Leucine derivatives, compositions comprising them, and their uses

Abstract

This invention relates to the use of compounds represented by Chemical Formula 1 and / or compounds represented by Chemical Formula 2, or salts of compounds represented by Chemical Formula 1 and / or salts of compounds represented by Chemical Formula 2, in the manufacture of compositions for the treatment, prevention, or improvement of menopausal symptoms in women, wherein the compositions comprise compounds represented by Chemical Formula 1 and / or compounds represented by Chemical Formula 2, or salts of compounds represented by Chemical Formula 1 and / or salts of compounds represented by Chemical Formula 2, Chemical Formula 1 Chemical Formula 2:

Description

[0001] This application is a divisional application of application number 201710824741.5, filed on September 14, 2017, entitled "Leucine derivatives, compositions comprising the same and uses thereof". Technical Field

[0002] This invention relates to leucine derivatives, compositions comprising them, and their uses. Specifically, this invention relates to novel cosmetic, pharmaceutical, and food applications of leucine derivatives, as well as cosmetic material compositions, pharmaceutical compositions, drug-like compositions, and food compositions comprising leucine derivatives. Background Technology

[0003] Aging is a life phenomenon, and all living organisms begin to age from birth. With the improvement of hygiene and health environment, human lifespan has been extended much compared to the past, and the proportion of elderly population has been increasing continuously (Kang Tae-jin, BioWave 2011, Vol.13, No.3(1)). Aging occurs throughout the body, such as the decline in overall bodily function and lifestyle diseases such as cardiovascular disease and diabetes, which lead to the decline in the physiological function of organs (Choi Hyun-chul, Korean Journal of Dermatology, 1997; 35: 292-302).

[0004] The skin is an important organ that not only prevents the loss of water, electrolytes, and proteins from the body on the outermost layer, but also performs multiple functions such as body temperature regulation, skin sensation, and immune function.

[0005] Like other organs, skin ages with time. Skin aging consists of intrinsic skin aging and photoaging. Intrinsic skin aging is characterized by functional changes rather than visible morphological changes. Photoaging, caused by chronic ultraviolet radiation, overlaps with intrinsic aging. Although it is not systemic or inevitable, it exhibits significant structural and physiological changes. On aging epidermis, not only do wrinkles, barrier loss, and immune abnormalities occur, leading to a loss of inherent functions, but it also affects the appearance of the body. While it's impossible to stop skin aging, various methods are tried to prevent and delay it.

[0006] Collagen is the main matrix protein produced in dermal fibroblasts and exists in the extracellular matrix. Its important functions include: maintaining the mechanical strength of the skin, resisting and binding tissues, supporting cell attachment, and inducing cell division and differentiation (during organismal growth or wound healing). Collagen decreases with age and due to photoaging caused by ultraviolet radiation, and this decrease is accelerated by the activity of collagenases, which break down collagen. This phenomenon is known to be closely related to the formation of wrinkles.

[0007] Furthermore, elastin is an important component of the skin involved in wrinkle formation related to skin elasticity. The deficiency and aggregation of elastin fibers, and the significant increase in the activity of elastase, an enzyme that breaks down elastin, are known to be contributing factors to wrinkle formation. Elastase is the only enzyme capable of breaking down elastin, and inhibiting elastase is known to fundamentally reduce wrinkles.

[0008] In addition, collagen and elastin are known to be matrix proteins that play an important role in moisturizing the dermis. These matrix proteins adsorb water and enhance the moisturizing capacity within the structures they form, so that the skin can maintain adequate moisture and thus affect the maintenance of skin elasticity.

[0009] Furthermore, elastic fibers generated in fibroblasts of the skin, which cross-link with collagen, are an important component of the skin in the formation of wrinkles related to skin elasticity. The lack and breakdown of elastic fibers are known to be one of the main factors contributing to the formation of wrinkles and decreased skin elasticity.

[0010] Currently, known cosmetic ingredients for improving wrinkles include retinoids, adenosine, proteins derived from animal placenta, and chlorella extract. Retinoids, the most typical example, promote collagen synthesis and inhibit elastase; however, they are unstable, and their use is limited due to safety concerns such as irritation and redness when applied to the skin. Chlorella extract and similar ingredients have minimal effects and are unlikely to provide substantial improvement in skin wrinkles.

[0011] Therefore, there is an urgent need to develop ingredients that are safe for organisms, have active ingredients that are safe and highly stable for the skin, and, more importantly, have better effects on improving skin elasticity, moisturizing, and preventing skin aging than known substances that have effects on improving elasticity, moisturizing, or anti-aging in the skin.

[0012] Furthermore, fatigue is one of the most prevalent ailments in modern society, referring to a state of low physical fitness (Uthayathas et al., 2007). Fatigue can be caused by excessive stress (Uthayathas et al., 2007) or abnormalities in immune function and the antioxidant system (Cleare, 2003). Blood biomarkers associated with fatigue include blood urea nitrogen (BUN), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and glucose (Klimas et al., 2012). LDH and creatine kinase (CK) levels are widely known as markers of muscle damage (Coombes and McNaughton, 2000). Malate dehydrogenase (MDH) is also associated with oxidative metabolism related to fatigue (Callister et al., 2004). Furthermore, the occurrence of immune symptoms in oxidative stress pathways and various inflammatory pathways can induce fatigue (Liu et al., 2000). Reactive oxygen species (ROS) produced after excessive exercise can exacerbate fatigue (Powers et al., 1999). To reduce ROS, superoxide dismutase (SOD) and catalase are generated (Liu et al., 2000). Nitric oxide (NO) is increased in patients with chronic fatigue (Suarez et al., 2010), and inflammatory cytokines are also associated with fatigue symptoms (Liburt et al., 2010).

[0013] Currently, many studies are being conducted to treat fatigue, but no suitable solution has been found so far.

[0014] The causes of fatigue can be categorized as follows: lack or inability to utilize stored energy, inflammatory responses caused by metabolism, abnormalities in the antioxidant system, accumulation of fatigue-causing substances, and imbalances in homeostasis. Most cases are known to be caused by the combined effects of multiple factors. Recovery from fatigue requires adequate energy supply and rest, as well as the inhibition and removal of fatigue-causing substances from the body. However, in today's busy society, it is practically impossible to meet these needs for sufficient nutrition and rest. Therefore, there is a need to explore functional materials for fatigue recovery.

[0015] Furthermore, amenorrhea in women refers to the genetically determined cessation of menstruation from birth until approximately 50 years of age when ovarian function reaches its lifespan. It signifies the loss of reproductive capacity and is not a pathological phenomenon but a physiological change. Currently, the average life expectancy for women in South Korea is 81.2 years (2011: Statistics Korea). Assuming the average age of menopause for South Korean women, as defined by the Korean Society of Obstetrics and Gynecology, is 50 years, this means that women live approximately one-third of their lives in a state of female hormone depletion (Pharmaceutical Information Service, Kim Sung-cheol).

[0016] Since menopause, women experience systemic changes due to hormonal imbalances and reductions, affecting the vascular, musculoskeletal, genitourinary, and neurological systems. These changes can manifest as vasomotor and psychological symptoms such as hot flashes, night sweats, sleep disturbances, fatigue, depression, anxiety, attention deficit, memory impairment, painful intercourse due to genitourinary atrophy, frequent urination, loss of skin elasticity due to collagen reduction, breast sagging, cardiovascular and musculoskeletal symptoms, and even dementia. While menopausal symptoms vary from person to person, it is generally believed that the more numerous, severe, and prolonged the menopausal symptoms, the lower the woman's quality of life. Furthermore, menopausal symptoms, along with overall aging, have a high probability of developing into chronic diseases.

[0017] Treatment for menopausal symptoms can include hormone therapy, drug therapy, exercise therapy, and diet therapy. However, the widely used female hormone therapy may increase the risk of breast cancer and other diseases. Long-term use may increase the rate of uterine cancer, thrombotic vascular disease, gallbladder disease, and hypertension.

[0018] Therefore, research is constantly being conducted on therapies that are both safe for the human body and can achieve excellent therapeutic effects. Summary of the Invention

[0019] Therefore, the problem to be solved by the present invention is to provide a new active ingredient that can solve the problems described above, has few side effects, is safe for the human body, and can achieve excellent effects such as improving skin elasticity, moisturizing or preventing skin aging, i.e., to provide such beneficial uses of the active ingredient.

[0020] In addition, the problem to be solved by the present invention is to provide a new active ingredient with few side effects, which is safe for the human body and has excellent effects on improving fatigue, inflammation or anti-oxidation, that is, to provide such beneficial uses of the active ingredient.

[0021] In addition, the problem to be solved by the present invention is to provide a new active ingredient with excellent effect on improving menopausal symptoms, that is, to provide this beneficial use of the active ingredient.

[0022] In other words, the problem to be solved by the present invention is to provide a composition containing the above-mentioned active ingredient with excellent efficacy, and further, to provide a composition containing the above-mentioned active ingredient with excellent efficacy as an effective ingredient or an indicator ingredient.

[0023] To address the aforementioned issues, the present invention provides compositions for improving skin elasticity, moisturizing skin, or combating skin aging. Preferably, these compositions are cosmetic material compositions, pharmaceutical compositions, quasi-drug compositions, or functional food compositions, wherein the active ingredients include glycyl-L-leucine peptides and / or L-leucylglycine peptides, or their salts.

[0024] Specifically, the present invention provides the use of glycyl-L-Leucine peptide and / or L-leucylglycine peptide, or their salts, as cosmetic material compositions, pharmaceutical compositions, drug-like compositions, or functional food compositions, as well as new uses for improving skin elasticity, moisturizing skin, or anti-aging skin.

[0025] This invention provides a method for improving skin elasticity, moisturizing skin, or preventing skin aging, wherein the method involves treating the skin with glycyl-L-leucine peptides and / or L-leucylglycine peptides, or their salts.

[0026] The aforementioned glycyl-L-leucine peptide and / or L-leucylglycine peptide can be active ingredients or indicator ingredients.

[0027] The inventors conducted careful research to develop peptides or natural materials containing such peptides that are effective in improving skin. The results confirmed that compositions containing glycyl-L-leucine peptide and / or L-leucylglycine as active ingredients are effective in improving skin elasticity, moisturizing skin, and preventing skin aging, thus completing this invention.

[0028] The inventors have experimentally confirmed that compositions containing glycyl-L-leucine and / or L-leucylglycine, or their salts, as active ingredients exhibit skin-improving effects by promoting the synthesis of hyaluronic acid and procollagen, and inhibiting elastase activity.

[0029] In this invention, "improved skin elasticity" refers to alleviating skin laxity. Furthermore, the aforementioned elasticity refers to maintaining skin elasticity when elastin and collagen are present in sufficient quantities.

[0030] In this invention, "skin moisturizing" refers to increasing the skin's hydration and maintaining a moist state. Skin moisturizing helps improve wrinkles and increase skin elasticity.

[0031] In this invention, "anti-skin aging" refers to preventing and mitigating the formation of wrinkles and loss of elasticity in the skin. For example, inhibiting the activity of elastase can suppress skin aging.

[0032] According to another embodiment of the present invention, compositions for improving fatigue, improving inflammation, or antioxidation are provided, preferably health food compositions, non-therapeutic cosmetic material compositions, pharmaceutical compositions, or pharmaceutical-like compositions, wherein the active ingredients of these compositions include glycyl-L-leucine peptides and / or L-leucylglycine peptides, or salts thereof.

[0033] Specifically, the present invention provides the use of glycyl-L-Leucine peptide and / or L-leucylglycine peptide, or their salts, as health functional food compositions, cosmetic material compositions, pharmaceutical compositions or pharmaceutical-like compositions, as well as novel uses for fatigue improvement, inflammation improvement or anti-oxidation.

[0034] This invention provides a method for improving fatigue, inflammation, or providing antioxidant effects, which involves treating glycyl-L-leucine peptides and / or L-leucylglycine peptides, or their salts.

[0035] The aforementioned glycyl-L-leucine peptide and / or L-leucylglycine peptide can be active ingredients or indicator ingredients.

[0036] The inventors of this invention have experimentally confirmed that glycyl-L-Leucine peptides and / or L-leucylglycine peptides, or their salts, prevent the accumulation of lactic acid and creatine kinase (CK) in muscles in a mouse model of exercise-induced fatigue, thus demonstrating a fatigue-improving effect, thereby completing this invention. Furthermore, experiments have confirmed that the aforementioned glycyl-L-Leucine peptides and / or L-leucylglycine peptides, or their salts, reduce the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in spleen cells, exhibiting an inflammation-improving effect. Experiments have also confirmed that they can increase the level of superoxide dismutase (SOD) in the blood, thus confirming an antioxidant effect.

[0037] The term "fatigue improvement" used in this manual refers to the elimination of accumulated fatigue or fatigue symptoms. Fatigue, as described above, refers to a temporary decrease in the strength or sensitivity of cells, muscles, or organs due to physical or mental activity. If mental and physical activity continues without restoring recovery from fatigue, fatigue will worsen, potentially leading to overwork or chronic fatigue, causing movement disorders, and even developing into health problems. Thus, excessive physical activity generates fatigue because energy sources such as glucose and glycogen are consumed during physical activity. The higher the intensity of exercise, the faster the depletion of muscle glycogen. Although consumption is relatively low at low intensity, it increases over time, contributing to fatigue. Other causes of fatigue include the accumulation of metabolic intermediates such as lactic acid and carbon dioxide in the body. It should be noted that the physiological mechanisms of fatigue are not limited to the above descriptions. Carbohydrates, used as energy sources in the human body, are broken down into glucose. The remaining portion is stored as glycogen in the liver and muscles and used as energy as needed. During exercise, glycogen breakdown occurs, and if sufficient oxygen is provided, it is completely broken down, generating a large amount of energy to sustain exercise. However, when oxygen supply is insufficient, lactic acid is generated and accumulates. When lactic acid accumulates, chemical reactions are disrupted, leading to fatigue. In this invention, experiments have confirmed that the accumulation of lactic acid and creatine kinase (CK) in muscles can be prevented in a mouse model of exercise-induced fatigue, thus confirming the fatigue-improving effect of the aforementioned peptides.

[0038] The term "inflammation improvement" as used in this manual refers not only to suppressing inflammation but also to eliminating and / or alleviating inflammation that has occurred. Inflammation is one of the body's defensive responses to certain stimuli, and it refers to a complex pathological condition involving three aspects: tissue deterioration, circulatory disturbances, and exudation and tissue proliferation. More specifically, inflammation is part of innate immunity. Like other animals, the human innate immune system can recognize patterns on the surface of cells specifically present with pathogens. Phages recognize cells with these patterns as foreign and attack pathogens. If pathogens break through the body's physical barriers and enter the body, an inflammatory response is triggered. The inflammatory response is an immunological defense mechanism that attempts to restore and regenerate the damaged site when the body is injured by various factors. However, if it occurs excessively or persistently, it can induce acute or chronic inflammatory diseases and may become a cause of future diseases such as circulatory disturbances, multiple sclerosis, and Parkinson's disease. Inflammation occurs through multiple mechanisms in the body, including hormone secretion, cytokines, and C-reactive protein (CRP), and many related substances are involved. Among them, various cytokines secreted by immune cells regulate the immune system, and some of them also promote inflammation. Therefore, the expression level of intracellular cytokines becomes an indicator of the activation of the inflammatory response. In this invention, experiments confirmed that in a mouse model of exercise-induced fatigue, the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in spleen cells can be reduced, thereby confirming the anti-inflammatory effect of the above-mentioned peptides.

[0039] The term "antioxidant" as used in this manual refers to the suppression of oxidative stress. Substances that contribute to oxidative stress are produced in the human body. When oxygen, used as energy in the body, is incompletely reduced, reactive oxygen species (ROS) are released into the cytoplasm. These ROS damage surrounding cells and accelerate tissue damage caused by inflammation. ROS are also produced under normal circumstances; without antioxidant defense mechanisms, we cannot survive. Therefore, the human body has various forms of antioxidant defense systems to combat normal oxidative stress. However, if oxidative stress exceeds normal levels, the body suffers severe damage, thus requiring countermeasures against oxidative stress. Antioxidants, whether natural or synthetic, function to prevent or suppress oxidative stress. To evaluate the antioxidant effects of these substances in the body, various methods are used to assess the levels of superoxide dismutase (SOD), catalase, peroxidase, and glutathione peroxidase (GPx), which are antioxidants that primarily function to remove ROS. In this invention, experiments confirmed that the peptide can increase the level of superoxide dismutase (SOD) in the blood in a mouse model of exercise-induced fatigue, thus confirming that the above-mentioned peptide exhibits antioxidant effects.

[0040] According to another embodiment of the present invention, the present invention provides a composition for relieving, improving or treating menopausal symptoms, preferably a pharmaceutical composition or a pharmaceutical-like composition, wherein the composition, as an active ingredient, comprises glycyl-L-leucine peptide and / or L-leucylglycine peptide, or a salt thereof.

[0041] Specifically, the present invention provides the use of glycyl-L-leucine peptide and / or L-leucylglycine peptide, or their salts, as pharmaceutical compositions or pharmaceutical-like compositions, as well as novel uses for relieving, improving, or treating menopausal symptoms.

[0042] The present invention provides a method for alleviating, improving or treating menopausal symptoms, the method comprising processing glycyl-L-leucine peptides and / or L-leucylglycine peptides, or salts thereof.

[0043] The aforementioned glycyl-L-leucine peptide and / or L-leucylglycine peptide can be active ingredients or indicator ingredients.

[0044] In this invention, "menopausal symptoms" refers to the general term for symptoms and diseases that occur in women before and after menopause due to decreased estrogen secretion caused by ovarian aging. It is also known as "menopausal syndrome" or "amenorrhea symptoms." For example, menopausal or amenorrhea symptoms include: hot flashes, sweating, nervousness, depression, dizziness, fatigue, joint pain, muscle pain, headache, palpitations, tingling sensations, night sweats, sleep disturbances, dry skin, vaginal dryness, vaginal atrophy, lower urinary tract atrophy, dyspareunia, vaginitis, cystitis, painful urination, urinary urgency, attention deficit, memory impairment, anxiety, nervousness, memory loss, dry skin, joint pain, or osteoporosis, but are not limited to these. Additionally, it may also be cardiovascular abnormalities such as heart disease, hypertension, or stroke.

[0045] In this invention, "prevention" refers to all behaviors that inhibit or delay the target symptoms by administering the composition of this invention.

[0046] In this invention, "treatment" refers to all actions that improve or eliminate target symptoms or diseases by administering the composition of this invention.

[0047] In this invention, "improvement" refers to all behaviors that result in a better or more favorable change in target symptoms compared to before administration, through administration of the composition of this invention.

[0048] In order to solve the problems described above and achieve the objectives of the present invention, the present invention provides a composition comprising a compound represented by Chemical Formula 1 and / or a compound represented by Chemical Formula 2, or a salt thereof.

[0049] The compound represented by the following chemical formula 1 and / or the compound represented by the following chemical formula 2 may be an active ingredient or an indicator ingredient.

[0050] Chemical Formula 1:

[0051]

[0052] The molecular formula of the compound represented by the above chemical formula 1 is C8H. 16 N2O3, with a molecular weight of 188.22, is named glycyl-L-Leucine. It is a dipeptide composed of two amino acids, glycine and L-leucine, and is abbreviated as Gly-Leu or GL.

[0053] This invention is not limited to the above-described method for obtaining glycyl-L-leucine; substances derived from natural sources, substances chemically synthesized using methods known in the art to which this invention pertains, or commercially available substances can be used. Substances extracted from the placenta of mammals such as pigs, cattle, sheep, and horses are preferred, and substances extracted from pig placentas are more preferred.

[0054] Chemical formula 2:

[0055]

[0056] The molecular formula of the compound represented by chemical formula 2 above is C8H. 16 N2O3, with a molecular weight of 188.22, is named L-Leucylglycine. It is a dipeptide composed of two amino acids, L-leucine and glycine, and is abbreviated as Leu-Gly or LG.

[0057] This invention is not limited to the above-described method for obtaining L-leucylglycine. Substances derived from natural sources, chemically synthesized using methods known in the art to which this invention pertains, or commercially available substances can be used. Preferably, substances extracted from the placenta of mammals such as pigs, cattle, sheep, and horses are used; more preferably, substances extracted from pig placenta are used.

[0058] In the cosmetic material composition, pharmaceutical composition, pharmaceutical-like composition, or food composition of the present invention, the content of the compound represented by the above chemical formula 1 and / or the compound represented by the above chemical formula 2, or their salts, is 0.00000001 to 100% by weight relative to the total weight of the cosmetic material composition, pharmaceutical composition, pharmaceutical-like composition, or food composition, that is, the compound represented by the above chemical formula 1 and / or the compound represented by the above chemical formula 2 can be ingested and used.

[0059] In this invention, when the active ingredient is a mixture of a compound represented by Chemical Formula 1 and a compound represented by Chemical Formula 2, the compound represented by Chemical Formula 1 and the compound represented by Chemical Formula 2 can be mixed at a weight ratio of 1:0.05 to 20, preferably at a weight ratio of 1:0.1 to 10. The inventors have experimentally confirmed not only the effects of each ingredient individually, but also the enhanced effect achieved by mixing the active ingredients at a weight ratio of 1:1, which is within the aforementioned range.

[0060] In this invention, the compounds represented by Chemical Formula 1 and / or Chemical Formula 2 used as active ingredients can be used in the form of salts. In this case, if the composition of this invention is a functional food, it can be used in the form of salts that do not cause health problems and achieve the intended effects of this invention; if the composition of this invention is a cosmetic material composition for non-therapeutic purposes, it can be used in the form of cosmetically acceptable salts; if the composition of this invention is a pharmaceutical composition or a pharmaceutical-like composition, it can be used in the form of pharmaceutically acceptable salts. The salts of the compounds represented by Chemical Formula 1 and / or Chemical Formula 2 mentioned above refer to salts produced by adding an acid (e.g., a non-toxic or low-toxic organic or inorganic acid) or a base to the compounds represented by Chemical Formula 1 and / or Chemical Formula 2.

[0061] For example, the salts mentioned above may include: sodium salts, salicylates, benzoates, calcium salts, citrates, sulfites and bisulfites, aluminum salts, hydrochlorides, permanganates, potassium salts, acetates, bromates, nitrates, carbonates, bicarbonates, thiocyanates, ferrous salts, thiosulfates, iron(III) salts, copper(II) salts, lactates, magnesium salts, sulfates, phosphates, bromides, zinc salts, iodinated salts, succinates, etc., but are not limited to these.

[0062] The compounds represented by Chemical Formula 1 and / or the compounds represented by Chemical Formula 2, or their salts, of the present invention can exist not only in a solvated form, including hydrates, ethanolates, etc., but also in an unsolvated form. The compounds represented by Chemical Formula 1 and / or the compounds represented by Chemical Formula 2, or their salts, of the present invention can exist in crystalline or amorphous forms; all such physical forms are included within the scope of the present invention.

[0063] This invention provides a functional food composition for improving skin elasticity, moisturizing skin, or anti-aging skin, wherein, as an active ingredient, it contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a salt thereof.

[0064] The invention provides a functional food composition for improving fatigue, inflammation, or anti-oxidation, wherein the composition, as an active ingredient, contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a salt thereof.

[0065] This invention provides a functional food composition for improving menopausal symptoms. The composition, as an active ingredient, contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a salt thereof. Preferably, the food composition can be a functional food.

[0066] The term "functional food" as used in this specification refers to a food made by adding the compounds represented by Chemical Formula 1 and / or Chemical Formula 2 to food ingredients such as beverages, teas, spices, chewing gum, and biscuits, or by making them into capsules, powders, suspensions, etc. Consuming such functional foods will bring specific health benefits. Because they are made from food ingredients, they have advantages different from general pharmaceuticals, namely, they do not cause the side effects that may occur with long-term use of pharmaceuticals. The functional food of the present invention obtained as described above can be consumed daily and is expected to have excellent effects in improving skin elasticity, moisturizing or anti-aging skin, improving fatigue, improving inflammation, anti-oxidation, and / or alleviating menopausal symptoms, and is therefore very useful.

[0067] When the compounds represented by Chemical Formula 1 and / or Chemical Formula 2, or their salts, are used as functional food additives, they can be added directly or used in conjunction with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredients can be suitably determined according to the intended use (health treatment). Generally, when manufacturing food or beverages, the amount of the composition of the present invention added relative to the raw materials is 15 parts by weight or less, preferably 10 parts by weight or less. However, when consumed long-term for health and hygiene purposes or for health regulation purposes, the above-mentioned amount can be below the above range, and since there are no safety concerns, the active ingredients can also be used in amounts above the above range. There are no special limitations on the types of food mentioned above. Foods that may contain the above-mentioned substances include: meat, sausage, bread, chocolate, sugar, pastries, biscuits, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, cooling agents, alcoholic beverages, and vitamin complexes, including all foods in the conventional sense.

[0068] When the food is a beverage, it can be contained in a ratio of 1 to 30g, preferably 3 to 20g, based on 100ml. Furthermore, the above composition may contain additional ingredients commonly used in functional food compositions to enhance aroma, taste, appearance, etc. For example, it may contain: vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folic acid, pantothenic acid, etc. Additionally, it may contain: minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), etc. Furthermore, it may contain: amino acids such as lysine, tryptophan, cysteine, valine, etc. In addition, the following food additives can be added: preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and bleaching agent, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG (monosodium glutamate), etc.), sweeteners (gallicin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium D-tartrate, etc.), reinforcing agents, emulsifiers, thickeners (paste), film-forming agents, gum bases, defoamers, solvents, modifiers, etc. The above additives can be selected according to the type of food and used in appropriate amounts. The ratio of the additive is not very important, but in general, the ratio of the additive is 0.01 to 0.8 parts by weight relative to 100 parts by weight of the composition of the present invention.

[0069] The functional foods of the present invention can be manufactured according to methods commonly used in the art. When manufacturing these functional foods, raw materials and ingredients commonly added in the art can be added. Furthermore, since they are made from food ingredients, unlike general pharmaceuticals, they have the advantage of not causing the side effects that may occur with long-term use of pharmaceuticals, and are extremely portable.

[0070] According to another embodiment of the present invention, a cosmetic material composition for non-therapeutic purposes, such as improving skin elasticity, moisturizing skin, or anti-aging skin, is provided, wherein the composition, as an active ingredient, contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a cosmetically acceptable salt thereof.

[0071] According to another embodiment of the present invention, a cosmetic material composition for non-therapeutic purposes, such as fatigue improvement, inflammation improvement, or antioxidant effects, is provided, wherein the composition, as an active ingredient, contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a salt thereof.

[0072] According to another embodiment of the present invention, a non-therapeutic cosmetic material composition for improving menopausal symptoms is provided, the composition containing, as an active ingredient, a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a salt thereof.

[0073] When the above-mentioned composition is a cosmetic material composition and is used as an active ingredient in a cosmetic product, it can be prepared in general emulsified and soluble formulations. For example, the formulation may be: toners such as softening lotions or nourishing lotions; lotions such as facial moisturizers and body lotions; creams such as nourishing creams, moisturizing creams, and eye creams; serums, cosmetic ointments, sprays, gels, masks, sunscreens, makeup bases, liquid or solid or spray foundations, powders, cleansing creams, cleansing milks, makeup removers such as cleansing oils, foaming cleansers, soaps, and shower gels, etc., but the present invention is not limited to these.

[0074] When the above-described composition is productized as a cosmetic, if it is a wash-off type cosmetic such as a makeup remover or cleanser where the active ingredients remain on the skin for a short period of time, it may contain a relatively high concentration of the composition of the present invention. Conversely, if it is a leave-on type cosmetic such as a toner, lotion, cream, or serum where the active ingredients remain on the skin for a longer period of time, it may contain a lower concentration of the composition of the present invention compared to wash-off cosmetics.

[0075] In addition to the compositions of the present invention, the cosmetics described above may also contain adjuvants commonly used in the field of cosmetics. Examples of such adjuvants include: fatty substances, organic solvents, solvents, concentrates and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion blocking agents and chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, liposomes, or any other ingredients commonly used in cosmetics.

[0076] In addition, the cosmetic material composition of the present invention may also contain one or more cosmetically acceptable carriers that are typically used in skin cosmetic materials.

[0077] Depending on the dosage form, the cosmetic material compositions of the present invention may contain a variety of cosmetically acceptable carriers. When the dosage form of the present invention is an ointment, paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin wax, starch, astragalus gum, cellulose derivatives, polyethylene glycol, organosilicon, bentonite, silica, talc, zinc oxide, or mixtures thereof may be used as carrier components.

[0078] When the dosage form of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or mixtures thereof may be used as the carrier component. In particular, when the dosage form is a spray, it may also contain propellants such as chlorofluorocarbons, propane / butane, or dimethyl ether.

[0079] When the dosage form of the present invention is a solution or emulsion, solvents, solubilizers or emulsifiers can be used as carrier components, such as: water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, polypropylene glycol, 1,3-butylethylene glycol oil, and especially: cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty acid esters, fatty acid esters of polyethylene glycol or sorbitol.

[0080] When the dosage form of the present invention is a suspension, the carrier component can be a liquid diluent such as water, ethanol or polypropylene glycol; a suspending agent such as ethoxyisooctadecyl alcohol, polyethylene oxide sorbitol ester and polyethylene oxide dehydrated sorbitol ester; or microcrystalline cellulose, aluminum hydroxide, bentonite, agar or astragalus gum.

[0081] When the dosage form of the present invention is soap, the carrier components may include alkali metal salts of fatty acids, fatty acid half-ester salts, fatty acid protein hydrolysates, hydroxyethanesulfonates, lanolin derivatives, fatty alcohols, vegetable oils, glycerin, sugars, etc.

[0082] As another embodiment of the present invention, the present invention provides a pharmaceutical composition for improving skin elasticity, moisturizing skin, or anti-aging skin, wherein the composition, as an active ingredient, contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a pharmaceutically acceptable salt thereof.

[0083] As another embodiment of the present invention, the present invention provides a pharmaceutical composition for fatigue improvement, inflammation improvement or anti-oxidation, wherein the composition, as an active ingredient, contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a salt thereof.

[0084] As another embodiment of the present invention, the present invention provides a pharmaceutical composition for improving, preventing or treating menopausal symptoms, the composition containing, as an active ingredient, a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a salt thereof.

[0085] The above composition may have one or more dosage forms selected from the group consisting of solutions, gels, emulsions, suspensions, microemulsions, capsules, liposomes, creams, lotions, ointments, aerosols, sprays, pastes, and patches.

[0086] The pharmaceutical compositions of the present invention can be used as single formulations, or can also be used as compound formulations containing drugs known to have anti-inflammatory or antioxidant effects, or can be manufactured by formulation using pharmaceutically acceptable carriers or excipients, in a unit volume form or injected into multi-volume containers.

[0087] As used in this specification, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate organisms and does not impede the biological activity and properties of the injected compound. There are no particular limitations on the types of carriers that can be used in this invention; any carrier that is commonly used in the field and is pharmaceutically acceptable may be used. Examples of such carriers include, but are not limited to, physiological saline, sterile water, Ringer's solution, buffered physiological saline, albumin injection, glucose solution, maltodextrin solution, glycerol, and ethanol. These can be used alone or in combination of two or more. The aforementioned carriers may include non-naturally occurring carriers.

[0088] In addition, depending on the need, other common additives such as antioxidants, buffers and / or antibacterial agents may be added. Alternatively, diluents, dispersants, surfactants, binders, lubricants, etc. may be added, and the product may be formulated into injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.

[0089] Furthermore, the pharmaceutical compositions of the present invention may comprise pharmaceutically effective amounts of compounds represented by Formula 1 and / or compounds represented by Formula 2, or pharmaceutically acceptable salts thereof. In this specification, the term "pharmaceutically effective amount" means: an amount sufficient to induce improved skin elasticity, skin moisturizing, or anti-aging effects at a reasonable benefit / risk ratio suitable for medical treatment; or an amount sufficient to induce improved fatigue, inflammation, or antioxidant effects; or an amount sufficient to alleviate, improve, or treat menopausal symptoms. Generally, an amount of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, more preferably 0.1 to 100 mg / kg, may be administered once daily or in divided doses daily. However, when considering the purpose of this invention, the specific therapeutic effective amount for a particular patient may preferably be determined based on various factors such as the type and degree of response to be achieved, the specific composition including whether other formulations are used as needed, the patient's age, weight, general health status, gender and diet, administration time, route of administration and the secretion rate of the composition, treatment period, drugs used with or concurrently with the specific composition, and similar factors known in the pharmaceutical field.

[0090] As another embodiment of the present invention, the present invention provides a pharmaceutical composition for improving skin elasticity, moisturizing skin, or anti-aging skin, wherein the composition, as an active ingredient, contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a pharmaceutically acceptable salt thereof.

[0091] As another embodiment of the present invention, the present invention provides a pharmaceutical composition for fatigue improvement, inflammation improvement or anti-oxidation, wherein the composition, as an active ingredient, contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a pharmaceutically acceptable salt thereof.

[0092] As another embodiment of the present invention, the present invention provides a pharmaceutical composition for relieving, improving or treating menopausal symptoms, wherein the composition, as an active ingredient, contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a pharmaceutically acceptable salt thereof.

[0093] In this specification, the term "medicinal product class" refers to: articles of fiber, rubber, or similar material used for the purpose of treating, alleviating, treating, or preventing diseases in humans or animals; articles that have a weak or no direct effect on the human body and are not appliances or machines or similar; preparations used for the prevention of infection as bactericides, insecticides, or similar purposes, and articles used for the diagnosis, treatment, alleviation, treatment, or prevention of diseases in humans or animals that are not appliances, machines, or devices; and articles used for the purpose of having a pharmacological effect on the structure and function of humans or animals, excluding articles that are not appliances, machines, or devices. Additionally, the above-mentioned medicinal products class may include topical skin agents and personal hygiene products. Preferably, these may be disinfectant cleaners, foaming shower gels, mouthwashes, wet wipes, laundry soaps, hand sanitizers, or ointments, but are not limited to these.

[0094] When the above-described compositions of the present invention are used as pharmaceutical additives, they can be added directly or used in conjunction with other pharmaceutical or pharmaceutical ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredients can be appropriately determined according to the intended use.

[0095] In addition, the present invention provides compositions for improving skin elasticity, moisturizing skin, and anti-aging skin, preferably cosmetic material compositions, pharmaceutical compositions, pharmaceutical-like compositions, or food compositions for non-therapeutic purposes, wherein the composition, as an indicator ingredient, contains glycyl-L-leucine peptides and / or L-leucylglycine peptides, or their salts.

[0096] As another embodiment of the present invention, the present invention provides a pharmaceutical composition for fatigue improvement, inflammation improvement or anti-oxidation, wherein the composition, as an indicator component, contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a pharmaceutically acceptable salt thereof.

[0097] As another embodiment of the present invention, the present invention provides a pharmaceutical composition for relieving, improving or treating menopausal symptoms, wherein the composition, as an indicator ingredient, contains a compound represented by chemical formula 1 and / or a compound represented by chemical formula 2, or a pharmaceutically acceptable salt thereof.

[0098] Invention Effects

[0099] The active ingredient of this invention comprises a composition of glycyl-L-leucine peptide and / or L-leucyl-glycine peptide, or their salts, which shows excellent effects on improving skin elasticity, moisturizing skin, and anti-aging skin.

[0100] The composition of the present invention comprising glycyl-L-leucine peptide and / or L-leucylglycine peptide, or salt thereof, exhibits excellent effects on fatigue improvement, inflammation reduction, or antioxidant activity.

[0101] The composition of the present invention comprising glycyl-L-leucine peptide and / or L-leucylglycine peptide, or salts thereof, shows excellent effects in alleviating menopausal symptoms.

[0102] The composition of the present invention comprising glycyl-L-leucine peptide and / or L-leucyl-glycine peptide, or its salt, can be used as a cosmetic ingredient, pharmaceutical ingredient, drug-like ingredient or food ingredient for non-therapeutic purposes that is safe for the skin and has excellent skin-improving effects.

[0103] The composition of the present invention comprising glycyl-L-leucine peptide and / or L-leucylglycine peptide, or salt thereof, can be used as a cosmetic ingredient, pharmaceutical ingredient, health food ingredient or feed ingredient for non-therapeutic purposes that is safe and has excellent effects in improving fatigue, inflammation or anti-oxidation.

[0104] The composition of the present invention comprising glycyl-L-leucine peptide and / or L-leucylglycine peptide, or salt thereof, can be used as a safe cosmetic ingredient, pharmaceutical ingredient, health food ingredient or feed ingredient with excellent menopausal symptom relief effect. Attached Figure Description

[0105] Figure 1A and Figure 1B This is a graph showing the promoting effect of glycyl-L-Leucine peptide and / or L-Leucylglycine peptide on hyaluronic acid synthesis.

[0106] Figure 2 This is a graph showing the promoting effect of glycyl-L-Leucine peptide and / or L-Leucylglycine peptide on procollagen synthesis.

[0107] Figure 3 This is a graph representing the elastase inhibition activity of glycyl-L-Leucine peptide and / or L-Leucylglycine peptide.

[0108] Figure 4 The graphs show the effects of inhibiting excessive secretion of prostaglandin E2 (PGE2) in the embodiments and comparative examples of the present invention on the improvement of inflammation and fatigue.

[0109] Figure 5 This is a graph illustrating the antioxidant effect of DPPH free radical scavenging activity in the embodiments and comparative examples of the present invention.

[0110] Figure 6 The graphs represent the fatigue-improving effects of preventing lactic acid accumulation in muscles in the embodiments and comparative examples of the present invention.

[0111] Figure 7 The graphs represent the fatigue-improving effects of preventing the accumulation of creatine kinase (CK) in muscles in the embodiments and comparative examples of the present invention.

[0112] Figure 8 This is a graph illustrating the inflammatory improvement effect resulting from the reduction of tumor necrosis factor-α (TNF-α) in the blood of the embodiments and comparative examples of the present invention.

[0113] Figure 9 This is a graph illustrating the effect of reducing blood interleukin-6 (IL-6) in the embodiments and comparative examples of the present invention on the improvement of inflammation.

[0114] Figure 10 This is a graph illustrating the antioxidant effect resulting from the increase in superoxide dismutase (SOD) in the blood of the embodiments and comparative examples of the present invention.

[0115] Figure 11 These are experimental results confirming whether estrogen receptor activity increases when treated with the embodiments and comparative examples of the present invention. It can be confirmed that estrogen receptor activity increases significantly when glycyl-L-leucine peptide and L-leucylglycine peptide are treated simultaneously. Based on these results, it can be confirmed that the glycyl-L-leucine peptide and / or L-leucylglycine peptide of the present invention can be effectively used for the treatment, improvement, and / or prevention of menopausal disorders.

[0116] Figure 12 This is a result confirming the inhibitory effect on osteoclast differentiation when treating the embodiments and comparative examples of the present invention. Through these results, it is confirmed that the glycyl-L-leucine peptide and / or L-leucyl-glycine peptide of the present invention inhibit osteoclast differentiation and can be effectively used in the treatment, improvement, and / or prevention of postmenopausal osteoporosis. Detailed Implementation

[0117] The following detailed description, including embodiments, is provided to aid in understanding the present invention. However, the embodiments of the present invention can be modified into various other forms and should not be construed as limiting the scope of the invention to the following embodiments. The embodiments of the present invention are provided to provide a more complete explanation of the invention to those skilled in the art.

[0118] Reference Example 1: Material information of glycyl-L-Leucine peptide

[0119] Chemical Formula 1:

[0120]

[0121] Substance Name: Glycyl-L-Leucine

[0122] CAS No.: 869-19-2

[0123] Molecular formula: C8H 16 N2O3

[0124] Molecular weight: 188.22

[0125] Purchaser: Bachem AG (Bubendorf, Switzerland)

[0126] Reference Example 2: Material information of L-Leucylglycine peptide

[0127] Substance Name: L-Leucylglycine

[0128] CAS No.: 686-50-0

[0129] Molecular formula: C8H 16 N2O3

[0130] Molecular weight: 188.22

[0131] Purchaser: Bachem AG (Bubendorf, Switzerland)

[0132] Experimental Example 1: Hyaluronic Acid Synthesis Promotion EffectAs skin ages, collagen and elastin, which form the basic support structure of the skin and are composed of fibrous tissue, solidify and become insoluble. The substance that fills the intercellular spaces and the spaces between collagen, elastin, and other fibrous tissues is called the matrix, which has a very strong ability to retain moisture. Representative components of this matrix include hyaluronic acid and mucopolysaccharides. Hyaluronic acid is a polysaccharide widely distributed in intracellular tissues such as synovial fluid and skin. As a polymer of hyaluronan, which is a combination of N-acetylglucosamine and glucuronic acid, it has moisturizing and viscous properties. When this substance decreases with aging, it causes xeroderma, and the skin's elasticity weakens (Kim Yoon-beom, Journal of Dongyi Medical Pathology Society, 2010; 24(4): 533-542).

[0133] To confirm the hyaluronic acid synthesis effect of glycine-L-leucine peptide or L-leucylglycine, the inventors cultured human epidermal keratinocytes and then treated the sample. After 24 hours, the culture medium was recovered, and the hyaluronic acid produced from the culture medium was measured using the Hyaluronan DuoSet ELISA Kit (DY3614), with the amount adjusted to protein content. Retinoic acid was used as a positive control. The untreated control group was used as 100%, and the experimental results are presented below. Figure 1A and Figure 1B middle.

[0134] The results confirmed that glycine-L-leucine peptide or L-leucylglycine peptide increased hyaluronic acid production even at low concentrations, implying its ability to improve skin condition through hydration. Furthermore, it was confirmed that treatment with glycine-L-leucine and L-leucylglycine together further increased hyaluronic acid production compared to treatment with either peptide alone.

[0135] Experimental Example 2: The promoting effect of procollagen synthesis

[0136] Procollagen, a component of collagen, is synthesized in fibroblasts, osteoblasts, and other cells, and is ultimately converted into collagen by procollagen peptidase. Increased procollagen levels are related to skin's adhesion and elasticity.

[0137] To confirm the procollagen-promoting effect of glycine-L-leucine peptide or L-leucylglycine peptide, human primary dermal fibroblasts (Neonatal) were cultured and treated. After 24 hours, the culture medium was recovered, and the amount of procollagen was measured using the Procollagen type I-C peptide (PIP) kit. Cells adhering to the substrate were washed with DPBS, dissolved in 1N NaOH, and the total protein content was determined using the BCA Protein Assay Kit. The amount of procollagen synthesized per unit of protein was calculated. TGF-β was used as a positive control. The untreated control group was used as 100%. The experimental results are shown below. Figure 2 middle.

[0138] The results confirmed that glycyl-L-leucine and L-leucylglycine peptides enhanced procollagen synthesis, thereby contributing to skin elasticity. Furthermore, it was confirmed that treatment with glycyl-L-leucine and L-leucylglycine together further enhanced procollagen synthesis compared to treatment with either peptide alone.

[0139] Experimental Example 3: Elastase Inhibition Activity

[0140] In the dermis of the skin, collagen and elastin form a network structure that maintains skin elasticity. However, due to internal and external stresses such as age and ultraviolet radiation, elasticity and moisture decrease. Furthermore, the network structure of elastin is destroyed by overexpressed elastase, leading to skin laxity, wrinkles, and skin aging. Therefore, by inhibiting the activity of elastin-degrading enzymes, one of the main causes of skin aging, namely by inhibiting the activity of elastase, skin aging can be suppressed (Lee Jae-won, Journal of Korean Society of Food and Nutrition, 2008, 21(2); 143-147).

[0141] The sample and 1 U / ml porcine pancreatic elastase (Sigma, E1250) dissolved in 50 mM Tris-HCl buffer (pH 8.0) were added to 96 wells and allowed to react at room temperature for 30 minutes. As a matrix, N-succinyl-(L-Ala)3-p-nitroaniline was added, and the amount of p-nitroaniline generated from the matrix was measured at 410 nm. The results are presented as a baseline against a control group without any treatment. Figure 3middle.

[0142] The results confirmed that glycine-L-leucine peptide, L-leucylglycine peptide, and a mixture of glycine-L-leucine peptide and L-leucylglycine peptide (1:1) inhibited the activity of elastase, thus confirming that skin aging can be inhibited by inhibiting the activity of elastase, the main cause of skin aging.

[0143] Experiment Example 4: Test Tube Experiment

[0144] Before evaluating the effects of compositions containing glycyl-L-leucine and / or L-leucylglycine on fatigue improvement, inflammation reduction, antioxidant effects, and menopausal symptoms improvement in women, the inventors conducted in vitro experiments. Glycyl-L-leucine or L-leucylglycine was supplied by Bachem AG (Bubendorf, Switzerland). Composite compositions were manufactured and used in the experiments as shown in Table 1 below.

[0145] Table 1

[0146]

[0147] Experimental Example 5: The effect of inhibiting excessive secretion of prostaglandin E2 (PGE2) on inflammation and fatigue improvement

[0148] Cytokines are proteins secreted primarily by monocytes, lymphocytes, T cells, B cells, natural killer cells, and fibroblasts during inflammatory responses. They act like neurotransmitters on cell membrane receptors or like hormones on intracellular receptors, transmitting intercellular information. Pro-inflammatory cytokines, such as interleukin-1β, -2, -6 (IL-1β, -2, -6), IFN-γ, and TNF-α, activate cyclooxygenase-2 (COX-2), increase prostaglandin E2 (PGE2), and activate inflammatory cells, thereby inducing the inflammatory process (Song Houlin et al., Korean Society of Psychopathology, 2013; 24: 5-10). The inventors evaluated the anti-inflammatory effect of inhibiting prostaglandin E2 (PGE2) secretion.

[0149] First, to determine prostaglandin E2 (PGE2), mouse macrophages RAW264.7 (Mousemacrophage cell line, KCLB no 40071, Korean Cell Line Bank, Seoul, Korea) were purchased and cultured at 2.5 × 10⁻⁶ cells / year. 6 Cell concentrations were measured at 100 μl / ml in 96-well microplates. Culture medium was DMEM (GibcoBRL, Grand Island, NY, USA) supplemented with 10% FBS (GibcoBRL, Grand Island, NY, USA) and 1% penicillin / streptomycin (Thermo Scientific Hyclone, Waltham, MA, USA), and cultured at 37°C with 5% CO2. Lipopolysaccharide (LPS) (Sigma, St. Louis, MO, US) at 1 μg / ml was added to the cell culture medium containing the RAW 264.7 cells to induce inflammation, as described in Experiment 1 above. Cells were then cultured for 24 hours, centrifuged at 900 rpm for 5 minutes at 4°C, and the cell culture medium was recovered to confirm the amount of prostaglandin E2 (PGE2) secreted in the culture medium. Prostaglandin E2 (PGE2) was measured using a commercially available prostaglandin E2 assay kit (PGE2 ELISA kit, MyBioSource Co., Ltd., San Diego, CA, USA) and in accordance with the manufacturer’s instructions.

[0150] The result, such as Figure 4As shown, in macrophages where inflammation was induced by lipopolysaccharide, a significant increase in prostaglandin E2 (PGE2) was observed compared to cells without induced inflammation; however, this increase was reversed in cells treated with the sample. Furthermore, compared to treatment with 1 μg / ml of glycyl-L-leucine peptide or 1 μg / ml of L-leucylglycine peptide (compared to Examples 1 and 3), the concentration of prostaglandin E2 (PGE2) was further reduced when treated with 10 μg / ml (Examples 2 and 4). In particular, it was confirmed that the secretion of prostaglandin E2 (PGE2) was further reduced in cells treated with the composite composition (Examples 5 and 6) compared to cells treated alone with glycyl-L-leucine peptide or L-leucylglycine peptide (Examples 1 to 4).

[0151] The results of Experiment 2 indicate that the composition containing glycyl-L-leucine and / or L-leucylglycine inhibits the secretion of prostaglandin E2 (PGE2), which promotes inflammation, in inflammation-induced macrophages, demonstrating an effective anti-inflammatory effect and thus showing a fatigue-improving function.

[0152] Experimental Example 6: Antioxidant Effect of DPPH Free Radical Scavenging Activity

[0153] DPPH (2,2-diphenyl-1-trinitrophenylhydrazine) is a stable, purple free radical that, upon reaction with antioxidants, accepts hydrogen and is reduced and decolorized. Using this principle, the DPPH free radical scavenging activity of samples can be compared to evaluate their antioxidant capacity. A higher percentage of free radical scavenging activity indicates a stronger antioxidant capacity.

[0154] The free radical scavenging activity of DPPH (1,1-Diphenyl-2-picryl-hydrazyl) was determined as follows. First, a DPPH solution was prepared by dissolving 4 mg of DPPH in 100 ml of methanol, and the composition of Experimental Example 1 was diluted in ethanol at different concentrations. After uniformly mixing 1 ml of ethanol and 1 ml of DPPH solution containing the composite composition, the mixture was allowed to stand at 37°C for 30 minutes, and then the absorbance was measured at 516 nm. Ethanol was used as a control group.

[0155] The antioxidant capacity derived from the DPPH free radical scavenging activity method was calculated using the following formula.

[0156] Mathematical formula 1:

[0157] DPPH free radical scavenging activity = 100 - (absorbance of sample / absorbance of control group) × 100

[0158] The result, such as Figure 5 As shown, the DPPH radical scavenging activity increased upon treatment of the samples. Furthermore, compared to treatment with 1 μg / ml of glycyl-L-Leucine peptide or 1 μg / ml of L-leucylglycine peptide (Examples 1 and 3), the DPPH radical scavenging activity was further enhanced when treated with 10 μg / ml (Examples 2 and 4). In particular, it was confirmed that the DPPH radical scavenging activity in the composite compositions (Examples 5 and 6) was further enhanced compared to individual samples of glycyl-L-Leucine peptide or L-leucylglycine peptide (Examples 1 to 4).

[0159] The results of Experiment 3 indicate that compositions containing glycyl-L-leucine and / or L-leucylglycine suppress oxidative stress and are therefore effectively used as antioxidant functional materials.

[0160] Experiment Example 7: Animal Experiment

[0161] For animal experiments, the inventors purchased male ICR mice (4 weeks old) from Dhbiolink (Daejeon, South Korea). The experimental animals were housed in cages of 5-10 mice at a temperature maintained at 22±1℃ and humidity at 55±10%. Glycyl-L-Leucine peptide or L-Leucylglycine peptide was provided by Bachem AG (Bubendorf, Switzerland), dissolved in distilled water before use. As a control group, mice were administered distilled water orally only. The glycyl-L-Leucine peptide or L-Leucylglycine peptide ingestion groups were administered orally once daily for 21 days using an oral probe (sonde). The types and concentrations of the compositions administered to each group are shown in Table 2 below. Fatigue was induced in each group by exercising for 30 minutes once a week for 3 weeks. The exercise speed was as follows: 10 m / min for the first 10 minutes, 16 m / min for the next 10 minutes, and 21 m / min for the last 10 minutes. On day 21, the mice were exercised at 10 m / min for the first 5 minutes, then the speed was increased by 3 m / min every 3 minutes for 30 minutes. After this exercise, the mice were sacrificed and used for analysis.

[0162] Table 2

[0163]

[0164] Experiment Example 8: Fatigue Improvement Effect of Preventing Lactic Acid Accumulation in Muscles

[0165] When oxygen is adequately supplied to cells, the TCA cycle proceeds smoothly, maintaining blood lactate concentration within the range of 0.56-2 mmol / L, preventing accumulation above this range. However, when the metabolic connection between the corresponding action and the TCA cycle is not carried out in an equivalence ratio, and the corresponding action is relatively active, or when the oxygen supply is less than the oxygen required by the cells, lactate is generated in the muscles. Furthermore, during high-intensity exercise, when the oxygen supply is less than the oxygen consumption of the muscles, the lactate concentration in muscle tissue increases. The lactate generated at this time diffuses into the blood and is processed in the heart and liver. Therefore, lactate accumulation due to exercise leads to acidification in the body, inducing fatigue. During exercise, the activity of phosphorylases involved in glucose metabolism decreases, resulting in the inhibition of glucose synthesis, which is the energy source for exercise under anaerobic conditions. In various fatigue improvement evaluations, the inventors confirmed the inhibition of lactate and creatine kinase (CK) accumulation in muscles, which indicates fatigue.

[0166] The result, such as Figure 6As shown, in a mouse model of induced fatigue, oral administration of glycyl-L-leucine peptide or L-leucylglycine peptide (Examples 7 to 10) significantly reduced the concentration of lactic acid accumulated in muscles compared to the control group (Comparative Example 2). In particular, it was confirmed that the lactic acid concentration was lower when 10 mg / kg / day of glycyl-L-leucine peptide or L-leucylglycine peptide was ingested (Examples 7 and 9) compared to the case of 1 mg / kg / day of glycyl-L-leucine peptide or L-leucylglycine peptide (Examples 8 and 10).

[0167] In other words, it was confirmed that in individuals with induced fatigue, intake of glycyl-L-leucine peptide or L-leucylglycine peptide could reduce the accumulation of lactic acid in muscles, thereby showing a fatigue-improving effect.

[0168] Experiment Example 9: Fatigue Improvement Effect of Preventing the Accumulation of Creatine Kinase (CK) in Muscles

[0169] When muscle cells are damaged, a large number of enzymes present in the muscles are released into the bloodstream to generate energy. Creatine kinase (CK) plays a role in synthesizing creatine phosphate, which is required for the resynthesis of ATP in anaerobic muscle cells during exercise (the process of synthesizing ATP from ADP and phosphocreatine).

[0170] To determine intramuscular creatine kinase (CK), as in Example 4, fatigue was induced in mice, and muscle tissue was collected from each group of mice after administration of the composition and used for analysis. Creatine kinase (CK) was determined using an analytical kit (Abcam, Cambridge, UK) and according to the manufacturer's instructions.

[0171] The result, such as Figure 7As shown, in a mouse model of induced fatigue, oral administration of glycyl-L-leucine peptide or L-leucylglycine peptide (Examples 7 to 10) significantly reduced the concentration of creatine kinase (CK) accumulated in muscle compared to the control group (Comparative Example 2). In particular, it was confirmed that the concentration of CK was further reduced when ingested at 10 mg / kg / day (Examples 8 and 10) compared to ingestion of 1 mg / kg / day of glycyl-L-leucine peptide or L-leucylglycine peptide (Examples 7 and 9).

[0172] Specifically, it was confirmed that in fatigue-induced individuals, the uptake of glycyl-L-leucine peptide or L-leucylglycine peptide reduced the accumulation of creatine kinase (CK) in muscles, thereby demonstrating a fatigue-improving effect.

[0173] Experiment Example 10: The effect of reducing inflammation caused by reducing blood cytokines

[0174] Typical cytokines include interleukins (IL), interferons (IFN), chemokines, tumor necrosis factor (TNF), and transforming growth factor (TGF). Among these, pro-inflammatory cytokines include interleukin-1β, -2, and -6 (IL-1β, -2, and -6), IFN-γ, and TNF-α; while anti-inflammatory cytokines include IL-4, IL-10, IL-11, IL-13, and TGF-β. In evaluating the effect of inflammation improvement, the inventors confirmed the inhibition of the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), proteins that promote inflammation.

[0175] First, to determine tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the blood, as in Example 4, fatigue was induced and blood samples were collected from each group of mice after administration of the composition. The collected blood was incubated at room temperature for 30 minutes and then centrifuged at 6000 rpm for 20 minutes to separate the serum. The separated serum was filtered through a 0.45 μm syringe filter and used for analysis. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured using a commercially available TNF-α and IL-6 ELISA kit (Thermo Scientific Co., Waltham, MA, USA) according to the manufacturer's instructions.

[0176] The result, such as Figure 8 As shown, in a mouse model induced by fatigue, oral administration of glycyl-L-leucine peptide or L-leucylglycine peptide (Examples 7 to 10) significantly reduced the concentration of tumor necrosis factor-α (TNF-α) in the blood compared to the control group (Comparative Example 2). In particular, it was confirmed that the concentration of tumor necrosis factor-α (TNF-α) was further reduced when the intake was 10 mg / kg / day (Examples 8 and 10) compared to the intake of glycyl-L-leucine peptide 1 mg / kg / day or L-leucylglycine peptide 1 mg / kg / day (Examples 7 and 9).

[0177] In addition, such as Figure 9 As shown, in a mouse model induced by fatigue, oral administration of glycyl-L-leucine peptide or L-leucylglycine peptide resulted in a significant reduction in blood interleukin-6 (IL-6) concentration compared to the control group (Comparative Example 2). In particular, it was confirmed that an intake of 10 mg / kg / day (Examples 8 and 10) further reduced the concentration of interleukin-6 (IL-6) compared to an intake of 1 mg / kg / day of glycyl-L-leucine peptide or L-leucylglycine peptide (Examples 7 and 9).

[0178] Specifically, it was confirmed that in fatigue-induced individuals, the uptake of glycyl-L-leucine peptide or L-leucylglycine peptide reduced the concentration of inflammatory cytokines in the blood, thereby demonstrating a fatigue-improving effect.

[0179] Experimental Example 11: Antioxidant Effects of Increased Superoxide Dismutase (SOD) in Blood

[0180] Superoxide dismutase (SOD) is an antioxidant enzyme that catalyzes the conversion of superoxide into oxygen and hydrogen peroxide (H2O2), playing a role in removing free radicals in the blood. Increased blood SOD levels can reduce muscle fatigue. To evaluate its antioxidant effects, the inventors confirmed the increase of superoxide dismutase (SOD), which performs antioxidant functions in vivo.

[0181] To determine the level of superoxide dismutase (SOD) in the blood, as in Experiment 4, fatigue was induced, and serum was obtained from each group of mice after administration of the composition for analysis. Superoxide dismutase (SOD) was measured using an analytical kit (Abcam, Cambridge, UK) according to the manufacturer's instructions.

[0182] The result, such as Figure 10 As shown, in a mouse model induced by fatigue, oral administration of glycyl-L-leucine peptide or L-leucylglycine peptide (Examples 7 to 10) significantly increased the concentration of superoxide dismutase (SOD) in the blood compared to the control group (Comparative Example 2). In particular, it was confirmed that the concentration of superoxide dismutase (SOD) was further increased when the intake was 10 mg / kg / day (Examples 8 and 10) compared to the intake of glycyl-L-leucine peptide 1 mg / kg / day or L-leucylglycine peptide 1 mg / kg / day (Examples 7 and 8).

[0183] Specifically, it was confirmed that in fatigue-induced individuals, the uptake of glycyl-L-leucine peptide or L-leucylglycine peptide increases the concentration of superoxide dismutase (SOD) in the blood, thereby exhibiting an antioxidant effect.

[0184] Experimental Example 12: Evaluation of Estrogen Receptor Activity

[0185] Menopausal symptoms refer to a variety of symptoms that women experience before and after menopause due to factors such as ovarian aging and decreased estrogen secretion.

[0186] To confirm the activity of two dipeptides of formulas 1 and 2 on the estrogen receptor, an ERE (estrogen response element) reporter assay was performed. 293T cells were cultured in 24-well plates for 24 hours in DMEM medium supplemented with 10% FBS. Each well was then exchanged with 500 μL of charcoal-stripped FBS containing phenol red-free medium (5% activated charcoal) and transfected. Liposome transfection reagent was used in each well. DNA was transfected with 0.1 μg ERa, 0.1 μg ERE, and 10 ng pRL-Tk using reagents (Thermo Fisher Scientific). Four hours after transfection, the composition of Experiment 1 was treated. As a positive control, 1 ppb (0.5 μL DMSO) of 17β-estradiol (E2) was used for treatment, and as a negative control (control, ctrl), an equal volume of DMSO (0.5 μL) was used for treatment. Twenty-four hours after treatment, the samples were analyzed using a dual-luciferase reporter assay system (…). The Reporter Assay System (Promega) was used to measure the luminescence intensity (cold light), with the values ​​of the negative control group (1.0) as a baseline. For the corresponding values, the firefly luciferase values ​​were corrected to the renilla luciferase values.

[0187] The result, such as Figure 11 As shown, treatment of the samples increased estrogen receptor activity. Furthermore, compared to treatment with 1 μg / ml of glycyl-L-Leucine peptide or 1 μg / ml of L-leucylglycine peptide (Examples 1 and 3), treatment with 10 μg / ml (Examples 2 and 4) further increased estrogen receptor activity. In particular, it was confirmed that the estrogen receptor activity was further enhanced in the composite compositions (Examples 5 and 6) compared to individual samples of glycyl-L-Leucine peptide or L-leucylglycine peptide (Examples 1 to 4).

[0188] Experimental Example 13: Effect of inhibiting osteoclast differentiation

[0189] During menopause, the secretion of female hormones, which play a crucial role in bone metabolism, from the ovaries ceases. This alters bone turnover; while osteoblasts produce new bone to fill the sites where osteoclasts have dissolved, this process is incomplete, leading to bone loss. Bone loss begins gradually after age 30. In men, bone density declines gradually, with a maximum loss of about 20-30% of bone mass. Conversely, in women, bone density declines sharply after age 50, approximately 10 years after menopause, before declining more slowly thereafter. Therefore, women develop osteoporosis faster and more severely than men.

[0190] In vitro, osteoclasts were generated from macrophage precursor cells by RANKL (RANKligand), an activator of the NF-κB ligand of a specific cytokine. In the control group without RANKL treatment, RAW264.7 cells proliferated while maintaining a spherical shape and showed a negative reaction to TRAP staining, appearing light brown or yellowish-brown. In contrast, in cells treated with RANKL, TRAP(+) multinucleated cells were observed, staining dark brown to reddish-brown.

[0191] To confirm the effects of the two dipeptides of Formula 1 and Formula 2 on osteoclast differentiation induced by the receptor activator of NF-κB ligand (RANKL), RAW264.7 cells cultured in Experiment 5 were used in the experiment. RAW264 cells were aliquoted at a concentration of 2.5 × 10⁶ cells / mL into each well of a 96-microplate (100 μL per well), cultured for 5 hours to allow cell adhesion, and then the culture medium was removed. A mixture of RANKL (50 ng / mL) and the composition from Experiment 1 (as an osteoclast differentiation factor) in α-MEM medium supplemented with 10% FBS was aliquoted and cultured for 7 days. To evaluate the effect on osteoclast differentiation, the cultured cells were stained with tartrate-resistant acid phosphatase (TRAP). Cells stained red were considered osteoclasts, and their number was determined.

[0192] The result, such as Figure 12As shown, osteoclast differentiation was inhibited upon treatment of the samples. Furthermore, compared to treatment with 1 μg / ml of glycyl-L-leucine peptide or 1 μg / ml of L-leucylglycine peptide (Examples 1, 3), treatment with 10 μg / ml (Examples 2, 4) further reduced osteoclast differentiation. In particular, it was confirmed that osteoclast differentiation was further inhibited in the composite composition samples (Examples 5, 6) compared to individual samples of glycyl-L-leucine peptide or L-leucylglycine peptide (Examples 1 to 4).

[0193] The results of Experiments 12 and 13 above indicate that the composition containing glycyl-L-leucine and / or L-leucylglycine increases the activity of estrogen receptors and can be effectively applied to female menopausal symptoms by inhibiting osteoclast differentiation, which is a trigger for postmenopausal osteoporosis.

[0194] Those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical concept and essential technical features. It should also be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be interpreted by the meaning and scope of the appended claims. It should be noted that all modifications and variations derived from the equivalent concepts of the claims are included within the scope of the present invention.

Claims

1. The use of a compound represented by Chemical Formula 1 and a compound represented by Chemical Formula 2, or a salt of a compound represented by Chemical Formula 1 and a salt of a compound represented by Chemical Formula 2, in the preparation of a pharmaceutical composition for the treatment, prevention, or improvement of menopausal symptoms in women, characterized in that, The menopausal symptoms in women are dry skin or osteoporosis. The pharmaceutical composition comprises a compound represented by chemical formula 1 and a compound represented by chemical formula 2 mixed in a 1:1 weight ratio, or a salt of a compound represented by chemical formula 1 and a salt of a compound represented by chemical formula 2 mixed in a 1:1 weight ratio. Chemical Formula 1 Chemical formula 2:

2. The use of a compound represented by Chemical Formula 1 and a compound represented by Chemical Formula 2, or a salt of a compound represented by Chemical Formula 1 and a salt of a compound represented by Chemical Formula 2, in the preparation of a functional food composition for improving dry skin or bone density during menopause in women, characterized in that, The functional food composition comprises a compound represented by Chemical Formula 1 and a compound represented by Chemical Formula 2 mixed in a 1:1 weight ratio, or a salt of a compound represented by Chemical Formula 1 and a salt of a compound represented by Chemical Formula 2 mixed in a 1:1 weight ratio. Chemical Formula 1 Chemical formula 2:

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