Composition for Anti-Inflammation, Antioxidant and Muscular Functions Improvement Comprising Plant Complex Extracts as Active Ingredient

Abstract

The present invention relates to a composition for anti-inflammatory, antioxidant, and muscle function improvement purposes comprising a complex extract of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow scaber* as an active ingredient. The complex extract of three components composed of *Achyranthes root*, *Sorghum*, and *Willow tree* of the present invention has no cytotoxicity and is effective in suppressing inflammation by inhibiting the production of nitric oxide (NO), an inflammatory mediator, and the expression of COX-2 and iNOS genes; it improves the survival rate of muscle cells, improves muscle atrophy by inhibiting the expression of atrogin-1 gene involved in protein breakdown, improves the exercise performance of muscle cells by reducing LDH gene expression, relieves muscle pain, effectively reduces the production of intracellular reactive oxygen species (ROS) to suppress oxidative stress, effectively inhibits the production of TSLP (Thymic stromal lymphopoietin), a factor in skin itching, and has excellent effects in strengthening the skin barrier by increasing the expression of genes such as filaggrin, so it can be usefully utilized for purposes such as health functional foods and cosmetics.

Description

Technology Field

[0001] The present invention relates to a composition for anti-inflammatory, antioxidant, and muscle function improvement containing a plant complex extract as an active ingredient. Background Technology

[0003] Some of the substances that play a crucial role in the process of maintaining homeostasis in living organisms are physiologically active substances derived from various organisms. Extensive research has been conducted on numerous physiologically active substances to date, and among them, the study of physiologically active substances isolated from microorganisms, plants, or animals is a very important area in the fields of life science and medicine.

[0004] Meanwhile, inflammation occurs when cells or tissues are damaged or when infected by external infectious agents such as bacteria, fungi, viruses, or various types of allergens. Various inflammatory mediators and immune cells in local blood vessels and body fluids are involved, manifesting a series of complex physiological reactions—including enzyme activation, secretion of inflammatory mediators, fluid infiltration, cell migration, and tissue destruction—as well as external symptoms such as erythema, edema, fever, and pain. Normal inflammatory responses serve to restore biological functions by eliminating external infectious agents and regenerating damaged tissues; however, if antigens are not eliminated or if the inflammatory response becomes excessive or prolonged due to internal substances, it actually promotes mucosal damage, which in some cases leads to diseases such as cancer.

[0005] It is known that various biochemical phenomena are involved in the causes of inflammation; in particular, nitric oxide synthase (NOS), an enzyme that generates nitric oxide (NO), and enzymes involved in the biosynthesis of prostaglandins are known to play the most important roles in mediating inflammatory responses. Therefore, blocking inflammation by inhibiting the activity of nitric oxide synthase (NOS), which generates NO from L-arginine, or cyclooxygenase (COX), an enzyme involved in the synthesis of prostaglandins from arachidonic acid, is a goal in the development of anti-inflammatory drugs.

[0006] According to recent research results, there are several types of NOS, including bNOS (brain NOS) present in the brain, nNOS (neuronal NOS) present in the nervous system, and eNOS (endothelial NOS) present in the vascular endothelial system, which are always expressed at a constant level in the body. While the small amount of nitric oxide (NO) produced by these plays an important role in maintaining normal bodily homeostasis, such as inducing neurotransmission or vasodilation, nitric oxide produced in rapid excess by iNOS (induced NOS), which is induced by various cytokines or external stimuli, is known to cause cytotoxicity or various inflammatory responses. Research results have also reported that chronic inflammation is associated with increased iNOS activity (Miller MJ et al., Mediators of inflammation, 4, pp387-396, 1995; Appleton L. et al., Adv. Pharmacol., 35, pp27-28, 1996).

[0007] In addition, there are two isoforms of cyclooxygenase: cyclooxygenase-1 (COX-1), which is always present in cells and synthesizes prostaglandins (PGs) necessary for cytoprotective action, whereas cyclooxygenase-2 (COX-2) is known to rapidly increase in cells when an inflammatory response occurs and plays a very important role in causing the inflammatory response (Weisz A., Biochem. J., 316, pp209-215, 1996).

[0008] Transcription factors involved in the expression of iNOS and COX-2 enzymes, which increase the secretion of NO and PGs, are involved in the pathogenesis of various sclerosis and chronic diseases. NF-κB, a transcription factor present in the nucleus, is a nuclear protein of the Rel gene family and exists in an inactive form in the cytoplasm by binding to I-κB; however, after I-κB kinase is activated by various stimuli such as reactive oxygen, chemokines like tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), or lipopolysaccharide (LPS), I-κB is removed through phosphorylation. As a result, it is known that NF-κB, composed of a heterodimer of p50 and p65, is activated, translocates to the nucleus, and promotes gene expression that induces an inflammatory response (Oh, GT et al., Atherosclerosis, 159(1), pp17-26, 2001). Additionally, externally secreted bacterial toxins, such as lipopolysaccharides (LPS), stimulate cellular inflammatory responses and induce the secretion of various inflammatory regulators, including nitric oxide (NO), cytokines, tumor necrosis factor (TNF-α), prostaglandin E2, and eicosanoid regulators that promote inflammatory responses (Chen YC, et al, Biochem. Pharmacol., 61, pp1417-1427, 2001).

[0009] Meanwhile, sarcopenia refers to the physiological decline in muscle mass and strength that accompanies increasing age. This causes significant impairment in the performance of normal daily activities and increases the risk of falls and fractures, ultimately restricting elderly patients from living independently. Sarcopenia is not merely a simple loss of muscle mass and strength; it serves as a precursor to frailty and acts as a predictor of increased mortality in chronic diseases. Importantly, sarcopenia increases the risk of physical disability, poor quality of life, and death. The causes of sarcopenia are diverse. A decrease in muscle mass with age implies either reduced protein synthesis, increased protein breakdown, or both. Many metabolic factors, including changes in anabolic hormones, catabolic stimulation caused by inflammation or disease, and nutritional factors such as low physical activity or insufficient protein intake, can contribute to this imbalance. Many clinical manifestations, chronic diseases, and aging cause a decrease in muscle mass, in which two important E3 ubiquitin ligases, muscle ring finger (MuRF1) and muscle atrophy F-box (MAFbx / atrogin-1), are activated, leading to muscle loss. Additionally, the use of high doses or continuous glucocorticoids (GCs) may cause muscle atrophy.Conversely, mTOR contributes to muscle mass increase by inducing muscle protein synthesis through the activation of two factors that initiate mRNA translation, 4E-binding protein (4E-BP1) and phosphorylated 70-kDa ribosomal S6 kinase (p70S6K) (The Korea Journal of Sports Science, 20(3): 1551-1561, 2011; The International Journal of Biochemistry and Cell Biology, 43(9): 1267-1276, 2011).

[0010] In addition, atopic dermatitis is a chronic, recurrent itchy skin condition that commonly occurs in infants and is accompanied by itching, dry skin, or characteristic eczema in patients or their family members. Typical symptoms of atopic dermatitis appear on the hands, scalp, face, neck, elbows, and knees, and include severe dryness of the skin, itching, inflammation, and scaling; when scratched severely, the skin thickens and becomes deeply wrinkled, a phenomenon known as lichenification.

[0011] Although the cause of atopic dermatitis has not yet been clearly identified, immunological factors such as an abnormal increase in IgE, a decrease in the number and dysfunction of T cells (which play a central role in cellular immunity), infiltration of monocytes and macrophages, an increase in the number of mast cells and eosinophils, and an increase in the number of CD4+ T lymphocytes have been reported (J Invest Dermatol., 96:523-526, 1991; J Invest Dermatol., 97:389-394, 1991; Immunol., 11:81-88, 1999; Curr Drug Targets Inflamm Allergy., 2:199-120, 2003; J Allergy Clin Immunol., 107:871-877, 2001; Adv Immunol., 78:57, 2001; International Immunology, 14(7):767-773, 2002; Pediatr Allergy Immunol., 19:605-613, 2008), in particular, Th1 / Th2 imbalance caused by an increase in the number of Th2 cells compared to Th1 cells is known to be an important factor (Kor J Pharmacogn, 43:59-65, 2012).

[0012] Currently, steroids that inhibit inflammatory responses and cytokine production are primarily used to treat atopic dermatitis; however, long-term administration can lead to various side effects, such as skin atrophy or growth retardation. Consequently, the use of non-steroidal agents has been increasing recently. Nevertheless, non-steroidal agents also carry various side effects, including symptoms such as erythema, itching, edema, maceration, and lichenification, as well as weakened immunity, making it difficult to achieve a fundamental cure for atopic dermatitis (Arellano FM et al., J Invest Dermatol. 2007 Apr;127(4):808-16.). Therefore, research is needed to identify substances with excellent therapeutic effects from natural products that offer relatively high safety.

[0013] Therefore, there is an urgent need for the development of natural materials that are safe for the body, have stable active ingredients, and, above all, are effective in improving existing inflammation, muscle function, and skin itching.

[0014] Meanwhile, cosmetic ingredients must strike a balance between safety, efficacy, and physical properties. Furthermore, as economic feasibility is a critical factor, cosmetic materials are developed using various methods to optimize skin penetration, anti-aging properties, biological activity, safety, and physical characteristics. As the boundaries between cosmetics, food, and pharmaceuticals blur, a diverse range of cosmeceutical products continues to experience high growth through complex functional ingredients and biomaterials that combine the safety of cosmetics with the efficacy of pharmaceuticals and health functional foods.

[0015] Against this backdrop, the inventors conducted research on combinations of natural materials that simultaneously have effects on the skin and muscles. As a result of diligent research efforts to find combinations of natural materials that are simultaneously effective for anti-inflammation, improvement of muscle function, and improvement of skin itching, they confirmed that a complex extract of three plants—Achyranthes root, sorghum, and white willow—can be usefully utilized for purposes such as anti-inflammation, improvement of skin itching, and strengthening of the skin barrier, and is also effective for improving muscle function, thereby completing the present invention. The problem to be solved

[0017] Accordingly, the main objective of the present invention is to provide a composition having a complex extract of three plants—Achyranthes root, sorghum, and white willow—as an active ingredient, which exhibits excellent effects in anti-inflammatory, antioxidant, and muscle function improvement.

[0018] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings. means of solving the problem

[0020] According to one aspect of the present invention, the present invention provides a composition for anti-inflammatory, antioxidant, and muscle function improvement comprising a complex extract of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow scaber* as an active ingredient.

[0021] The inventors conducted research on a combination of plant extracts with excellent effects in improving skin condition and confirmed that a complex extract of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow scaber* does not exhibit toxicity to the human body, is effective in suppressing inflammatory responses by inhibiting the production of nitric oxide (NO), an inflammatory mediator, and the expression of COX-2 and iNOS genes, effectively inhibits the production of TSLP (Thymic stromal lymphopoietin), a factor in skin itching, and exhibits excellent activity in strengthening the skin barrier, and confirmed that it can be used for the purpose of improving skin condition.

[0022] In addition, the inventors were able to confirm that the complex extract of the above-mentioned *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow scaber* has excellent effects in effectively reducing the production of intracellular reactive oxygen species (ROS) to suppress oxidative stress, improving the survival rate of muscle cells, improving muscle atrophy by suppressing the expression of the atrogin-1 gene involved in protein breakdown, improving the exercise performance of muscle cells by reducing the expression of the LDH gene, and alleviating muscle pain.

[0023] The "Usul" of the present invention is a perennial herbaceous plant belonging to the Amaranthaceae family, *Soseumureup* ( Achyranthes bidentata It means the root of ) Also, "sorghum ( Sorghum bicolor "" refers to an annual grass plant belonging to the Poaceae family, and in this invention, sorghum seeds are used. In addition, "white willow ( Salix alba "" refers to a plant belonging to the genus Salix in the family Salicaceae, order Salicales, and in this invention, the bark of the white willow is used.

[0024] In the composition of the present invention, the complex extract may be extracted by mixing Achyranthes root, sorghum, and white willow in a weight ratio of 1:0.8 to 1.2:0.8 to 1.2, although this is not limited thereto.

[0025] The term "extract" as used in the present invention includes the extract itself and all formulations of extracts that can be formed using the extract, such as an extract obtained by extraction treatment of a natural product, a distillate, diluted solution, or concentrated solution of said extract, a dried product obtained by drying said extract, a modified or purified product of said extract, or a mixture thereof. Preferably, said extract of the present invention may be prepared and used in the form of a dried powder after heated reflux extraction.

[0026] The extract of *Achyranthes japonica*, *Sorghum sorghum*, or *Willow sapling* of the present invention can be extracted from natural, hybrid, or variant plants, and can also be extracted from plant tissue cultures.

[0027] In the extraction of the above-mentioned *Achyranthes bidentata*, *Sorghum*, or *Willow* according to the present invention, the method of extracting the extract is not particularly limited and can be performed according to methods commonly used in the relevant technical field. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction.

[0028] In the present invention, the type of extraction solvent used to extract the above-mentioned *Achyranthes bidentata*, *Sorghum*, or *Willow* is not particularly limited, and any solvent known in the relevant technical field may be used. Non-limiting examples of the extraction solvent may include purified water; lower alcohols having 1 to 4 carbon atoms, such as methanol, ethanol, propyl alcohol, and butyl alcohol; polyhydric alcohols, such as glycerin, butylene glycol, and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; or mixtures thereof. Preferably, purified water, lower alcohols, 1,3-butylene glycol, and ethyl acetate may be used alone or in a mixture of two or more types.

[0029] In the present invention, the extract obtained by hot water extraction or cold maceration extraction can be used as is or dried using freeze-drying, hot air drying, spray drying, etc., after filtering to remove suspended solid particles by filtering particles using, for example, nylon, etc.

[0030] The term "improvement of skin condition" used in the present invention refers to an action that improves the overall skin condition by inhibiting the production of nitric oxide (NO), exhibiting anti-inflammatory activity by inhibiting the expression of COX-2 and iNOS genes, improving skin itching by inhibiting the expression of TLSP genes, and strengthening the skin barrier by increasing the expression of genes such as filaggrin. Additionally, the term "improvement of muscle function" used in the present invention refers to an action that improves muscle function overall by promoting the survival of muscle cells, inhibiting muscle atrophy caused by the use of glucocorticoids (GCs), protecting muscle cells by inhibiting the expression of atrogin-1 genes involved in the breakdown of muscle proteins, improving the exercise performance of muscle cells by reducing LDH gene expression, and exhibiting activity that reduces muscle pain.

[0031] The complex extract of the present invention is characterized by having anti-inflammatory activity by inhibiting the production of nitric oxide (NO) and inhibiting the expression of COX-2 and iNOS genes. In a preferred embodiment of the present invention, it has been confirmed that the complex extract of *Achyranthes bidentata*, *Sorghum sorghum*, or *Willow scaber* effectively blocks the intracellular signaling pathway related to inflammation by inhibiting the production of nitric oxide (NO) in the signaling pathway mediated by LPS, an inflammation-inducing substance (see FIG. 2), and exhibits excellent anti-inflammatory activity by showing a synergistic effect in inhibiting the expression of COX-2 and iNOS genes, which play a key role in inflammatory mediation responses (see FIG. 3 and FIG. 4).

[0032] In addition, the complex extract of the present invention is characterized by inhibiting the expression of the atrogin-1 gene and the LDH gene, and alleviating muscle pain. In a preferred embodiment of the present invention, it has been confirmed that a complex extract of *Achyranthes bidentata*, sorghum, or white willow increases the survival rate of muscle cells (see FIGS. 5 and 6), improves muscle atrophy by inhibiting the expression of the atrogin-1 gene involved in the breakdown of muscle proteins (see FIGS. 7), significantly improves the exercise performance of muscle cells by inhibiting the expression of the LDH gene (see FIGS. 8), and is also effective in improving muscle pain (see FIGS. 10 and 11).

[0033] In addition, the complex extract of the present invention is characterized by inhibiting the generation of reactive oxygen species (ROS). In a preferred embodiment of the present invention, it has been confirmed that a complex extract of *Achyranthes bidentata*, sorghum, or white willow effectively reduces the generation of reactive oxygen species (ROS) within cells, thereby inhibiting oxidative stress and protecting cells (see FIG. 12).

[0034] In addition, the complex extract of the present invention is characterized by inhibiting the expression of the TLSP gene, which is the cause of skin itching. In a preferred embodiment of the present invention, it has been confirmed that a complex extract of *Achyranthes root*, *Sorghum*, or *Willow tree* can be usefully used for skin health by inhibiting the expression of the TLSP gene, which is the cause of skin itching (see FIG. 13).

[0035] In addition, the complex extract of the present invention is characterized by strengthening the skin barrier by increasing filaggrin gene expression. In a preferred embodiment of the present invention, it has been confirmed that a complex extract of Achyranthes root, sorghum, or white willow shows excellent effects in improving skin condition by contributing to strengthening the skin barrier by increasing the expression of the profilaggrin gene (see FIG. 14).

[0036] According to one aspect of the present invention, the composition may be used in the form of a cosmetic composition. The complex extract of the present invention is derived from nature, is harmless to the human body, and does not impair the quality of the cosmetic product upon addition, making it suitable for inclusion in cosmetic compositions. In particular, since it has effects related to anti-inflammatory, antioxidant, skin itching relief, and skin barrier strengthening, or muscle function improvement, it is preferable to include it in functional cosmetic compositions.

[0037] The cosmetic composition of the present invention may be prepared in any one formulation selected from skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, mask pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, hair treatment, soap, shampoo, and rinse, but is not limited thereto.

[0038] The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are incorporated into general skin cosmetics, and may appropriately incorporate, for example, oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, colorants, preservatives, fragrances, etc., as conventional ingredients, but is not limited thereto.

[0039] Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation of the cosmetic composition.

[0040] When the formulation of the present invention is an ointment, paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used as a carrier component, but is not limited thereto. These may be used alone or in a mixture of two or more types.

[0041] When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included, but is not limited thereto. These may be used alone or in a mixture of two or more types.

[0042] When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be 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, etc., may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan may be used, but are not limited thereto. These may be used alone or in a mixture of two or more.

[0043] When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used as carrier components, but are not limited thereto. These may be used alone or in a mixture of two or more.

[0044] When the formulation of the present invention is a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components, but are not limited thereto. These may be used alone or in a mixture of two or more types.

[0045] In the cosmetic composition of the present invention, the complex extract of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow scaber* may be included specifically in an amount of 0.0001% to 50% by weight of the total weight of the cosmetic composition in terms of dry weight, and more specifically in an amount of 0.0005% to 10% by weight. Within the above range, there is an advantage of exhibiting excellent efficacy regarding functions related to anti-inflammatory, antioxidant, improvement of skin itching, and strengthening of the skin barrier or improvement of muscle function, and there is an advantage of stabilizing the formulation of the composition.

[0046] According to another aspect of the present invention, the composition may be used in the form of a food composition. The complex extract of three components composed of *Achyranthes root*, sorghum, and white willow of the present invention exhibits excellent effects in anti-inflammatory, antioxidant, skin itching relief, and skin barrier strengthening. At the same time, it exhibits excellent effects in inhibiting the expression of atrogin-1 and LDH genes and alleviating muscle pain to improve muscle function; therefore, it can be usefully utilized as a food composition for improving skin condition and muscle function. The complex extract of three components composed of *Achyranthes root*, sorghum, and white willow of the present invention, and their effects on anti-inflammatory, antioxidant, and muscle function improvement, as well as skin itching relief and skin barrier strengthening, are as described above. The food composition may be used in the form of a health functional food, but is not limited thereto.

[0047] The food composition of the present invention may be included in the form of a complex extract of the above-mentioned Achyranthes root, sorghum, and white willow, and processed products thereof. In addition, the composition may include a food-grade acceptable food additive in addition to the active ingredient.

[0048] In the present invention, "food auxiliary additive" refers to a component that can be added to food as an auxiliary component, and can be appropriately selected and used by a person skilled in the art as an additive for manufacturing health functional foods of each formulation. Examples of food auxiliary additives include various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food auxiliary additives of the present invention are not limited by the above examples.

[0049] The food composition of the present invention may include a health functional food. In the present invention, "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients having functional properties useful to the human body. Here, "functionality" means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body; in the present invention, it means obtaining effects related to the improvement of inflammation, antioxidant properties, improvement of muscle function, improvement of skin itching, and strengthening of the skin barrier. The health functional food of the present invention can be manufactured by methods commonly used in the art, and during such manufacturing, it can be manufactured by adding raw materials and ingredients commonly added in the art.

[0050] In addition, the formulation of the above-mentioned health functional food may be manufactured without restriction as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various forms of formulations. Unlike general pharmaceuticals, it has the advantage of being extracted from edible natural materials, thus avoiding side effects that may occur during long-term consumption of the food. It is highly portable and can be consumed as an adjuvant to enhance effects such as inflammation improvement, antioxidant effects, muscle function improvement, skin itching relief, and skin barrier strengthening.

[0051] There are no restrictions on the forms that the health functional food of the present invention may take, and it may include all foods in the conventional sense and may be used interchangeably with terms known in the art, such as functional food. Furthermore, the health functional food of the present invention may be manufactured by mixing known additives with other appropriate auxiliary ingredients that may be included in food, at the choice of a person skilled in the art. Examples of foods to which it may be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. It may also be manufactured by adding it to juices, teas, jellies, and juices prepared using the complex extract of *Achyranthes root*, sorghum, and white willow according to the present invention as the main ingredient. Additionally, foods used as animal feed are also included. Effects of the invention

[0053] The features and advantages of the present invention are summarized as follows:

[0054] (1) The present invention relates to a composition for anti-inflammatory, antioxidant, and muscle function improvement containing a complex extract of *Achyranthes root*, *Sorghum*, and *Willow tree* as an active ingredient.

[0055] (2) The complex extract of three components of the present invention, composed of *Achyranthes root*, *Sorghum*, and *Willow*, has no cytotoxicity and is effective in suppressing inflammation by inhibiting the production of nitric oxide (NO), an inflammatory mediator, and the expression of COX-2 and iNOS genes. It also improves the survival rate of muscle cells, improves muscle atrophy by inhibiting the expression of atrogin-1 gene involved in protein breakdown, improves the exercise performance of muscle cells by reducing LDH gene expression, relieves muscle pain, effectively reduces the production of intracellular reactive oxygen species (ROS) to suppress oxidative stress, effectively inhibits the production of TSLP (Thymic stromal lymphopoietin), a factor in skin itching, and has excellent effects in strengthening the skin barrier by increasing the expression of genes such as filaggrin. Therefore, it can be usefully utilized for purposes such as health functional foods and cosmetics.

[0056] (3) In addition, the composition of the present invention is not only very safe for the human body, but also has excellent stability. Brief explanation of the drawing

[0058] Figure 1 is a graph showing the cell viability of RAW 264.7 cells treated with single extracts of Achyranthes root, sorghum, and white willow, or a complex extract thereof. Figure 2 is a graph showing the change in the amount of nitric oxide (NO) produced after treating RAW 264.7 cells with single extracts of Achyranthes root, sorghum, and white willow, or a combination extract thereof. Figure 3 is a graph showing the change in COX-2 gene expression after treating RAW 264.7 cells with single extracts of Achyranthes root, sorghum, and white willow, or a combination extract thereof. Figure 4 is a graph showing the change in iNOS gene expression after treating RAW 264.7 cells with single extracts of Achyranthes root, sorghum, and white willow, or a combination extract thereof. Figure 5 is a graph showing the cell viability of C2C12 cells after treatment with single extracts of Achyranthes root, sorghum, and white willow, or a complex extract thereof. Figure 6 is a graph showing the change in cell viability of C2C12 cells after treatment with dexamethasone and single extracts or complex extracts of Achyranthes root, sorghum, and white willow. Figure 7 is a graph showing the change in atrogin-1 gene expression after treatment with a complex extract of Achyranthes root, sorghum, and white willow. Figure 8 is a graph showing the change in LDH gene expression after treatment with a complex extract of Achyranthes root, sorghum, and white willow. Figure 9 is a diagram summarizing the pain areas of subjects who responded to the muscle pain questionnaire. Figure 10 is a figure summarizing the degree of pain improvement in subjects who applied a patch containing the three types of complex extracts of the present invention to muscle pain. Figure 11 is a figure summarizing the intention of a subject to recommend to an acquaintance after applying a patch containing the three types of complex extracts of the present invention to muscle pain. Figure 12 is a graph showing the change in the amount of reactive oxygen species (ROS) produced after treating human keratinocytes (HaCaT) with single extracts of Achyranthes root, sorghum, and white willow, or a complex extract thereof. Figure 13 is a graph showing the change in TSLP gene expression after treating human keratinocytes (HaCaT) with single extracts of Achyranthes root, sorghum, and white willow or a combination extract thereof. Figure 14 is a graph showing the change in profilagrin mRNA expression after treating human keratinocytes (HaCaT) with single extracts of Achyranthes root, sorghum, and white willow, or a combination extract thereof. Specific details for implementing the invention

[0059] The present invention will be described in more detail below through examples. These examples are merely illustrative of the present invention, and therefore the scope of the present invention should not be interpreted as being limited by these examples.

[0061] Example 1. Preparation of complex extract

[0062] 1-1. Preparation of Achyranthes Root Extract

[0063] A ground Achyranthes root powder with a dry weight of 100g was reflux-extracted at room temperature for 24 hours with 20 times its weight in 50% ethanol, cold-macerated, and then filtered using filter paper (Glassfiber, Sartorius, USA) with a permeability size of 0.45㎛. The filtered extract was concentrated under reduced pressure at 50℃ or below, and then completely dried using a vacuum dryer (Eyela Rotary evaporator, Tokyo Rikakika Co., LTD, Japan) to obtain an Achyranthes root extract (Preparation Example 1).

[0065] 1-2. Preparation of Sorghum Extract

[0066] 100 g of ground sorghum powder with a dry weight was reflux-extracted at room temperature for 24 hours with 20 times its weight of 50% ethanol, cold-macerated, and then filtered using filter paper (Glassfiber, Sartorius, USA) with a permeability size of 0.45 µm. The filtered extract was concentrated under reduced pressure at 50°C or below, and then completely dried using a vacuum dryer (Eyela Rotary evaporator, Tokyo Rikakika Co., LTD, Japan) to obtain a sorghum extract (Preparation Example 2).

[0068] 1-3. Preparation of White Willow Extract

[0069] 100 g of ground white willow bark powder with a dry weight was reflux-extracted at room temperature for 24 hours with 20 times its weight of 50% ethanol, cold-macerated, and then filtered using filter paper (Glassfiber, Sartorius, USA) with a permeability size of 0.45 µm. The filtered extract was concentrated under reduced pressure at 50°C or below, and then completely dried using a vacuum dryer (Eyela Rotary evaporator, Tokyo Rikakika Co., LTD, Japan) to obtain a white willow extract (Preparation Example 3).

[0071] 1-4. Preparation of a trio complex extract

[0072] A complex extract of three components (Preparation Example 4) was obtained in the same manner as in Example 1-1, except that the powder of Achyranthes root in Example 1-1 was a mixed powder of Achyranthes root, sorghum, and white willow (stem) (weight ratio of 1:1:1).

[0074] Example 2. Confirmation of skin cell protective effect through cytotoxicity test (MTT Assay)

[0075] The cytotoxicity of the single extracts of Achyranthes root, sorghum, and white willow prepared in Example 1, or their complex extracts, was tested on RAW 264.7 macrophage cell line.

[0076] Raw 264.7 mouse macrophage cells were obtained from the Korean Cell Line Bank (KTCC, Seoul, Korea) and subcultured in the laboratory for use. Raw 264.7 cells were attached to a culture dish and cultured in DMEM (PAA, Canada) medium supplemented with 1% antibacterial-antifungal solution (PAA, Canada) containing penicillin and streptomycin and 10% FBS (PAA, Canada).

[0077] To investigate the cytoprotective effects of single extracts of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow suffruticosa* or their complex extracts on LPS-treated RAW 264.7 cells, LPS was administered at a concentration of 1 μg / ml, and then the samples prepared in Examples 1-1 to 1-4 were treated at concentrations of 10 μg / ml, 100 μg / ml, and 250 μg / ml, respectively, and cultured in a 37°C 5% CO2 incubator. After 24 hours of culture, 20 μl of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reagent was added to each well and cultured at 37°C for 3 hours. Subsequently, the amount of MTT degraded into formazan was measured by absorbance at 460 nm using an ELISA reader.

[0078] Referring to [Figure 1] which summarizes the experimental results, it was confirmed that single extracts of *Achyranthes root*, *Sorghum*, and *Willow tree*, or their combined extracts, did not inhibit cell survival of the macrophage cell line RAW 264.7 cells, but rather promoted cell growth in a concentration-dependent manner.

[0079] Specifically, compared to the control group treated with LPS (1 μg / ml), the cell viability of the test group treated with the single extract of *Achyranthes root* increased by 102% (10 μg / ml), 106% (100 μg / ml), and 109% (250 μg / ml), respectively; the cell viability of the test group treated with the single extract of *Sorghum* increased by 101% (250 μg / ml); and the cell viability of the test group treated with the single extract of *Willow tree* increased by 103% (10 μg / ml), 105% (100 μg / ml), and 108% (250 μg / ml), respectively, confirming that the effect of the *Achyranthes root* extract was the best among the single extract treatment groups.

[0080] In addition, in the groups treated with the complex extract of Achyranthes root, sorghum, and white willow, cell viability increased by 105% (10 μg / ml), 109% (100 μg / ml), and 113% (250 μg / ml), respectively, compared to the LPS-treated control group, showing superior effects compared to single extracts. Through the above experiment, it was confirmed that the complex extract composed of Achyranthes root, sorghum, and white willow of the present invention did not inhibit cell viability of RAW 264.7 macrophage cells but promoted cell growth.

[0082] Example 3. Anti-inflammatory activity

[0083] 3-1. Inhibition of Nitric Oxide Production

[0084] To confirm the anti-inflammatory effects of single extracts of *Achyranthes root*, *Sorghum*, and *Willow tree*, as well as their complex extracts, the inhibition rate of nitric oxide (NO) production, a representative cytotoxic substance involved in inducing inflammation, was measured.

[0085] For the experiment, macrophage cell line RAW 264.7 was obtained from the Korea Cell Line Bank (KTCC, Seoul, Korea) and used. The cells were inoculated into DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal bovine serum) supplemented with 10% FBS, 100 µg / ml penicillin, and 100 µg / ml streptomycin, and cultured at 37°C and 5% CO2.

[0086] Specifically, the above RAW 264.7 cells are 1×10 5After inoculating a 96-well plate with cells / mL (in DMEM) and culturing for 24 hours, the single extracts of Achyranthes root, sorghum, and white willow prepared in Example 1 and their complex extracts (Preparation Examples 1 to 4) were diluted to concentrations of 10 μg / mL, 100 μg / mL, and 250 μg / mL, respectively, and a new medium containing LPS (1 μg / mL), known as an endotoxin, was simultaneously treated and cultured for 24 hours. Subsequently, 100 μl of the cell culture supernatant and 100 μl of Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphtylethylenediamine in 2.5% (v / v) phosphoric acid] were mixed and reacted in a 96-well plate for 10 minutes, and the amount of nitric oxide produced was measured by measuring the absorbance at 540 nm using an ELISA reader.

[0087] The concentration of generated nitrite was calculated using a standard curve prepared by dissolving sodium nitrite in DMEM medium. The nitric oxide production inhibitory activity of each sample was determined based on the difference in the amount of nitrite produced between the control group treated with LPS and the control group not treated with LPS. For comparative experiments, dexamethasone (Dex, 10 μg / ml) was used as a positive control.

[0088] Referring to [Figure 2] which summarizes the experimental results, it was confirmed that the complex extract of Achyranthes root, sorghum, and white willow had a superior effect in inhibiting nitric oxide production compared to the single extracts.

[0089] Specifically, compared to the negative control group treated with LPS (1 μg / ml), the test group treated with a single extract of *Achyranthes bidentata* showed a decrease in nitric oxide production of 3% (10 μg / ml), 16% (100 μg / ml), and 24% (250 μg / ml), respectively; the test group treated with a single extract of *Sorghum sorghum* showed a decrease in nitric oxide production of 4% (10 μg / ml), 12% (100 μg / ml), and 19% (250 μg / ml), respectively; and the test group treated with a single extract of *Willow scaber* showed a decrease in nitric oxide production of 11% (10 μg / ml), 25% (100 μg / ml), and 39% (250 μg / ml), respectively. In contrast, the test group treated with a complex extract of three types of *Achyranthes japonica*, *Sorghum sorghum*, and *Willow scaber* showed a decrease in nitric oxide production of 35% (10 µg / ml), 51% (100 µg / ml), and 62% (250 µg / ml), respectively, and at a concentration of 100 µg / ml, it was confirmed to exhibit superior anti-inflammatory activity compared to the positive control group treated with dexamethasone (36% decrease).

[0090] Through the results above, it was confirmed that the three-component complex extract of the present invention, composed of Achyranthes root, sorghum, and white willow, exhibits a synergistic effect in inhibiting nitric oxide production and demonstrates excellent anti-inflammatory activity.

[0092] 3-2. Changes in Expression of COX-2 and iNOS Genes

[0093] The COX-2 (cyclooxygenase type 2) enzyme is activated by nitric oxide and produces PGE2 (prostaglandin E2), which is involved in immune responses. In addition, nitric oxide is synthesized by the iNOS (inducible macrophage-type nitric oxide synthase) enzyme, which is activated for immune responses.

[0094] To evaluate the effect of the three-component complex extract of the present invention, composed of *Achyranthes root*, *Sorghum*, and *Willow tree*, on PGE2-mediated immune responses, changes in the expression of COX-2 and iNOS genes were measured.

[0095] The experiment involved placing RAW 264.7 cells in a 100π dish at a ratio of 1 × 106 After dispensing at a concentration of cell / ml, the cells were cultured in a cell culture incubator for 24 hours, and the samples prepared in Examples 1-1 to 1-4 were treated at concentrations of 50 and 250 μg / ml, respectively.

[0096] Cells were harvested 24 hours after sample treatment, mRNA was extracted using a total RNA extraction kit (iNtRON Biotechnology, Korea) according to the manufacturer's protocol, and mRNA concentration was measured using a nanodrop spectrophotometer.

[0097] 2 µg of isolated and purified RNA was synthesized into cDNA using a cDNA synthesis kit (Takara, Japan), and the cDNA was stored at -20°C until the experiment. The mRNA expression levels were measured using the reverse transcription PCR method on the cDNA obtained from RNA isolation. In this experiment, a maxime PCR PreMix instrument (iNtRON, Korea) was used. 1 µl each of the primer sets for the COX-2 and iNOS genes to be analyzed, along with 1 µl of the previously obtained cDNA, were placed into separate tubes and mixed thoroughly. Subsequently, PCR was performed using a gene amplifier (BIO-RAD, USA) under the following conditions: denture at 94°C for 45 seconds, annealing at 58°C for 35 seconds, and extension at 72°C for 45 seconds, for 26 cycles. Each primer was designed by Cosmogenetech (Korea), and the primer sequences used are as follows.

[0098] - COX-2

[0099] Forward: 5′―GA GAG ACT ATC AAG ATA GT ―3′

[0100] Reverse: 5′―TG GTC AGT AGA CTT TTA CA ―3′

[0101] - iNOS

[0102] Forward: 5′—AT GGC AAC ATC AGG TCG GCC ATC ACT —3′

[0103] Reverse: 5′―CT GTG TGT CAC AGA AGT CTC GAA CTC ―3′

[0104] - GAPDH

[0105] Forward: 5′—AG GGG CCA TCC ACA GTC TTC —3′

[0106] Reverse: 5′―AT CAC CAT CTT CCA GGA GCG ―3′

[0107] Samples obtained through RT-PCR were mixed with loading STAR and loaded onto a 1% agarose gel for electrophoresis, and the results were checked by irradiating with UV light.

[0108] Referring to [Figure 3] and [Figure 4], which summarize the changes in expression of the COX-2 gene activated by nitric oxide and the iNOS gene that produces nitric oxide, it was confirmed that the combined extract of Achyranthes root, sorghum, and white willow had a superior effect in inhibiting the expression of COX-2 and iNOS genes compared to the single extracts.

[0109] Specifically, compared to the negative control group treated with LPS (1 μg / ml), the test group treated with a single extract of *Achyranthes bidentata* showed a decrease in COX-2 gene expression of 19% (50 μg / ml) and 30% (250 μg / ml), the test group treated with a single extract of *Sorghum sorghum* showed a decrease of 9% (50 μg / ml) and 17% (250 μg / ml), and the test group treated with a single extract of *Willow sparsufolia* showed a decrease of 11% (50 μg / ml) and 19% (250 μg / ml), respectively. In contrast, the test group treated with a complex extract of three types of *Achyranthes bidentata*, *Sorghum sparsufolia*, and *Willow sparsufolia* showed a decrease in COX-2 gene expression of 29% (50 μg / ml) and 42% (250 μg / ml), respectively, confirming that it exhibited superior COX-2 gene expression inhibitory activity compared to the single extract treatment groups.

[0110] In addition, compared to the negative control group treated with LPS (1 μg / ml), the iNOS gene expression in the test group treated with a single extract of *Achyranthes root* decreased by 19% (50 μg / ml) and 33% (250 μg / ml), respectively; the test group treated with a single extract of *Sorghum* decreased by 6% (50 μg / ml) and 15% (250 μg / ml), respectively; and the test group treated with a single extract of *Willow tree* decreased by 22% (50 μg / ml) and 39% (250 μg / ml), respectively. In contrast, the iNOS gene expression in the test group treated with a complex extract of three types of *Achyranthes root*, *Sorghum*, and *Willow tree* was observed to decrease by 43% (50 μg / ml) and 69% (250 μg / ml), respectively, confirming that it exhibited superior iNOS gene expression inhibitory activity compared to the single extract treatment groups.

[0111] Through the results described above, it was confirmed that the three-component complex extract of the present invention, composed of *Achyranthes root*, *Sorghum*, and *Willow tree*, exhibits a synergistic effect in suppressing the expression of COX-2 and iNOS genes, which play a key role in inflammatory mediation reactions, and demonstrates excellent anti-inflammatory activity.

[0113] Example 4. Muscle cell protective activity

[0114] Sarcopenia refers to the decrease in muscle mass and strength associated with aging, which makes it difficult to perform normal daily activities. The use of high doses or continuous glucocorticoids (GCs) may cause muscle atrophy.

[0115] The protective effect of the complex extract of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow sapling* of the present invention on C2C12 cells following treatment with dexamethasone, a type of glucocorticoid (GCs), in muscle cells was evaluated.

[0116] The cell viability of the samples for C2C12 cells was evaluated using the MTT reagent. Specifically, C2C12 cells were 1×10⁶ on the day before the experiment. 6The cells were seeded into a 96-well plate at a concentration of cells / mL, and the single extracts of *Achyranthes root* (Preparation Example 1), *Sorghum* (Preparation Example 2), and *Willow tree* (Preparation Example 3) prepared in Example 1, and the three-component extract of these (Preparation Example 4) were treated at concentrations of 10, 100, and 250 μg / mL, respectively, and cultured for 24 hours.

[0117] After 24 hours of incubation, 20 µl of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reagent was added to each well and incubated at 37°C for 3 hours, after which the amount of MTT decomposed into formazan was measured by absorbance at 460 nm using an ELISA reader.

[0118] Referring to [Figure 5], which summarizes the changes in cell viability of C2C12 cells (root cells) according to sample treatment, it was confirmed that single extracts of *Achyranthes root*, *Sorghum*, and *Willow tree*, or their complex extracts, did not inhibit cell viability of C2C12 muscle cell lines, but rather promoted cell growth in a concentration-dependent manner.

[0119] Specifically, based on the untreated negative control group, the cell viability of the test group treated with a single extract of *Achyranthes bidentata* increased by 104% (100 µg / ml) and 111% (250 µg / ml), respectively; the cell viability of the test group treated with a single extract of *Sorghum sorghum* increased by 101% (10 µg / ml), 106% (100 µg / ml), and 105% (250 µg / ml); and the cell viability of the test group treated with a single extract of *Willow scaber* increased by 101% (10 µg / ml), 108% (100 µg / ml), and 112% (250 µg / ml), respectively. In contrast, in the group treated with the complex extract of Achyranthes root, sorghum, and white willow, cell viability increased by 113% (10 μg / ml), 119% (100 μg / ml), and 127% (250 μg / ml), respectively, compared to the untreated negative control group, showing a superior effect in promoting muscle cell growth compared to the single extract.

[0120] Meanwhile, referring to [Figure 6], which summarizes the changes in cell viability of differentiated C2C12 cells (Myotubes) treated with dexamethasone, the viability of C2C12 cells decreased to approximately 2 / 3 of the level with treatment with dexamethasone (10 μM), and it was confirmed that the complex extract of *Achyranthes root*, *Sorghum*, and *Willow tree* promoted cell growth of differentiated C2C12 cells (Myotubes) in a concentration-dependent manner.

[0121] Specifically, based on the group treated with dexamethasone (10 μM), the cell viability of the test group treated with a single extract of *Achyranthes root* increased by 107% (10 μg / ml), 109% (100 μg / ml), and 112% (250 μg / ml), respectively; the cell viability of the test group treated with a single extract of *Sorghum* increased by 105% (10 μg / ml), 104% (100 μg / ml), and 106% (250 μg / ml); and the cell viability of the test group treated with a single extract of *Willow tree* increased by 104% (10 μg / ml), 107% (100 μg / ml), and 109% (250 μg / ml), respectively, indicating that the protective effect of these single extracts on muscle cells was minimal.

[0122] In contrast, in the group treated with a complex extract of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow sapling*, cell viability increased by 116% (10 μg / ml), 122% (100 μg / ml), and 139% (250 μg / ml), respectively, compared to the group treated with dexamethasone (10 μM), showing a superior protective effect on muscle cells compared to the single extract.

[0123] Through the above experiment, it was confirmed that the complex extract of *Achyranthes root*, *Sorghum*, and *Willow tree* of the present invention promotes the survival of muscle cell line C2C12 cells and protects muscle cells by inhibiting muscle atrophy caused by the use of glucocorticoids (GCs).

[0125] Example 5. Inhibition of atrogin-1 genetic expression

[0126] Changes in the expression of the atrogin-1 gene, which is involved in the breakdown and synthesis of muscle proteins, were measured following treatment with the complex extract composed of *Achyranthes root*, *Sorghum*, and *Willow tree* of the present invention.

[0127] The experiment involved placing C2C12 cells in a 100π dish at a ratio of 1 × 10 6 After dispensing at a concentration of cell / ml, the cells were cultured in a cell culture incubator for 24 hours, and the complex extract samples prepared in Examples 1-4 were treated at concentrations of 50 and 250 μg / ml, respectively.

[0128] Cells were harvested 24 hours after sample treatment to check for changes in atrogin-1 gene expression. The antibodies used in the experiment were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Referring to [Figure 7] which summarizes the experimental results, it was confirmed that atrogin-1 gene expression increased by more than twofold with treatment with dexamethasone (10 μM), and that atrogin-1 gene expression decreased in a concentration-dependent manner with treatment with the complex extract of *Achyranthes root*, *Sorghum*, and *Willow tree* of the present invention.

[0129] Specifically, compared to the negative control group treated with dexamethasone (10 μM), the test group treated with a complex extract of three types of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow sparsu* showed a decrease in atrogin-1 gene expression of 42% (50 μg / ml) and 69% (250 μg / ml), respectively.

[0130] Through the above experiment, it was confirmed that the complex extract of *Achyranthes root*, *Sorghum*, and *Willow tree* of the present invention improves muscle atrophy by inhibiting the expression of the atrogin-1 gene, which is involved in the breakdown of muscle proteins.

[0132] Example 6. Inhibition of LDH gene expression

[0133] Changes in the expression of the LDH (Lactate dehydrogenase) gene were measured following treatment with the complex extract composed of Achyranthes root, sorghum, and white willow of the present invention.

[0134] Glucose must undergo glycolysis to synthesize final ATP. One of the factors determining this glucose metabolism is the oxygen utilization capacity of muscle cells. If oxygen is insufficient, the amount of ATP synthesized decreases, and lactic acid is produced as a byproduct.

[0135] We evaluated whether the exercise performance of muscle cells improved by confirming the expression of the LDH (Lactate dehydrogenase) enzyme, which regulates the action of lactic acid by controlling pyruvate during oxygen deficiency in muscles, and assessed that the exercise performance of muscle cells improved when LDH gene expression decreased.

[0136] In this experiment, differentiated C2C12 cells were cultured after being treated with dexamethasone (10 μM) and single or complex extracts of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow suffruticosa* at concentrations of 10, 100, and 250 μg / ml, respectively. After 24 hours, the supernatant was centrifuged at 7,500 rpm, 5 min, and 4°C, and the volume was recovered to approximately 400-800 μl and transferred to a new e-tube. The supernatant from each well was then quantified according to the manufacturer's protocol using a human LDH ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA).

[0137] Referring to [Figure 8] which summarizes the changes in LDH gene expression, treatment with single extracts of Achyranthes root, sorghum, and white willow was observed not to induce significant changes in LDH gene expression, whereas treatment with a complex extract of Achyranthes root, sorghum, and white willow was observed to significantly reduce LDH gene expression.

[0138] Specifically, compared to the dexamethasone (10 μM) treatment group, the LDH expression levels in the single extract treatment group of Achyranthes root were found to decrease by 5% (10 μg / ml), 6% (100 μg / ml), and 9% (250 μg / ml), respectively; in the single extract treatment group of sorghum root, the LDH expression levels were found to decrease by 2% (10 μg / ml) and 1% (100 μg / ml), respectively; and in the single extract treatment group of willow tree, the LDH expression levels were found to decrease by 2% (10 μg / ml), 4% (100 μg / ml), and 5% (250 μg / ml), respectively, indicating that the effect of the single extracts was minimal. In contrast, in the group treated with the three types of plant complex extract of the present invention, the LDH expression levels decreased by 5% (10 μg / ml), 28% (100 μg / ml), and 48% (250 μg / ml), respectively, compared to the group treated with dexamethasone (10 μM), thereby further inhibiting LDH expression compared to the single extract.

[0139] Through the results above, it was confirmed that the three-plant complex extract of the present invention, composed of *Achyranthes root*, *Sorghum*, and *Willow tree*, inhibits LDH gene expression and significantly improves the exercise performance of muscle cells.

[0141] Example 7. Improvement of muscle pain

[0142] In order to confirm whether the three-plant complex extract of the present invention exhibits a pain-relieving effect, a three-plant complex extract composed of *Achyranthes root*, *Sorghum*, and *Willow tree* was dissolved in purified water at a concentration of 250 μg / ml, and then applied to a muscle pain area to test the ability to improve pain.

[0143] The above test evaluated the quality of the Simple Monadic Test method through a survey asking about the degree of pain improvement after applying the sample for more than one month. The survey was conducted on 220 adult men and women visiting golf courses (see Table 1), and the areas with symptoms before and after golf rounds for the subjects are summarized in [Figure 9].

[0144] [Table 1]

[0145]

[0146] The survey was conducted on two items regarding the degree of pain improvement and the doctor recommended by acquaintances, according to the criteria in [Table 2] below.

[0147] [Table 2]

[0148]

[0149] Referring to [Fig. 10] and [Fig. 11], which summarize the test results, it was analyzed that when a patch containing the three types of complex extract of the present invention is applied to muscle pain, 62% of subjects experienced significant pain improvement and 75% were willing to recommend it to acquaintances, and through this, it was confirmed that the three types of complex extract of the present invention has excellent muscle pain improvement.

[0151] Example 8. Reactive Oxygen Species (ROS) Scavenging Ability

[0152] Changes in the production of intracellular reactive oxygen species (ROS) following treatment with the complex extract composed of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow scaber* of the present invention were analyzed.

[0153] UVB (UVB Peak range: 312nm, UV dose: 150 mJ / cm²) to HaCaT cells, a human-derived skin keratinocyte cell line 2 ) was investigated to induce an increase in the concentration of reactive oxygen species (ROS) within the cells. Subsequently, single extracts of *Achyranthes root* (Preparation Example 1), *Sorghum* (Preparation Example 2), and *Willow tree* (Preparation Example 3) prepared in Example 1, and a complex extract of these three (Preparation Example 4) were diluted and treated at concentrations of 10, 100, and 250 μg / ml, respectively, and the reactive oxygen species (ROS) scavenging activity within HaCaT cells was measured.

[0154] After dispensing 100 μl of 30 μM DCFH-DA (Dichlorofluorescein diacetate) into each well of an experimental plate, the samples were treated at 37°C for 30 minutes, and the fluorescence values ​​were measured and compared at 485 / 538 nm (excitation / emission) using a fluorescence reader (SpectraMax M5).

[0155] Referring to [Figure 12] which summarizes the experimental results, the amount of reactive oxygen species (ROS) produced in HaCaT cells increased by approximately twofold due to UVB irradiation, but a concentration-dependent decrease in the production of reactive oxygen species (ROS) in RAW 264.7 cells was observed due to treatment with single extracts of Achyranthes root, sorghum, and white willow, as well as their complex extracts.

[0156] Specifically, compared to the UVB irradiation treatment group, the test group treated with a single extract of *Achyranthes bidentata* showed reduced levels of reactive oxygen species (ROS) by 15% (10 μg / ml), 20% (100 μg / ml), and 21% (250 μg / ml), respectively; the test group treated with a single extract of *Sorghum sorghum* showed reduced levels of reactive oxygen species (ROS) by 6% (10 μg / ml), 11% (100 μg / ml), and 17% (250 μg / ml), respectively; and the test group treated with a single extract of *Willow scaber* showed reduced levels of reactive oxygen species (ROS) by 9% (10 μg / ml), 13% (100 μg / ml), and 18% (250 μg / ml), indicating that the reactive oxygen species production inhibitory activity of the single components was negligible.

[0157] In contrast, the test group treated with a complex extract of three plant ingredients showed reduced levels of reactive oxygen species (ROS) compared to the UVB irradiation group, with reductions of 28% (10 μg / ml), 36% (100 μg / ml), and 49% (250 μg / ml), respectively, and it was confirmed that the combination of the three ingredients showed a synergistic effect.

[0158] Through the above results, it was confirmed that the three-component complex extract of the present invention effectively reduces the production of reactive oxygen species (ROS) within cells, thereby suppressing oxidative stress and protecting cells.

[0160] Example 9. Improvement of skin itching

[0161] The cytokine TSLP (thymic stromal lymphopoietin) is known to induce a Th2 inflammatory response by inducing IL-17, IL-6, and CD11c+ myeloid dendritic cells, and atopic dermatitis is also known as a Th2-mediated immune disease.

[0162] In addition, TSLP (Thymic stromal lymphopoietin) has recently been identified as being associated with the severity of dermatitis and is known to be a cause of itching, so it is being used as a marker for skin itching (Wilson SR et al., The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell. 2013;155(2):285-95).

[0163] To verify the improvement effect of single extracts of *Achyranthes root*, *Sorghum*, and *Willow tree*, or their complex extracts, on skin itching, changes in the expression of the TSLP gene in human keratinocytes (HaCaT) were analyzed.

[0164] The experiment was conducted on human keratinocytes (HaCaT, 1×10⁻⁶ 6After incubating the wells in FBS DMEM medium for 24 hours, single extracts or complex extracts of *Achyranthes root*, *Sorghum*, and *Willow* were added to FBS-free DMEM medium at concentrations of 10 µg / ml, 100 µg / ml, and 250 µg / ml, respectively. Diluted TNF-α and IFN-γ (10 ng / ml) were then added, followed by further incubation. After 24 hours, the supernatant was centrifuged at 7,500 rpm, 5 min, and 4°C to recover a volume of approximately 400-800 µl, which was then transferred to a new e-tube. The supernatant from each well was then quantified using a human TSLP ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol. For comparative experiments, a test group treated with dexamethasone (10 µg / ml) was used as a positive control.

[0165] Referring to [Figure 13] which summarizes the experimental results, it was confirmed that the production of TSLP protein overexpressed by treatment with TNF-α and IFN-γ (10 ng / ml) was inhibited in a concentration-dependent manner by treatment with single extracts of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow suffruticosa* or their complex extracts, and it was confirmed that the complex extract had a superior inhibitory effect on TSLP protein expression compared to the single extracts.

[0166] Specifically, compared to the TNF-α and IFN-γ treatment groups, the TLSP expression levels in the single extract treatment group of Achyranthes root decreased by 8% (10 µg / ml), 21% (100 µg / ml), and 25% (250 µg / ml), respectively; in the single extract treatment group of sorghum root, the TLSP expression levels decreased by 2% (10 µg / ml), 9% (100 µg / ml), and 13% (250 µg / ml), respectively; and in the single extract treatment group of willow tree, the TLSP expression levels decreased by 9% (10 µg / ml), 16% (100 µg / ml), and 22% (250 µg / ml), respectively, indicating that the effect of the single extracts was minimal. In contrast, in the group treated with the three types of plant complex extract of the present invention, the TLSP expression levels were found to be reduced by 19% (10 μg / ml), 35% (100 μg / ml), and 42% (250 μg / ml), respectively, compared to the TNF-α and IFN-γ treatment groups, and the TLSP expression level was observed to be even more improved compared to the positive control group treated with dexamethasone in the treatment group with a dose of 250 μg / ml.

[0167] Through the results above, it was confirmed that the three-plant complex extract of the present invention, composed of *Achyranthes root*, *Sorghum*, and *Willow tree*, can be usefully used for skin health by suppressing the expression of the TLSP gene, which causes skin itching.

[0169] Example 10. Skin barrier strengthening activity

[0170] Changes in filaggrin (FLG, stratum corneum formation factor) gene expression following treatment with a complex extract composed of *Achyranthes bidentata*, sorghum, and white willow of the present invention were analyzed.

[0171] For the experiment, human keratinocytes (HaCaT) were used as the cell line, and cells were cultured at a rate of 1×10⁶ in DMEM medium supplemented with 10% FBS in 100 mm cell culture dishes. 6The cells were inoculated at a specific concentration and cultured for 24 hours in a 37°C, 5% CO2 incubator. After culture, diluted solutions prepared by diluting the single extracts of *Achyranthes root*, *Sorghum*, and *Willow tree*, as well as their complex extracts, in DMEM medium to concentrations of 50, 100, and 250 μg / ml, respectively, were added, and the cells were cultured for an additional 24 hours under the same conditions. Subsequently, cells were harvested using Trizol reagent (Invitrogen, USA), and RNA was isolated. RT-PCR was performed using a PCR instrument (Step One Plus, Applied Biosystems, USA), and the CyberGreen (SYBR Green supermix, Applied Biosystems, USA) reagent was added along with profilaggrin, GAPDH, and cDNA. The polymerization reaction was carried out for 40 cycles, starting with polymerase activation at 94°C for 5 minutes, followed by 95°C for 30 seconds, 54°C for 1 minute, and 72°C for 1 minute.

[0172] The primers used are as follows:

[0173] - Propilaggrin

[0174] Forward: 5′-AAG CTT CAT GGT GAT GCG AC-3′

[0175] Reverse: 5′-TCA AGC AGA AGA GGA AGG CA-3′

[0176] - GAPDH

[0177] Forward: 5'-ACC ACA GTC CAT GCC ATC AC-3'

[0178] Reverse: 5'-CCA CCA CCC TGT TGC TGT AG-3'

[0179] RT-PCR was performed using the above primers, and the experimental results are summarized in [Figure 14].

[0180] Filaggrin exists in the form of profilaggrin, a protein that forms keratohyalin granules in the granular layer of the epidermis, and then, after being broken down into filaggrin during the final differentiation process of keratinocytes, aggregates keratin filaments to form a hard and flat structure of keratinocytes when forming the membrane of keratinocytes, thereby performing the role of a brick in the skin barrier.

[0181] Referring to [Figure 14] which summarizes the experimental results, it was found that the mRNA expression of profilaggrin increased with treatment with single extracts of Achyranthes root, sorghum, and white willow, or with a complex extract thereof, and the increase in profilaggrin expression was observed more distinctly in the complex extract treatment group compared to the single extract treatment group.

[0182] Specifically, compared to the untreated group, the mRNA expression of propyllagrin increased by 102% (100 µg / ml) and 109% (250 µg / ml) in the test group treated with a single extract of *Achyranthes bidentata*, and by 106% (50 µg / ml), 103% (100 µg / ml), and 111% (250 µg / ml) in the test group treated with a single extract of *Sorghum sorghum*, and by 104% (50 µg / ml), 105% (100 µg / ml), and 107% (250 µg / ml) in the group treated with a single extract of *Willow scaber*. In contrast, in the test group treated with a complex extract of three plants—Achyranthes root, sorghum, and white willow—the mRNA expression of profilaggrin was observed to increase by 122% (50 μg / ml), 147% (100 μg / ml), and 156% (250 μg / ml), respectively, compared to the untreated group. This indicates a significant increase in profilaggrin mRNA expression compared to a single extract, and a synergistic effect resulting from the combination of the three components was observed. In other words, it was confirmed that the complex extract of three plants—Achyranthes root, sorghum, and white willow—shows excellent effects in improving skin condition by increasing the expression of the profilaggrin gene and contributing to the strengthening of the skin barrier.

[0183] Through the results described above, it was confirmed that the three-plant complex extract of *Achyranthes bidentata*, *Sorghum sorghum*, and *Willow scaber* of the present invention has i) no cytotoxicity, ii) suppresses inflammatory responses by inhibiting the production of nitric oxide (NO), an inflammatory mediator, and the expression of COX-2 and iNOS genes, iii) improves muscle cell viability and improves muscle atrophy by inhibiting the expression of atrogin-1 genes involved in protein breakdown, iv) improves the exercise performance of muscle cells by reducing LDH gene expression, v) has excellent effects in relieving muscle pain, vi) suppresses oxidative stress by effectively reducing the production of intracellular reactive oxygen species (ROS), vii) effectively inhibits the production of TSLP (Thymic stromal lymphopoietin), a factor in skin itching, and viii) strengthens the skin barrier by increasing the expression of genes such as filaggrin.

[0184] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A composition for relieving muscle pain, comprising a complex extract of *Achyranthes root*, *Sorghum*, and *Willow tree* as an active ingredient, wherein the complex extract is an extract obtained by mixing *Achyranthes root*, *Sorghum*, and *Willow tree* in a weight ratio of 1:0.8~1.2:0.8~1.2, and wherein the complex extract is characterized by relieving muscle pain. Claim 2 delete Claim 3 A composition for relieving muscle pain according to claim 1, characterized in that the complex extract inhibits the production of nitric oxide (NO) and inhibits the expression of COX-2 and iNOS genes, thereby having anti-inflammatory activity. Claim 4 A composition for relieving muscle pain according to claim 1, characterized in that the complex extract exhibits muscle function improvement activity by inhibiting the expression of the atrogin-1 gene and the LDH gene. Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 A composition for relieving muscle pain according to claim 1, characterized in that the composition is a food composition. Claim 9 delete

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