. A composition according to claim 15, wherein the supercritical extract is extracted from fleaweed under the following (1) to (3) conditions: (1) Temperature conditions of 30 to 70℃; (2) pressure conditions of 300 to 500 Bar; and (3) Time conditions of 90 to 150 minutes.

19. A composition according to claim 18, wherein the extract is prepared by extracting using CO2 as a supercritical fluid.

20. A composition having one or more characteristics selected from the group consisting of (1) and (2) below, in any one of claims 15 to 19: (1) Tyrosinase inhibitory activity; and (2) Elastase inhibitory activity.

21. A composition having one or more effects selected from the group consisting of skin whitening, anti-aging, skin elasticity improvement, antioxidant, skin barrier improvement, exfoliation improvement, and skin moisturization, in any one of claims 15 to 19.

22. An anti-inflammatory composition comprising an ultrasonic extract or supercritical extract of Styrax japonica.

23. A composition according to claim 22, wherein the ultrasonic extract is extracted under the conditions of (1) to (4) below: (1) Frequency conditions of 20 to 100 KHz; (2) Output conditions of 100 to 1000W; (3) Time conditions of 30 to 120 minutes; and (4) Temperature conditions of 20 to 50℃.

24. A composition according to claim 23, wherein the extract is prepared by extraction using water, a straight-chain or branched-chain alcohol having 1 to 4 carbon atoms, or a mixture thereof as an auxiliary solvent.

25. A composition according to claim 22, wherein the supercritical extract is extracted from fleaweed under the following (1) to (3) conditions: (1) Temperature conditions of 30 to 70℃; (2) pressure conditions of 300 to 500 Bar; and (3) Time conditions of 90 to 150 minutes.

26. A composition according to claim 25, wherein the extract is prepared by extracting using CO2 as a supercritical fluid.

27. A composition having NO generation inhibitory activity in any one of claims 22 to 26.

28. A composition having an improving effect on one or more selected from the group consisting of acne, atopic dermatitis, seborrheic dermatitis, dry dermatitis, contact dermatitis, nummular dermatitis, and allergic dermatitis, in any one of claims 22 to 26.

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